Cancers

18 pages, 5843 KiB

Open AccessArticle

Detecting and Tracking Circulating Tumour DNA Copy Number Profiles during First Line Chemotherapy in Oesophagogastric Adenocarcinoma

by Michael Davidson, Louise J. Barber, Andrew Woolston, Catherine Cafferkey, Sonia Mansukhani, Beatrice Griffiths, Sing-Yu Moorcraft, Isma Rana, Ruwaida Begum, Ioannis Assiotis, Nik Matthews, Sheela Rao, David Watkins, Ian Chau, David Cunningham, Naureen Starling and Marco Gerlinger

Cancers 2019, 11(5), 736; https://doi.org/10.3390/cancers11050736 - 27 May 2019

Cited by 15 | Viewed by 5200

Abstract

DNA somatic copy number aberrations (SCNAs) are key drivers in oesophagogastric adenocarcinoma (OGA). Whether minimally invasive SCNA analysis of circulating tumour (ct)DNA can predict treatment outcomes and reveal how SCNAs evolve during chemotherapy is unknown. We investigated this by low-coverage whole genome sequencing [...] Read more.

DNA somatic copy number aberrations (SCNAs) are key drivers in oesophagogastric adenocarcinoma (OGA). Whether minimally invasive SCNA analysis of circulating tumour (ct)DNA can predict treatment outcomes and reveal how SCNAs evolve during chemotherapy is unknown. We investigated this by low-coverage whole genome sequencing (lcWGS) of ctDNA from 30 patients with advanced OGA prior to first-line chemotherapy and on progression. SCNA profiles were detectable pretreatment in 23/30 (76.7%) patients. The presence of liver metastases, primary tumour in situ, or of oesophageal or junctional tumour location predicted for a high ctDNA fraction. A low ctDNA concentration associated with significantly longer overall survival. Neither chromosomal instability metrics nor ploidy correlated with chemotherapy outcome. Chromosome 2q and 8p gains before treatment were associated with chemotherapy responses. lcWGS identified all amplifications found by prior targeted tumour tissue sequencing in cases with detectable ctDNA as well as finding additional changes. SCNA profiles changed during chemotherapy, indicating that cancer cell populations evolved during treatment; however, no recurrent SCNA changes were acquired at progression. Tracking the evolution of OGA cancer cell populations in ctDNA is feasible during chemotherapy. The observation of genetic evolution warrants investigation in larger series and with higher resolution techniques to reveal potential genetic predictors of response and drivers of chemotherapy resistance. The presence of liver metastasis is a potential biomarker for the selection of patients with high ctDNA content for such studies. Full article

(This article belongs to the Special Issue Liquid Biopsy for Cancer)

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(A) No correlation between circulating free (cf)DNA concentration and the tumour-derived cfDNA fraction in 30 plasma samples from patients with treatment naïve metastatic gastro-oesophageal cancers. (B) Correlation between selected clinical features and circulating tumour (ct)DNA fraction (line denotes median; p-value Mann–Whitney test). (C) Kaplan–Meier survival analyses of pretreatment samples grouping by high/intermediate/low cfDNA yield ng/mL plasma, (D) ichorCNA ctDNA fraction, and (E) ctDNA concentration ng/mL plasma (p-values Log-rank (Mantel–Cox) test). Full article ">Figure 2
(A) Integer copy number profiles (500 kb bins) for pretreatment samples, grouped by subsequent response or (B) nonresponse to treatment. Red = gain, blue = loss, and black = ploidy. (C) Frequency plots showing the number of cases that show segment gains (red) or losses (blue) in the responder and (D) nonresponder groups. (E) Frequency plots showing segment gains and losses that are unique to the responder group or (F) nonresponder group. (G) Frequency of gain (red) and loss (blue) segments of chromosome 8p in the responder group (top) and nonresponder group (bottom). The most frequent region of unique 8p gain is indicated, bounded by dotted lines. The locations of MCPH1 and GATA4 are delineated with a blue dashed line. Two additional nonresponder cases showed focal amplifications (orange) of GATA4, which were identified with the 50 kb bin method but not the 500 kb ichorCNA analysis. Full article ">Figure 3
(A) Association of pretreatment chromosomal instability (CIN) metrics with subsequent treatment response by comparing analysis of genomic change relative to ploidy using weighted genomic instability index (wGII), (B) nonploidy segment number, and (C) ploidy between responder and nonresponder groups (line denotes median and interquartile range; p-value Mann–Whitney test). (D) Kaplan–Meier progression free survival analyses grouping by high/low wGII, (E) nonploidy segment number, and (F) ploidy. (G) Kaplan–Meier overall survival analyses grouping by high/low wGII, (H) nonploidy segment number, and (I) ploidy. (J) Heatmap showing focal gene amplifications (50 kb bins) detected by cfDNA lcWGS at pretreatment (orange) or by archival target sequencing (purple) in each case. Black dots indicate cases classed as HER2+ by immunohistochemistry. Green = responder group, blue = stable group, and red = primary progressor group. Full article ">Figure 4
(A) Frequency plots showing the number of cases (n = 17) that show segment gains (red) or losses (blue) at pretreatment (top) and at progression (bottom). (B) For 7 pairs where both samples had >10% ctDNA fractions, comparative plots show absolute copy number gains and losses at progression relative to pretreatment, ordered by the extent of genomic change. The percent genomic change for each sample is indicated to the right of each plot. Red = gain, blue = loss, and black = no change. A minimum of 0.8 copy number change was required to score a gain or a loss. (C) Frequency plot showing the number of cases (n = 7) that show segment gains (red) or losses (blue) at progression relative to pretreatment. Full article ">Figure A1
Integer copy number profiles for the 17 paired non-zero ctDNA cases at progression. ichorCNA ctDNA fraction is indicated for each sample. Full article ">

13 pages, 3281 KiB

Open AccessArticle

miR551b Regulates Colorectal Cancer Progression by Targeting the ZEB1 Signaling Axis

by Kwang Seock Kim, Dongjun Jeong, Ita Novita Sari, Yoseph Toni Wijaya, Nayoung Jun, Sanghyun Lee, Ying-Gui Yang, Sae Hwan Lee and Hyog Young Kwon

Cancers 2019, 11(5), 735; https://doi.org/10.3390/cancers11050735 - 27 May 2019

Cited by 5 | Viewed by 3784

Abstract

Our current understanding of the role of microRNA 551b (miR551b) in the progression of colorectal cancer (CRC) remains limited. Here, studies using both ectopic expression of miR551b and miR551b mimics revealed that miR551b exerts a tumor suppressive effect in CRC cells. Specifically, miR551b [...] Read more.

Our current understanding of the role of microRNA 551b (miR551b) in the progression of colorectal cancer (CRC) remains limited. Here, studies using both ectopic expression of miR551b and miR551b mimics revealed that miR551b exerts a tumor suppressive effect in CRC cells. Specifically, miR551b was significantly downregulated in both patient-derived CRC tissues and CRC cell lines compared to normal tissues and non-cancer cell lines. Also, miR551b significantly inhibited the motility of CRC cells in vitro, including migration, invasion, and wound healing rates, but did not affect cell proliferation. Mechanistically, miR551b targets and inhibits the expression of ZEB1 (Zinc finger E-box-binding homeobox 1), resulting in the dysregulation of EMT (epithelial-mesenchymal transition) signatures. More importantly, miR551b overexpression was found to reduce the tumor size in a xenograft model of CRC cells in vivo. Furthermore, bioinformatic analyses showed that miR551b expression levels were markedly downregulated in the advanced-stage CRC tissues compared to normal tissues, and ZEB1 was associated with the disease progression in CRC patients. Our findings indicated that miR551b could serve as a potential diagnostic biomarker and could be utilized to improve the therapeutic outcomes of CRC patients. Full article

(This article belongs to the Special Issue Colorectal Cancers)

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miR551b-3p is downregulated in CRC patient-derived samples and CRC cell lines. (A) and (B), The expression levels of miR551b-3p and miR551b-5p were determined by RT-qPCR in CRC cell lines (A) and patient-derived samples (n = 10; (B)). Data were shown as mean ± SEM of three independent experiments. *** p < 0.001. N.S means not significant. Full article ">Figure 2
miR551boverexpression suppresses the motility of CRC cell lines. (A and B), The expression levels of miR551b-3p and miR551b-5p were determined by RT-qPCR in SW620 (A) and HCT116 (B). (C and D), SW620 and HCT116 cells were transduced with either control vector (pZeo) or miR551b, and subsequently incubated for 72 h to determine cell proliferation rates by the MTT assay. N.S means not significant. (E–J), Control (pZeo) or miR551b-transduced CRC cell lines were seeded in a transwell non-coated or coated with Matrigel, followed by incubation for 24 h for evaluation of cell migration and invasion, respectively. Cells that had migrated to the lower surface of the transwell were stained and quantified. Images were taken using an inverted microscope and representative images were shown (E, migration; H, invasion). Data were presented as mean ± SEM of three independent experiments (E–G, migration; H–J, invasion). * p < 0.05, *** p < 0.001. (K–N), Control (pZeo) or miR551b-transduced SW620 (K and L) and HCT116 (M and N) cells were seeded and analyzed by in vitro scratch assays. Images were captured at 0, 1, and 2 days after incubation. The dotted lines defined the areas lacking cells. Representative images were shown (K and M). Data were shown as mean ± SEM of three independent experiments. * p < 0.05. Full article ">Figure 3
miR551b-3p mimic, but not miR551b-5p mimic,suppresses the motility of CRC cell lines. (A and B), SW620 cells were transiently transfected with either control, miR551b-3p mimic, or miR551b-5p mimic, and the expression levels of miR551b-3p and miR551b-5p were determined by RT-qPCR. (C and D), SW620 cells transfected with either control, miR551b-3p mimic, or miR551b-5p mimic were incubated for 72 h to determine the proliferation rate by MTT assay. N.S means not significant. (E–J), SW620 cells transfected with either control, miR551b-3p mimic, or miR551b-5p mimic were seeded in a transwell non-coated or coated with Matrigel, followed by incubation for 24 h for evaluating cell migration and invasion, respectively. Images were obtained using an inverted microscope. Representative images were shown (E, migration; H, invasion). Data were presented as mean ± SEM of three independent experiments (E–G, migration; H–J, invasion). *** p < 0.001. N.S means not significant. (K–N), SW620 cells transduced with control, miR551b-3p mimic, or miR551b-5p mimic were seeded and analyzed by to in vitro scratch assays. Images were captured at 0, 1, and 2 days after incubation. The dotted lines defined the areas lacking cells. Representative images were shown (K and M). Data are presented as mean ± SEM of three independent experiments. * p < 0.05, *** p < 0.001. N.S means not significant. Full article ">Figure 4
ZEB1 was a direct target of miR551b. (A), Sequence alignment of miR551b with the 3′ UTR of ZEB1 binding site was shown. (B), 293T cells were transfected with either control vector or miR551b together with psiCheck2; samples were analyzed by dual luciferase reporter assay. (C–E), ZEB1 expression was evaluated in control (pZeo)- or miR551b- transduced SW620 cells by RT-qPCR (C) and immunoblotting (D,E). ACTIN was used as the loading control. Representative images were shown (D). Relative protein expression of ZEB1 was quantified using Image J (E). Data were presented as mean ± SEM of three independent experiments. * p < 0.05, *** p < 0.001. (F–I), RNA was isolated from control vector (pZeo)- or miR551b-transduced SW620 cells; expression levels of E-CAD (F), N-CAD (G), SNAIL (H), and VIMENTIN (I) were determined by RT-qPCR. The data shown were mean ± SEM of three independent experiments. * p < 0.05, *** p < 0.001. Full article ">Figure 5
miR551b suppressed the xenograft of SW620 cells in vivo. (A), Control (pZeo)- or miR551b- transduced SW620 cells were subcutaneously injected into NSG mice. The mice were then monitored for tumor growth. Tumor volume was measured every 3 days up to 1 month (n = 6). Data were presented as mean ± SEM. * p < 0.05. (B), Representative images of tumors were shown. (C), RNA was isolated from each tumor, and miR551b-3p and miR551b-5p expression levels were analyzed by RT-qPCR. The data shown were mean ± SEM (n = 5). * p < 0.05. (D), The tumors were isolated from each mouse and weighed. Data were presented as mean ± SEM (n = 6). * p < 0.05. Full article ">Figure 6
miR551b is inversely correlated with progression of CRC patients. (A) The expression level of hsa-miR-551b in normal and CRC patient samples was analyzed using microRNA datasets GSE81582, GSE41655, GSE18392, GSE98406, and GSE30454 (normal, n = 64; CRC, n = 208; mean ± s.e.m.). *** p < 0.001. (B) Dataset GSE41655 was analyzed to determine the correlation of hsa-miR-551b expression with CRC stage (normal, n = 15; low-grade dysplasia n = 27; high-grade dysplasia n = 15; adenocarcinoma, n = 22). *** p < 0.001 by one-way ANOVA. (C) Kaplan-Meier curves of relapse-free survival probability in CRC patients (GSE39582) based on the expression of ZEB1 (high, n = 140; low, n = 417). p = 0.01. Full article ">

10 pages, 6047 KiB

Open AccessReview

Deciphering The Potential Role of Hox Genes in Pancreatic Cancer

by Tzu-Lei Kuo, Kuang-Hung Cheng, Li-Tzong Chen and Wen-Chun Hung

Cancers 2019, 11(5), 734; https://doi.org/10.3390/cancers11050734 - 27 May 2019

Cited by 21 | Viewed by 4942

Abstract

The Hox gene family plays an important role in organogenesis and animal development. Currently, 39 Hox genes that are clustered in four chromosome regions have been identified in humans. Emerging evidence suggests that Hox genes are involved in the development of the pancreas. [...] Read more.

The Hox gene family plays an important role in organogenesis and animal development. Currently, 39 Hox genes that are clustered in four chromosome regions have been identified in humans. Emerging evidence suggests that Hox genes are involved in the development of the pancreas. However, the expression of Hox genes in pancreatic tumor tissues has been investigated in only a few studies. In addition, whether specific Hox genes can promote or suppress cancer metastasis is not clear. In this article, we first review the recent progress in studies on the role of Hox genes in pancreatic cancer. By comparing the expression profiles of pancreatic cancer cells isolated from genetically engineered mice established in our laboratory with three different proliferative and metastatic abilities, we identified novel Hox genes that exhibited tumor-promoting activity in pancreatic cancer. Finally, a potential oncogenic mechanism of the Hox genes was hypothesized. Full article

(This article belongs to the Special Issue HOX Genes in Cancer)

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8 pages, 736 KiB

Open AccessArticle

Clinical Evaluation of CA72-4 for Screening Gastric Cancer in a Healthy Population: A Multicenter Retrospective Study

by Ping-Jen Hu, Ming-Yao Chen, Ming-Shun Wu, Ying-Chin Lin, Ping-Hsiao Shih, Chih-Ho Lai and Hwai-Jeng Lin

Cancers 2019, 11(5), 733; https://doi.org/10.3390/cancers11050733 - 27 May 2019

Cited by 37 | Viewed by 14316

Abstract

Early detection is important for improving the survival rate of patients with gastric cancer (GC). Serum tumor markers have been widely used for detecting GC. However, their clinical values remain controversial. This study aims to investigate the role of serum cancer antigen 72-4 [...] Read more.

