Cancers

17 pages, 2439 KiB

Open AccessFeature PaperArticle

A Novel Inhibitor of STAT5 Signaling Overcomes Chemotherapy Resistance in Myeloid Leukemia Cells

by Marie Brachet-Botineau, Margaux Deynoux, Nicolas Vallet, Marion Polomski, Ludovic Juen, Olivier Hérault, Frédéric Mazurier, Marie-Claude Viaud-Massuard, Gildas Prié and Fabrice Gouilleux

Cancers 2019, 11(12), 2043; https://doi.org/10.3390/cancers11122043 - 17 Dec 2019

Cited by 17 | Viewed by 4061

Abstract

Signal transducers and activators of transcription 5A and 5B (STAT5A and STAT5B) are crucial downstream effectors of tyrosine kinase oncogenes (TKO) such as BCR-ABL in chronic myeloid leukemia (CML) and FLT3-ITD in acute myeloid leukemia (AML). Both proteins have been shown to promote [...] Read more.

Signal transducers and activators of transcription 5A and 5B (STAT5A and STAT5B) are crucial downstream effectors of tyrosine kinase oncogenes (TKO) such as BCR-ABL in chronic myeloid leukemia (CML) and FLT3-ITD in acute myeloid leukemia (AML). Both proteins have been shown to promote the resistance of CML cells to tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM). We recently synthesized and discovered a new inhibitor (17f) with promising antileukemic activity. 17f selectively inhibits STAT5 signaling in CML and AML cells by interfering with the phosphorylation and transcriptional activity of these proteins. In this study, the effects of 17f were evaluated on CML and AML cell lines that respectively acquired resistance to IM and cytarabine (Ara-C), a conventional therapeutic agent used in AML treatment. We showed that 17f strongly inhibits the growth and survival of resistant CML and AML cells when associated with IM or Ara-C. We also obtained evidence that 17f inhibits STAT5B but not STAT5A protein expression in resistant CML and AML cells. Furthermore, we demonstrated that 17f also targets oncogenic STAT5B N642H mutant in transformed hematopoietic cells. Full article

(This article belongs to the Special Issue Targeting STAT3 and STAT5 in Cancer)

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17 pages, 4499 KiB

Open AccessArticle

The Benzimidazole-Based Anthelmintic Parbendazole: A Repurposed Drug Candidate That Synergizes with Gemcitabine in Pancreatic Cancer

by Rosalba Florio, Serena Veschi, Viviana di Giacomo, Sara Pagotto, Simone Carradori, Fabio Verginelli, Roberto Cirilli, Adriano Casulli, Antonino Grassadonia, Nicola Tinari, Amelia Cataldi, Rosa Amoroso, Alessandro Cama and Laura De Lellis

Cancers 2019, 11(12), 2042; https://doi.org/10.3390/cancers11122042 - 17 Dec 2019

Cited by 42 | Viewed by 14028

Abstract

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and [...] Read more.

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC₅₀) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy. Full article

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Parbendazole is more effective than fenbendazole, mebendazole and oxibendazole on PC cell viability. (A) Chemical structures of the four tested benzimidazole-based anthelmintics. (B) Pancreatic cancer cell viability was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) after treatment of AsPC-1 and Capan-2 cell lines with fenbendazole, mebendazole, oxibendazole, parbendazole or with vehicle control at the indicated concentrations for 72 h. (C) Cell viability IC50 values for fenbendazole, mebendazole, oxibendazole and parbendazole. Data shown are means ± SD of two independent experiments with quintuplicate determinations. * Statistically significant differences as compared to vehicle (control) (* p < 0.05; ** p < 0.01; *** p < 0.001). Full article ">Figure 2
Parbendazole abolishes growth and clonogenicity of PC cell lines. (A) Cell growth was assessed by trypan blue exclusion test over a 72-h time course treatment with 0.2 μM and 0.7 μM parbendazole, or with vehicle control. Data shown are the means (±SD) of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001). (B) Representative plates of colony formation assays for AsPC-1 and Capan-2 (top). Values represented in the histograms (bottom) are the means (±SD) of three independent experiments (*** p < 0.001). PE: plating efficiency [(# of colonies formed/# of cells plated) × 100]; SF: surviving fraction [# of colonies formed × 100/(# of cells plated × PE of control vehicle)]. Full article ">Figure 3
Parbendazole alters mitotic spindles formation. Immunofluorescence of PC cells (AsPC-1, left panels; Capan-2, right panels) stained using anti-α-tubulin antibody (green) and 1,5-bis{[2-(dimethylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) (blue, nuclear staining). Both cell lines were treated for 24 h with 0.2 µM and 0.7 µM parbendazole, or with vehicle control. Representative pictures of two independent experiments are shown (scale bar = 20 µm). Full article ">Figure 4
Parbendazole affects cell cycle, ploidy and size of PC cells. (A) Representative DNA distribution histograms of AsPC-1 and Capan-2 cell lines incubated with 0.2 μM, 0.7 μM parbendazole or with vehicle control for 24, 48 and 72 h, as measured by flow cytometry. (B) Histograms show the percentages of 8N and 16N cells after parbendazole treatment for 24, 48 and 72 h, as determined by flow cytometry. Data shown are the means (±SD) of two independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001). (C) Histogram plots showing cell size shifts after parbendazole treatment for 24, 48 and 72 h, as detected by flow cytometry analysis of forward scatter. (D) Representative western blots showing cyclin B1 expression in AsPC-1 and Capan-2 treated with 0 μM, 0.2 μM or 0.7 μM parbendazole for 24, 48 and 72 h. β-actin was included as loading control. Numbers below blots refer to the densitometric analysis of the immunoreactive bands and represent the fold change in protein expression normalized to β-actin. Full article ">Figure 5
Parbendazole induces apoptosis and DNA damage in PC cell lines. (A) Cells were treated with parbendazole, or with vehicle control for 24, 48 and 72 h. Values represented in the histograms (left) are the means (±SD) of at least three independent flow cytometry experiments (* p < 0.05; *** p < 0.001). Representative dot plots (right) of flow cytometry experiments after a 72-h treatment with parbendazole, or with vehicle control. (B) Representative western blots showing PARP and cleaved PARP protein expression in AsPC-1 and Capan-2 treated with 0 μM, 0.2 μM or 0.7 μM parbendazole. Ratios of cleaved: uncleaved PARP are indicated. (C) Representative western blots showing levels of H2AX phosphorylation at Ser139 in AsPC-1 and Capan-2 treated with 0 μM, 0.2 μM or 0.7 μM parbendazole. β-actin was included as loading control. Numbers below blots refer to the densitometric analysis of the immunoreactive bands and represent the fold change in protein expression normalized to β-actin. Full article ">Figure 6
Parbendazole affects PC cell migration. Representative wound-healing assay pictures for AsPC-1 and Capan-2 treated with parbendazole, or with vehicle control (upper panels). Pictures of PC cells were taken at 0, 6 and 24 h post-scratching to analyze the dynamics of wound closure (vertical black lines indicate wound edges). Histograms (bottom panels) represent cell migration in two independent experiments expressed as relative scratch gap. * Statistically significant differences as compared to vehicle (* p < 0.05; *** p < 0.001). Full article ">Figure 7
Effect of parbendazole and gemcitabine combinations on PC cell viability. Cell viability was assessed by MTT after a 72-h incubation of AsPC-1 and Capan-2 with parbendazole and gemcitabine, as single agents or in combination, at the indicated concentrations. Combination indexes (CIs) were calculated by CompuSyn software. Synergistic combinations (CI < 1) are boxed and in bold. Values represented in the histograms are the means (±SD) of two independent experiments with quintuplicate determinations. Full article ">

21 pages, 6482 KiB

Open AccessReview

A Review of Key Biological and Molecular Events Underpinning Transformation of Melanocytes to Primary and Metastatic Melanoma

by Louise A. Jackett and Richard A. Scolyer

Cancers 2019, 11(12), 2041; https://doi.org/10.3390/cancers11122041 - 17 Dec 2019

Cited by 17 | Viewed by 4427

Abstract

Melanoma is a major public health concern that is responsible for significant morbidity and mortality, particularly in countries such as New Zealand and Australia where it is the commonest cause of cancer death in young adults. Until recently, there were no effective drug [...] Read more.

Melanoma is a major public health concern that is responsible for significant morbidity and mortality, particularly in countries such as New Zealand and Australia where it is the commonest cause of cancer death in young adults. Until recently, there were no effective drug therapies for patients with advanced melanoma however significant advances in our understanding of the biological and molecular basis of melanoma in recent decades have led to the development of revolutionary treatments, including targeted molecular therapy and immunotherapy. This review summarizes our current understanding of the key events in the pathway of melanomagenesis and discusses the role of genomic analysis as a potential tool for improved diagnostic evaluation, prognostication and treatment strategies. Ultimately, it is hoped that a continued deeper understanding of the mechanisms of melanomagenesis will lead to the development of even more effective treatments that continue to provide better outcomes for patients with melanoma. Full article

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22 pages, 11627 KiB

Open AccessArticle

Magnetic Silica-Coated Iron Oxide Nanochains as Photothermal Agents, Disrupting the Extracellular Matrix, and Eradicating Cancer Cells

by Jelena Kolosnjaj-Tabi, Slavko Kralj, Elena Griseti, Sebastjan Nemec, Claire Wilhelm, Anouchka Plan Sangnier, Elisabeth Bellard, Isabelle Fourquaux, Muriel Golzio and Marie-Pierre Rols

Cancers 2019, 11(12), 2040; https://doi.org/10.3390/cancers11122040 - 17 Dec 2019

Cited by 25 | Viewed by 4067

Abstract

Cancerous cells and the tumor microenvironment are among key elements involved in cancer development, progression, and resistance to treatment. In order to tackle the cells and the extracellular matrix, we herein propose the use of a class of silica-coated iron oxide nanochains, which [...] Read more.

Cancerous cells and the tumor microenvironment are among key elements involved in cancer development, progression, and resistance to treatment. In order to tackle the cells and the extracellular matrix, we herein propose the use of a class of silica-coated iron oxide nanochains, which have superior magnetic responsiveness and can act as efficient photothermal agents. When internalized by different cancer cell lines and normal (non-cancerous) cells, the nanochains are not toxic, as assessed on 2D and 3D cell culture models. Yet, upon irradiation with near infrared light, the nanochains become efficient cytotoxic photothermal agents. Besides, not only do they generate hyperthermia, which effectively eradicates tumor cells in vitro, but they also locally melt the collagen matrix, as we evidence in real-time, using engineered cell sheets with self-secreted extracellular matrix. By simultaneously acting as physical (magnetic and photothermal) effectors and chemical delivery systems, the nanochain-based platforms offer original multimodal possibilities for prospective cancer treatment, affecting both the cells and the extracellular matrix. Full article

(This article belongs to the Special Issue Cancer Nanomedicine)

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18 pages, 4749 KiB

Open AccessArticle

Simultaneous Multi-Organ Metastases from Chemo-Resistant Triple-Negative Breast Cancer Are Prevented by Interfering with WNT-Signaling

by Iram Fatima, Ikbale El-Ayachi, Hilaire C. Playa, Jackelyn A. Alva-Ornelas, Aysha B. Khalid, William L. Kuenzinger, Peter Wend, Jackelyn C. Pence, Lauren Brakefield, Raisa I. Krutilina, Daniel L. Johnson, Ruth M. O’Regan, Victoria Seewaldt, Tiffany N. Seagroves, Susan A. Krum and Gustavo A. Miranda-Carboni

Cancers 2019, 11(12), 2039; https://doi.org/10.3390/cancers11122039 - 17 Dec 2019

Cited by 28 | Viewed by 4196

Abstract

Triple-negative breast cancers (TNBCs), which lack specific targeted therapy options, evolve into highly chemo-resistant tumors that metastasize to multiple organs simultaneously. We have previously shown that TNBCs maintain an activated WNT10B-driven network that drives metastasis. Pharmacologic inhibition by ICG-001 decreases β-catenin-mediated proliferation of [...] Read more.