Early detection is important for improving the survival rate of patients with gastric cancer (GC). Serum tumor markers have been widely used for detecting GC. However, their clinical values remain controversial. This study aims to investigate the role of serum cancer antigen 72-4 (CA72-4) in the diagnosis of GC in a healthy population. A total of 7757 adults who underwent upper gastrointestinal endoscopy and serum CA72-4 level measurement in multicenters in Taiwan from January 2006 to August 2016 were recruited in this retrospective study. Risk factors for GC, serum tumor markers, and esophagogastroduodenoscopy (EGD) findings were evaluated. High serum levels of CA72-4 were found in 7.2% of healthy adults. CA72-4 level showed lower sensitivity (33.3%) but higher specificity (92.8%); however, the positive predictive value was quite low (0.18%). After adjustment of clinical risk factors for GC using EGD findings, gastric ulcer (adjusted odds ratio (aOR) = 2.11), gastric polyps (aOR = 1.42), and atrophic gastritis (aOR = 1.27) were significantly associated with high serum CA72-4 levels. Furthermore, both age (OR = 1.01) and Helicobacter pylori infection (OR = 1.44) exhibited a significant association with high serum CA72-4 levels. These results indicate that routine screening of CA72-4 levels for diagnosing GC in asymptomatic patients may be ineffective due to low sensitivity and low positive predictive value. The clinical utility of EGD findings along with serum CA72-4 level for screening healthy individuals with GC is warranted. Full article

(This article belongs to the Special Issue New Biomarkers in Cancers)

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18 pages, 921 KiB

Open AccessReview

Targeting Cancer Stem Cells: A Strategy for Effective Eradication of Cancer

by Masahiro Shibata and Mohammad Obaidul Hoque

Cancers 2019, 11(5), 732; https://doi.org/10.3390/cancers11050732 - 27 May 2019

Cited by 141 | Viewed by 10736

Abstract

Cancer stem cells (CSCs) are subpopulations of tumor cells with the ability to self-renew, differentiate, and initiate and maintain tumor growth, and they are considered to be the main drivers of intra- and inter-tumoral heterogeneity. While conventional chemotherapy can eradicate the majority of [...] Read more.

Cancer stem cells (CSCs) are subpopulations of tumor cells with the ability to self-renew, differentiate, and initiate and maintain tumor growth, and they are considered to be the main drivers of intra- and inter-tumoral heterogeneity. While conventional chemotherapy can eradicate the majority of non-CSC tumor cells, CSCs are often drug-resistant, leading to tumor recurrence and metastasis. The heterogeneity of CSCs is the main challenge in developing CSC-targeting therapy; therefore, we and other investigators have focused on developing novel therapeutic strategies that combine conventional chemotherapy with inhibitors of CSC-regulating pathways. Encouraging preclinical findings have suggested that CSC pathway blockade can indeed enhance cellular sensitivity to non-targeted conventional therapy, and this work has led to several ongoing clinical trials of CSC pathway inhibitors. Our studies in bladder cancer and lung adenocarcinoma have demonstrated a crucial role of YAP1, a transcriptional regulator of genes that promote cell survival and proliferation, in regulating CSC phenotypes. Moreover, using cell lines and patient-derived xenograft models, we showed that inhibition of YAP1 enhances the efficacy of conventional therapies by attenuating CSC stemness features. In this review, we summarize the therapeutic strategies for targeting CSCs in several cancers and discuss the potential and challenges of the approach. Full article

(This article belongs to the Special Issue Advances in Cancer Stem Cell Research)

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15 pages, 623 KiB

Open AccessReview

STAT3: A Promising Therapeutic Target in Multiple Myeloma

by Phyllis S. Y. Chong, Wee-Joo Chng and Sanjay de Mel

Cancers 2019, 11(5), 731; https://doi.org/10.3390/cancers11050731 - 26 May 2019

Cited by 64 | Viewed by 7063

Abstract

Multiple myeloma (MM) is an incurable plasma cell malignancy for which novel treatment options are required. Signal Transducer and Activator of Transcription 3 (STAT3) overexpression in MM appears to be mediated by a variety of factors including interleukin-6 signaling and downregulation of Src [...] Read more.

Multiple myeloma (MM) is an incurable plasma cell malignancy for which novel treatment options are required. Signal Transducer and Activator of Transcription 3 (STAT3) overexpression in MM appears to be mediated by a variety of factors including interleukin-6 signaling and downregulation of Src homology phosphatase-1 (SHP-1). STAT3 overexpression in MM is associated with an adverse prognosis and may play a role in microenvironment-dependent treatment resistance. In addition to its pro-proliferative role, STAT3 upregulates anti-apoptotic proteins and leads to microRNA dysregulation in MM. Phosphatase of regenerating liver 3 (PRL-3) is an oncogenic phosphatase which is upregulated by STAT3. PRL-3 itself promotes STAT-3 phosphorylation resulting in a positive feedback loop. PRL-3 is overexpressed in a subset of MM patients and may cooperate with STAT3 to promote survival of MM cells. Indirectly targeting STAT3 via JAK (janus associated kinase) inhibition has shown promise in early clinical trials. Specific inhibitors of STAT3 showed in vitro efficacy but have failed in clinical trials while several STAT3 inhibitors derived from herbs have been shown to induce apoptosis of MM cells in vitro. Optimising the pharmacokinetic profiles of novel STAT3 inhibitors and identifying how best to combine these agents with existing anti-myeloma therapy are key questions to be addressed in future clinical trials. Full article

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8 pages, 4642 KiB

Open AccessCase Report

Living Donor Liver Re-Transplantation for Recurrent Hepatoblastoma in the Liver Graft following Complete Eradication of Peritoneal Metastases under Indocyanine Green Fluorescence Imaging

by Nobuhiro Takahashi, Yohei Yamada, Ken Hoshino, Miho Kawaida, Teizaburo Mori, Kiyotomo Abe, Takumi Fujimura, Kentaro Matsubara, Taizo Hibi, Masahiro Shinoda, Hideaki Obara, Kyohei Isshiki, Haruko Shima, Hiroyuki Shimada, Kaori Kameyama, Yasushi Fuchimoto, Yuko Kitagawa and Tatsuo Kuroda

Cancers 2019, 11(5), 730; https://doi.org/10.3390/cancers11050730 - 26 May 2019

Cited by 29 | Viewed by 4966

Abstract

The curability of chemotherapy-resistant hepatoblastoma (HB) largely depends on the achievement of radical surgical resection. Navigation techniques utilizing indocyanine green (ICG) are a powerful tool for detecting small metastatic lesions. We herein report a patient who underwent a second living donor liver transplantation [...] Read more.

The curability of chemotherapy-resistant hepatoblastoma (HB) largely depends on the achievement of radical surgical resection. Navigation techniques utilizing indocyanine green (ICG) are a powerful tool for detecting small metastatic lesions. We herein report a patient who underwent a second living donor liver transplantation (LDLTx) for multiple recurrent HBs in the liver graft following metastasectomy for peritoneal dissemination with ICG navigation. The patient initially presented with ruptured HB at 6 years of age and underwent 3 liver resections followed by the first LDLTx with multiple sessions of chemotherapy at 11 years of age. His alpha-fetoprotein (AFP) level increased above the normal limit, and metastases were noted in the transplanted liver and peritoneum four years after the first LDLTx. The patient underwent metastasectomy of the peritoneally disseminated HBs with ICG navigation followed by the second LDLTx for multiple metastases in the transplanted liver. The patient has been recurrence-free with a normal AFP for 30 months since the second LDLTx. To our knowledge, this report is the first successful case of re-LDLTx for recurrent HBs. Re-LDLTx for recurrent HB can be performed in highly select patients, and ICG navigation is a powerful surgical tool for achieving tumor clearance. Full article

(This article belongs to the Special Issue Hepatoblastoma and Pediatric Liver Tumors)

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The clinical course of the patient. The years after the initial presentation are plotted on the horizontal axis, and the values of alpha-fetoprotein (AFP) are plotted on the vertical axis. Blue arrowheads indicate laparotomies other than living donor liver transplantation (LDLTx). Bars on the bottom represent the chemotherapeutic regimen. CITA: cisplatin 80 mg/m2 and tetrahydropyranyl adriamycin (THP-ADR) 30 mg/m2, C5V: cisplatin 90 mg/m2, 5-fluorouracil 600 mg/m2 and vincristine 1.5 mg/m2, CPT-11: Irinotecan 20 mg/m2. Full article ">Figure 2
(A,B,C) The pathological findings of the metastatic nodule in the liver, which were compatible with wholly epithelial-type (fetal subtype) hepatoblastoma (HB) with vascular invasion. (A,B; H.E. stain, A; 100×, B; 200×, C; Elastica van Gieson (EVG) stain, 200×). (D) The arrowhead represents the peritoneal nodule adjacent to the transplanted liver, which was noted in the first laparotomy after the first LDLTx procedure. (E) The pathological findings of the peritoneal nodule is shown in Figure (H.E. stain, 100×). Full article ">Figure 3
White-light mode (A,C) and corresponding near-infrared mode (B,D) findings in the peritoneal cavity at the time of the second laparotomy after the first LDLTx procedure are shown, macroscopic image. Full article ">Figure 4
(A) The sliced explanted liver in white-light mode and (B) near-infrared mode. The hot spots in near-infrared mode were compatible with hepatoblastomas in histology, macroscopic image. Full article ">Figure 5
A schematic illustration of the hypothesized mechanism of recurrence in this patient. Multiple metastases in the liver graft are assumed to be derived from disseminated nodules through the portal vein. Full article ">

17 pages, 2854 KiB

Open AccessReview

Interplay Between LOX Enzymes and Integrins in the Tumor Microenvironment

by Pier Giorgio Amendola, Raphael Reuten and Janine Terra Erler

Cancers 2019, 11(5), 729; https://doi.org/10.3390/cancers11050729 - 26 May 2019

Cited by 53 | Viewed by 7492

Abstract

Members of the lysyl oxidase (LOX) family are secreted copper-dependent amine oxidases that catalyze the covalent crosslinking of collagens and elastin in the extracellular matrix (ECM), an essential process for the structural integrity of all tissues. LOX enzymes can also remodel the tumor [...] Read more.

Members of the lysyl oxidase (LOX) family are secreted copper-dependent amine oxidases that catalyze the covalent crosslinking of collagens and elastin in the extracellular matrix (ECM), an essential process for the structural integrity of all tissues. LOX enzymes can also remodel the tumor microenvironment and have been implicated in all stages of tumor initiation and progression of many cancer types. Changes in the ECM can influence several cancer cell phenotypes. Integrin adhesion complexes (IACs) physically connect cells with their microenvironment. This review article summarizes the main findings on the role of LOX proteins in modulating the tumor microenvironment, with a particular focus on how ECM changes are integrated by IACs to modulate cells behavior. Finally, we discuss how the development of selective LOX inhibitors may lead to novel and effective therapies in cancer treatment. Full article

(This article belongs to the Special Issue The Role of Integrins in Cancer)

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12 pages, 10040 KiB

Open AccessArticle

TCam-2 Cells Deficient for SOX2 and FOXA2 Are Blocked in Differentiation and Maintain a Seminoma-Like Cell Fate In Vivo

by Daniel Nettersheim, Saskia Vadder, Sina Jostes, Alena Heimsoeth and Hubert Schorle

Cancers 2019, 11(5), 728; https://doi.org/10.3390/cancers11050728 - 25 May 2019

Cited by 13 | Viewed by 5584

Abstract

Testicular germ cell tumors (GCTs) are very common in young men and can be stratified into seminomas and non-seminomas. While seminomas share a similar gene expression and epigenetic profile with primordial germ cells, the stem cell population of the non-seminomas, the embryonal carcinoma [...] Read more.

Testicular germ cell tumors (GCTs) are very common in young men and can be stratified into seminomas and non-seminomas. While seminomas share a similar gene expression and epigenetic profile with primordial germ cells, the stem cell population of the non-seminomas, the embryonal carcinoma (EC), resembles malignant embryonic stem cells. Thus, ECs are able to differentiate into cells of all three germ layers (teratomas) and even extra-embryonic-tissue-like cells (yolk-sac tumor, choriocarcinoma). In the last years, we demonstrated that the cellular microenvironment considerably influences the plasticity of seminomas (TCam-2 cells). Upon a microenvironment-triggered inhibition of the BMP signaling pathway in vivo (murine flank or brain), seminomatous TCam-2 cells reprogram to an EC-like cell fate. We identified SOX2 as a key factor activated upon BMP inhibition mediating the reprogramming process by regulating pluripotency, reprogramming and epigenetic factors. Indeed, CRISPR/Cas9 SOX2-deleted TCam-2 cells were able to maintain a seminoma-cell fate in vivo for about six weeks, but after six weeks in vivo still small sub-populations initiated differentiation. Closer analyses of these differentiated clusters suggested that the pioneer factor FOXA2 might be the driving force behind this induction of differentiation, since many FOXA2 interacting genes and differentiation factors like AFP, EOMES, CDX1, ALB, HAND1, DKK, DLK1, MSX1 and PITX2 were upregulated. In this study, we generated TCam-2 cells double-deficient for SOX2 and FOXA2 using the CRISPR/Cas9 technique and xenografted those cells into the flank of nude mice. Upon loss of SOX2 and FOXA2, TCam-2 maintained a seminoma cell fate for at least twelve weeks, demonstrating that both factors are key players in the reprogramming to an EC-like cell fate. Therefore, our study adds an important piece to the puzzle of GCT development and plasticity, providing interesting insights in what can be expected in a patient, when GCT cells are confronted with different microenvironments. Full article

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HE and IHC staining of SOX2 and FOXA2 in TCam-2-ΔSOX2/FOXA2 tumor tissues six and twelve weeks after xenografting. TCam-2-ΔSOX2 tumor tissue served as control (six weeks in vivo). Scale bars: 200 μm. Full article ">Figure 2
IHC staining of the pluripotency and seminoma markers OCT4, SOX17, TFAP2C and PRDM1 in TCam-2-ΔSOX2/FOXA2 tumor tissues six and twelve weeks after xenografting. TCam-2-ΔSOX2 tumor tissue served as control (six weeks in vivo). Scale bars: 200 μm. Full article ">Figure 3
IHC staining of the differentiation markers AFP and EOMES and the proliferation marker Ki67 in TCam-2-ΔSOX2/FOXA2 tumor tissues six and twelve weeks after xenografting. TCam-2-ΔSOX2 tumor tissue served as control (six weeks in vivo). Scale bars: 200 μm. Full article ">Figure 4
qRT-PCR analysis of indicated marker genes in TCam-2-ΔSOX2/FOXA2 (six (n = 4) and twelve weeks (n = 5)) and TCam-2-ΔSOX2 (six weeks (n = 4)). In vivo reprogrammed TCam-2 (TCam-2 6w) and in vivo grown 2102EP (2102EP 8w) as well as in vitro cultivated TCam-2-ΔSOX2/FOXA2, TCam-2-ΔSOX2 and parental TCam-2 cells served as controls. Expression levels were normalized against GAPDH. Full article ">Figure 5
(A) Model summarizing the influence of the microenvironment on the cell fate of TCam-2 cells and the role of SOX2 and FOXA2 in these processes (based on [<a href="#B9-cancers-11-00728" class="html-bibr">9</a>]). (B) Illustration of the molecular effects associated with the switch of SOX17 from a pluripotency to a differentiation-inducing factor during in vivo growth of TCam-2-ΔSOX2. Full article ">

21 pages, 5199 KiB

Open AccessArticle

Acute Skin Damage and Late Radiation-Induced Fibrosis and Inflammation in Murine Ears after High-Dose Irradiation

by Annique C. Dombrowsky, Jannis Schauer, Matthias Sammer, Andreas Blutke, Dietrich W. M. Walsh, Benjamin Schwarz, Stefan Bartzsch, Annette Feuchtinger, Judith Reindl, Stephanie E. Combs, Günther Dollinger and Thomas E. Schmid

Cancers 2019, 11(5), 727; https://doi.org/10.3390/cancers11050727 - 25 May 2019

Cited by 16 | Viewed by 6184

Abstract

The use of different scoring systems for radiation-induced toxicity limits comparability between studies. We examined dose-dependent tissue alterations following hypofractionated X-ray irradiation and evaluated their use as scoring criteria. Four dose fractions (0, 5, 10, 20, 30 Gy/fraction) were applied daily to ear [...] Read more.