Triple-negative breast cancers (TNBCs), which lack specific targeted therapy options, evolve into highly chemo-resistant tumors that metastasize to multiple organs simultaneously. We have previously shown that TNBCs maintain an activated WNT10B-driven network that drives metastasis. Pharmacologic inhibition by ICG-001 decreases β-catenin-mediated proliferation of multiple TNBC cell lines and TNBC patient-derived xenograft (PDX)-derived cell lines. In vitro, ICG-001 was effective in combination with the conventional cytotoxic chemotherapeutics, cisplatin and doxorubicin, to decrease the proliferation of MDA-MB-231 cells. In contrast, in TNBC PDX-derived cells doxorubicin plus ICG-001 was synergistic, while pairing with cisplatin was not as effective. Mechanistically, cytotoxicity induced by doxorubicin, but not cisplatin, with ICG-001 was associated with increased cleavage of PARP-1 in the PDX cells only. In vivo, MDA-MB-231 and TNBC PDX orthotopic primary tumors initiated de novo simultaneous multi-organ metastases, including bone metastases. WNT monotherapy blocked multi-organ metastases as measured by luciferase imaging and histology. The loss of expression of the WNT10B/β-catenin direct targets HMGA2, EZH2, AXIN2, MYC, PCNA, CCND1, transcriptionally active β-catenin, SNAIL and vimentin both in vitro and in vivo in the primary tumors mechanistically explains loss of multi-organ metastases. WNT monotherapy induced VEGFA expression in both tumor model systems, whereas increased CD31 was observed only in the MDA-MB-231 tumors. Moreover, WNT-inhibition sensitized the anticancer response of the TNBC PDX model to doxorubicin, preventing simultaneous metastases to the liver and ovaries, as well as to bone. Our data demonstrate that WNT-inhibition sensitizes TNBC to anthracyclines and treats multi-organ metastases of TNBC. Full article

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

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Determination of differential IC50 (DIC50) of ICG-001 on multiple TNBC cell lines. (A) Results of WST-1 proliferation assays following 48 h of treatment with ICG-001 at various dosages ranging from 0.2 μM–30 μM using MDA-MB-231, MDA-MB-157, HCC38, MDA-MB-468 and TNBC PDX patient-derived cHCI-2 and cHCI-10 cells. All experiments in panels (B–E) used the cells’ specific IC50s as shown in <a href="#app1-cancers-11-02039" class="html-app">Figure S1C</a>. (B,C) qPCR for AXIN2, HMGA2, CCND1, MYC and PCNA in some of the same cells from panel A. (D,E) Immunoblot analysis for AXIN2, HMGA2, MYC, CCND1 and PCNA. (F) Immunoblot for non-phosphorylated Active-β-CATENIN (ABC) and total-β-CATENIN and are shown. β-ACTIN serves as the loading control. Results are expressed as mean ± SE, n = 3; unpaired Student’s t-test, two-tailed unequal variance used to calculate p values; *** p = 0.001, ** p ≤ 0.01 and * p ≤ 0.05 vs. control. Details of western blot can be viewed at the <a href="#app1-cancers-11-02039" class="html-app">supplementary materials</a>. Full article ">Figure 2
ICG-001 is able to synergize with doxorubicin, but not cisplatin, to repress tumor cell proliferation and to increase cytotoxicity in the doxorubicin chemoresistant HCI-10 PDX TNBC cells. MDA-MB-231Luc (Ai,ii) and cHCI-10Luc (Bi,ii) cells were analyzed by WST-1 assays at 96 h, following exposure to ICG-001 (1 µM and 5 µM concentration, MDA-MB-231 or 5 µM or 10 µM concentrations for cHCI-10 cells) in combination with cisplatin or DOX at various increasing concentrations, demonstrating inhibition of tumor cell proliferation. IncuCyte® Cytox Green reagent was used to measure cytotoxicity in MDA-MB-231 (Ci,ii) and cHCI-10 (Di,ii) cells at various combinatorial concentrations over 48 h, showing an increased cytotoxicity response with ICG plus DOX. (E) MDA-MB-231 and cHCI-10 cells were exposed to ICG-001 (MDA−MB−231 = 5 µM; cHCI-10 at 10 µM) alone or in combination with DOX (0.5 µM) or with cisplatin (0.5 µM) for 48 h. Immunoblotting for total PARP, cleaved PARP, and BAX was conducted. ACTIN and TUBULIN served as loading controls. Results are expressed as mean ± SEM, n = 3. Details of western blot can be viewed at the <a href="#app1-cancers-11-02039" class="html-app">supplementary materials</a>. Full article ">Figure 3
WNT inhibition interferes with simultaneous multi-organ and bone metastases in vivo in MDA-MB-231 cells. MDA-MB-231 stably transduced with lentivector-luciferase was used to track cells by bio-imaging after surgical transplantation into the mammary fat pad of NSG mice, beginning one week after initiation of ICG-001 therapy (200 mg/kg, IP every other day for two weeks). (Ai) Tumor volume was tracked over time using calipers (n = 10 mice). (Aii) Representative images of tumors from the vehicle and ICG-001-treated mice. (Bi) Total bioluminescence flux (photons/sec, p/s) was quantified longitudinally in the primary tumors. Standard deviation is shown. (Bii) Ex vivo bioluminescence images of tumors. (C) Kaplan–Meier Survival curve from the vehicle and ICG-001-treated mice, n = 9/group. (D) Immunoblot analysis of HMGA2, EZH2, AXIN2, PCNA, CCND1 and MYC (i) and EMT markers VIMENTIN and SNAI from the primary tumors and (ii), β-ACTIN serves as the loading control. (E) Representative whole-body luciferase reporter images from vehicle or ICG-001 treated mice. (F) Quantification of whole-body metastasis as measured by the total bioluminescence flux in (photons/sec, p/s’ n = 6 mice/group). (G) Ex vivo bioluminescence of the organs. P-values were generated by one-way ANOVA followed by pairwise Student’s t-tests (* p < 0.05, ** p < 0.01 and *** p < 0.001). Total light flux was compared in the whole-body of the mice after therapy. Details of western blot can be viewed at the <a href="#app1-cancers-11-02039" class="html-app">supplementary materials</a>. Full article ">Figure 4
WNT inhibition interferes with de novo whole-body metastasis in highly chemoresistant TNBC PDX model. The TNBC PDX tumor model HCI-10, which is stably transduced with luciferase to track cells in vivo was bilaterally transplanted into the mammary fat pad of NSG mice. Three weeks after transplantation, ICG-001 therapy was initiated at a dose of either 100 or 200 mg/kg (IP, every other day for two weeks). (A) Bioluminescence images of both the primary tumors and either the lungs or lymph nodes at 6-weeks post-transplant. (B) Representative images of mice with whole-body metastases at 8 weeks after transplantation. (C) Ex vivo bioluminescence of lung, liver, ovary, kidney bone and brain harvested from the vehicle and ICG-001-treated mice. (D) Total light flux was compared in the whole body of the mice after therapy. (E) Immunoblot analysis of AXIN2, MYC (i) and VIMENTIN and SNAIL (ii) in primary tumors (ICG-001 at 200 mg/kg). β-ACTIN serves as the loading control. (F) Anti-metastatic effects of ICG-001 are shown, along with micrographs of H&E staining and anti-human mitochondria antibody staining of harvested tissue to confirm the presence of human tumor cells in rodent organs. (G) IHC was performed for CD31 and/or VEGF-A in the vehicle or ICG-001 treated cohorts for both the cHCI-10 or MDA-MB-231 primary tumors. p-Values were generated by one-way ANOVA followed by pairwise Student’s t-tests (** p < 0.01). Details of western blot can be viewed at the <a href="#app1-cancers-11-02039" class="html-app">supplementary materials</a>. Full article ">Figure 5
Impact of bone metastases on the bones as analyzed by high-resolution micro-computed tomography (μCT). (A) 3D images of a femur and a tibia from a control non-tumor-baring NSG mouse at 12 weeks of age, showing normal bone phenotype in both femoral and proximal tibial trabeculae bone. (B) Bones bearing metastases derived from MDA-MD-231Luc cells show the femoral and proximal tibial trabeculae; two representative mice are shown from the vehicle and ICG-001-treated mice. Red arrows (the pores) highlight loss of bone mass only in the tibial trabeculae. (C) Representative ex vivo bioluminescence images of bones from the vehicle and ICG-001-treated mice (i) and the quantification of flux-units (ii; n = 5 mice) at study endpoint. Inserts show the ex vivo bioluminescence images obtained for the same bones that were analyzed by μCT. p-Values generated by one-way ANOVA followed by pairwise Student’s t-tests (* p = 0.0225). Scale Bar of 1 mm or 100 μm (A,B). Full article ">Figure 6
ICG-001 sensitizes chemoresistant TNBC PDX tumor cells to doxorubicin, preventing liver, bone and ovarian metastasis. cHCI-10 cells (1.25 × 106) that were freshly isolated from primary PDX HCI-10Luc2 tumors were tail vein injected into NSG females. One day after tail vein injection, mice were treated with either DOX alone (1.4 mg/kg, IP) or DOX in combination with ICG-001 (50 mg/kg, IP) using the dosing schedules outlined in the materials and methods. Total flux (p/s) was quantified by ex vivo bioluminescence imaging of the liver (A), bone (B) and ovaries (C); p-Values generated by unpaired Student’s t-test; two-tailed unequal variance (* p < 0.018). Full article ">

18 pages, 3203 KiB

Open AccessArticle

GSTO1*CC Genotype (rs4925) Predicts Shorter Survival in Clear Cell Renal Cell Carcinoma Male Patients

by Tanja Radic, Vesna Coric, Zoran Bukumiric, Marija Pljesa-Ercegovac, Tatjana Djukic, Natasa Avramovic, Marija Matic, Smiljana Mihailovic, Dejan Dragicevic, Zoran Dzamic, Tatjana Simic and Ana Savic-Radojevic

Cancers 2019, 11(12), 2038; https://doi.org/10.3390/cancers11122038 - 17 Dec 2019

Cited by 8 | Viewed by 3528

Abstract

Omega class glutathione transferases, GSTO1-1 and GSTO2-2, exhibit different activities involved in regulation of inflammation, apoptosis and redox homeostasis. We investigated the the prognostic significance of GSTO1 (rs4925) and GSTO2 (rs156697 and rs2297235) polymorphisms in clear cell renal cell carcinoma (ccRCC) patients. GSTO1-1 [...] Read more.

Omega class glutathione transferases, GSTO1-1 and GSTO2-2, exhibit different activities involved in regulation of inflammation, apoptosis and redox homeostasis. We investigated the the prognostic significance of GSTO1 (rs4925) and GSTO2 (rs156697 and rs2297235) polymorphisms in clear cell renal cell carcinoma (ccRCC) patients. GSTO1-1 and GSTO2-2 expression and phosphorylation status of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ /mammalian target of rapamycin (mTOR) and Raf/MEK/extracellular signal-regulated kinase (ERK) signaling pathways in non-tumor and tumor ccRCC tissue, as well as possible association of GSTO1-1 with signaling molecules were also assessed. GSTO genotyping was performed by quantitative PCR in 228 ccRCC patients, while expression and immunoprecipitation were analyzed by Western blot in 30 tissue specimens. Shorter survival in male carriers of GSTO1*C/C wild-type genotype compared to the carriers of at least one variant allele was demonstrated (p = 0.049). GSTO1*C/C genotype independently predicted higher risk of overall mortality among male ccRCC patients (p = 0.037). Increased expression of GSTO1-1 and GSTO2-2 was demonstrated in tumor compared to corresponding non-tumor tissue (p = 0.002, p = 0.007, respectively), while GSTO1 expression was correlated with interleukin-1β (IL-1β)/pro-interleukin-1β (pro-IL-1β) ratio (r = 0.260, p = 0.350). Interaction of GSTO1 with downstream effectors of investigated pathways was shown in ccRCC tumor tissue. This study demonstrated significant prognostic role of GSTO1 polymorphism in ccRCC. Up-regulated GSTO1-1 and GSTO2-2 in tumor tissue might contribute to aberrant ccRCC redox homeostasis. Full article

(This article belongs to the Special Issue Renal Cell Carcinoma)

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(a) Overall survival of clear cell renal cell carcinoma (ccRCC) patients stratified by GSTO1 (rs4925) and GSTO2 (rs156697 and rs2297235) polymorphisms; (b) overall survival of male ccRCC patients stratified by GSTO1 (rs4925) and GSTO2 (rs156697 and rs2297235) polymorphisms; (c) overall survival of female ccRCC patients stratified by GSTO1 (rs4925) and GSTO2 (rs156697 and rs2297235) polymorphisms. Full article ">Figure 2
(a) Expression of GSTO1 (28 kDa) in ccRCC tumor (T) and corresponding non-tumor (nT) tissue samples; (b) expression of GSTO2 (28 kDa) in ccRCC tumor (T) and corresponding non-tumor (nT) tissue samples; (c) expression of GSTO1 and GSTO2 (28 kDa) in tumor ccRCC tissue samples according to pT stage and Fuhrman nuclear grade of ccRCC; early-stage ccRCC- pT1 and pT2; late-stage ccRCC- pT3 and pT4; (d) expression of GSTO1 stratified according to GSTO1 polymorphism; correlation between GSTO1 and IL-1β/ pro-IL-1β ratio in tumor ccRCC tissue samples. Full article ">Figure 2 Cont.
(a) Expression of GSTO1 (28 kDa) in ccRCC tumor (T) and corresponding non-tumor (nT) tissue samples; (b) expression of GSTO2 (28 kDa) in ccRCC tumor (T) and corresponding non-tumor (nT) tissue samples; (c) expression of GSTO1 and GSTO2 (28 kDa) in tumor ccRCC tissue samples according to pT stage and Fuhrman nuclear grade of ccRCC; early-stage ccRCC- pT1 and pT2; late-stage ccRCC- pT3 and pT4; (d) expression of GSTO1 stratified according to GSTO1 polymorphism; correlation between GSTO1 and IL-1β/ pro-IL-1β ratio in tumor ccRCC tissue samples. Full article ">Figure 3
Phosphorylation status of downstream effectors of PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways in ccRCC tumor (T) and corresponding non-tumor (nT) tissue samples; RSK1p90-90 kDa ribosomal protein S6 kinase 1; Akt—protein kinase B; ERK—extracellular signal-regulated kinase; RPS6—ribosomal protein S6. Full article ">Figure 4
Immunoprecipitation of GSTO1 and associated proteins in tumor ccRCC tissue samples; RSK1p90- 90 kDa ribosomal protein S6 kinase 1; Akt—protein kinase B; RPS6—ribosomal protein S6. Their cytosolic expression was shown in <a href="#cancers-11-02038-f003" class="html-fig">Figure 3</a>. Full article ">

18 pages, 958 KiB

Open AccessReview

Pain Management in Patients with Multiple Myeloma: An Update

by Flaminia Coluzzi, Roman Rolke and Sebastiano Mercadante

Cancers 2019, 11(12), 2037; https://doi.org/10.3390/cancers11122037 - 17 Dec 2019

Cited by 43 | Viewed by 7661

Abstract

Most patients with multiple myeloma (MM) suffer from chronic pain at every stage of the natural disease process. This review focuses on the most common causes of chronic pain in MM patients: (1) pain from myeloma bone disease (MBD); (2) chemotherapy-induced peripheral neuropathy [...] Read more.