The use of different scoring systems for radiation-induced toxicity limits comparability between studies. We examined dose-dependent tissue alterations following hypofractionated X-ray irradiation and evaluated their use as scoring criteria. Four dose fractions (0, 5, 10, 20, 30 Gy/fraction) were applied daily to ear pinnae. Acute effects (ear thickness, erythema, desquamation) were monitored for 92 days after fraction 1. Late effects (chronic inflammation, fibrosis) and the presence of transforming growth factor beta 1 (TGFβ1)-expressing cells were quantified on day 92. The maximum ear thickness displayed a significant positive correlation with fractional dose. Increased ear thickness and erythema occurred simultaneously, followed by desquamation from day 10 onwards. A significant dose-dependency was observed for the severity of erythema, but not for desquamation. After 4 × 20 and 4 × 30 Gy, inflammation was significantly increased on day 92, whereas fibrosis and the abundance of TGFβ1-expressing cells were only marginally increased after 4 × 30 Gy. Ear thickness significantly correlated with the severity of inflammation and fibrosis on day 92, but not with the number of TGFβ1-expressing cells. Fibrosis correlated significantly with inflammation and fractional dose. In conclusion, the parameter of ear thickness can be used as an objective, numerical and dose-dependent quantification criterion to characterize the severity of acute toxicity and allow for the prediction of late effects. Full article

(This article belongs to the Special Issue New Discoveries in Radiation Science: Selection of Papers from the 44th Congress of the European Radiation Research Society (Pécs, Hungary))

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17 pages, 1023 KiB

Open AccessReview

Epigenetic Reprogramming of TGF-? Signaling in Breast Cancer

by Sudha Suriyamurthy, David Baker, Peter ten Dijke and Prasanna Vasudevan Iyengar

Cancers 2019, 11(5), 726; https://doi.org/10.3390/cancers11050726 - 24 May 2019

Cited by 58 | Viewed by 9952

Abstract

The Transforming Growth Factor-β (TGF-β) signaling pathway has a well-documented, context-dependent role in breast cancer development. In normal and premalignant cells, it acts as a tumor suppressor. By contrast, during the malignant phases of breast cancer progression, the TGF-β signaling pathway elicits tumor [...] Read more.

The Transforming Growth Factor-β (TGF-β) signaling pathway has a well-documented, context-dependent role in breast cancer development. In normal and premalignant cells, it acts as a tumor suppressor. By contrast, during the malignant phases of breast cancer progression, the TGF-β signaling pathway elicits tumor promoting effects particularly by driving the epithelial to mesenchymal transition (EMT), which enhances tumor cell migration, invasion and ultimately metastasis to distant organs. The molecular and cellular mechanisms that govern this dual capacity are being uncovered at multiple molecular levels. This review will focus on recent advances relating to how epigenetic changes such as acetylation and methylation control the outcome of TGF-β signaling and alter the fate of breast cancer cells. In addition, we will highlight how this knowledge can be further exploited to curb tumorigenesis by selective targeting of the TGF-β signaling pathway. Full article

(This article belongs to the Special Issue Epigenetic Dysregulation in Cancer: From Mechanism to Therapy)

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Figure 1
Schematic representation of the TGF-β (Transforming growth factor- β)/SMAD (SMA and MAD related protein)-induced transcriptional response mediated by coactivators and corepressors. The extracellular TGF-β signals via heteromeric complex of transmembrane TβR1 and TβR2 (TGF-β receptors 1 and 2). Upon TβR1 activation, R-SMADs (Regulatory SMADs) become phosphorylated and form heteromeric complexes with SMAD4. R-SMAD/SMAD4 complexes can act as transcription factors in concert with coactivators such as p300/CBP (CREB-binding protein) and p/CAF (p300/CBP associated factor), as well as corepressors such as c-SKI/HDAC (Histone deacetylase). ‘P’ in yellow circles indicates phosphorylation. Arrows denote addition of a modification or transfer of a protein complex and dotted arrow represents the reverse of this. ‘Ac’ indicates acetylation. Full article ">Figure 2
Methylation of genomic DNA. (A) unmethylated regions of DNA can allow binding of SMAD components and other transcription factors to enhance gene expression; (B) DNA methyltransferases (DNMTs) methylate genomic DNA, which inhibits binding of transcription factors thereby silencing certain genes. Ten-eleven translocation (TETs) antagonize DNMTs by removing methyl groups from DNA. ‘P’ in yellow circles indicates phosphorylation, ‘Me’ in green circles indicates methylation. Full article ">Figure 3
Representation of Histone methyltransferases and demethylases acting on histones. (A) PRMT5 (Protein arginine methyltransferase) (di-) methylates H3R2 (histone 3, arginine 2) and H4R3 which leads to enhanced transcription. (B) SETDB1 (Set domain bifurcated 1) tri-methylates H3K9 to repress SNAI1 transcription. (C) JARID1B (Jumonji/ARID domain-containing protein 1B) de-methylates H3K4 to promote growth in breast cancer cells. (D) PHF8 (PHD finger protein 8) recognizes and demethylates H3K9me2, H3K29me2 and H4K20me leading to enhanced gene expression. Arrows indicate addition and dotted lines indicate removal of methyl groups. ‘Me’ in green circles indicates methylation. Full article ">Figure 4
Representation of the action of the histone acetyltransferase, GCN5 (General control non-repressed protein 5) (Left); SND1 (Staphylococcal nuclease domain-containing 1) recruits GCN5 to the promoter regions of SMAD2, 3 and 4. GCN5 acetylates at H3K9 which results in transcriptional activation. (Right) The SMAD complex can further enhance transcription of SND1 creating a positive feedback loop. ‘Ac’ in orange circles indicates acetylation, ‘Me’ in green circles indicates methylation and ‘P’ in yellow circles indicates phosphorylation. Full article ">Figure 5
Representation of SMAD-mediated transcription of lncATB (Long non-coding RNA, activated by TGF-β). LncATB acts as sponge to soak up miRNA200c, which is a negative regulator of TWIST1 (Twist family bHLH transcription factor 1), this ultimately leads to its enhanced levels. ‘P’ in yellow circles indicates phosphorylation. Full article ">

10 pages, 1430 KiB

Open AccessArticle

Western-Type Helicobacter pylori CagA are the Most Frequent Type in Mongolian Patients

by Tegshee Tserentogtokh, Boldbaatar Gantuya, Phawinee Subsomwong, Khasag Oyuntsetseg, Dashdorj Bolor, Yansan Erdene-Ochir, Dashdorj Azzaya, Duger Davaadorj, Tomohisa Uchida, Takeshi Matsuhisa and Yoshio Yamaoka

Cancers 2019, 11(5), 725; https://doi.org/10.3390/cancers11050725 - 24 May 2019

Cited by 19 | Viewed by 4101

Abstract

Helicobacter pylori infection possessing East-Asian-type CagA is associated with carcinogenesis. Mongolia has the highest mortality rate from gastric cancer. Therefore, we evaluated the CagA status in the Mongolian population. High risk and gastric cancer patients were determined using endoscopy and histological examination. H. [...] Read more.

Helicobacter pylori infection possessing East-Asian-type CagA is associated with carcinogenesis. Mongolia has the highest mortality rate from gastric cancer. Therefore, we evaluated the CagA status in the Mongolian population. High risk and gastric cancer patients were determined using endoscopy and histological examination. H. pylori strains were isolated from different locations in Mongolia. The CagA subtypes (East-Asian-type or Western-type, based on sequencing of Glu-Pro-Ile-Tyr-Ala (EPIYA) segments) and vacA genotypes (s and m regions) were determined using PCR-based sequencing and PCR, respectively. In total, 368 patients were examined (341 gastritis, 10 peptic ulcer, and 17 gastric cancer). Sixty-two (16.8%) strains were cagA-negative and 306 (83.1%) were cagA-positive (293 Western-type, 12 East-Asian-type, and one hybrid type). All cagA-negative strains were isolated from gastritis patients. In the gastritis group, 78.6% (268/341) had Western-type CagA, 2.9% (10/341) had East-Asian-type, and 18.2% (61/341) were cagA-negative. However, all H. pylori from gastric cancer patients possessed Western-type CagA. Histological analyses showed that East-Asian-type CagA was the most virulent strains, followed by Western-type and cagA-negative strains. This finding agreed with the current consensus. CagA-positive strains were the most virulent type. However, the fact that different CagA types can explain the high incidence of gastric cancer might be inapplicable in Mongolia. Full article

(This article belongs to the Special Issue Helicobacter pylori Associated Cancer)

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16 pages, 799 KiB

Open AccessReview

Malignant Pheochromocytomas/Paragangliomas and Ectopic Hormonal Secretion: A Case Series and Review of the Literature

by Anna Angelousi, Melpomeni Peppa, Alexandra Chrisoulidou, Krystallenia Alexandraki, Annabel Berthon, Fabio Rueda Faucz, Eva Kassi and Gregory Kaltsas

Cancers 2019, 11(5), 724; https://doi.org/10.3390/cancers11050724 - 24 May 2019

Cited by 19 | Viewed by 4671

Abstract

Malignant pheochromocytomas (PCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasms defined by the presence of distant metastases. There is currently a relatively paucity of data regarding the natural history of PCs/PGLs and the optimal approach to their treatment. We retrospectively analyzed the clinical, [...] Read more.

Malignant pheochromocytomas (PCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasms defined by the presence of distant metastases. There is currently a relatively paucity of data regarding the natural history of PCs/PGLs and the optimal approach to their treatment. We retrospectively analyzed the clinical, biochemical, imaging, genetic and histopathological characteristics of fourteen patients with metastatic PCs/PGLs diagnosed over 15 years, along with their response to treatment. Patients were followed-up for a median of six years (range: 1–14 years). Six patients had synchronous metastases and the remaining developed metastases after a median of four years (range 2–10 years). Genetic analysis of seven patients revealed that three harbored succinate dehydrogenase subunit B/D gene (SDHB/D) mutations. Hormonal hypersecretion occurred in 70% of patients; normetanephrine, either alone or with other concomitant hormones, was the most frequent secretory component. Patients were administered multiple first and subsequent treatments including surgery (n = 12), chemotherapy (n = 7), radionuclide therapy (n = 2) and radiopeptides (n = 5). Seven patients had stable disease, four had progressive disease and three died. Ectopic hormonal secretion is rare and commonly encountered in benign PCs. Ectopic secretion of interleukin-6 in one of our patients, prompted a literature review of ectopic hormonal secretion, particularly from metastatic PCs/PGLs. Only four cases of metastatic PC/PGLs with confirmed ectopic secretion of hormones or peptides have been described so far. Full article

(This article belongs to the Special Issue Pheochromocytoma (PHEO) and Paraganglioma (PGL))

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23 pages, 3176 KiB

Open AccessCommunication

Profiling of Epigenetic Features in Clinical Samples Reveals Novel Widespread Changes in Cancer

by Roberta Noberini, Camilla Restellini, Evelyn Oliva Savoia, Francesco Raimondi, Lavinia Ghiani, Maria Giovanna Jodice, Giovanni Bertalot, Giuseppina Bonizzi, Maria Capra, Fausto Antonio Maffini, Marta Tagliabue, Mohssen Ansarin, Michela Lupia, Marco Giordano, Daniela Osti, Giuliana Pelicci, Susanna Chiocca and Tiziana Bonaldi

Cancers 2019, 11(5), 723; https://doi.org/10.3390/cancers11050723 - 24 May 2019

Cited by 20 | Viewed by 5645

Abstract

Aberrations in histone post-translational modifications (PTMs), as well as in the histone modifying enzymes (HMEs) that catalyze their deposition and removal, have been reported in many tumors and many epigenetic inhibitors are currently under investigation for cancer treatment. Therefore, profiling epigenetic features in [...] Read more.

Aberrations in histone post-translational modifications (PTMs), as well as in the histone modifying enzymes (HMEs) that catalyze their deposition and removal, have been reported in many tumors and many epigenetic inhibitors are currently under investigation for cancer treatment. Therefore, profiling epigenetic features in cancer could have important implications for the discovery of both biomarkers for patient stratification and novel epigenetic targets. In this study, we employed mass spectrometry-based approaches to comprehensively profile histone H3 PTMs in a panel of normal and tumoral tissues for different cancer types, identifying various changes, some of which appear to be a consequence of the increased proliferation rate of tumors, while others are cell-cycle independent. Histone PTM changes found in tumors partially correlate with alterations of the gene expression profiles of HMEs obtained from publicly available data and are generally lost in culture conditions. Through this analysis, we identified tumor- and subtype-specific histone PTM changes, but also widespread changes in the levels of histone H3 K9me3 and K14ac marks. In particular, H3K14ac showed a cell-cycle independent decrease in all the seven tumor/tumor subtype models tested and could represent a novel epigenetic hallmark of cancer. Full article

(This article belongs to the Collection Histone Modification in Cancer)

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Figure 1
Comparison of histone H3 post-translational modifications (PTMs) in tumor and normal tissues. (A,C) Heatmap display and hierarchical clustering of the log2 transformed ratios obtained for the indicated histone H3 (H3) PTMs for different tumor types. Matched normal and tumor tissues are indicated by the same sample number. L/H (light/heavy) relative abundances ratios obtained with the super SILAC (Stable Isotope Labeling with Amino acids in Cell culture) strategy (light channel: Patient sample, heavy channel: Spike-in super-SILAC standard), normalized over the average value across the samples belonging to the same tumor type are shown. In (C) only the differentially modified forms of histone H3 peptides 9–17 are shown. The average % relative abundance (%RA) across samples is indicated by shades of grey and numbers on the left side (see <a href="#app1-cancers-11-00723" class="html-app">Figure S1</a> for a histogram representation of %RAs). The grey color indicates those peptides that were not quantified. (B,D) Modified peptides were compared by unpaired t-test in tumors compared with normal tissues. The red color indicates an increase in tumors, the blue color a decrease (p < 0.05 for darker colors, p < 0.1 for lighter colors). The grey color indicates those peptides that could not be quantified in formalin-fixed paraffin-embedded (FFPE) tissues or for which enough data points to obtain a p-value were not available. (E) Boxplot representation of the L/H ratios for total H3K14ac (given by the sum of H3K14ac, H3K9me1/K14ac, H3K9me2/K14ac, H3K9me3/K14ac, and H3K9ac/K14ac) and total H3K9me3 (given by the sum of H3K9me3 and H3K9me3/K14ac) for all the tumor types tested. For luminal A-like and triple negative breast cancers, a comparison with samples where tumor cells were isolated by laser microdissection (n = 3 for each subtype) is also shown. Normal and tumor samples were compared by t-test (* p < 0.05, ** p < 0.01). BC: breast cancer; LuA: Luminal A-like; LuB: Luminal B-like; TN: triple negative; OC: ovarian cancer; HNC: head and neck cancer; PC: prostate cancer; GBM: glioblastoma; LMD: laser micro-dissected. Full article ">Figure 2
Correlation of histone post-translational modifications (PTMs) and proliferation rate. (A) L/H (light/heavy) ratios for the indicated peptides were plotted against the Ki-67 proliferation index for the frozen breast cancer patient samples analyzed in this study and are shown in <a href="#cancers-11-00723-f001" class="html-fig">Figure 1</a>A, C (left panel), or breast cancer FFPE samples analyzed in [<a href="#B15-cancers-11-00723" class="html-bibr">15</a>] (right panel, containing luminal A-like, luminal B-like, HER2 (human epidermal growth factor receptor 2) positive, and triple negative subtypes). The Pearson’s correlation score (r) is shown when above 0.4 and an asterisk indicates a correlation with a p value < 0.05. (B) Heatmap display of the log2 transformed ratios obtained for the indicated histone H3 PTMs for the MDA-MB-231 and MCF7 breast cancer cell lines and the MCF10A normal breast cell line. “Ratios of ratios” are shown, which were obtained by dividing the L/H ratios for nocodazole-treated cells (synchronized in G2-M phase) by the L/H ratio for thymidine treated cells (synchronized in G1-S phase). The grey color indicates peptides that were not quantified. Peptides containing K14ac are highlighted in blue. (C) Modified peptides in nocodazole- and thymidine-synchronized cells were compared by paired t-test. The red color indicates an increase in G2-M phase, the blue color a decrease (p < 0.05 for darker colors, p < 0.1 for lighter colors). The grey color indicates peptides for which enough data points to obtain a p-value were not available. (D) Histograms representing the ratios of nocodazole- and thymidine-treated cells for total H3K14ac (given by the sum of H3K14ac, H3K9me1/K14ac, H3K9me2/K14ac, H3K9me3/K14ac, and H3K9ac/K14ac), total H3K9me3 (given by the sum of H3K9me3 and H3K9me3/K14ac), and total H3K27me3 (given by the sum of H3K27me3 and H3K27me3/K36me1) for the three cell lines tested. Changes in nocodazole-compared with thymidine-treated cells are indicated by an asterisk (*: p < 0.1). The red color indicates an increase in G2-M phase, while the blue color indicates a decrease. Full article ">Figure 3
Differential expression of histone modifying enzymes in normal and tumor tissues. Only significantly deregulated genes (adjusted p-value < 0.01) are displayed. Histone modifying enzyme (HME) specificity for common histone H3 PTMs is marked. BLCA: Bladder Urothelial Carcinoma; BRCA: Breast invasive carcinoma; COAD: Colon adenocarcinoma; ESCA: Esophageal carcinoma; HNSC: Head and Neck squamous cell carcinoma; KICH: Kidney Chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; PRAD: Prostate adenocarcinoma; READ: Rectum adenocarcinoma; STAD: Stomach adenocarcinoma; THCA: Thyroid carcinoma; UCEC: Uterine Corpus Endometrial Carcinoma. Full article ">Figure 4
Functional interaction network of histone modifying enzymes (HMEs). (A) Functional interaction network of HMEs, generated through ReactomeFIViz [<a href="#B45-cancers-11-00723" class="html-bibr">45</a>] and colored on the basis of node clustering, which is achieved by optimizing network modularity. (B) HME interaction networks, where red and blue colors indicate up- or down-regulation in the tumors, compared with normal tissues, and node diameters are proportional to RPKM (reads per kilobase million) base mean from DESEQ2 (differential gene expression analysis based on the negative binomial distribution) analysis. BRCA; Breast invasive carcinoma. Full article ">Figure 5
Mutational analysis of HMEs in cancer. (A) Stacked bar plot summarizing the frequency (minimum % of altered cases = 1) and type of mutations of HMEs in the TCGA PanCan 2018 cohort [<a href="#B47-cancers-11-00723" class="html-bibr">47</a>]. Different colors correspond to different types of mutations. (B) Same representation as in A for the SETDB1 and SETDB2 genes. (C) Horizontal stacked bar plot for significantly mutated HME genes (MutSig2CV adjusted p-value < 0.01). The bar widths are proportional to the number of samples mutated divided by the cohort size. When a gene is significantly mutated in multiple cancer types, the resulting cohort is given by the sum of the total samples of the individual cohorts. The right panel indicates whether the genes have been reported as oncogenes and/or tumor suppressor genes (TSG) in at least one tumor type in the Cancer Census Genes [<a href="#B48-cancers-11-00723" class="html-bibr">48</a>]. AML: Acute Myeloid Leukemia; ACC: Adrenocortical carcinoma; BLCA: Bladder Urothelial Carcinoma; LGG: Brain Lower Grade Glioma; BRCA: Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma (1) and endocervical adenocarcinoma (2); CHOL: Cholangiocarcinoma; CML: Chronic Myelogenous Leukemia; COAD: Colon adenocarcinoma; ESCA: Esophageal squamous cell carcinoma (1) and esophagogastric adenocarcinoma (2); GBM: Glioblastoma multiforme; HNSC: Head and Neck squamous cell carcinoma; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LIHC: Liver hepatocellular carcinoma; LUAD; Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; DLBC: Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; MESO: Mesothelioma; MISC: Miscellaneous; OV: Ovarian serous cystadenocarcinoma; PAAD: Pancreatic adenocarcinoma; PCPG: Pheochromocytoma and Paraganglioma; PRAD: Prostate adenocarcinoma; SARC: Sarcoma; SKCM: Skin Cutaneous Melanoma; STAD: Stomach adenocarcinoma; TGCT: Testicular Germ Cell Tumors; THYM: Thymoma; THCA: Thyroid carcinoma; UCS: Uterine Carcinosarcoma; UCEC; Uterine Corpus Endometrial Carcinoma; UVM: Uveal Melanoma. Full article ">Figure 6
Histone post-translational modifications (PTM) profiling of normal and tumoral cell lines. (A) Principal component analysis of normal and tumoral tissues and cell lines for the luminal A-like and triple negative breast cancer and prostate cancer models. The grey color indicates peptides that were not quantified. (B) Heatmap display of the log2 transformed ratios obtained for the indicated histone PTMs in normal and tumor breast cell lines. Tumoral cell lines were divided in luminal A (LuA) and triple negative (TN) and were analyzed separately. L/H (light/heavy) relative abundances ratios obtained with the super-SILAC strategy (light channel: Normal/tumor cell line, heavy channel: Spike-in super-SILAC standard), normalized over the average value across all the samples. Some of the data for histone H3 PTMs is from [<a href="#B13-cancers-11-00723" class="html-bibr">13</a>] (see dataset S1). Right panel: Modified peptides were compared in LuA/TN tumor cells and normal cell lines by unpaired t-test. The red color indicates an increase in tumors, the blue color a decrease (p < 0.05 for darker colors, p < 0.1 for lighter colors). (C) L/H ratios for the differentially modified versions of the H3 9–17 peptide in the indicated tumor models. *: p < 0.05 by Student’s t-test. (D) Boxplot representation of the L/H ratios for total H3K14ac (given by the sum of H3K14ac, H3K9me1/K14ac, H3K9me2/K14ac, H3K9me3/K14ac, and H3K9ac/K14ac) and total H3K9me3 (given by the sum of H3K9me3 and H3K9me3/K14ac) for all the tumor models tested. Normal and tumor samples were compared by t-test (*p < 0.05). BC: Breast cancer; PC: Prostate cancer; GBM: Glioblastoma, N: Normal; T: Tumor. In A–D, the “normal” prostate cell lines were infected with HPV. Full article ">

18 pages, 8559 KiB

Open AccessArticle

POLQ Overexpression Is Associated with an Increased Somatic Mutation Load and PLK4 Overexpression in Lung Adenocarcinoma

by Kazuya Shinmura, Hisami Kato, Yuichi Kawanishi, Katsuhiro Yoshimura, Kazuo Tsuchiya, Yoshiyuki Takahara, Seiji Hosokawa, Akikazu Kawase, Kazuhito Funai and Haruhiko Sugimura

Cancers 2019, 11(5), 722; https://doi.org/10.3390/cancers11050722 - 24 May 2019

Cited by 24 | Viewed by 5686

Abstract

DNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs). In the present study, we examined whether abnormal POLQ expression may be involved in the pathogenesis of lung adenocarcinoma (LAC). [...] Read more.

DNA Polymerase Theta (POLQ) is a DNA polymerase involved in error-prone translesion DNA synthesis (TLS) and error-prone repair of DNA double-strand breaks (DSBs). In the present study, we examined whether abnormal POLQ expression may be involved in the pathogenesis of lung adenocarcinoma (LAC). First, we found overexpression of POLQ at both the mRNA and protein levels in LAC, using data from the Cancer Genome Atlas (TCGA) database and by immunohistochemical analysis of our LAC series. POLQ overexpression was associated with an advanced pathologic stage and an increased total number of somatic mutations in LAC. When H1299 human lung cancer cell clones overexpressing POLQ were established and examined, the clones showed resistance to a DSB-inducing chemical in the clonogenic assay and an increased frequency of mutations in the supF forward mutation assay. Further analysis revealed that POLQ overexpression was also positively correlated with Polo Like Kinase 4 (PLK4) overexpression in LAC, and that PLK4 overexpression in the POLQ-overexpressing H1299 cells induced centrosome amplification. Finally, analysis of the TCGA data revealed that POLQ overexpression was associated with an increased somatic mutation load and PLK4 overexpression in diverse human cancers; on the other hand, overexpressions of nine TLS polymerases other than POLQ were associated with an increased somatic mutation load at a much lower frequency. Thus, POLQ overexpression is associated with advanced pathologic stage, increased somatic mutation load, and PLK4 overexpression, the last inducing centrosome amplification, in LAC, suggesting that POLQ overexpression is involved in the pathogenesis of LAC. Full article

(This article belongs to the Special Issue Molecular Profiling of Lung Cancer)

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Figure 1
Overexpression of POLQ mRNA and protein in primary lung adenocarcinoma (LAC). (a) Detection of POLQ mRNA overexpression in LAC determined using data from the TCGA database (ID: LUAD). A Mann–Whitney U test was used for statistical comparison of the findings between non-cancerous tissue (N) and cancerous tissue (T); the p-value and median expression levels are shown. (b) Overexpression of POLQ protein in LAC determined by IHC analysis using rabbit anti-POLQ polyclonal antibody in cases of our hospital (HUH). A Mann–Whitney U test was used for statistical comparison of the findings between non-cancerous lung alveolar tissue and LAC tissue; the p-value and median expression levels are shown. (c) Representative IHC results of POLQ protein expression in primary LAC. The leftmost panel represents the results in non-cancerous lung tissue, while the remaining panels show the results in LAC tissue. The lower panels show a part of the upper panels at a higher magnification. Scale bar = 50 μm (upper); 20 μm (lower). Full article ">Figure 2
Association of increased POLQ expression with the somatic mutation load in LAC, determined using the data (n = 513) from the TCGA database (ID: LUAD). (a) Comparison of the total number of somatic mutations between a group of cancers showing high POLQ expression levels and another group showing low POLQ expression levels among cases of LAC. A box-plot analysis showed a statistically significant difference in the number of somatic mutations between the two groups (p < 0.0001, Mann–Whitney U test). The median values are shown. (b) Scatterplot showing a positive correlation between the POLQ mRNA expression level and the total number of somatic mutations in LAC. The Spearman rank correlation coefficient (ρ) and p-value are shown; a bivariate normal ellipse (p = 0.95) was obtained. Full article ">Figure 3
Comparison of the sensitivities to DNA-damaging agent and mutation frequency between lung cancer cells showing different POLQ expression levels. (a) Detection of FLAG-POLQ proteins in cumate-inducible stable H1299 lung cancer cell lines (POLQ-1 and -2) designed to express FLAG-POLQ in the presence of cumate; the POLQ proteins were detected by Western blot analysis. Empty vector-transposed cells (Empty-1 and -2) were used as control. GAPDH expression was also analyzed as the internal control. (b) Clonogenic survival assay following treatment with etoposide. The survival fraction was compared between the empty vector-transposed H1299 clones and H1299 clones showing inducible POLQ expression. Data are means + standard deviation (SD) of three independent experiments. (c) Measurement of the mutation frequency of the supF gene in the pMY189 shuttle plasmid, using a supF forward mutation assay with pMY189 treated or not treated with UV light in the parental H1299 or H1299-derived stable clones. Data are means + SD of >3 independent experiments. (d) Measurement of the mutation frequency of the supF gene in pMY189, using a supF forward mutation assay with Tg-containing pMY189 in parental H1299 or H1299-derived stable clones. Data shown are means + SD of ≥3 independent experiments. Statistical analyses were performed using an unpaired t-test and the asterisks (*) show a statistically significant difference (b–d). Full article ">Figure 4
Concurrent overexpression of POLQ and PLK4 in primary LAC. (a) PLK4 mRNA overexpression detected in LAC using data from the TCGA database (ID: LUAD). Statistical comparison was performed using a Mann–Whitney U test between non-cancerous tissue (N) and cancerous tissue (T); median expression levels and the p-value are shown. (b) A significant positive correlation between the POLQ and PLK4 mRNA expression levels in LAC. The Spearman rank correlation coefficient (ρ) and p-value are provided. A bivariate normal ellipse (p = 0.95) was observed. (c) Overexpression of PLK4 protein in LAC, determined by IHC analysis using rabbit anti-PLK4 polyclonal antibody in cases of our hospital (HUH). A Mann–Whitney U test was used for statistical comparison between non-cancerous lung alveolar tissue (N) and LAC tissue (T); the p-value and median expression levels are shown. (d) A significant positive correlation was detected between the POLQ and PLK4 protein expression levels in LAC. The data on the POLQ protein expression level were derived from the data shown in <a href="#cancers-11-00722-f001" class="html-fig">Figure 1</a>b,c. The Spearman rank correlation coefficient (ρ) and p-value are shown. A bivariate normal ellipse (p = 0.95) was obtained. (e) Representative IHC results of co-overexpression of PLK4 and POLQ proteins in a case of LAC. The leftmost panel represents the results in non-cancerous lung tissue, while the remaining show the results in LAC from the same patient. The lower panels show a part of the upper panels at a higher magnification. Scale bar = 50 μm (upper); 20 μm (lower). Another set of representative results is shown in <a href="#app1-cancers-11-00722" class="html-app">Supplementary Figure S1</a>. Full article ">Figure 5
Centrosome amplification in the lung cancer cells with concurrent overexpression of POLQ and PLK4. The H1299 lung cancer cell line-derived cells (Empty-1, Empty-2, POLQ-1, and POLQ-2) were transiently transfected with plasmid for expression of GFP or GFP-PLK4 (green) and 48 h post-transfection, the cells were immunostained with anti-γ-tubulin antibody (red) and the nuclei were stained with DAPI (blue). The percentage of cells with ≥3 centrosomes was measured among the GFP-positive (or GFP-PLK4-positive) cells, and is shown in the upper bar graph. Data shown are as the means and standard errors derived from three independent experiments. Statistical analyses were performed using an unpaired t-test and the asterisks (*) denote a statistically significant difference. Representative immunostained images are shown in the lower panels. The arrows show the positions of the centrosomes, and the insets show magnified images of the areas indicated by the arrows. Full article ">Figure 6
Association of POLQ overexpression with the somatic mutation load and PLK4 overexpression in diverse human cancers, determined using data from the TCGA database. (a) Comparison of the number of cancer types showing increased expression of the TLS polymerase gene associated with an increased number of somatic mutations. Ten TLS polymerase genes were examined for the number in 18 cancer types, with Mann–Whitney U test used to compare the total number of somatic mutations between a group of cancers showing low TLS polymerase expression and a group of cancers showing high TLS polymerase gene expression. (b) Results of box-plot analyses of cancer types showing increased expression of the POLQ gene associated with an increased number of somatic mutations in (a). Among the 12 cancer types which showed a significant association, the results for LAC is already shown in <a href="#cancers-11-00722-f002" class="html-fig">Figure 2</a>, and the results for the remaining 11 cancer types are shown. The median mutation number in each group and the p-values are shown in the graph. Results of the box-plot analyses of cancer types showing increased expression of the TLS polymerase gene other than POLQ associated with an increased number of somatic mutations in (a) are shown in <a href="#app1-cancers-11-00722" class="html-app">Supplementary Figure S2</a>. (c) Concurrent overexpression of POLQ and PLK4 in diverse human cancers. Cancer types in which statistical significance was observed in relation to POLQ overexpression and PLK4 overexpression, and a positive correlation was observed between the expression levels of POLQ and PLK4, which are marked with filled circles; other cancer types are marked with clear circles. Detailed data are shown in <a href="#app1-cancers-11-00722" class="html-app">Supplementary Figure S3</a>. Full article ">