Most patients with multiple myeloma (MM) suffer from chronic pain at every stage of the natural disease process. This review focuses on the most common causes of chronic pain in MM patients: (1) pain from myeloma bone disease (MBD); (2) chemotherapy-induced peripheral neuropathy as a possible consequence of proteasome inhibitor therapy (i.e., bortezomib-induced); (3) post-herpetic neuralgia as a possible complication of varicella zoster virus reactivation because of post-transplantation immunodepression; and (4) pain in cancer survivors, with increasing numbers due to the success of antiblastic treatments, which have significantly improved overall survival and quality of life. In this review, non-pain specialists will find an overview including a detailed description of physiopathological mechanisms underlying central sensitization and pain chronification in bone pain, the rationale for the correct use of analgesics and invasive techniques in different pain syndromes, and the most recent recommendations published on these topics. The ultimate target of this review was to underlie that different types of pain can be observed in MM patients, and highlight that only after an accurate pain assessment, clinical examination, and pain classification, can pain be safely and effectively addressed by selecting the right analgesic option for the right patient. Full article

(This article belongs to the Special Issue Latest Development in Multiple Myeloma)

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13 pages, 3061 KiB

Open AccessArticle

Pathways, Processes, and Candidate Drugs Associated with a Hoxa Cluster-Dependency Model of Leukemia

by Laura M. Kettyle, Charles-Étienne Lebert-Ghali, Ivan V. Grishagin, Glenda J. Dickson, Paul G. O’Reilly, David A. Simpson, Janet J. Bijl, Ken I. Mills, Guy Sauvageau and Alexander Thompson

Cancers 2019, 11(12), 2036; https://doi.org/10.3390/cancers11122036 - 17 Dec 2019

Cited by 4 | Viewed by 3058

Abstract

High expression of the HOXA cluster correlates with poor clinical outcome in acute myeloid leukemias, particularly those harboring rearrangements of the mixed-lineage-leukemia gene (MLLr). Whilst decreased HOXA expression acts as a readout for candidate experimental therapies, the necessity of the HOXA [...] Read more.

High expression of the HOXA cluster correlates with poor clinical outcome in acute myeloid leukemias, particularly those harboring rearrangements of the mixed-lineage-leukemia gene (MLLr). Whilst decreased HOXA expression acts as a readout for candidate experimental therapies, the necessity of the HOXA cluster for leukemia maintenance has not been fully explored. Primary leukemias were generated in hematopoietic stem/progenitor cells from Cre responsive transgenic mice for conditional deletion of the Hoxa locus. Hoxa deletion resulted in reduced proliferation and colony formation in which surviving leukemic cells retained at least one copy of the Hoxa cluster, indicating dependency. Comparative transcriptome analysis of Hoxa wild type and deleted leukemic cells identified a unique gene signature associated with key pathways including transcriptional mis-regulation in cancer, the Fanconi anemia pathway and cell cycle progression. Further bioinformatics analysis of the gene signature identified a number of candidate FDA-approved drugs for potential repurposing in high HOXA expressing cancers including MLLr leukemias. Together these findings support dependency for an MLLr leukemia on Hoxa expression and identified candidate drugs for further therapeutic evaluation. Full article

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

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Development of MLL-AF9 (MA9) leukemias. (A) Donor hematopoietic stem/progenitor cells (HSPCs) were spinoculated with MA9 retroviral particles and serially passaged in methylcellulose prior to transplantation into recipient mice. (B) Kaplan–Meier plot demonstrating survival of transplanted mice receiving MA9 leukemic cells derived from Hoxaflox/flox (AFF), MxCre+/Hoxaflox/flox (MAFF) or control CD45.1 donor HSPCs. Primary transplants (1°) are denoted as solid lines, secondary transplants (2°) as dotted lines. (C) Microscope images of modified Wright’s stained touch preps and peripheral blood smears of age-matched control (Normal) or leukemic (MLL-AF9) tissues derived from Hoxaflox/flox mice. Scale bar 20 µm. (D) Digital image of spleens removed from an age-matched control mouse (upper) and leukemic (MLL-AF9) Hoxaflox/flox mouse (lower). Full article ">Figure 2
Overexpression of Hoxa cluster genes in MA9 leukemias. Bar chart of relative gene expression of Hoxa cluster genes in normal bone marrow (NBM) and MA9 leukemias derived from wild type (CD45.1-MA9), Hoxaflox/flox (AFF-MA9) and MxCre+/Hoxaflox/flox (MAFF-MA9) genetic backgrounds. The mean values from triplicate experiments are plotted. Significance as calculated by 1 way ANOVA compared to control bone marrow is denoted as * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001. Full article ">Figure 3
In vitro deletion of the Hoxa cluster in MxCre+/Hoxaflox/flox (MAFF) derived leukemic cells. (A) Digital images of leukemic colonies derived from MAFF-MA9 and wild type (CD45.1-MA9) mice compared to MAFF derived normal bone marrow controls (MAFF-NBM). Colonies were treated with interferon-alpha (IFN-α) at the indicated dose units (U) or with PBS vehicle control (upper panel), counted after 7 days using the GelCount™ analyzer and plotted on a bar chart (lower panel). Not-significant (ns); ** p ≤ 0.01. (B) Digital images of electrophoresed agarose gels containing PCR amplicons of expected size (arrows) derived from bulk CD45.1 or MAFF-derived leukemic colonies (left panel) or representative MAFF-MA9-derived individual colonies (right panel). (C) A schematic of the aligned Hoxadel amplicon sequence with the mouse chromosome 6 tract (UCSC). The sequencing read (−600 bp) encompassed a portion of the 3ʹ UTR of mHoxa1 (blue), the recombined loxP site (black) and apportion of the 5ʹ UTR of mHoxa13 (red). Full article ">Figure 4
In vivo treatment of MAFF-MA9 leukemia. (A) Schematic of the treatment of MAFF-MA9 leukemias prior to and following transplantation into recipient mice. Interferon-alpha (IFN-α), Polyinosinic:polycytidylic acid (PolyI:C), vehicle (PBS). (B) Kaplan–Meier plot demonstrating survival of transplanted mice receiving MAFF-MA9 leukemia cells following treatment. * p ≤ 0.05; ** p ≤ 0.01. (C) In Vivo Imaging Systems (IVIS) derived images of luciferase expressing MAFF-MA9 transplanted into NOD-scid IL2rγnull (NSG) recipient mice. Mice were treated with PBS or PolyI:C and live images taken as indicated (n = 5 per group). Full article ">Figure 5
Differential gene expression in Hoxadel MA9 cells. Left panel; Volcano plot of significance for each of 24,326 probes from Cre-GFP treated (Hoxadel) AFF-MA9 cells. Negative log10 (Adjusted P-value) versus log2 (fold change) compared to GFP-treated AFF-MA9 cells. Genes with –log10 (Adjusted P-value) > 2 and log2 (fold change) > 0.5 (annotated and highlighted in red) were considered differentially expressed. Right panel; sample transcripts induced or repressed at log2 (fold change) of 0.5 or more (p ≤ 0.05) were subjected to unsupervised hierarchical agglomerative clustering by treatment with Cre based on Euclidean distance and linkage. Full article ">Figure 6
Gene set enrichment analysis of the Hoxadel signature. Summary bar charts demonstrating association of the Hoxadel signature with databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Gene Ontology (GO) of Molecular Function and Biological processes and the NCBI drug signatures database for gene set analysis (DSigDB) using Enrichr [<a href="#B30-cancers-11-02036" class="html-bibr">30</a>,<a href="#B31-cancers-11-02036" class="html-bibr">31</a>]. Combined scores (based on the log Fisher exact test p-value and z-score for deviation from expected rank) are plotted against the key pathway, process or drug interactions. Full article ">

16 pages, 1066 KiB

Open AccessReview

Companion Animals as Models for Inhibition of STAT3 and STAT5

by Matthias Kieslinger, Alexander Swoboda, Nina Kramer, Barbara Pratscher, Birgitt Wolfesberger and Iwan A. Burgener

Cancers 2019, 11(12), 2035; https://doi.org/10.3390/cancers11122035 - 17 Dec 2019

Cited by 3 | Viewed by 4827

Abstract

The use of transgenic mouse models has revolutionized the study of many human diseases. However, murine models are limited in their representation of spontaneously arising tumors and often lack key clinical signs and pathological changes. Thus, a closer representation of complex human diseases [...] Read more.

The use of transgenic mouse models has revolutionized the study of many human diseases. However, murine models are limited in their representation of spontaneously arising tumors and often lack key clinical signs and pathological changes. Thus, a closer representation of complex human diseases is of high therapeutic relevance. Given the high failure rate of drugs at the clinical trial phase (i.e., around 90%), there is a critical need for additional clinically relevant animal models. Companion animals like cats and dogs display chronic inflammatory or neoplastic diseases that closely resemble the human counterpart. Cat and dog patients can also be treated with clinically approved inhibitors or, if ethics and drug safety studies allow, pilot studies can be conducted using, e.g., inhibitors of the evolutionary conserved JAK-STAT pathway. The incidence by which different types of cancers occur in companion animals as well as mechanisms of disease are unique between humans and companion animals, where one can learn from each other. Taking advantage of this situation, existing inhibitors of known oncogenic STAT3/5 or JAK kinase signaling pathways can be studied in the context of rare human diseases, benefitting both, the development of drugs for human use and their application in veterinary medicine. Full article

(This article belongs to the Special Issue Targeting STAT3 and STAT5 in Cancer)

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Figure 1
Advantages and disadvantages of different models during drug discovery. (A) Companion animals can be used as an intermediate step between the mechanistic work in murine models and clinical studies in humans, particularly with regard to comparative aspects of tumor biology. (B) Advantages and disadvantages of the individual models for translation into human clinical studies. Full article ">Figure 2
Cross-species conservation of STAT protein domains. (A) STAT1, STAT3, STAT5a and STAT5b from dog, cat and mouse are analyzed for their overall homology compared to the respective human protein (grey boxes, left). In the schematic representation of STAT protein domains, the amino acid positions are indicated above. All proteins share the same domain positions, except for murine STAT1, which has a five amino acid insertion in the DNA binding domain (numbers below the scheme indicate the aa position in this case). Percentages in the domain boxes of dog, cat and mouse STAT proteins show the homology of each domain to the human counterpart. Analyses were carried out using ClustalX. (B) Comparison of key phosphorylation sites in the transactivation domain of STAT1, STAT3, STAT5a and STAT5b from dog, cat and mouse to the human sequence. Amino acid sequence is shown, with phosphorylation sites in green and position indicated; positive amino acid exchanges (conserving protein function) are indicated in yellow, other exchanges in red. (STAT1: human NP_009330.1, dog XP_848353.1, cat XP_006935505.1, mouse NP_001192242.1; STAT3: human NP_644805.1, dog XP_005624514.1, cat XP_003996930.1, mouse NP_998824.1; STAT5a: human NP_001275647.1, dog XP_548091.2, cat XP_023099834.1, mouse NP_001157534.1; STAT5b: human NP_036580.2, dog XP_548092.1, cat XP_023100377.1, mouse NP_035619.3). Full article ">

18 pages, 2218 KiB

Open AccessArticle

Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied by Immunogenic Cell Death in Murine Models of Lymphoma and Colorectal Cancer

by Alessandra Rossi, Olga N. Pakhomova, Peter A. Mollica, Maura Casciola, Uma Mangalanathan, Andrei G. Pakhomov and Claudia Muratori

Cancers 2019, 11(12), 2034; https://doi.org/10.3390/cancers11122034 - 17 Dec 2019

Cited by 40 | Viewed by 5161

Abstract

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric [...] Read more.