17 pages, 446 KiB

Open AccessEditor’s ChoiceReview

Integrins, CAFs and Mechanical Forces in the Progression of Cancer

by Imjoo Jang and Karen A. Beningo

Cancers 2019, 11(5), 721; https://doi.org/10.3390/cancers11050721 - 24 May 2019

Cited by 127 | Viewed by 12817

Abstract

Cells respond to both chemical and mechanical cues present within their microenvironment. Various mechanical signals are detected by and transmitted to the cells through mechanoreceptors. These receptors often contact with the extracellular matrix (ECM), where the external signals are converted into a physiological [...] Read more.

Cells respond to both chemical and mechanical cues present within their microenvironment. Various mechanical signals are detected by and transmitted to the cells through mechanoreceptors. These receptors often contact with the extracellular matrix (ECM), where the external signals are converted into a physiological response. Integrins are well-defined mechanoreceptors that physically connect the actomyosin cytoskeleton to the surrounding matrix and transduce signals. Families of ? and ? subunits can form a variety of heterodimers that have been implicated in cancer progression and differ among types of cancer. These heterodimers serve as the nexus of communication between the cells and the tumor microenvironment (TME). The TME is dynamic and composed of stromal cells, ECM and associated soluble factors. The most abundant stromal cells within the TME are cancer-associated fibroblasts (CAFs). Accumulating studies implicate CAFs in cancer development and metastasis through their remodeling of the ECM and release of large amounts of ECM proteins and soluble factors. Considering that the communication between cancer cells and CAFs, in large part, takes place through the ECM, the involvement of integrins in the crosstalk is significant. This review discusses the role of integrins, as the primary cell-ECM mechanoreceptors, in cancer progression, highlighting integrin-mediated mechanical communication between cancer cells and CAFs. Full article

(This article belongs to the Special Issue The Role of Integrins in Cancer)

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22 pages, 5428 KiB

Open AccessArticle

Tumor Mesenchymal Stromal Cells Regulate Cell Migration of Atypical Teratoid Rhabdoid Tumor through Exosome-Mediated miR155/SMARCA4 Pathway

by Yi-Ping Yang, Phan Nguyen Nhi Nguyen, Hsin-I Ma, Wen-Jin Ho, Yi-Wei Chen, Yueh Chien, Aliaksandr A. Yarmishyn, Pin-I Huang, Wen-Liang Lo, Chien-Ying Wang, Yung-Yang Liu, Yi-Yen Lee, Chien-Min Lin, Ming-Teh Chen and Mong-Lien Wang

Cancers 2019, 11(5), 720; https://doi.org/10.3390/cancers11050720 - 24 May 2019

Cited by 23 | Viewed by 4705

Abstract

Atypical teratoid/rhabdoid tumor (ATRT) is a rare pediatric brain tumor with extremely high aggressiveness and poor prognosis. The tumor microenvironment is regulated by a complex interaction among distinct cell types, yet the crosstalk between tumor-associated mesenchymal stem cells (tMSCs) and naïve ATRT cells [...] Read more.

Atypical teratoid/rhabdoid tumor (ATRT) is a rare pediatric brain tumor with extremely high aggressiveness and poor prognosis. The tumor microenvironment is regulated by a complex interaction among distinct cell types, yet the crosstalk between tumor-associated mesenchymal stem cells (tMSCs) and naïve ATRT cells are unclear. In this study, we sought to identify the secretory factor(s) that is responsible for the tMSC-mediated regulation of ATRT migration. Comparing with ATRT cell alone, co-culture of tMSCs or addition of its conditioned medium (tMSC-CM) promoted the migration of ATRT, and this effect could be abrogated by exosome release inhibitor GW4869. The exosomes in tMSC-CM were detected by transmission electron microscope and flow cytometry. ATRT naïve cell-derived conditioned media (ATRT-CM) also enhanced the exosome secretion from tMSCs, indicating the interplay between ATRT cells and tMSCs. Microarray analysis revealed that, compared with that in bone marrow-derived MSCs, microRNA155 is the most upregulated microRNA in the tMSC-CM. Tracing the PK67-labeled exosomes secreted from tMSCs confirmed their incorporation into naïve ATRT cells. After entering ATRT cells, miR155 promoted ATRT cell migration by directly targeting SMARCA4. Knockdown of SMARCA4 mimicked the miR155-driven ATRT cell migration, whereas SMARCA4 overexpression or the delivery of exosomes with miR155 knockdown suppressed the migration. Furthermore, abrogation of exosome release with GW4869 reduced the tumorigenesis of the xenograft containing naïve ATRT cells and tMSCs in immunocompromised recipients. In conclusion, our data have demonstrated that tMSCs secreted miR155-enriched exosomes, and the exosome incorporation and miR155 delivery further promoted migration in ATRT cells via a SMARCA4-dependent mechanism. Full article

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Figure 1
Tumor-associated mesenchymal stromal cells enhanced the migratory ability of ATRT cell lines through an exosome-dependent mechanism. (A,B) A wound healing migration assay was performed with ATRT cells co-cultured with different types of stromal cells. The ATRT-1 and ATRT-2 cell lines were seeded in the silica chamber attached in 12-well plates. The silica chambers were removed after 24 h to create the gap for cell migration, and the indicated stromal cells were seeded on the 0.2 µm filter trans-well chambers inserted in the 12-well plates for an indirect co-culture system. The cell migration was observed under a microscope for up to 18 h. Ctrl: no cells seeded in the upper chamber; Mock: the upper chambers were seeded with the same ATRT cells as the lower chamber; tMSC: tumor-associated mesenchymal stem cell; hUVEC: human umbilical vein endothelial cell, THP1: human monocytic leukemia; U937: human myeloid leukaemia (A). The area covered by migrated cells was calculated by Image J software and presented as a percentage in the xenografts (B). This experiment was done with three distinct biological replicates. * p < 0.05. (C–D) ATRT cells were subjected to a wound-healing migration assay in the presence of conditioned medium derived from different types of stromal cells. The cell migration was observed under a microscope for up to 24 h (C). The area covered by migrated cells was calculated by Image J software and presented as percentage in the grafts (D). This experiment was done with three distinct biological replicates. * p < 0.05. (E) Vesicles in tMSC conditioned medium were isolated and stained with anti-CD63 antibodies using the Exosome-Human CD63 Isolation/Detection Reagent (Thermo Fisher Scientific). The CD63-positive exosomes were analyzed by Flow Cytometry. IgG: Immunoglobulin G (F) Left: Exosomes in the tMSC conditioned medium were observed under transmission electron microscopy (TEM). The scale bar in the top picture represents 100 nm, while the scale bar in the bottom picture represents 50 nm. Right: control medium and tMSCs conditioned medium were subjected to quantification of vesicles/particles by nanoparticle tracking analysis (NTA). These experiments were done with three distinct biological replicates. * p < 0.05. (G) ATRT cells cultured in conditioned media derived from tMSC (tMSC-CM) treated with or without GW4869 were subjected to a wound-healing migration assay. The migrated cells were photographed at 24 h (top) and the area covered by migrated cells were calculated and presented as a percentage in the graft (bottom). This experiment was done with three distinct biological replicates. * p < 0.05. Full article ">Figure 2
ATRT cells promote exosome released from tMSCs via a paracrine mechanism. (A) PKH67 green fluorescent-labeled tMSCs were indirectly co-cultured with ATRT-1 and ATRT-2 cells in the 0.2 µm filter trans-well system. Right: 24 h after the co-culture, the ATRT cells were observed under a fluorescence microscope to investigate the exosome uptake. Left: schematic presentation of the PKH67-labeling and indirect co-culture system. (B,C) PKH67 green fluorescent-labeled tMSCs-derived exosomes were cultured with ATRT-1 and ATRT-2 cells and cells were observed in a time-course manner for the exosome update of ATRT cells. ATRT cells co-cultured under −4 °C condition for 24 h served as a negative control. The fluorescent intensity was quantified by Image J and presented in the chart in (C). (D) Schematic illustration of the experimental design to investigate the effect on ATRT educated tMSCs. (E) Conditioned media from tMSCs pretreated with indicated media were collected and the exosome in each condition was visualized by TEM (left). Both the naïve culture medium for tMSC and ATRT cells serve as background controls. The number of exosomes released from tMSCs was quantified by Image J based on TEM photo and presented in the charts (right). This experiment was done with three distinct biological replicates. * p < 0.05. (F) tMSCs were pre-treated with low (CM-1X) and high (CM-2X) concentration of ATRT conditioned media for 24 h. Media were then replaced with tMSC culture medium for another 24 h. Naïve medium for ATRT culture was served as background control. Exosomes released from tMSC were observed under TEM and quantified by Image J based on TEM photo and presented in the charts (right). Full article ">Figure 3
Exosomal miR155 suppressed the protein expression level of SMARCA4 in ATRT cells. (A) Exosomes in the conditioned medium of ATRT-educated tMSCs and bone marrow MSCs were collected by centrifugation. The total RNA was extracted and subjected to a miRNA microarray to detect the expressional changes of miRNA. (B) RNA extracted from tMSC (left) and tMSC-derived exosomes (right) were subjected to Taq-man quantitative real-time PCR analysis to evaluate the expression levels of indicated miRNAs. miR155 was the highest expressed within the three miRNAs. (C) The cellular (left) and exosomal (right) levels of miR155 in tMSC, ATRT-1, and ATRT-2 cells were detected by quantitative real-time PCR. Both cellular and exosomal levels of miR155 were higher in tMSC than in ATRT-1 and ATRT-2. (D) ATRT-1 (left) and ATRT-2 (right) were co-cultured with different types of stromal cells for 24 h. ATRT cells were harvested and total RNA was extracted for the evaluation of miR155 expression level by quantitative real-time PCR. The miR155 level in ATRT cells co-cultured with tMSCs was significantly higher than those co-cultured with other types of stromal cells. (E) MSCs were transfected with sponge miR155 (spg-155) or sponge scramble (spg-scr) before subjected to cellular miR155 level assessment by qRT-PCR (left). ATRT-1 (middle) and ATRT-2 (right) co-cultured with spg-scr or spg-155 transfected tMSCs were subjected to RT-PCR to analyze the cellular miR155 expression levels. (F) tMSC (left), ATRT-1 (middle) and ATRT-2 (right) were treated with either PBS or heparin for 24 h before subjected to qRT-PCR to analyzed cellular miR155 expression levels. (G) Search for miR155 targets by micro-RNA binding site database (microrna.org). SMARCA4 and SOCS1 were selected as strong candidates for exosomal miR155 targeting in ATRT cells. (H) ATRT-2 cultured in 40 mL medium contained a dose-course of tMSC conditioned medium (from 10 mL to 40 mL) were subjected to a Western blot analysis to evaluate the protein expression levels of miR155 targets, SMARCA4 and SOCS1, and ATRT biomarker, SNF5. Ctrl: 40 mL of fresh DMEM medium. (I) ATRT-1 (left) and ATRT-2 (right) transfected with wildtype or mutated SMARCA4- three prime untranslated region (3’UTR) reporter plasmids in the presence or absence of miR155 expression plasmids were subjected to luciferase assay. Full article ">Figure 4
The exosomal-miR155/SMARCA4 pathway regulates ATRT migration ability. (A) ATRT-1 and ATRT-2 cells were subjected to a wound-healing migration assay in the presence or absence of a dose-course manner from 10 to 40 µg of purified tMSC-exosomes. The migrated cells were observed under a microscope for 24 h (A); the area covered by migrated cells were quantified by Image J and presented in the charts (B). This experiment was done with three distinct biological replicates. * p < 0.05. (C) ATRT-2 cells were treated by purified tMSC-exosome in a dose-course manner and then subjected to a Western blot to analyze SMARCA4 protein expression levels (left). The intensity of SMARCA4 blot was quantified and standardized with that of GAPDH and presented in the bar chart (right). (D) ATRT-2 cells were transfected with scrambled shRNA (Scr) or shRNA against SMARCA4 (shSMARCA4) and subjected to Western blot analysis to assess the knockdown efficiency of shSMARCA4. A non-transfected ATRT-2 served as a background control (Ctrl). (E,F) ATRT-2 cells transfected with shScr or shSMARCA4 were subjected to a wound-healing migration assay for 24 h (E). The area covered by migrated cells were calculated by Image J and presented in the chart (F). This experiment was done with three distinct biological replicates. * p < 0.05. (G) ATRT-2 cells were transfected with empty vector (Ctrl) or Flag-tagged SMARCA4 and subjected to Western blot analysis to assess the expression of exogenous SMARCA4. (H,I) ATRT-2 cells transfected with Ctrl or Flag-tagged SMARCA4 were subjected to a wound-healing migration assay for 36 h (H). The area covered by migrated cells were calculated by Image J and presented in the chart (I). This experiment was done with three distinct biological replicates. * p < 0.05. Full article ">Figure 5
Blocking of the exosomal miR155/SMARCA4 signaling suppressed ATRT migration. (A) The tMSC was stably transfected with plasmids expressing scrambled or miR155 sponge (Spg-Scr and Spg-155, respective). ATRT-2 cells were treated with conditioned media collected from tMSC-transfected Spg-Scr and Spg-155 and subjected to a Western blot to assess the protein expression levels of SMARCA4 (top). The intensity of each blot was quantified by Image J and presented as relative levels in the chart (bottom). (B–C) ATRT-1 and ATRT-2 treated with conditioned media derived from tMSC which were transfected with plasmids expressing scrambled or miR155 sponge (SPG-Scr and SPG-155, respective) for 24 h were subjected to a wound-healing migration assay (B). The migrated cells covered area was calculated by Image J and presented as percentages relative to the initial area (C). (D) tMSC was transfected with either scrambled (Spg-Scr) or sponge miR155 (Spg-155), respectively. The tMSC was pre-incubated with Heparin or PBS for 2 h before the collection of the tMSC-CM. ATRT-2 cells were treated with tMSC-CM from various conditions for 24 h and analyzed by Western blot analysis to assess the protein levels of SMARCA4 (top). The intensity of each blot was quantified by presented as relative levels in the chart (bottom). (E) ATRT-2 cells with the same treatment as D were subjected to a wound-healing migration assay for 24 h (top), as well as a qRT-PCR for the expression levels of miR155 (bottom). (F) Immunocompromised mice were intracranially transplanted with ATRT-2 cells along with tMSC-CM in the presence or absence of GW4689 (10 μM). (G) Tumors were allowed to grow for 42 days and the tumor lesion area is measured by functional magnetic resonance imaging (fMRI). Full article ">Figure 6
Correlation of miR155 levels and ATRT recurrence in clinical samples. (A) The percentage of miR155 + AT/RT cells (1st surgery: nine patients) was dramatically elevated in the tumor relapse samples (2nd surgery: six patients). (B) Comparison of the tumor samples from the first and second surgeries in the six patients whose tumors relapsed. ** p < 0.001. Full article ">Figure 7
Schematic demonstration of paracrine interaction between MSCs and ATRT tumors. Briefly, tMSC-derived highly expressed miR-155- containing exosomes are transferred to ATRT cells. The abundant expression of exosomal miR-155 in ATRT leads to downregulation of SMARCA4, a direct target gene of miR-155. Hence, the migratory ability of ATRT increases. On the other hand, ATRT cells educate/stimulate tMSCs to release a higher amount of exosomes, and thus improve migration of ATRT cells. This malignant property of ATRT is reduced when miR-155 or exosome inhibitors are introduced into tMSCs. Full article ">

12 pages, 2784 KiB

Open AccessArticle

Changes in Ultraviolet Radiation Exposure to the Ocular Region: A Population-Based Study

by Ezekiel Weis, Sebastian Q. Vrouwe, David B. LeBaron, Matthew B. Parliament, Jerry Shields and Carol L. Shields

Cancers 2019, 11(5), 719; https://doi.org/10.3390/cancers11050719 - 24 May 2019

Cited by 7 | Viewed by 3631

Abstract

In contrast to the well-established association between ultraviolet radiation (UVR) exposure and skin cancers, the relationship between UVR and uveal malignant melanoma (UM) remains controversial. To address this controversy, we evaluated the incidence rates of cutaneous malignancies in the eyelids as a proxy [...] Read more.

In contrast to the well-established association between ultraviolet radiation (UVR) exposure and skin cancers, the relationship between UVR and uveal malignant melanoma (UM) remains controversial. To address this controversy, we evaluated the incidence rates of cutaneous malignancies in the eyelids as a proxy for UVR exposure in the ocular region using a population-based cancer registry. Overall, 74,053 cases of eyelid basal cell carcinoma (BCC) and 7890 cases of melanoma over a 26-year period (1982–2007) were analyzed. The incidence of eyelid basal cell carcinoma and uveal melanoma remained stable, whereas other cutaneous areas demonstrated an increase in the rates. A comparability test demonstrated that BCC incidence trends were significantly different between the eyelid versus both chronically exposed (males p = 0.001; females p = 0.01) and intermittently exposed skin (males and females, p = 0.0002), as well as the skin of the face (males p = 0.002; females p = 0.02). Similarly, melanoma trends were significantly different between the UM group versus both chronically exposed cutaneous melanoma (CM) (males p = 0.001; females p = 0.04) and intermittently exposed CM (males p = 0.005), as well as facial skin CM (males and females p = 0.0002). The discrepancy of cancer incidence between tumors in the peri-ocular region versus the rest of the body suggests that the peri-ocular region might have a different or unique exposure pattern to ultraviolet radiation. Full article

(This article belongs to the Special Issue Uveal Melanoma)

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16 pages, 1893 KiB

Open AccessArticle

ROR1 Potentiates FGFR Signaling in Basal-Like Breast Cancer

by Gaurav Pandey, Nicholas Borcherding, Ryan Kolb, Paige Kluz, Wei Li, Sonia Sugg, Jun Zhang, Dazhi A. Lai and Weizhou Zhang

Cancers 2019, 11(5), 718; https://doi.org/10.3390/cancers11050718 - 24 May 2019

Cited by 11 | Viewed by 5845 | Correction

Abstract

Among all breast cancer types, basal-like breast cancer (BLBC) represents an aggressive subtype that lacks targeted therapy. We and others have found that receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in BLBC and other types of cancer and that ROR1 is [...] Read more.

Among all breast cancer types, basal-like breast cancer (BLBC) represents an aggressive subtype that lacks targeted therapy. We and others have found that receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in BLBC and other types of cancer and that ROR1 is significantly correlated with patient prognosis. In addition, using primary patient-derived xenografts (PDXs) and ROR1-knockout BLBC cells, we found that ROR1⁺ cells form tumors in immunodeficient mice. We developed an anti-ROR1 immunotoxin and found that targeting ROR1 significantly kills ROR1⁺ cancer cells and slows down tumor growth in ROR1⁺ xenografts. Our bioinformatics analysis revealed that ROR1 expression is commonly associated with the activation of FGFR-mediated signaling pathway. Further biochemical analysis confirmed that ROR1 stabilized FGFR expression at the posttranslational level by preventing its degradation. CRISPR/Cas9-mediated ROR1 knockout significantly reduced cancer cell invasion at cellular levels by lowering FGFR protein and consequent inactivation of AKT. Our results identified a novel signaling regulation from ROR1 to FGFR and further confirm that ROR1 is a potential therapeutic target for ROR1⁺ BLBC cells. Full article

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Figure 1
Higher expression of ROR1 is correlated with poor overall survival in several cancers. (a) Cox proportional hazard regression summary of hazard ratios and –log10 (p-value) across the TCGA Pan Can cohort. Labeled points indicate significance of p < 0.05, which is also marked by the horizontal dotted line. (b) Z-score transformed mRNA for ROR1 across PAM50 subtypes in the breast cancer TCGA cohort. Samples compared using one-way ANOVA test. (c) KM plotter was used to analyze ROR1 expression and prognosis. Using ROR1 max probe: 205805_s_at and optimal cutoff, ROR1 mRNA is inversely correlated with distal metastasis free survival (DMFS) in all breast cancer patients (n = 1311 in the ROR1-high group and n = 435 in the low group, p = 0.0036). Full article ">Figure 2
ROR1+ cells are tumorigenic. (a–c) One clinical TNBC specimen was stained with cytokeratin-5 (KRT-5) for defining basal cancer type and with ROR1 for its positivity (a). A fresh TNBC with confirmed basal type was digested into single cell suspensions, following with flow cytometry to separate into ROR1+ and ROR1− epithelial (labeled by EpCAM+) populations (b). 5000 ROR1+ or ROR1- TNBC cells were orthotopically injected into 7-week old immunodeficient female NSG mice for tumor growth, with one #4 fatpad injected with ROR1+ and the other #4 fatpad with ROR1− cells for stringent comparison. Tumors were monitored (c, shown as mean ± SD, n = 4). (d,e) CRISPR/Cas9-mediated KO of ROR1 in MDA-MB-231 cells with two clones shown as negative of surface ROR1 staining by flow cytometry (d) and 2 million of WT and KO MDA-MB-231 cells were injected into #4 fatpad of NSG mice and tumor growth were measured (e, shown as mean ± SD, n = 7–8, p < 0.001, 2-way ANOVA). Full article ">Figure 3
Targeting ROR1+ cancer cells with immunotoxin. (a,b) Construction and purification of anti-ROR1-immunotoxin by fusing variable region of anti-human ROR1 antibody (clone 2A2) with modified exotoxin A (PElo10) from Pseudomonas aeruginosa (a), using variable region of MOPC21 (a non-specific mouse antibody)-fused with PElo10 as control. Plasmids encoding ROR1-IT or MOPC21-IT were used to transform E. coli for purification (b). (c) A panel of breast epithelial or cancer cells were screened for ROR1 expression by flow cytometry, including immortalized HMLE cells, three ROR1-negative (BT-474 and AU-565) or low (MDA-MB-436), and two ROR1-positive (MDA-MB-468 and Hs578T) cells. (d) Cells in c. were treated with different doses of ROR1-IT or MOPC-21-IT (0, 40, 200, 1000, 5000 ng/mL). 48 h later, cells were collected and stained with 7-AAD for viability and quantitated by flow cytometry (n = 3, only data for ROR1-IT was shown here). (e) WT or KO cells of MDA-MB-231, ROR1+ Hs578T, or ROR1− AU-565 cells were treated with 400 ng/mL of ROR1-IT or MOPC-21-IT for 48 h, following with counting of viable cells. ROR1-IT-treated cells were normalized to their corresponding MOPC-21-IT-treated cells (n = 3). * p < 0.05; ** p < 0.001. (f,g) ROR1+ Hs578T cells were injected orthotopically into NSG mice and treated with MOPC21-IT or ROR1-IT daily at 100 µg/injection for 4 weeks. Body weight (f) and tumor volume (g) were monitored weekly (n = 3). Full article ">Figure 4
ROR1 is associated with FGFR signaling across different cancers. (a–c) Stomach adenocarcinoma (STAD: data from TCGA), triple-negative breast cancer (BrCa: data from GSE76275), ovarian (OV: data from TCGA) and prostate adenocarcinoma (PRAD: data from TCGA) were separated into low and high ROR1 expressing groups (77 samples with the lowest expression vs. 77 samples with the highest expression) and gene set enrichment analysis was performed. (a) Venn diagram of genesets significantly enriched (FDR < 0.25) in the ROR1 high group from each dataset. (b) Genesets related to FGFR and PDGFR receptor tyrosine kinase signaling pathways from the common 377 genesets in (a) are indicated. (c) Enrichment plot for FGFR geneset showing enrichment in ROR1-high expressing group in the BrCa TNBC dataset. (d) Ingenuity Pathway Analysis (IPA) for upstream regulators comparing the ROR1high versus ROR1low tertiles in the BLBC samples in the TCGA. Labeled regulators have a –log10 (p-value) > 1.3, the equivalent of p < 0.05, and bias-corrected Z-scores > 1.2. Full article ">Figure 5
ROR1 stabilizes FGFR1 protein level. (a) CRISPR/Cas9-mediated KO of ROR1 in MDA-MB-231 cells with two clones shown as ROR1-KO clones. Representative lanes are shown from immunoblots of cell lysate probed with the antibody against EGFR, FGFR1, ROR1, and β-ACTIN was used as loading control. (b) Real-time PCR analysis of FGFR1, EGFR1, and ERBB3 (HER3) in MDA-MB-231 ROR1-WT or KO cells. (c) ROR1− cells were transfected with ROR1 plasmid for 48 h. Representative lanes are shown from immunoblots of cell lysate probed with the antibody against ROR1, FGFR1, and β-ACTIN was used as loading control. (d) Cells were transfected with scrambled control siRNA and Cav-1 (CAV1) siRNA for 48 h. Representative lanes are shown from immunoblots of cell lysate probed with the antibody against CAV-1, FGFR1, p-AKT and β-ACTIN was used as loading control. (e,f) Cells were treated with MG-132 (e) or chloroquine (f, CHL) for 4 h, or mock-treated with DMSO (e,f). Representative lanes are shown from immunoblots of cell lysate probed with the antibody against FGFR1 and β-ACTIN was used as loading control. For all western blots, summary of band densities, normalized to β-ACTIN (n = 3). (g,h) Listed cell lines were treated with 200 ng/mL of ROR1-IT or MOPC21-IT for the indicated periods. The 0 h control were lysates from MOPC21-IT-treated cells for 24 h. Cell lysates were collected in, following with SDS-PAGE and immunoblotting of pFGFR1, FGFR1, ROR1, pAKT, AKT and β-ACTIN was used as loading control. Con or ROR1 in MCF7 groups refers to MCF7 cells that stably express empty plasmid or ROR1-encoding plasmid. MDA-468 refers to MDA-MB-468 cells. Full article ">Figure 6
ROR1 regulates invasion through FGFR1 in BLBC cells. (a) Cells in <a href="#cancers-11-00718-f005" class="html-fig">Figure 5</a>a were used for invasion assay. Scale bar represents 200 μm. (b) The graph represents the average cell number/microscopic field (n = 3). (c,d) WT or KO Cells were plated for invasion assay and treated with control DMSO, SB-431542 (5 µM), PD173074 (50 nM), ruxolitinib (10 µM), or lapatinib (100 nM) for 24 h. Representative images were shown (c) and quantitated (d, shown as mean ± SD, n = 3–4). (e) WT or KO Cells were treated with same set of inhibitors for 2 hours in the complete media. Cells lysates were collected for immunoblotting with pAKT and AKT, with β-ACTIN as a loading control. (f) WT or KO Cells were plated for invasion assay and treated with control DMSO or AKT inhibitor MK2206 for 24 h and quantitated. Data shown as mean ± SD (n = 3), * p < 0.05, ** p < 0.01. Full article ">

15 pages, 989 KiB

Open AccessArticle

A Multidisciplinary Team Guided Approach to the Management of cT3 Laryngeal Cancer: A Retrospective Analysis of 104 Cases

by Filippo Marchi, Marta Filauro, Francesco Missale, Giampiero Parrinello, Fabiola Incandela, Almalina Bacigalupo, Stefania Vecchio, Cesare Piazza and Giorgio Peretti

Cancers 2019, 11(5), 717; https://doi.org/10.3390/cancers11050717 - 24 May 2019

Cited by 14 | Viewed by 4457

Abstract

The optimal treatment for T3 laryngeal carcinoma (LC) is still a matter of debate. Different therapeutic options are available: Transoral laser microsurgery (TLM), open partial horizontal laryngectomies (OPHLs), total laryngectomy (TL), and organ preservation protocols (radiation therapy (RT) or chemo-radiation (CRT)). This study [...] Read more.