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of caspase-3/7 was increased fourteen-fold as compared with four-fold in EL-4 cells. Moreover, while nsPEF treatments induced the release of the ICD hallmark HMGB1 in both cell lines, extracellular ATP was detected only in CT-26. Finally, in vaccination assays, CT-26 cells treated with nsPEF or doxorubicin equally impaired the growth of tumors at challenge sites eliciting a protective anticancer immune response in 78% and 80% of the animals, respectively. As compared to CT-26, both nsPEF- and mitoxantrone-treated EL-4 cells had a less pronounced effect and protected 50% and 20% of the animals, respectively. These results support our conclusion that nsPEF induce ER stress, accompanied by bona fide ICD. Full article

(This article belongs to the Special Issue Electric Field Based Therapies in Cancer Treatment: a Selection of Studies Presented at the 3rd World Congress on Electroporation)

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Figure 1
Effect of nsPEF on the activation of the endoplasmic reticulum (ER) stress sensors IRE1 (A) and PERK (B). EL-4 cells (top panels) and CT26 cell (bottom panels) were treated with iso-effective doses of 100 and 300 pulses, respectively (200 ns, 7 kV/cm, 10 Hz). Samples were collected at 5 h post treatment. In (A) the expression level of XBP1 in both EL-4 and CT26 was measured by real-time quantitative PCR. The gene mRNA level was normalized to the housekeeping HPRT gene mRNA and is shown as relative expression. In (B) phosphorylation of eIF2α was measured by Western blot using an anti-phospho-eIF2α (Serine 51) antibody. Left panels show a representative image for both EL-4 (top panel) and CT26 cells (bottom panel) with eIF2α (phosphorylated and total) and the housekeeping Vinculin protein seen as a 38 and 140 kDa band, respectively. Graphs on the right are the quantifications of the p-eIF2α expressed as fold to sham. 1 µM thaspigargin (Thaps.) was used as a positive control for ER stress induction. Mean +/− s.e. n = 3 for both A and B. * p < 0.001 for the difference of nsPEF from sham. Full article ">Figure 2
nsPEF trigger apoptotic cell death in both CT-26 and EL-4 cells but not in B16F10 melanoma cells. Cells were exposed in cuvettes to increasing numbers of 200 ns pulses (7 kV/cm, 10 Hz). Both caspase-3/7 activation (A) and cell viability (B) were measured at 4 h (black) and 24 h (red) post treatment. Caspase activity is shown as relative luminescence units (RLU) per live cell while cell viability is expressed in % to sham-exposed parallel control. In (C) cells were treated with 10 µM staurosporin for 24 h. The left y-axis refers to cell viability (grey), and the right y-axis is the caspase activity (blue). Mean +/− s.e. n = 3–5. * p < 0.05, ** p < 0.01 for caspase activity of nsPEF from sham. Full article ">Figure 3
nsPEF induce CRT externalization on the cell surface of both EL-4 (A) and CT26 cells (B). EL-4 and CT26 cells were treated with iso-effective doses of 100 and 300 pulses, respectively (200 ns, 7 kV/cm, 10 Hz). 1 µM mitoxantrone (MTX) was used as a positive control for CRT exposure. At 24 h post-treatment, surface CRT was measured by FACS analysis. Left and middle panels show representative FACS histograms. Bar graphs on the right are quantifications of the surface CRT expressed as mean fluorescence intensity (MFI). Mean +/− s.e. n = 4 for both (A) and (B). * p < 0.01, ** p < 0.001 for the difference of nsPEF from sham. Full article ">Figure 4
Effect of nsPEF on HMGB1 (A) and ATP (B) release. EL-4 and CT26 cells were treated with (A) the indicated number of pulses or (B) iso-effective doses of 100 and 300 pulses, respectively (200 ns, 7 kV/cm, 10 Hz). In (A) 24 h post treatment, supernatants were assessed for HMGB1 by ELISA. In (B) ATP release in the supernatant was measured at 18 h post treatment using a luciferase-based assay. Doxorubicin (10 µM) was used as a positive control for both HMGB1 and ATP release. Mean +/− s.e. n = 4–5 and 3–5 for (A) and (B), respectively. * p < 0.05, ** p < 0.01, *** p < 0.001 for the difference of nsPEF from sham. Full article ">Figure 5
nsPEF-treated CT26 cells vaccinated mice from tumor challenge. CT26 tumor cells were treated with nsPEF (600, 200 ns, 7 kV/cm, 10 Hz) and immediately injected into the flank of syngeneic BalbC mice (0.6 × 106 cells/mouse). Control groups were vaccinated with either PBS or with CT-26 cells treated with doxorubicin (Doxo, 25 µM) for 24 h. After 7 days, animals were challenged with live cells (0.1 × 106 cells/mouse) into the opposite flank. Panel (A) shows the tumor growth curves and (B) the % of tumor free animals. Graphs show the effects measured in animals that did not develop tumor at the vaccination site. Mean +/− s.e., n = 10, 10, and 9 for PBS, Doxo and nsPEF groups, respectively. Full article ">Figure 6
Immunogenicity of killed EL-4 cells: nsPEF vs. mitoxantrone. In (A) EL-4 tumor cells were treated with nsPEF (200, 200 ns, 7 kV/cm, 10 Hz) and immediately injected into the flank of syngeneic C57BL6 mice (0.6 × 106 cells/mouse). In (B) cells were treated with either 0.5 µM (B, left graphs) or 1 µM (B, right graphs) mitoxantrone (MTX) for 24 h. Control groups were vaccinated with PBS. After 7 days, animals were challenged with live cells (0.03 × 106 cells/mouse) into the opposite flank. Top graphs show the tumor growth curves and bottom graphs show % of tumor free animals. Graphs show the effects measured in animals that did not develop tumor at the vaccination site. Mean +/− s.e., n = 9 and 6 for PBS, and nsPEF groups in (A), respectively. Mean +/− s.e., n = 9, 9, 8, and 8 for PBS, 0.5 µM MTX, PBS, and 1 µM MTX groups in (B), respectively. * p < 0.01. Full article ">

11 pages, 992 KiB

Open AccessArticle

Clinicopathologic and Imaging Features of Non-Small-Cell Lung Cancer with MET Exon 14 Skipping Mutations

by Subba R. Digumarthy, Dexter P. Mendoza, Eric W. Zhang, Jochen K. Lennerz and Rebecca S. Heist

Cancers 2019, 11(12), 2033; https://doi.org/10.3390/cancers11122033 - 17 Dec 2019

Cited by 26 | Viewed by 3571

Abstract

MET exon 14 (METex14) skipping mutations are an emerging potentially targetable oncogenic driver mutation in non-small-cell lung cancer (NSCLC). The imaging features and patterns of metastasis of NSCLC with primary METex14 skipping mutations (METex14-mutated NSCLC) are not well [...] Read more.

MET exon 14 (METex14) skipping mutations are an emerging potentially targetable oncogenic driver mutation in non-small-cell lung cancer (NSCLC). The imaging features and patterns of metastasis of NSCLC with primary METex14 skipping mutations (METex14-mutated NSCLC) are not well described. Our goal was to determine the clinicopathologic and imaging features that may suggest the presence of METex14 skipping mutations in NSCLC. This IRB-approved retrospective study included NSCLC patients with primary METex14 skipping mutations and pre-treatment imaging data between January 2013 and December 2018. The clinicopathologic characteristics were extracted from electronic medical records. The imaging features of the primary tumor and metastases were analyzed by two thoracic radiologists. In total, 84 patients with METex14-mutated NSCLC (mean age = 71.4 ± 10 years; F = 52, 61.9%, M = 32, 38.1%; smokers = 47, 56.0%, nonsmokers = 37, 44.0%) were included in the study. Most tumors were adenocarcinoma (72; 85.7%) and presented as masses (53/84; 63.1%) that were peripheral in location (62/84; 73.8%). More than one in five cancers were multifocal (19/84; 22.6%). Most patients with metastatic disease had only extrathoracic metastases (23/34; 67.6%). Fewer patients had both extrathoracic and intrathoracic metastases (10/34; 29.4%), and one patient had only intrathoracic metastases (1/34, 2.9%). The most common metastatic sites were the bones (14/34; 41.2%), the brain (7/34; 20.6%), and the adrenal glands (7/34; 20.6%). Four of the 34 patients (11.8%) had metastases only at a single site. METex14-mutated NSCLC has distinct clinicopathologic and radiologic features. Full article

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20 pages, 5801 KiB

Open AccessArticle

Gigantol Targets Cancer Stem Cells and Destabilizes Tumors via the Suppression of the PI3K/AKT and JAK/STAT Pathways in Ectopic Lung Cancer Xenografts

by Nattanan Losuwannarak, Arnatchai Maiuthed, Nakarin Kitkumthorn, Asada Leelahavanichkul, Sittiruk Roytrakul and Pithi Chanvorachote

Cancers 2019, 11(12), 2032; https://doi.org/10.3390/cancers11122032 - 17 Dec 2019

Cited by 35 | Viewed by 4584

Abstract

Lung cancer has long been recognized as an important world heath concern due to its high incidence and death rate. The failure of treatment strategies, as well as the regrowth of the disease driven by cancer stem cells (CSCs) residing in the tumor, [...] Read more.

Lung cancer has long been recognized as an important world heath concern due to its high incidence and death rate. The failure of treatment strategies, as well as the regrowth of the disease driven by cancer stem cells (CSCs) residing in the tumor, lead to the urgent need for a novel CSC-targeting therapy. Here, we utilized proteome alteration analysis and ectopic tumor xenografts to gain insight on how gigantol, a bibenzyl compound from orchid species, could attenuate CSCs and reduce tumor integrity. The proteomics revealed that gigantol affected several functional proteins influencing the properties of CSCs, especially cell proliferation and survival. Importantly, the PI3K/AKT/mTOR and JAK/STAT related pathways were found to be suppressed by gigantol, while the JNK signal was enhanced. The in vivo nude mice model confirmed that pretreatment of the cells with gigantol prior to a tumor becoming established could decrease the cell division and tumor maintenance. The results indicated that gigantol decreased the relative tumor weight with dramatically reduced tumor cell proliferation, as indicated by Ki-67 labeling. Although gigantol only slightly altered the epithelial-to-mesenchymal and angiogenesis statuses, the gigantol-treated group showed a dramatic loss of tumor integrity as compared with the well-grown tumor mass of the untreated control. This study reveals the effects of gigantol on tumor initiation, growth, and maintain in the scope that the cells at the first step of tumor initiation have lesser CSC property than the control untreated cells. This study reveals novel insights into the anti-tumor mechanisms of gigantol focused on CSC targeting and destabilizing tumor integrity via suppression of the PI3K/AKT/mTOR and JAK/STAT pathways. This data supports the potential of gigantol to be further developed as a drug for lung cancer. Full article

(This article belongs to the Special Issue Role of Natural Bioactive Compounds in the Rise and Fall of Cancers)

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Figure 1
(A) Gigantol structure and the plant specimen, Dendrobium draconis Rchb.f. (B) Graphs showing the percentages of H460 and BEAS-2B cells viability. Both cell lines were treated with 0 to 200 µM of gigantol or vehicle for 24 to 48 h and then analyzed by MTT assay for cell viability. The percentages of viable cells were compared to their untreated controls. (C) Graph showing the percentages of apoptotic cell death after 24 h of gigantol exposure. Necrotic cells could not be detected. (D) Photographs of Hoechst 33342, Propidium iodide (PI), and phase contrast fields showing cancer cells morphologies after 24 h of gigantol treatment, the scale bars represent 100 µm and the magnification is 100×. Each experiment was performed in biological triplicates, * indicates p < 0.05 as compared with untreated group of H460, # indicates p < 0.05 as compared with untreated group of BEAS-2B (one-way ANOVA, Dunnett’s test). Full article ">Figure 2
H460 cells were treated with 20 µM of gigantol or its vehicle (0.004% DMSO) for 24 h before the whole-cell lysates were collected. The experiment was performed in biological triplicates. (A) Venn diagram showing the difference in proteins expressions between the control and gigantol-treated H460 cells. Three functional classifications of the 2373 down- and 1767 upregulated proteins affected by gigantol treatment using Panther software: (B) molecular function, (C) biological process, and (D) cellular component. Full article ">Figure 3
Networks presenting the functional protein-protein interactions of the (A) 97 down- and (C) 67 upregulated proteins related to the GO term “protein phosphorylation” (GO:0006468). The significant nodes of each network are identified and rebuilt as a network of CSC linked pathways. (B) According to the KEGG pathways database, significant nodes of the downregulated proteins were labeled with red for the PI3K-AKT signaling pathway (hsa04151) with FDR 4.08e−13 and blue for the JAK-STAT signaling pathway (hsa04630) with false discovery rate (FDR) 2.80e−09. (D) Significant nodes of upregulated proteins were labeled with red for the ErbB signaling pathway (hsa04012) with FDR 1.28 × 10−9 Full article ">Figure 4
(A) Heatmap representing the levels of proteins associated with the signaling pathways regulating the pluripotency of stem cells in the control and gigantol-treated H460 cells (left and right columns of the heatmap, respectively). Proteins belonging to each pathway are listed to the right. (B) CSC markers and key kinases of AKT and STAT3 were determined by Western blotting and (C) the immunoblot signal intensities were quantified by densitometry. The uncropped protein bands are in <a href="#app1-cancers-11-02032" class="html-app">Figure S2</a> (S2A: The protein bands from <a href="#cancers-11-02032-f004" class="html-fig">Figure 4</a>B; S2B: The protein bands from <a href="#cancers-11-02032-f004" class="html-fig">Figure 4</a>D). (D) The effects of gigantol on AKT, STATS3, and CSC markers were confirmed in two other NSCLC cell lines, A549 and H292, and (E) the relative protein levels were quantified. The mean data from each experiment was normalized to the GAPDH results. The experiment was performed biologically triplicated. Data represent the means ± SD (n = 3) * indicates p < 0.05 as compared with the control group (student’s t-test). Full article ">Figure 5
(A) Graph showing mice body weights starting at the day of cancer cell inoculation. There was no significant change of the body weights until the day of termination. (B) Untreated (upper row) and gigantol-treated (lower row) tumors were dissected and photographed at day 13 after inoculation. Scale bars represent 10 mm in length. (C) Graph showing grouped means of the control and gigantol tumor weights. The 5 different markers represent each pair of tumors. The gigantol-treated tumors had lower tumor weights as compared with their own control tumors, except for mouse 3. (D) Graph presenting the mean growth rate of the control and gigantol groups. (E) Four graphs demonstrating the individual tumor growth rate of each mouse (tumor growth of mouse 5 could not be accomplished because the gigantol-treated tumor was not palpable and measured until the day of termination). (F) Tumor density was calculated as weight by volume. The horizon lines represent means of each group. Full article ">Figure 6
Hematoxylin and eosin staining showing intratumor morphology (20×). Percentages of necrotic areas as compared with their total areas are shown at the lower-right edge of each picture. Scale bars at the lower-left edge of each picture represent 500 µm lengths. Full article ">Figure 7
(A) Immunohistochemistry (IHC) staining demonstrating 200-fold magnified pictures of hot spots and cold spots from the control and gigantol-treated tumors. The percentages of Ki-67 positive cells as compared with total cells are displayed under their pictures. (B) Graph showing the means of %Ki-67 positive cells. The gigantol-treated tumors have lower Ki-67 positive cells than the control tumors. * indicates p < 0.05 as compared with the control group (Student’s t-test). (C) α-SMA IHC staining of cancer cells in both edge and center areas of tumors showing no difference of signal levels (200×). The numbers of mature tumor vessels per areas between the control and gigantol groups were not different. (D) Pictures showing vessel distribution among the tumor mass (100×). Arrow indicates a vessel (400×). Full article ">Figure 8
Scheme showing the in vivo experimental procedures. Full article ">

18 pages, 3256 KiB

Open AccessArticle

Aberrant DNA Methylation Predicts Melanoma-Specific Survival in Patients with Acral Melanoma

by Dinesh Pradhan, George Jour, Denái Milton, Varshini Vasudevaraja, Michael T. Tetzlaff, Priyadharsini Nagarajan, Jonathan L. Curry, Doina Ivan, Lihong Long, Yingwen Ding, Ravesanker Ezhilarasan, Erik P. Sulman, Adi Diab, Wen-Jen Hwu, Victor G. Prieto, Carlos Antonio Torres-Cabala and Phyu P. Aung

Cancers 2019, 11(12), 2031; https://doi.org/10.3390/cancers11122031 - 16 Dec 2019

Cited by 14 | Viewed by 3765

Abstract

Acral melanoma (AM) is a rare, aggressive type of cutaneous melanoma (CM) with a distinct genetic profile. We aimed to identify a methylome signature distinguishing primary acral lentiginous melanoma (PALM) from primary non-lentiginous AM (NALM), metastatic ALM (MALM), primary non-acral CM (PCM), and [...] Read more.