The optimal treatment for T3 laryngeal carcinoma (LC) is still a matter of debate. Different therapeutic options are available: Transoral laser microsurgery (TLM), open partial horizontal laryngectomies (OPHLs), total laryngectomy (TL), and organ preservation protocols (radiation therapy (RT) or chemo-radiation (CRT)). This study aimed to retrospectively evaluate oncologic outcomes of 104 T3 LCs treated by surgery or non-surgical approaches from January 2011 to December 2016 at a single academic tertiary referral center. Each case was evaluated by a multidisciplinary team (MDT) devoted to the management of head and neck cancers. We divided the cohort into two subgroups: Group A, surgical treatment (TLM, OPHLs, TL) and Group B, non-surgical treatment (RT, CRT). For the entire cohort, two- and five-year overall survival (OS) rates were 83% and 56%, respectively. The two- and five-year disease-free survival (DFS) rates were 75% and 65%, and disease-specific survival rates were 93% and 70%, respectively. The N category was a significant independent prognosticator for OS (p = 0.02), whereas Group B was significantly and independently associated with DFS (HR 4.10, p = 0.006). Analyzing laryngo-esophageal dysfunction-free survival as an outcome, it was found that this was significantly lower in higher N categories (p = 0.04) and in cases that underwent non-surgical treatments (p = 0.002). Optimization of oncologic outcomes in T3 LCs may be obtained only by a comprehensive MDT approach, considering that different treatment options have heterogenous toxicity profiles and indications. Full article

(This article belongs to the Special Issue Emerging Concepts in Treatment of Laryngeal Cancer)

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21 pages, 4687 KiB

Open AccessArticle

On the Impact of Chemo-Mechanically Induced Phenotypic Transitions in Gliomas

by Pietro Mascheroni, Juan Carlos López Alfonso, Maria Kalli, Triantafyllos Stylianopoulos, Michael Meyer-Hermann and Haralampos Hatzikirou

Cancers 2019, 11(5), 716; https://doi.org/10.3390/cancers11050716 - 24 May 2019

Cited by 11 | Viewed by 4605

Abstract

Tumor microenvironment is a critical player in glioma progression, and novel therapies for its targeting have been recently proposed. In particular, stress-alleviation strategies act on the tumor by reducing its stiffness, decreasing solid stresses and improving blood perfusion. However, these microenvironmental changes trigger [...] Read more.

Tumor microenvironment is a critical player in glioma progression, and novel therapies for its targeting have been recently proposed. In particular, stress-alleviation strategies act on the tumor by reducing its stiffness, decreasing solid stresses and improving blood perfusion. However, these microenvironmental changes trigger chemo–mechanically induced cellular phenotypic transitions whose impact on therapy outcomes is not completely understood. In this work we analyze the effects of mechanical compression on migration and proliferation of glioma cells. We derive a mathematical model of glioma progression focusing on cellular phenotypic plasticity. Our results reveal a trade-off between tumor infiltration and cellular content as a consequence of stress-alleviation approaches. We discuss how these novel findings increase the current understanding of glioma/microenvironment interactions and can contribute to new strategies for improved therapeutic outcomes. Full article

(This article belongs to the Collection Targeting Solid Tumors)

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13 pages, 5205 KiB

Open AccessReview

Integrin Regulation of CAF Differentiation and Function

by C. Michael DiPersio and Livingston Van De Water

Cancers 2019, 11(5), 715; https://doi.org/10.3390/cancers11050715 - 24 May 2019

Cited by 21 | Viewed by 5361

Abstract

Extensive remodeling of the extracellular matrix, together with paracrine communication between tumor cells and stromal cells, contribute to an “activated” tumor microenvironment that supports malignant growth and progression. These stromal cells include inflammatory cells, endothelial cells, and cancer-associated fibroblasts (CAFs). Integrins are expressed [...] Read more.

Extensive remodeling of the extracellular matrix, together with paracrine communication between tumor cells and stromal cells, contribute to an “activated” tumor microenvironment that supports malignant growth and progression. These stromal cells include inflammatory cells, endothelial cells, and cancer-associated fibroblasts (CAFs). Integrins are expressed on all tumor and stromal cell types where they regulate both cell adhesion and bidirectional signal transduction across the cell membrane. In this capacity, integrins control pro-tumorigenic cell autonomous functions such as growth and survival, as well as paracrine crosstalk between tumor cells and stromal cells. The myofibroblast-like properties of cancer-associated fibroblasts (CAFs), such as robust contractility and extracellular matrix (ECM) deposition, allow them to generate both chemical and mechanical signals that support invasive tumor growth. In this review, we discuss the roles of integrins in regulating the ability of CAFs to generate and respond to extracellular cues in the tumor microenvironment. Since functions of specific integrins in CAFs are only beginning to emerge, we take advantage of a more extensive literature on how integrins regulate wound myofibroblast differentiation and function, as some of these integrin functions are likely to extrapolate to CAFs within the tumor microenvironment. In addition, we discuss the roles that integrins play in controlling paracrine signals that emanate from epithelial/tumor cells to stimulate fibroblasts/CAFs. Full article

(This article belongs to the Special Issue The Role of Integrins in Cancer)

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17 pages, 674 KiB

Open AccessReview

Targeting the Interplay between Epithelial-to-Mesenchymal-Transition and the Immune System for Effective Immunotherapy

by Rama Soundararajan, Jared J. Fradette, Jessica M. Konen, Stacy Moulder, Xiang Zhang, Don L. Gibbons, Navin Varadarajan, Ignacio I. Wistuba, Debasish Tripathy, Chantale Bernatchez, Lauren A. Byers, Jeffrey T. Chang, Alejandro Contreras, Bora Lim, Edwin Roger Parra, Emily B. Roarty, Jing Wang, Fei Yang, Michelle Barton, Jeffrey M. Rosen and Sendurai A. Mani Show full author list Hide full author list

Cancers 2019, 11(5), 714; https://doi.org/10.3390/cancers11050714 - 24 May 2019

Cited by 88 | Viewed by 7304

Abstract

Over the last decade, both early diagnosis and targeted therapy have improved the survival rates of many cancer patients. Most recently, immunotherapy has revolutionized the treatment options for cancers such as melanoma. Unfortunately, a significant portion of cancers (including lung and breast cancers) [...] Read more.

Over the last decade, both early diagnosis and targeted therapy have improved the survival rates of many cancer patients. Most recently, immunotherapy has revolutionized the treatment options for cancers such as melanoma. Unfortunately, a significant portion of cancers (including lung and breast cancers) do not respond to immunotherapy, and many of them develop resistance to chemotherapy. Molecular characterization of non-responsive cancers suggest that an embryonic program known as epithelial-mesenchymal transition (EMT), which is mostly latent in adults, can be activated under selective pressures, rendering these cancers resistant to chemo- and immunotherapies. EMT can also drive tumor metastases, which in turn also suppress the cancer-fighting activity of cytotoxic T cells that traffic into the tumor, causing immunotherapy to fail. In this review, we compare and contrast immunotherapy treatment options of non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC). We discuss why, despite breakthrough progress in immunotherapy, attaining predictable outcomes in the clinic is mostly an unsolved problem for these tumors. Although these two cancer types appear different based upon their tissues of origin and molecular classification, gene expression indicate that they possess many similarities. Patient tumors exhibit activation of EMT, and resulting stem cell properties in both these cancer types associate with metastasis and resistance to existing cancer therapies. In addition, the EMT transition in both these cancers plays a crucial role in immunosuppression, which exacerbates treatment resistance. To improve cancer-related survival we need to understand and circumvent, the mechanisms through which these tumors become therapy resistant. In this review, we discuss new information and complementary perspectives to inform combination treatment strategies to expand and improve the anti-tumor responses of currently available clinical immune checkpoint inhibitors. Full article

(This article belongs to the Special Issue Towards New Promising Discoveries for Lung Cancer Patients: A Selection of Papers from the First Joint Meeting on Lung Cancer of the FHU OncoAge (Nice, France) and the MD Anderson Cancer Center (Houston, TX, USA))

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11 pages, 1174 KiB

Open AccessArticle

Potential Prognostic Role of ¹⁸F-FDG PET/CT in Invasive Epithelial Ovarian Cancer Relapse. A Preliminary Study

by Anna Myriam Perrone, Giulia Dondi, Giacomo Maria Lima, Paolo Castellucci, Marco Tesei, Sara Coluccelli, Giuseppe Gasparre, Anna Maria Porcelli, Cristina Nanni, Stefano Fanti and Pierandrea De Iaco

Cancers 2019, 11(5), 713; https://doi.org/10.3390/cancers11050713 - 23 May 2019

Cited by 11 | Viewed by 4337

Abstract

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with relapse occurring in about 70% of advanced cases with poor prognosis. Fluorine-18-2-fluoro-2-deoxy-d-glucose PET/CT (¹⁸F-FDGPET/CT) is the most specific radiological imaging used to assess recurrence. Some intensity-based and volume-based PET parameters, [...] Read more.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with relapse occurring in about 70% of advanced cases with poor prognosis. Fluorine-18-2-fluoro-2-deoxy-d-glucose PET/CT (¹⁸F-FDGPET/CT) is the most specific radiological imaging used to assess recurrence. Some intensity-based and volume-based PET parameters, maximum standardized uptake values (SUV_max), metabolic tumor volume (MTV) and total lesion glycolysis (TLG), are indicated to have a correlation with treatment response. The aim of our study is to correlate these parameters with post relapse survival (PRS) and overall survival (OS) in Epithelial Ovarian Cancer (EOC) relapse. The study included 50 patients affected by EOC relapse who underwent ¹⁸F-FDGPET/CT before surgery. All imaging was reviewed and SUV_max, MTV and TLG were calculated and correlated to PRS and OS. PRS and OS were obtained from the first relapse and from the first diagnosis to the last follow up or death, respectively. SUV_max, MTV and TLG were tested in a univariate logistic regression analysis, only SUV_max demonstrated to be significantly associated to PRS and OS (p = 0.005 and p = 0.024 respectively). Multivariate analysis confirmed the results. We found a cut-off of SUV_max of 13 that defined worse or better survival (p = 0.003). In the first relapse of EOC, SUV_max is correlated to PRS and OS, and when SUV_max is greater than 13, it is an unfavorable prognostic factor. Full article

(This article belongs to the Special Issue Role of Medical Imaging in Cancers)

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13 pages, 2278 KiB

Open AccessArticle

Huzhangoside A Suppresses Tumor Growth through Inhibition of Pyruvate Dehydrogenase Kinase Activity

by Choong-Hwan Kwak, Jung-Hee Lee, Eun-Yeong Kim, Chang Woo Han, Keuk-Jun Kim, Hanna Lee, MyoungLae Cho, Se Bok Jang, Cheorl-Ho Kim, Tae-Wook Chung and Ki-Tae Ha

Cancers 2019, 11(5), 712; https://doi.org/10.3390/cancers11050712 - 23 May 2019

Cited by 15 | Viewed by 5050

Abstract

Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1α subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for [...] Read more.

Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1α subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for cancer treatment. Here, we report that huzhangoside A (Hu.A), a triterpenoid glycoside compound isolated from several plants of the Anemone genus, acts as a novel PDHK inhibitor. Hu.A was found to decrease the cell viability of human breast cancer MDA-MB-231, hepatocellular carcinoma Hep3B, colon cancer HT-29, DLD-1, and murine lewis lung carcinoma LLC cell lines. The activity of PDHK1 was decreased by Hu.A in both in vitro assays and in vivo assays in DLD-1 cells. Hu.A significantly increased the oxygen consumption and decreased the secretory lactate levels in DLD-1 cells. In addition, Hu.A interacted with the ATP-binding pocket of PDHK1 without affecting the interaction of PDHK1 and pyruvate dehydrogenase complex (PDC) subunits. Furthermore, Hu.A significantly induced mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in DLD-1 cells. Consistently, when Hu.A was intraperitoneally injected into LLC allograft mice, the tumor growth was significantly decreased. In conclusion, Hu.A suppressed the growth of tumors in both in vitro and in vivo models via inhibition of PDHK activity. Full article

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Huzhangoside A (Hu.A) decreased the cell viability of several cancer cell lines. (A) Chemical structure of Hu.A was indicated. (B) The indicated cells were treated with Hu.A for 24 h and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. The cell viability is shown as mean ± standard deviations (SD). **, p < 0.01 and ***, p < 0.001 compared with control. Full article ">Figure 2
Hu.A reduced the PDHK1 activity and promoted oxidative phosphorylation (OXPHOS) in DLD-1 cells. (A) In vitro PDHK1 kinase assay was performed. (B, C) DLD-1 cells were treated with Hu.A in serum-free medium for 4 h. The levels of phosphorylated pyruvate dehydrogenase E1α subunit (PDHA) (B), and PDHK1-4 (C) were analyzed using western blot assay. PDHA (B) and GAPDH (C) were used as loading controls. (D) The intensity of bands (PDHK1-4/GAPDH) from three independent experiments was measured and indicated by mean ± SD. (E) DLD-1 cells were treated with Hu.A in serum-free medium for 6 h. O2 consumption rate was measured by using commercially available Oxygen Consumption Rate Assay Kit. (F) Lactate production was measured by lactate fluorometric assay kit (right panel). The data are shown as mean ± SD, respectively. **, p < 0.01 compared with control. Full article ">Figure 3
Hu.A interacted with the ATP-binding pocket of PDHK1. (A) The modeled structure of PDHK1 (PDB ID: 2Q8F) with Hu.A (CID: 73347426) around the ATP-binding domain is shown as a ribbon representation. The interaction residues between PDHK1 and Hu.A are shown, and hydrogen bonds are shown as black dotted lines. (B) The complex structure of PDHK1 with Hu.A was predicted as a surface representation. The relative distribution of the electrostatic surface of PDHK1 is shown with the acidic region in red, basic region in blue, and neutral region in white. (C) ATP-binding assay of PDHK1 in the presence or absence of Hu.A is shown. [α-32P]ATP-bound values were measured using scintillation counter. The results were calculated as percentage values in comparison to control and shown as mean ± SD. ***, p < 0.01 compared with positive control group (2nd lane). (D) PDC subunits and PDHK-binding assay was performed. (E) The bands intensity of panel (D) was indicated. The data are indicated as mean ± SD. NS, not significant. Full article ">Figure 4
Hu.A induced mitochondrial reactive oxygen species (ROS) production and mitochondrial damage in DLD-1 cells. DLD-1 cells were treated with Hu.A for indicated time points in serum-free condition. The cells were stained with MitoSOX™ (Thermo Fisher Scientific, Rockford, IL, USA) Red (A and B), and tetramethylrhodamine methyl ester (TMRM) (C and D). The fluorescence for both assays was measured by FACS analysis. The relative mitochondrial ROS and TMRM fluorescence levels were calculated to determine the fold difference in comparison to control and are shown as mean ± SD. ***, p < 0.001. (E) In the serum-free condition, the cells were pre-treated with MitoTEMPO (Sigma-Aldrich, St. Louis, MO, USA) (50 μM) for 1 h and treated with 3 μM of Hu.A for 6 h. MTT assay was then performed. Data are indicated as mean ± SD. ***, p < 0.001 compared with the only Hu.A-treated group and MitoTEMPO with Hu.A co-treated group. Full article ">Figure 5
Hu.A induced apoptosis in DLD-1 cells. (A, and B) The cells were stained with annexin V-PI and were analyzed using FACS. The data are shown as mean ± SD, respectively. ***, p < 0.01 compared with control. (C) The expression of caspase-3, caspase-9, and poly ADP-ribose polymerase (PARP) in the cells was analyzed by western blot assay. GAPDH was used as a loading control. Full article ">Figure 6
Hu.A reduced the tumor growth in vivo in LLC cell allograft mice model. The LLC cells were injected into the dorsal subcutaneous skin of C57BL/6 mice. On the 14th day, Hu.A at concentrations of 0.1, 0.5 and 1 mg/kg/day were intraperitoneally injected into the mice. (A) The representative picture of the tumors is shown. The tumor volume (B) and weight (C) were measured. Tumor volume and weight are indicated as mean ± SD. **, p < 0.01 and ***, p < 0.001 compared with the control group. (D) The tumor tissues were stained with the Ki-67 antibody and examined at ×200 magnification. (E) Ki-67 expression index was determined with Aperio Image scope software for three separate tumors. The data are shown as mean ± SD, respectively. *, p < 0.05 compared with control. (F) The expression of cleaved-caspase-3, caspase-9, and PARP in the tumor tissues were analyzed by western blot assay. GAPDH was used as a loading control. (G) The levels of phosphor-PDHA and total PDHA in the representative tumors were analyzed by western blot assay. Full article ">

19 pages, 10627 KiB

Open AccessArticle

Novel Thienopyrimidine Derivative, RP-010, Induces ?-Catenin Fragmentation and Is Efficacious against Prostate Cancer Cells

by Haneen Amawi, Noor Hussein, Sai H. S. Boddu, Chandrabose Karthikeyan, Frederick E. Williams, Charles R. Ashby, Jr., Dayanidhi Raman, Piyush Trivedi and Amit K. Tiwari

Cancers 2019, 11(5), 711; https://doi.org/10.3390/cancers11050711 - 23 May 2019

Cited by 17 | Viewed by 4760

Abstract

Thienopyrimidines containing a thiophene ring fused to pyrimidine are reported to have a wide-spectrum of anticancer efficacy in vitro. Here, we report for the first time that thieno[3,2-d]pyrimidine-based compounds, also known as the RP series, have efficacy in prostate cancer cells. [...] Read more.