Acral melanoma (AM) is a rare, aggressive type of cutaneous melanoma (CM) with a distinct genetic profile. We aimed to identify a methylome signature distinguishing primary acral lentiginous melanoma (PALM) from primary non-lentiginous AM (NALM), metastatic ALM (MALM), primary non-acral CM (PCM), and acral nevus (AN). A total of 22 PALM, nine NALM, 10 MALM, nine PCM, and three AN were subjected to genome-wide methylation analysis using the Illumina Infinium Methylation EPIC array interrogating 866,562 CpG sites. A prominent finding was that the methylation profiles of PALM and NALM were distinct. Four of the genes most differentially methylated between PALM and NALM or MALM were HHEX, DIPK2A, NELFB, and TEF. However, when primary AMs (PALM + NALM) were compared with MALM, IFITM1 and SIK3 were the most differentially methylated, highlighting their pivotal role in the metastatic potential of AMs. Patients with NALM had significantly worse disease-specific survival (DSS) than patients with PALM. Aberrant methylation was significantly associated with aggressive clinicopathologic parameters and worse DSS. Our study emphasizes the importance of distinguishing the two epigenetically distinct subtypes of AM. We also identified novel epigenetic prognostic biomarkers that may serve to risk-stratify patients with AM and may be leveraged for the development of targeted therapies. Full article

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Figure 1
Disease-specific survival by melanoma subtype among (A) the four subtypes (p = 0.014) and (B) three subtypes (p = 0.024); (C–H) Disease-specific survival by specific methylome probes showing significant correlation between aberrant methylation of (A) HHEX, (B) NELFB, (C) DIPK2A, (D) TEF, (E) IFITM1, and (F) SIK3 and worse disease-specific survival. Full article ">Figure 2
Results of methylation analyses. (A,C,E,G): Unsupervised hierarchical clustering heatmaps with different β-scores for the 50 most significantly differentially methylated gene-coding and non-gene-coding CpG islands (i.e., lowest p and q values [p < 0.05; q < 0.01]). The heatmap shows distinct methylation profiles between (A) PALM and NALM, (C) PALM and MALM, (E) PALM + NALM and MALM and (G) NALM and MALM. Loci hypermethylated in one tumor are hypomethylated in the other and vice versa. (In the heatmap: RED corresponds to hypermethylation or low gene expression and BLUE corresponds to hypomethylation or high gene expression) (B,D,F,H): Three-dimensional T-distributed stochastic neighbor embedding (t-SNE) showing the distribution of cases classified on the basis of differentially methylated probes adjusted for age and sex. The algorithm calculates the similarity of the patient samples in the two compared groups in a 3-dimensional space, in this case labeled as t-SNE1; t-SNE2; and t-SNE3. The numbers in the three different axes do not have units; they represent the approximate distance between the two different groups/clusters and reflect whether they are truly distinct or not. (B) NALM (red dots) and PALM (green dots) show no neighboring and discrete clusters. (D) MALM (red dots) and PALM (green dots) showing discrete clusters. Note that case MALM 5 (circle) is an outlier that clusters with the PALM group. The blue arrows indicate paired samples from the same patient. (F) MALM (red dots) and PALM + NALM (green dots). Note that case MALM5 (circle) is an outlier that clusters with the PALM + NALM group. (H) NALM (green dots) and MALM (red dots) show discrete clusters with the exception of MALM5 (circle) that clusters with the NALM group. Full article ">Figure 3
Prevalence and degree of hypomethylation (using the raw β score) of the probes of interest across the studied samples. (A) Significant probes associated with PALM vs. NALM: cg26732804 (HHEX), cg07088959 (DIPK2A), cg21298070 (TEF), and cg14397361 (NELFB). (B) Significant probes associated with metastasis of AM: cg11694510 (IFITM1) and cg09923443 (SIK3). (C) Significant probes associated with metastasis of AM: cg24666276 (MYC). In each panel, each column represents one sample; the top row indicates the clinical outcome of the patient that the sample came from; and each row indicates the degree of hypomethylation of the corresponding probe/gene in that sample. Full article ">

16 pages, 710 KiB

Open AccessProject Report

Standardization of Somatic Variant Classifications in Solid and Haematological Tumours by a Two-Level Approach of Biological and Clinical Classes: An Initiative of the Belgian ComPerMed Expert Panel

by Guy Froyen, Marie Le Mercier, Els Lierman, Karl Vandepoele, Friedel Nollet, Elke Boone, Joni Van der Meulen, Koen Jacobs, Suzan Lambin, Sara Vander Borght, Els Van Valckenborgh, Aline Antoniou and Aline Hébrant

Cancers 2019, 11(12), 2030; https://doi.org/10.3390/cancers11122030 - 16 Dec 2019

Cited by 30 | Viewed by 6714

Abstract

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human [...] Read more.

In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software. Full article

(This article belongs to the Collection Targeting Solid Tumors)

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13 pages, 1472 KiB

Open AccessArticle

Expression of GP88 (Progranulin) Protein Is an Independent Prognostic Factor in Prostate Cancer Patients

by Amer Abdulrahman, Markus Eckstein, Rudolf Jung, Juan Guzman, Katrin Weigelt, Ginette Serrero, Binbin Yue, Carol Geppert, Robert Stöhr, Arndt Hartmann, Bernd Wullich, Sven Wach, Helge Taubert and Verena Lieb

Cancers 2019, 11(12), 2029; https://doi.org/10.3390/cancers11122029 - 16 Dec 2019

Cited by 9 | Viewed by 2547

Abstract

Prostate cancer, the second most common cancer, is still a major cause of morbidity and mortality among men worldwide. The expression of the survival and proliferation factor progranulin (GP88) has not yet been comprehensively studied in PCa tumors. The aim of this study [...] Read more.

Prostate cancer, the second most common cancer, is still a major cause of morbidity and mortality among men worldwide. The expression of the survival and proliferation factor progranulin (GP88) has not yet been comprehensively studied in PCa tumors. The aim of this study was to characterize GP88 protein expression in PCa by immunohistochemistry and to correlate the findings to the clinico-pathological data and prognosis. Immunohistochemical staining for GP88 was performed by TMA with samples from 442 PCa patients using an immunoreactive score (IRS). Altogether, 233 cases (52.7%) with negative GP88 staining (IRS < 2) and 209 cases (47.3%) with positive GP88 staining (IRS ≥ 2) were analyzed. A significant positive correlation was found for the GP88 IRS with the PSA value at prostatectomy and the cytoplasmic cytokeratin 20 IRS, whereas it was negatively associated with follow-up times. The association of GP88 staining with prognosis was further studied by survival analyses (Kaplan–Meier, univariate and multivariate Cox’s regression analysis). Increased GP88 protein expression appeared as an independent prognostic factor for overall, disease-specific and relapse-free survival in all PCa patients. Interestingly, in the subgroup of younger PCa patients (≤65 years), GP88 positivity was associated with a 3.8-fold (p = 0.004), a 6.0-fold (p = 0.008) and a 3.7-fold (p = 0.003) increased risk for death, disease-specific death and occurrence of a relapse, respectively. In the PCa subgroup with negative CK20 staining, GP88 positivity was associated with a 1.8-fold (p = 0.018) and a 2.8-fold increased risk for death and disease-specific death (p = 0.028). Altogether, GP88 protein positivity appears to be an independent prognostic factor for PCa patients. Full article

(This article belongs to the Special Issue Prostate Cancer: Past, Present, and Future)

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20 pages, 3331 KiB

Open AccessArticle

Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin

by Julia Biedermann, Matthias Preussler, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Khalil Abou-El-Ardat, Alexander Krüger, Matthias Meinhardt, Gabriele Schackert, William P. Leenders, Christel Herold-Mende, Simone P. Niclou, Rolf Bjerkvig, Graeme Eisenhofer, Achim Temme, Michael Seifert, Leoni A. Kunz-Schughart, Evelin Schröck and Barbara Klink

Cancers 2019, 11(12), 2028; https://doi.org/10.3390/cancers11122028 - 16 Dec 2019

Cited by 20 | Viewed by 6009

Abstract

IDH1^R132H (isocitrate dehydrogenase 1) mutations play a key role in the development of low-grade gliomas. IDH1^wt converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP⁺), whereas IDH1^R132H uses α-ketoglutarate and NADPH to generate the oncometabolite 2-hydroxyglutarate [...] Read more.

IDH1^R132H (isocitrate dehydrogenase 1) mutations play a key role in the development of low-grade gliomas. IDH1^wt converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP⁺), whereas IDH1^R132H uses α-ketoglutarate and NADPH to generate the oncometabolite 2-hydroxyglutarate (2-HG). While the effects of 2-HG have been the subject of intense research, the 2-HG independent effects of IDH1^R132H are still ambiguous. The present study demonstrates that IDH1^R132H expression but not 2-HG alone leads to significantly decreased tricarboxylic acid (TCA) cycle metabolites, reduced proliferation, and enhanced sensitivity to irradiation in both glioblastoma cells and astrocytes in vitro. Glioblastoma cells, but not astrocytes, showed decreased NADPH and NAD⁺ levels upon IDH1^R132H transduction. However, in astrocytes IDH1^R132H led to elevated expression of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT). These effects were not 2-HG mediated. This suggests that IDH1^R132H cells utilize NAD⁺ to restore NADP pools, which only astrocytes could compensate via induction of NAMPT. We found that the expression of NAMPT is lower in patient-derived IDH1-mutant glioma cells and xenografts compared to IDH1-wildtype models. The Cancer Genome Atlas (TCGA) data analysis confirmed lower NAMPT expression in IDH1-mutant versus IDH1-wildtype gliomas. We show that the IDH1 mutation directly affects the energy homeostasis and redox state in a cell-type dependent manner. Targeting the impairments in metabolism and redox state might open up new avenues for treating IDH1-mutant gliomas. Full article

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IDH1R132H influences intracellular TCA (tricarboxylic acid) cycle metabolite levels: Concentrations of TCA metabolites were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and results are shown relatively to the empty vector control for (a) cells transduced with IDH1R132H, (b) cells transduced with IDH1wt, or (c) empty vector controls cells treated with 1 mM D-2-Hydroxyglutarate. For better overview, Y-values were converted to fractions of control. The control of each cell line was defined as a 1.0 baseline and the Y-values were divided by the baseline. All statistical analyses were performed on untransformed data comparing IDH1R132H, IDH1wt, or empty vector+ 2-HG to empty vector cells using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc t-test. (* p < 0.05, ** p < 0.01). Full article ">Figure 2
IDH1R132H reduces growth and increases radio-sensitivity: (a) Cell viability was determined using a WST-1 based colorimetric assay after 48 h culture in adherent condition. Values were normalized to empty vector cells and means from all experiments performed with different transductions were compared (* p < 0.01; one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc t-test). (b) Quantification of cell numbers counted using CASY® TTC 72 h after seeding. (c) Clonogenic survival assays showed that IDH1R132H significantly enhanced the capacity of glioblastoma cells to form colonies, but not of the astrocytes. (d) To analyze cell growth under 3-D conditions, U87-MG control and IDH-mutant cells were seeded in liquid overlay and cultured for up to 50 days. Spheroid size and volume were routinely monitored. In the example shown here, 2 × 103 cells/well were seeded. Data are expressed as the mean volume ± SD. Representative phase contrast microscopic images of the same spheroids are shown at day 4, 11, and 18 (scale bare = 400 µm). (e) Radiation-dose response curves were derived from clonogenic survival assays. Data are expressed as mean of three biological experiments ± SD with n ≥4 wells per experiment and treatment condition. Dose response curves were fitted using a linear-quadratic model (surviving fraction = exp − (αD + βD2)). Full article ">Figure 3
IDH1R132H and not 2-HG alone leads to a drop in NADPH and NAD+ concentrations and sirtuin activity in glioblastoma cells but not in astrocytes: Concentrations of NADPH/t and NAD+/t were measured in cell lysates of stably transduced cell lines from three different transductions and in triplicates using the NAD+/NADH and NADP+/NADPH Quantification Kit (MBL). The activity of NAD+ dependent sirtuins was measured using the HDAC Fluorimetric Cellular Activity Assay Kit (Enzo Life Science). The values were normalized to the mean value of the empty vector cells and the means of normalized values were compared (* p < 0.05; ** p < 0.01; one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc t-test). (a) Normalized values of NADPH levels (top) and NADPt levels (middle) as well as NADPH/NADPt ratios (bottom) are given. (b) IDH1R132H led to a significant drop in NADt (top) and NAD+ (middle) levels in glioblastoma cells, but not in astrocytes. Sirtuins showed less enzymatic activity in IDH1R132H glioblastoma cells compared to the empty vector cells (bottom). Full article ">Figure 4
IDH1-mutation influences the expression of the NAD+ synthesis enzyme NAMPT: (a) NAD synthesis and salvage pathway (adapted from [<a href="#B27-cancers-11-02028" class="html-bibr">27</a>]). NAD is synthesized de novo from tryptophan or from nicotinic acid, nicotinamide riboside and nicotinamide via a salvage pathway. NAD-Kinase (NADK) generates NADPH from NAD+ and ATP. NAPRT: nicotinicacid phosphorybosiltransferse, NMRK1: nicotinamide riboside kinase, NAMPT: nicotinaminde phosphoribosyltransferase, QPRT: quinolinic acid phosphoribosyltransferase, 3-HAO: quniolinicacid-synthesis-enzyme 3-hydroxyanthranilate 3,4-dioxygenase. (b) Representative Western Blot showing the expression of NAD-Synthesis enzymes NAMPT, NMRK1, NAPRT, QRPT and 3-HAO in our cell models. GAPDH was applied to determine protein loading. (c) Proteins expression of NAD-Synthesis enzymes in patient-derived cell lines and xenografts (PDX) tissues of IDH1R132H-mutant and IDH-wildtype gliomas. To quantify NAMPT protein levels, density per sample was divided by the loading-control (GAPDH) and relative density for that lane is given. (d) Mean values of NAMPT protein expression normalized to GAPDH and the empty vector controls from four Western Blots performed with protein extracts of different transductions are shown. (e,f) NAMPT RNA expression levels were measured using reverse transcription quantitative PCR (RT-qPCR). Gene expression was normalized to the expression of reference genes GAPDH and ARF1 (E-ΔCT) and thereafter to the expression in astrocytes or commercially available RNA from normal brain control tissue (E-ΔΔCT). (g) Box plots showing gene expression of NAMPT in IDH1-wildtype (GBM IV) and IDH1-mutant (AS III and AS II) glioma patients based on The Cancer Genome Atlas (TCGA) RNA-seq normalized read count data. Box limits indicate 25th and 75th percentiles, whiskers extend at most to 1.5 times the interquartile range of the box, dots represent outliers. NAMPT was significantly higher expressed in GBM IV versus AS II and III (Wilcoxon rank sum test: p = 1.456541 × 10−26 and p = 1.240315 × 10−36, respectively). (** p < 0.01, *** p < 0.001 one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc t-test). Full article ">Figure 5
Proposed hypothesis of different effects of the IDH1R132H mutation in glial and tumor cells: We found that the IDH1R132H mutation differently affects the redox state of glial cells and tumor cells. Wildtype IDH1 provides essential amounts of NADPH for the cell whereas IDH1R132H consumes α-KG and NAPDH, leading to abnormally high concentrations of 2-HG, reduced concentrations of α-KG and downstream TCA cycle metabolites, as well as an imbalance between NADPH and NADP+ levels. Based on our observations, we hypothesize that in astrocytes, the increased NADPH consumption by IDH1R132H can still be compensated for by elevating the total NADP pool via the induction of NADK and the NAD+ synthesizing enzyme NAMPT. Malignant, proliferating cells, however, cannot compensate for the imbalance of NADPH/NADP+ due to IDH1R132H, leading to decreased NADPH and NAD+ levels. This could be due to the increased requirement of NAD+ and/or NADPH in proliferating cells or insufficient upregulation of NAD synthesis pathways, potentially accompanied by additional inhibition of NAMPT expression due to the IDH1R132H. Abbreviations: NAD = nicotinamide adenine dinucleotide, NADP = nicotinamide adenine dinucleotide phosphate, NMRK1 = nicotinamide riboside kinase, NAMPT = nicotinaminde phosphoribosyltransferase, QPRT = quinolinic acid phosphoribosyltransferase, NADK = NAD-Kinase. Full article ">

19 pages, 6009 KiB

Open AccessFeature PaperArticle

Inhibition of Histone Demethylases LSD1 and UTX Regulates ERα Signaling in Breast Cancer

by Rosaria Benedetti, Carmela Dell’Aversana, Tommaso De Marchi, Dante Rotili, Ning Qing Liu, Boris Novakovic, Serena Boccella, Salvatore Di Maro, Sandro Cosconati, Alfonso Baldi, Emma Niméus, Johan Schultz, Urban Höglund, Sabatino Maione, Chiara Papulino, Ugo Chianese, Francesco Iovino, Antonio Federico, Antonello Mai, Hendrik G. Stunnenberg, Angela Nebbioso and Lucia Altucci Show full author list Hide full author list

Cancers 2019, 11(12), 2027; https://doi.org/10.3390/cancers11122027 - 16 Dec 2019

Cited by 36 | Viewed by 6351

Abstract

In breast cancer, Lysine-specific demethylase-1 (LSD1) and other lysine demethylases (KDMs), such as Lysine-specific demethylase 6A also known as Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), are co-expressed and co-localize with estrogen receptors (ERs), suggesting the potential use of hybrid (epi)molecules to target [...] Read more.

In breast cancer, Lysine-specific demethylase-1 (LSD1) and other lysine demethylases (KDMs), such as Lysine-specific demethylase 6A also known as Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), are co-expressed and co-localize with estrogen receptors (ERs), suggesting the potential use of hybrid (epi)molecules to target histone methylation and therefore regulate/redirect hormone receptor signaling. Here, we report on the biological activity of a dual-KDM inhibitor (MC3324), obtained by coupling the chemical properties of tranylcypromine, a known LSD1 inhibitor, with the 2OG competitive moiety developed for JmjC inhibition. MC3324 displays unique features not exhibited by the single moieties and well-characterized mono-pharmacological inhibitors. Inhibiting LSD1 and UTX, MC3324 induces significant growth arrest and apoptosis in hormone-responsive breast cancer model accompanied by a robust increase in H3K4me2 and H3K27me3. MC3324 down-regulates ERα in breast cancer at both transcriptional and non-transcriptional levels, mimicking the action of a selective endocrine receptor disruptor. MC3324 alters the histone methylation of ERα-regulated promoters, thereby affecting the transcription of genes involved in cell surveillance, hormone response, and death. MC3324 reduces cell proliferation in ex vivo breast cancers, as well as in breast models with acquired resistance to endocrine therapies. Similarly, MC3324 displays tumor-selective potential in vivo, in both xenograft mice and chicken embryo models, with no toxicity and good oral efficacy. This epigenetic multi-target approach is effective and may overcome potential mechanism(s) of resistance in breast cancer. Full article

(This article belongs to the Collection Targeting Solid Tumors)

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41 pages, 5674 KiB

Open AccessReview

Liver Cancer: Current and Future Trends Using Biomaterials

by Sue Anne Chew, Stefania Moscato, Sachin George, Bahareh Azimi and Serena Danti

Cancers 2019, 11(12), 2026; https://doi.org/10.3390/cancers11122026 - 16 Dec 2019

Cited by 29 | Viewed by 7288

Abstract

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer diagnosed and the second leading cause of death worldwide. Despite advancement in current treatments for HCC, the prognosis for this cancer is still unfavorable. This comprehensive review article focuses on all the [...] Read more.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancer diagnosed and the second leading cause of death worldwide. Despite advancement in current treatments for HCC, the prognosis for this cancer is still unfavorable. This comprehensive review article focuses on all the current technology that applies biomaterials to treat and study liver cancer, thus showing the versatility of biomaterials to be used as smart tools in this complex pathologic scenario. Specifically, after introducing the liver anatomy and pathology by focusing on the available treatments for HCC, this review summarizes the current biomaterial-based approaches for systemic delivery and implantable tools for locally administrating bioactive factors and provides a comprehensive discussion of the specific therapies and targeting agents to efficiently deliver those factors. This review also highlights the novel application of biomaterials to study HCC, which includes hydrogels and scaffolds to tissue engineer 3D in vitro models representative of the tumor environment. Such models will serve to better understand the tumor biology and investigate new therapies for HCC. Special focus is given to innovative approaches, e.g., combined delivery therapies, and to alternative approaches—e.g., cell capture—as promising future trends in the application of biomaterials to treat HCC. Full article

(This article belongs to the Special Issue Models of Experimental Liver Cancer)

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16 pages, 2698 KiB

Open AccessArticle

Discovering Rare Genes Contributing to Cancer Stemness and Invasive Potential by GBM Single-Cell Transcriptional Analysis

by Lin Pang, Jing Hu, Feng Li, Huating Yuan, Min Yan, Gaoming Liao, Liwen Xu, Bo Pang, Yanyan Ping, Yun Xiao and Xia Li

Cancers 2019, 11(12), 2025; https://doi.org/10.3390/cancers11122025 - 16 Dec 2019

Cited by 7 | Viewed by 3623

Abstract

Single-cell RNA sequencing presents the sophisticated delineation of cell transcriptomes in many cancer types and highlights the tumor heterogeneity at higher resolution, which provides a new chance to explore the molecular mechanism in a minority of cells. In this study, we utilized publicly [...] Read more.

Single-cell RNA sequencing presents the sophisticated delineation of cell transcriptomes in many cancer types and highlights the tumor heterogeneity at higher resolution, which provides a new chance to explore the molecular mechanism in a minority of cells. In this study, we utilized publicly available single-cell RNA-seq data to discover and comprehensively dissect rare genes existing in few glioblastoma (GBM) cells. Moreover, we designed a framework to systematically identify 51 rare protein-coding genes (PCGs) and 47 rare long non-coding RNAs (lncRNAs) in GBM. Patients with high expression levels of rare genes like CYB5R2 and TPPP3 had worse overall survival and disease-free survival, implying their potential implication in GBM progression and prognosis. We found that these rare genes tended to be specifically expressed in GBM cancer stem cells, which emphasized their ability to characterize stem-like cancer cells and implied their contribution to GBM growth. Furthermore, rare genes were enriched in a 17-cell subset, which was located in an individual branch of the pseudotime trajectory of cancer progression and exhibited high cell cycle activity and invasive potential. Our study captures the rare genes highly expressed in few cells, deepens our understanding of special states during GBM tumorigenesis and progression such as cancer stemness and invasion, and proposes potential targets for cancer therapy. Full article

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11 pages, 649 KiB

Open AccessReview

Chimeric Antigen Receptor T-Cell Therapy for Multiple Myeloma

by Naoki Hosen

Cancers 2019, 11(12), 2024; https://doi.org/10.3390/cancers11122024 - 15 Dec 2019

Cited by 14 | Viewed by 6664

Abstract

CD19 Chimeric antigen receptor (CAR) T cell therapy has been shown to be effective for B cell leukemia and lymphoma. Many researchers are now trying to develop CAR T cells for various types of cancer. For multiple myeloma (MM), B-cell maturation antigen (BCMA) [...] Read more.

CD19 Chimeric antigen receptor (CAR) T cell therapy has been shown to be effective for B cell leukemia and lymphoma. Many researchers are now trying to develop CAR T cells for various types of cancer. For multiple myeloma (MM), B-cell maturation antigen (BCMA) has been recently proved to be a promising target. However, cure of MM is still difficult, and several other targets, for example immunoglobulin kappa chain, SLAM Family Member 7 (SLAMF7), or G-protein coupled receptor family C group 5 member D (GPRC5D), are being tested as targets for CAR T cells. We also reported that the activated integrin β7 can serve as a specific target for CAR T cells against MM, and are preparing a clinical trial. In this review, we summarized current status of CAR T cell therapy for MM and discussed about the future perspectives. Full article

(This article belongs to the Special Issue Latest Development in Multiple Myeloma)

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20 pages, 4224 KiB

Open AccessArticle

Identification of a Subtype of Hepatocellular Carcinoma with Poor Prognosis Based on Expression of Genes within the Glucose Metabolic Pathway

by Xiaoli Zhang, Jin Li, Kalpana Ghoshal, Soledad Fernandez and Lang Li

Cancers 2019, 11(12), 2023; https://doi.org/10.3390/cancers11122023 - 14 Dec 2019

Cited by 16 | Viewed by 4368

Abstract

Hepatocellular carcinoma (HCC) is the most prevalent primary cancer and a highly aggressive liver malignancy. Liver cancer cells reprogram their metabolism to meet their needs for rapid proliferation and tumor growth. In the present study, we investigated the alterations in the expression of [...] Read more.

Hepatocellular carcinoma (HCC) is the most prevalent primary cancer and a highly aggressive liver malignancy. Liver cancer cells reprogram their metabolism to meet their needs for rapid proliferation and tumor growth. In the present study, we investigated the alterations in the expression of the genes involved in glucose metabolic pathways as well as their association with the clinical stage and survival of HCC patients. We found that the expressions of around 30% of genes involved in the glucose metabolic pathway are consistently dysregulated with a predominant down-regulation in HCC tumors. Moreover, the differentially expressed genes are associated with an advanced clinical stage and a poor prognosis. More importantly, unsupervised clustering analysis with the differentially expressed genes that were also associated with overall survival (OS) revealed a subgroup of patients with a worse prognosis including reduced OS, disease specific survival, and recurrence-free survival. This aggressive subtype had significantly increased expression of stemness-related genes and down-regulated metabolic genes, as well as increased immune infiltrates that contribute to a poor prognosis. Collectively, this integrative study indicates that expressions of the glucose metabolic genes could be used as potential prognostic markers and/or therapeutic targets, which might be helpful in developing precise treatment for patients with HCC. Full article

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18 pages, 923 KiB

Open AccessReview

Immunotherapy in Pediatric Solid Tumors—A Systematic Review

by Raoud Marayati, Colin H. Quinn and Elizabeth A. Beierle

Cancers 2019, 11(12), 2022; https://doi.org/10.3390/cancers11122022 - 14 Dec 2019

Cited by 13 | Viewed by 4761

Abstract

Despite advances in the treatment of many pediatric solid tumors, children with aggressive and high-risk disease continue to have a dismal prognosis. For those presenting with metastatic or recurrent disease, multiple rounds of intensified chemotherapy and radiation are the typical course of action, [...] Read more.