Thienopyrimidines containing a thiophene ring fused to pyrimidine are reported to have a wide-spectrum of anticancer efficacy in vitro. Here, we report for the first time that thieno[3,2-d]pyrimidine-based compounds, also known as the RP series, have efficacy in prostate cancer cells. The compound RP-010 was efficacious against both PC-3 and DU145 prostate cancer (PC) cells (IC₅₀ < 1 µM). The cytotoxicity of RP-010 was significantly lower in non-PC, CHO, and CRL-1459 cell lines. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in G2 phase of the cell cycle, and induced mitotic catastrophe and apoptosis in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) affected the wingless-type MMTV (Wnt)/β-catenin signaling pathway, in association with β-catenin fragmentation, while also downregulating important proteins in the pathway, including LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the Wnt pathway. In addition, RP-010 (0.5, 1, 2 and 4 µM) significantly decreased the migration of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations of up to 6 µM. In conclusion, RP-010 may be an efficacious and relatively nontoxic anticancer compound for prostate cancer. Future mechanistic and in vivo efficacy studies are needed to optimize the hit compound RP-010 for lead optimization and clinical use. Full article

(This article belongs to the Special Issue Targeting Wnt Signaling in Cancer)

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RP-010 cytotoxicity to prostate cancer cells. (a) An illustration of the chemical structures of the thirteen RP compounds. (b) RP-010 cytotoxicity to prostate cancer cells (DU145 and PC-3), as represented by survival curves (lower panel), and IC50 values, compared to non-malignant CRL-1459 cells (upper panel). (c) Representative pictures of the morphological changes in cells incubated with RP-010 (0.1, 0.3, or 1 µM), or vehicle, for 72 h. (d) Colony formation assay showing the effect of RP-010 or vehicle (1 or 2 µM) on the colony density (10×) and size (20×) of DU145 cells. All results are presented as the means ± SDs of three independent experiments. *** p < 0.001. Full article ">Figure 2
The changes induced by RP-010 on the cell cycle and nuclear events. (a) Analysis of RP-010 (0, 0.5, 1, or 2 µM)-induced changes on the cell cycle, using a flow cytometry assay (propidium iodide, “PI,” on the ordinate, and cell count on the abcissa). A graph showing the percent change for each phase, following incubation with RP-010, is shown on the right. In (b) and (c) the effects of RP-010 (1, 2 or 4 µM) and vehicle on events in the nuclei of DU145 cells, at 24 and 48 h, respectively, are shown. Both chromatin condensation and mitotic catastrophe can be seen. Full article ">Figure 3
RP-010 activates the intrinsic apoptosis pathway. (Left) The effect of RP-010 (1 or 2 µM, at 24 h incubation) or vehicle on the expression levels of the apoptotis-regulating proteins Bak, Bcl2, caspase-3, cleaved caspase-3, cytosolic (“Cyto”) and nuclear (“Nu”) poly ADP-ribose polymerase (PARP), cytosolic and nuclear cleaved PARP, and cytochrome c, are shown. β-actin and histone were used as reference proteins. (Right) The histograms represent a quantitative summary of the results. (R) = relative. ** p < 0.01, *** p < 0.001. Full article ">Figure 4
RP-010 significantly reduces cell migration in DU145 cells. (a) Results of wound healing assays following incubation with RP-010 (0.5, 1, 2, or 4 µM) or vehicle, with the histogram (right panel) representing a quantitative summary of the results; (b) Cell migration following incubation with RP-010 (0.5-, 1- or 2-μM) or vehicle. The histogram (right panel) represents a summary of the results. ** p < 0.01, *** p < 0.001. The micrograph images were taken at 4× (scale bar: 1/3rd width/box is 1000 μm). Full article ">Figure 5
RP-010 induces significant changes in Wnt/ β-catenin signaling proteins. (Left) The effect of RP-010 (1 or 2 µM, after 24 h of incubation), or vehicle, on the expression levels of the following proteins: Wnt 5a, DVL3, LRP-6, P-LRP-6, cytosolic and nuclear Naked1 and 2, cytosolic and nuclear cyclin B, c-Myc, and β-catenin (bottom panel). β-actin and histone were used as reference proteins. The histograms (right panels) represent quantitative summaries of the results. (R) = relative. * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 6
RP-010 toxicity in an in vivo zebrafish model. The effect of RP-010 (0, 0.3, 1, 3, 6, or 10 µM), or vehicle treatment, on the bodies (left) and tails (right) of zebrafish, after (a) 24- or (b) 48-h drug exposure. Scale bar: Images were taken at 4× (scale bar: 1000 μm) and zoomed to 10× (scale bar: 400 μm). Full article ">

14 pages, 1725 KiB

Open AccessFeature PaperReview

Detection Rate of ¹⁸F-Labeled PSMA PET/CT in Biochemical Recurrent Prostate Cancer: A Systematic Review and a Meta-Analysis

by Giorgio Treglia, Salvatore Annunziata, Daniele A. Pizzuto, Luca Giovanella, John O. Prior and Luca Ceriani

Cancers 2019, 11(5), 710; https://doi.org/10.3390/cancers11050710 - 23 May 2019

Cited by 83 | Viewed by 6704

Abstract

Background: The use of radiolabeled prostate-specific membrane antigen positron emission tomography/computed tomography (PSMA PET/CT) for biochemical recurrent prostate cancer (BRPCa) is increasing worldwide. Recently, ¹⁸F-labeled PSMA agents have become available. We performed a systematic review and meta-analysis regarding the detection rate [...] Read more.

Background: The use of radiolabeled prostate-specific membrane antigen positron emission tomography/computed tomography (PSMA PET/CT) for biochemical recurrent prostate cancer (BRPCa) is increasing worldwide. Recently, ¹⁸F-labeled PSMA agents have become available. We performed a systematic review and meta-analysis regarding the detection rate (DR) of ¹⁸F-labeled PSMA PET/CT in BRPCa to provide evidence-based data in this setting. Methods: A comprehensive literature search of PubMed/MEDLINE, EMBASE, and Cochrane Library databases through 23 April 2019 was performed. Pooled DR was calculated on a per-patient basis, with pooled proportion and 95% confidence interval (95% CI). Furthermore, pooled DR of ¹⁸F-PSMA PET/CT using different cut-off values of prostate-specific antigen (PSA) was obtained. Results: Six articles (645 patients) were included in the meta-analysis. The pooled DR of ¹⁸F-labeled PSMA PET/CT in BRPCa was 81% (95% CI: 71–88%). The pooled DR was 86% for PSA ≥ 0.5 ng/mL (95% CI: 78–93%) and 49% for PSA < 0.5 ng/mL (95% CI: 23–74%). Statistical heterogeneity was found. Conclusions: ¹⁸F-labeled PSMA PET/CT demonstrated a good DR in BRPCa. DR of ¹⁸F-labeled PSMA PET/CT is related to PSA values with significant lower DR in patients with PSA < 0.5 ng/mL. Prospective multicentric trials are needed to confirm these findings. Full article

(This article belongs to the Special Issue Role of Medical Imaging in Cancers)

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13 pages, 1229 KiB

Open AccessArticle

Continued Weight Loss and Sarcopenia Predict Poor Outcomes in Locally Advanced Pancreatic Cancer Treated with Chemoradiation

by Patrick Naumann, Jonathan Eberlein, Benjamin Farnia, Thilo Hackert, Jürgen Debus and Stephanie E. Combs

Cancers 2019, 11(5), 709; https://doi.org/10.3390/cancers11050709 - 23 May 2019

Cited by 35 | Viewed by 4205

Abstract

Background: Surgical resection offers the best chance of survival in patients with pancreatic cancer, but those with locally advanced disease (LAPC) are usually not surgical candidates. This cohort often receives either neoadjuvant chemotherapy or chemoradiation (CRT), but unintended weight loss coupled with [...] Read more.

Background: Surgical resection offers the best chance of survival in patients with pancreatic cancer, but those with locally advanced disease (LAPC) are usually not surgical candidates. This cohort often receives either neoadjuvant chemotherapy or chemoradiation (CRT), but unintended weight loss coupled with muscle wasting (sarcopenia) can often be observed. Here, we report on the predictive value of changes in weight and muscle mass in 147 consecutive patients with LAPC treated with neoadjuvant CRT. Methods: Clinicopathologic data were obtained via a retrospective chart review. The abdominal skeletal muscle area (SMA) at the third lumbar vertebral body was determined via computer tomographic (CT) scans as a surrogate for the muscle mass and skeletal muscle index (SMI) calculated. Uni- and multi-variable statistical tests were performed to assess for impact on survival. Results: Weight loss (14.5 vs. 20.3 months; p = 0.04) and loss of muscle mass (15.1 vs. 22.2 months; p = 0.007) were associated with poor outcomes. The highest survival was observed in patients who had neither cachectic weight loss nor sarcopenia (27 months), with improved survival seen in those who ultimately received a resection (23 vs. 10 months; p < 0.001). Cox regression revealed that either continued weight loss or continued muscle wasting (SMA reduction) was predictive of poor outcomes, whereas a sarcopenic SMI was not. Conclusions: Loss of weight and lean muscle in patients with LAPC is prognostic when persistent. Therefore, both should be assessed longitudinally and considered before surgery. Full article

(This article belongs to the Special Issue Advances in Pancreatic Cancer Research)

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Absolute (A) and relative (B) changes in weight and skeletal muscle area (SMA) measured prior to treatment initiation, and at the time of planning CT for radiation (RT) and first follow-up (FU). (C) Changes in relative weight loss and (D) skeletal muscle index (SMI) according to gender. Full article ">Figure 2
Distribution and combination of weight loss and sarcopenia observed between planning CT and first follow-up. Full article ">Figure 3
Distribution of National Comprehensive Cancer Network (NCCN) Common Toxicity Criteria (CTC) grades for nausea, emesis and diarrhea during chemoradiation (CRT) grouped according to the presence or absence of cachectic weight loss and sarcopenia, respectively. Full article ">Figure 4
(A) Average overall survival according to status of cachectic weight loss and/or sarcopenia. (B) Kaplan–Meier survival curves grouped according to the extent of surgical resection and (C) in combination with or without cachexia as well as by gender. Full article ">Figure 5
Uni- and multivariable Cox regression analysis, with hazard ratios (HR) and a 95% confidence interval (CI). Factors with p-values < 0.15 in univariable cox regression were selected for multivariable analysis. Full article ">

14 pages, 3714 KiB

Open AccessArticle

Down-Regulation of Cannabinoid Type 1 (CB1) Receptor and its Downstream Signaling Pathways in Metastatic Colorectal Cancer

by Valeria Tutino, Maria Gabriella Caruso, Valentina De Nunzio, Dionigi Lorusso, Nicola Veronese, Isabella Gigante, Maria Notarnicola and Gianluigi Giannelli

Cancers 2019, 11(5), 708; https://doi.org/10.3390/cancers11050708 - 22 May 2019

Cited by 21 | Viewed by 4743

Abstract

Changes in the regulation of endocannabinoid production, together with an altered expression of their receptors are hallmarks of cancer, including colorectal cancer (CRC). Although several studies have been conducted to understand the biological role of the CB1 receptor in cancer, little is known [...] Read more.

Changes in the regulation of endocannabinoid production, together with an altered expression of their receptors are hallmarks of cancer, including colorectal cancer (CRC). Although several studies have been conducted to understand the biological role of the CB1 receptor in cancer, little is known about its involvement in the metastatic process of CRC. The aim of this study was to investigate the possible link between CB1 receptor expression and the presence of metastasis in patients with CRC, investigating the main signaling pathways elicited downstream of CB1 receptor in colon cancer. Fifty-nine consecutive patients, with histologically proven colorectal cancer, were enrolled in the study, of which 30 patients with synchronous metastasis, at first diagnosis and 29 without metastasis. A low expression of CB1 receptor were detected in primary tumor tissue of CRC patients with metastasis and consequently, we observed an alteration of CB1 receptor downstream signaling. These signaling routes were also altered in intestinal normal mucosa, suggesting that, normal mucosa surrounding the tumor provides a realistic picture of the molecules involved in tissue malignant transformation. These observations contribute to the idea that drugs able to induce CB1 receptor expression can be helpful in order to set new anticancer therapeutic strategies. Full article

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12 pages, 417 KiB

Open AccessArticle

NF1 Patients Receiving Breast Cancer Screening: Insights from The Ontario High Risk Breast Screening Program

by Nika Maani, Shelley Westergard, Joanna Yang, Anabel M. Scaranelo, Stephanie Telesca, Emily Thain, Nathan F. Schachter, Jeanna M. McCuaig and Raymond H. Kim

Cancers 2019, 11(5), 707; https://doi.org/10.3390/cancers11050707 - 22 May 2019

Cited by 19 | Viewed by 5552

Abstract

Neurofibromatosis Type I (NF1) is caused by variants in neurofibromin (NF1). NF1 predisposes to a variety of benign and malignant tumor types, including breast cancer. Women with NF1 <50 years of age possess an up to five-fold increased risk of developing [...] Read more.

Neurofibromatosis Type I (NF1) is caused by variants in neurofibromin (NF1). NF1 predisposes to a variety of benign and malignant tumor types, including breast cancer. Women with NF1 <50 years of age possess an up to five-fold increased risk of developing breast cancer compared with the general population. Impaired emotional functioning is reported as a comorbidity that may influence the participation of NF1 patients in regular clinical surveillance despite their increased risk of breast and other cancers. Despite emphasis on breast cancer surveillance in women with NF1, the uptake and feasibility of high-risk screening programs in this population remains unclear. A retrospective chart review between 2014–2018 of female NF1 patients seen at the Elizabeth Raab Neurofibromatosis Clinic (ERNC) in Ontario was conducted to examine the uptake of high-risk breast cancer screening, radiologic findings, and breast cancer characteristics. 61 women with pathogenic variants in NF1 enrolled in the high-risk Ontario breast screening program (HR-OBSP); 95% completed at least one high-risk breast screening modality, and four were diagnosed with invasive breast cancer. Our findings support the integration of a formal breast screening programs in clinical management of NF1 patients. Full article

(This article belongs to the Special Issue New Insights into Neurofibromatosis)

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Cancers, Volume 11, Issue 5 (May 2019) – 149 articles

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