Despite advances in the treatment of many pediatric solid tumors, children with aggressive and high-risk disease continue to have a dismal prognosis. For those presenting with metastatic or recurrent disease, multiple rounds of intensified chemotherapy and radiation are the typical course of action, but more often than not, this fails to control the progression of the disease. Thus, new therapeutics are desperately needed to improve the outcomes for these children. Recent advances in our understanding of both the immune system’s biology and its interaction with tumors have led to the development of novel immunotherapeutics as alternative treatment options for these aggressive malignancies. Immunotherapeutic approaches have shown promising results for pediatric solid tumors in early clinical trials, but challenges remain concerning safety and anti-tumor efficacy. In this review, we aim to discuss and summarize the main classes of immunotherapeutics used to treat pediatric solid tumors. Full article

(This article belongs to the Special Issue Immunotherapy, Tumor Microenvironment and Survival Signaling)

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18 pages, 3756 KiB

Open AccessArticle

Inhibitor of DNA-Binding Protein 4 Suppresses Cancer Metastasis through the Regulation of Epithelial Mesenchymal Transition in Lung Adenocarcinoma

by Chi-Chung Wang, Yuan-Ling Hsu, Chi-Jen Chang, Chia-Jen Wang, Tzu-Hung Hsiao and Szu-Hua Pan

Cancers 2019, 11(12), 2021; https://doi.org/10.3390/cancers11122021 - 14 Dec 2019

Cited by 11 | Viewed by 3658

Abstract

Metastasis is a predominant cause of cancer death and the major challenge in treating lung adenocarcinoma (LADC). Therefore, exploring new metastasis-related genes and their action mechanisms may provide new insights for developing a new combative approach to treat lung cancer. Previously, our research [...] Read more.

Metastasis is a predominant cause of cancer death and the major challenge in treating lung adenocarcinoma (LADC). Therefore, exploring new metastasis-related genes and their action mechanisms may provide new insights for developing a new combative approach to treat lung cancer. Previously, our research team discovered that the expression of the inhibitor of DNA binding 4 (Id4) was inversely related to cell invasiveness in LADC cells by cDNA microarray screening. However, the functional role of Id4 and its mechanism of action in lung cancer metastasis remain unclear. In this study, we report that the expression of Id4 could attenuate cell migration and invasion in vitro and cancer metastasis in vivo. Detailed analyses indicated that Id4 could promote E-cadherin expression through the binding of Slug, cause the occurrence of mesenchymal-epithelial transition (MET), and inhibit cancer metastasis. Moreover, the examination of the gene expression database (GSE31210) also revealed that high-level expression of Id4/E-cadherin and low-level expression of Slug were associated with a better clinical outcome in LADC patients. In summary, Id4 may act as a metastatic suppressor, which could not only be used as an independent predictor but also serve as a potential therapeutic for LADC treatment. Full article

(This article belongs to the Special Issue Cancer Invasion and Metastasis)

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Full article ">Figure 1
Inhibitor of DNA binding 4 (Id4) expression inversely correlates with lung cancer metastasis in vitro and in vivo. (a) Id4 mRNA and protein expression levels in different lung cancer cell lines were detected by RT-PCR (left, Id4) and immunoblotting (left, ID4). The numbers under the images of bands indicate the quantification of mRNA and protein expressions, both of which were calculated by ImageJ software and normalized to the internal control, Gβ-like or β-actin, of each cell line. The invasive ability of each cell line was evaluated by a modified Boyden chamber invasion assay in vitro. The images of the invasion assay (original magnification, ×100) were presented (middle) and the numbers of invasive cells were calculated (bottom left; p < 0.05 by one-way ANOVA). The correlation of Id4 expressions and cell invasiveness in different lung cancer cells was calculated by linear regression (top right: the correlation of Id4 mRNA expression and cell invasiveness; bottom right: the correlation of Id4 protein expression and cell invasiveness; p < 0.05). (b) Expressions of Id4 interfere with cell invasiveness. Id4 expressions and images of invasive cells (original magnification, ×100) are shown for CL1-0 or H1975/Id4-silencing (up, left) and CL1-5 or H1299/Id4-overexpressing (up, right) stable cell lines. The protein expression levels and the invasive abilities of Id4 stable cells were quantified. The relative fold changes compared with the control cells (* p < 0.05) are displayed. (c) The effects of Id4 expression in cancer metastasis in vivo were examined by a tail vein metastasis assay with H1299/Id4-overexpressing stable cells. The numbers of metastatic tumor nodules were calculated from five mice per group (* p < 0.05). Histology of the metastatic pulmonary nodules was confirmed as lung adenocarcinoma (LADC) by H&E staining; the arrows indicated the distribution of tumors, and the area of black rectangles was zoomed and presented at the bottom. Full article ">Figure 2
Id4 is involved in the regulation of epithelial–mesenchymal transition (EMT). (a) Cell morphology of CL1-0/Id4-silencing (left), CL1-5/Id4-overexpressing (middle), and H1299/Id4-overexpressing (right) cells was pictured, and the mRNA and protein expressions of two well-known EMT markers, E-cad and N-cad, were detected by RT-PCR and immunoblotting (bottom). The mRNA and protein expression levels in Id4-overexpressing or silencing cells were quantified, and the relative fold changes compared with the scramble or vector control cells are displayed. The arrow heads in the images indicate that the cells presented mesenchymal (blue arrows) or epithelial-like phenotypes (red arrows). (b) Biological functional analysis of differentially expressed genes by Ingenuity Pathway Analysis (red: the number of genes which was displayed changed at least twofold between CL1-5/Id4-overexpressing and CL1-5/vector control cell lines). (c) Top 10 genes significantly down-regulated in CL1-5/ Id4-overexpressing stable cells compared with those in CL1-5/vector control cells. (d) The mRNA and protein expression levels of Slug in CL1-0/Id4-silencing (left), CL1-5/Id4-overexpressing (middle), and H1299/Id4-overexpressing stable cell lines (right) were detected by RT-PCR and immunoblotting. The mRNA and protein expression levels in Id4-overexpressing or silencing cells were quantified and displayed the relative fold changes compared with the scramble or vector control cells. Full article ">Figure 3
Id4 binds to Slug and inhibits the transcriptional repression activity of Slug in cells. (a,b) HEK293T cell lines were co-transfected with V5-tagged Id4 and Flag-tagged Slug (a) or different Slug mutants, which involved amino acid residues 1–106, 107–158, 159–212, and 213–268 (b), and the protein–protein interaction between Id4 and Slug or Slug mutants was recognized by immunoprecipitation with anti-V5 or anti-Flag antibodies (as indicated). In total, 30 μg total cell lysates were taken as the input. Black arrow: the molecular weight of ID4. (c,d) HEK293T cells were co-transfected with the indicated plasmids and a reporter vector driven by SBS-Gal4 (c) or by the E-cadherin promoter (d). Luciferase activities and immunoblotting were evaluated 24 h after transfection. The activity induced by Gal4-VP16 alone (c) or basal activity (d) was normalized to 100%. All data were reported as mean values ± SEMs, and p-values were calculated via Student’s t-test. The asterisk represents a p value of < 0.05 compared to the group stimulated with Gal4-VP16 alone or the basal activity level. The protein expression levels in cells were quantified and displayed the relative fold changes compared with the control cells, which were only transfected with promoter-construction. (e) Right: The IgG and Slug antibodies were used to pull down protein-DNA complexes in HEK293-Slug/Id4 and H1299/Id4 cell lines, and the E-cadherin promoter level in each sample was determined by PCR using a gene-specific primer set (bottom, p < 0.05). Input: an aliquot of each sample was prepared and used as a template for PCR to examine the level of the E-cadherin promoter before immunoprecipitation (IP). Left: Immunoblot analysis of the indicated proteins was performed on the products of the chromatin immunoprecipitation assay (ChIP). The protein expression levels in Id4-overexpressing cells were quantified and displayed the relative fold changes compared with the vector control cells. Full article ">Figure 4
Low-level Id4 expression is associated with a poor clinical outcome in lung adenocarcinoma (LADC) patients. The data of Id4, Slug, and E-cadherin expression was obtained from 168 pathological stage I LADC patients in the public database GSE31210 [<a href="#B51-cancers-11-02021" class="html-bibr">51</a>]. (a,b) The patients were divided into the high- (red line) and low-expression (blue line) of each gene using the median value as the cutoff, and Kaplan-Meier estimates of overall survival and relapse-free survival in these patients. The combined effects of Id4, Slug, and E-cadherin on the overall survival and relapse-free survival of non-small cell lung cancer (NSCLC) patients were analyzed. p-values were calculated using the log rank test. (c) A tree diagram showing the proportions of patients with high and low Id4/Slug/E-cadherin expression levels in patients. Full article ">Figure 5
Inhibitor of DNA binding 4 (Id4) induces mesenchymal-epithelial transition (MET) through regulating the Slug/E-cadherin axis. Up: Slug protein can bind to the E-box motif in the promoter region and suppress the expression of E-cadherin in the nucleus. Bottom: When Id4 is overexpressed in cells, it can bind to Slug, remove the Slug protein from the E-box motif in the promoter region, promote the expression of E-cadherin, and induce cell-processed MET. Full article ">

12 pages, 578 KiB

Open AccessArticle

Low Systemic Levels of Chemokine C-C Motif Ligand 3 (CCL3) are Associated with a High Risk of Venous Thromboembolism in Patients with Glioma

by Pegah Mir Seyed Nazari, Christine Marosi, Florian Moik, Julia Riedl, Öykü Özer, Anna Sophie Berghoff, Matthias Preusser, Johannes A. Hainfellner, Ingrid Pabinger, Gerhard J. Zlabinger and Cihan Ay

Cancers 2019, 11(12), 2020; https://doi.org/10.3390/cancers11122020 - 14 Dec 2019

Cited by 13 | Viewed by 3102

Abstract

A tight interplay between inflammation and hemostasis has been described as a potential driver for developing venous thromboembolism (VTE). Here, we investigated the association of systemic cytokine levels and risk of VTE in patients with glioma. This analysis was conducted within the prospective, [...] Read more.

A tight interplay between inflammation and hemostasis has been described as a potential driver for developing venous thromboembolism (VTE). Here, we investigated the association of systemic cytokine levels and risk of VTE in patients with glioma. This analysis was conducted within the prospective, observational Vienna Cancer and Thrombosis Study. Patients with glioma were included at time of diagnosis or progression and were observed for a maximum of two years. Primary endpoint was objectively confirmed VTE. At study entry, a single blood draw was performed. A panel of nine cytokines was measured in serum samples with the xMAP technology developed by Luminex. Results: Overall, 76 glioma patients were included in this analysis, and 10 (13.2%) of them developed VTE during the follow-up. Chemokine C-C motif ligand 3 (CCL3) levels were inversely associated with risk of VTE (hazard ratio [HR] per double increase, 95% confidence interval [CI]: 0.385, 95% CI: 0.161–0.925, p = 0.033), while there was no association between the risk of VTE and serum levels of interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-11, tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF), respectively. In conclusion, low serum levels of CCL3 were associated with an increased risk of VTE. CCL3 might serve as a potential biomarker to predict VTE risk in patients with glioma. Full article

(This article belongs to the Special Issue Treatment of Cancer-Associated Thrombosis)

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14 pages, 1895 KiB

Open AccessArticle

Tumor Suppressor Function of miR-127-3p and miR-376a-3p in Osteosarcoma Cells

by Joerg Fellenberg, Burkhard Lehner, Heiner Saehr, Astrid Schenker and Pierre Kunz

Cancers 2019, 11(12), 2019; https://doi.org/10.3390/cancers11122019 - 14 Dec 2019

Cited by 27 | Viewed by 3256

Abstract

Since the introduction of high-dose chemotherapy about 35 years ago, survival rates of osteosarcoma patients have not been significantly improved. New therapeutic strategies replacing or complementing conventional chemotherapy are therefore urgently required. MicroRNAs represent promising targets for such new therapies, as they are [...] Read more.

Since the introduction of high-dose chemotherapy about 35 years ago, survival rates of osteosarcoma patients have not been significantly improved. New therapeutic strategies replacing or complementing conventional chemotherapy are therefore urgently required. MicroRNAs represent promising targets for such new therapies, as they are involved in the pathology of multiple types of cancer, and aberrant expression of several miRNAs has already been shown in osteosarcoma. In this study, we identified silencing of miR-127-3p and miR-376a-3p in osteosarcoma cell lines and tissues and investigated their role as potential tumor suppressors in vitro and in vivo. Transfection of osteosarcoma cells (n = 6) with miR-127-3p and miR-376a-3p mimics significantly inhibited proliferation and reduced the colony formation capacity of these cells. In contrast, we could not detect any influence of miRNA restoration on cell cycle and apoptosis induction. The effects of candidate miRNA restoration on tumor engraftment and growth in vivo were analyzed using a chicken chorioallantoic membrane (CAM) assay. Cells transfected with mir-127-3p and miR-376a-3p showed reduced tumor take rates and tumor volumes and a significant decrease of the cumulative tumor volumes to 41% and 54% compared to wildtype cells. The observed tumor suppressor function of both analyzed miRNAs indicates these miRNAs as potentially valuable targets for the development of new therapeutic strategies for the treatment of osteosarcoma. Full article

(This article belongs to the Special Issue Bone and Soft Tissue Tumors)

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Silencing of miR-127-3p and miR-376a-3p in osteosarcoma cell lines and tissue. Quantitative real-time PCR analysis of (A) miR-127-3p and (B) miR-376a-3p expression in osteosarcoma cell lines (n = 7), osteosarcoma tissue (n = 8), and primary human osteoblasts (hOBs) (n = 5). Expression values were normalized to the expression of the small nuclear RNA U6 pseudogene (RNU6B). The white lines indicate the medians, the lower boundary of the box of the 25th percentile and the upper boundary of the box of the 75th percentile. The whiskers indicate the highest and lowest values. p-values were determined by the Mann–Whitney U test. (** p < 0.01). (C) Upregulation of miRNA expression by epigenetic modifiers. The osteosarcoma cell line 143B was treated for seven days with the indicated concentrations of 5′-Aza-2′-deoxycytidine (AZA) followed by a further three days of culture with or without the addition of phenylbutyric acid (PBA). After the incubation period, the expression of miR-127-3p and miR-376a-3p was quantified and normalized to the expression of RNU6B. Full article ">Figure 2
Restoration of endogenous levels of miR-127-3p and miR-376a-3p inhibits proliferation of osteosarcoma cells. Six osteosarcoma cell lines were transfected with miR-127-3p and miR-376a-3p mimics before cells were counted at the indicated time points. Non-transfected wildtype cells (WT) and cells transfected with a negative control miRNA (NC) served as controls. Experiments were done in triplicates. * p < 0.05, ** p < 0.01 compared to wildtype cells. Full article ">Figure 3
Mir-127-3p and miR-376a-3p reduce the colony forming capacity of osteosarcoma cells. (A) Cells transfected with miRNA mimics, a negative control miRNA (NC), or untreated wildtype cells (WT) were cultured for 10 days in soft agar, photographed, and colonies >500 µm2 were counted using ImageJ software (* p < 0.05 compared to WT group). (B) Mean colony size of all five osteosarcoma cell lines normalized to the wildtype cells. (C) Representative photographs of colonies formed by MG-63 cells. Full article ">Figure 4
Cell cycle analysis of osteosarcoma cells with restored miRNA expression. The percentage of cells in the subG, G0/G1-, S-, and G2/M-phase was determined 96 h after transfection by flow cytometry. (A) Representative graphs of the cell line CAL-72. (B) Summary of the mean cell counts and standard deviations obtained from three independent experiments with six cell lines. (C) Percentage of apoptotic cells determined by NucView 488 staining 96 h after miRNA restoration. Positive control cells were treated with 2mM H2O2 for 4 h and further incubated for 24 h without the addition of H2O2. Full article ">Figure 5
Restoration of miR-127-3p and miR-376a-3p inhibits tumor growth in vivo. Osteosarcoma cells were transiently transfected with miRNA mimics, a negative control miRNA (NC), or left untreated (WT). Then, 72 h after transfection, cells were transplanted onto the chorioallantoic membrane (CAM) of fertilized chicken eggs (n = 15 per group). Tumors were resected on day 16 and the tumor volumes (A), ** p < 0.01, the tumor take rates (B), and the cumulative tumor volumes (C) were calculated (* p < 0.05). (D) Representative photographs of tumor xenografts from each treatment group. Full article ">

29 pages, 1836 KiB

Open AccessReview

What Is the Role of Interleukins in Breast Cancer Bone Metastases? A Systematic Review of Preclinical and Clinical Evidence

by Francesca Salamanna, Veronica Borsari, Deyanira Contartese, Viviana Costa, Gianluca Giavaresi and Milena Fini

Cancers 2019, 11(12), 2018; https://doi.org/10.3390/cancers11122018 - 13 Dec 2019

Cited by 16 | Viewed by 3362

Abstract

Breast cancer cells produce stimulators of bone resorption known as interleukins (ILs). However, data on the functional roles of ILs in the homing of metastatic breast cancer to bone are still fragmented. A systematic search was carried out in three databases (PubMed, Scopus, [...] Read more.

Breast cancer cells produce stimulators of bone resorption known as interleukins (ILs). However, data on the functional roles of ILs in the homing of metastatic breast cancer to bone are still fragmented. A systematic search was carried out in three databases (PubMed, Scopus, Web of Science Core Collection) to identify preclinical reports, and in three clinical registers (ClinicalTrials.gov, World Health Organization (WHO) International Clinical Trials Registry Platform, European Union (EU) Clinical Trials Register) to identify clinical trials, from 2008 to 2019. Sixty-seven preclinical studies and 11 clinical trials were recognized as eligible. Although preclinical studies identified specific key ILs which promote breast cancer bone metastases, which have pro-metastatic effects (e.g., IL-6, IL-8, IL-1β, IL-11), and whose inhibition also shows potential preclinical therapeutic effects, the clinical trials focused principally on ILs (IL-2 and IL-12), which have an anti-metastatic effect and a potential to generate a localized and systemic antitumor response. However, these clinical trials are yet to post any results or conclusions. This inconsistency indicates that further studies are necessary to further develop the understanding of cellular and molecular relations, as well as signaling pathways, both up- and downstream of ILs, which could represent a novel strategy to treat tumors that are resistant to standard care therapies for patients affected by breast cancer bone disease. Full article

(This article belongs to the Special Issue Targeting Bone Metastasis in Cancers)

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25 pages, 6318 KiB

Open AccessArticle

Aberrant Protein Phosphorylation in Cancer by Using Raman Biomarkers

by Halina Abramczyk, Anna Imiela, Beata Brożek-Płuska, Monika Kopeć, Jakub Surmacki and Agnieszka Śliwińska

Cancers 2019, 11(12), 2017; https://doi.org/10.3390/cancers11122017 - 13 Dec 2019

Cited by 35 | Viewed by 6079

Abstract

(1) Background: Novel methods are required for analysing post-translational modifications of protein phosphorylation by visualizing biochemical landscapes of proteins in human normal and cancerous tissues and cells. (2) Methods: A label-free Raman method is presented for detecting spectral changes that arise in proteins [...] Read more.

(1) Background: Novel methods are required for analysing post-translational modifications of protein phosphorylation by visualizing biochemical landscapes of proteins in human normal and cancerous tissues and cells. (2) Methods: A label-free Raman method is presented for detecting spectral changes that arise in proteins due to phosphorylation in the tissue of human breasts, small intestines, and brain tumours, as well as in the normal human astrocytes and primary glioblastoma U-87 MG cell lines. Raman spectroscopy and Raman imaging are effective tools for monitoring and analysing the vibrations of functional groups involved in aberrant phosphorylation in cancer without any phosphorecognition of tag molecules. (3) Results: Our results based on 35 fresh human cancer and normal tissues prove that the aberrant tyrosine phosphorylation monitored by the unique spectral signatures of Raman vibrations is a universal characteristic in the metabolic regulation in different types of cancers. Overexpressed tyrosine phosphorylation in the human breast, small intestine and brain tissues and in the human primary glioblastoma U-87 MG cell line was monitored by using Raman biomarkers. (4) We showed that the bands at 1586 cm⁻¹ and 829 cm⁻¹, corresponding to phosphorylated tyrosine, play a pivotal role as a Raman biomarker of the phosphorylation status in aggressive cancers. We found that the best Raman biomarker of phosphorylation is the 1586/829 ratio showing the statistical significance at p Values of ≤ 0.05. (5) Conclusions: Raman spectroscopy and imaging have the potential to be used as screening functional assays to detect phosphorylated target proteins and will help researchers to understand the role of phosphorylation in cellular processes and cancer progression. The abnormal and excessive high level of tyrosine phosphorylation in cancer samples compared with normal samples was found in the cancerous human tissue of breasts, small intestines and brain tumours, as well as in the mitochondria and lipid droplets of the glioblastoma U-87 MG cell line. Detailed insights are presented into the intracellular oncogenic metabolic pathways mediated by phosphorylated tyrosine. Full article

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10 pages, 228 KiB

Open AccessArticle

Dose-Limiting Organs at Risk in Carbon Ion Re-Irradiation of Head and Neck Malignancies: An Individual Risk-Benefit Tradeoff

by Thomas Held, Semi B. Harrabi, Kristin Lang, Sati Akbaba, Paul Windisch, Denise Bernhardt, Stefan Rieken, Klaus Herfarth, Jürgen Debus and Sebastian Adeberg

Cancers 2019, 11(12), 2016; https://doi.org/10.3390/cancers11122016 - 13 Dec 2019

Cited by 7 | Viewed by 2526

Abstract

Background: Carbon ion re-irradiation (CIR) was evaluated to investigate treatment planning and the consequences of individual risk-benefit evaluations concerning dose-limiting organs at risk (OAR). Methods: A total of 115 consecutive patients with recurrent head and neck cancer (HNC) were analyzed after [...] Read more.

Background: Carbon ion re-irradiation (CIR) was evaluated to investigate treatment planning and the consequences of individual risk-benefit evaluations concerning dose-limiting organs at risk (OAR). Methods: A total of 115 consecutive patients with recurrent head and neck cancer (HNC) were analyzed after initial radiotherapy and CIR at the same anatomical site. Toxicities were evaluated in line with the Common Terminology Criteria for Adverse Events 4.03. Results: The median maximum cumulative equivalent doses applied in fractions of 2 Gy (EQD2) to the brainstem, optic chiasm, ipsilateral optic nerve, and spinal cord were 56.8 Gy (range 0.94–103.9), 51.4 Gy (range 0–120.3 Gy), 63.6 Gy (range 0–146.1 Gy), and 28.8 Gy (range 0.2–87.7 Gy). The median follow up after CIR was 24.0 months (range 2.5–72.0 months). The cumulative rates of acute and late severe (≥grade III) side effects after CIR were 1.8% and 14.3%. Conclusion: In recurrent HNC, an individual risk-benefit tradeoff is frequently inevitable due to unfavorable location of tumors in close proximity to vital OAR. There are uncertainties about the dose tolerance of OAR after CIR, which warrant increased awareness about the potential treatment toxicity and further studies on heavy ion re-irradiation. Full article

(This article belongs to the Special Issue New Horizons in Particle Therapy)

38 pages, 600 KiB

Open AccessReview

Pursuing a Curative Approach in Multiple Myeloma: A Review of New Therapeutic Strategies

by Mattia D'Agostino, Luca Bertamini, Stefania Oliva, Mario Boccadoro and Francesca Gay

Cancers 2019, 11(12), 2015; https://doi.org/10.3390/cancers11122015 - 13 Dec 2019

Cited by 26 | Viewed by 7295

Abstract

Multiple myeloma (MM) is still considered an incurable hematologic cancer and, in the last decades, the treatment goal has been to obtain a long-lasting disease control. However, the recent availability of new effective drugs has led to unprecedented high-quality responses and prolonged progression-free [...] Read more.

Multiple myeloma (MM) is still considered an incurable hematologic cancer and, in the last decades, the treatment goal has been to obtain a long-lasting disease control. However, the recent availability of new effective drugs has led to unprecedented high-quality responses and prolonged progression-free survival and overall survival. The improvement of response rates has prompted the development of new, very sensitive methods to measure residual disease, even when monoclonal components become undetectable in patients’ serum and urine. Several scientific efforts have been made to develop reliable and validated techniques to measure minimal residual disease (MRD), both within and outside the bone marrow. With the newest multidrug combinations, a good proportion of MM patients can achieve MRD negativity. Long-lasting MRD negativity may prove to be a marker of “operational cure”, although the follow-up of the currently ongoing studies is still too short to draw conclusions. In this article, we focus on results obtained with new-generation multidrug combinations in the treatment of high-risk smoldering MM and newly diagnosed MM, including the potential role of MRD and MRD-driven treatment strategies in clinical trials, in order to optimize and individualize treatment. Full article

(This article belongs to the Special Issue Latest Development in Multiple Myeloma)

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17 pages, 2544 KiB

Open AccessArticle

HuR Reduces Radiation-Induced DNA Damage by Enhancing Expression of ARID1A

by Daniel Andrade, Meghna Mehta, James Griffith, Sangphil Oh, Joshua Corbin, Anish Babu, Supriyo De, Allshine Chen, Yan D. Zhao, Sanam Husain, Sudeshna Roy, Liang Xu, Jeffrey Aube, Ralf Janknecht, Myriam Gorospe, Terence Herman, Rajagopal Ramesh and Anupama Munshi

Cancers 2019, 11(12), 2014; https://doi.org/10.3390/cancers11122014 - 13 Dec 2019

Cited by 25 | Viewed by 4245

Abstract

Tumor suppressor ARID1A, a subunit of the chromatin remodeling complex SWI/SNF, regulates cell cycle progression, interacts with the tumor suppressor TP53, and prevents genomic instability. In addition, ARID1A has been shown to foster resistance to cancer therapy. By promoting non-homologous end joining (NHEJ), [...] Read more.

Tumor suppressor ARID1A, a subunit of the chromatin remodeling complex SWI/SNF, regulates cell cycle progression, interacts with the tumor suppressor TP53, and prevents genomic instability. In addition, ARID1A has been shown to foster resistance to cancer therapy. By promoting non-homologous end joining (NHEJ), ARID1A enhances DNA repair. Consequently, ARID1A has been proposed as a promising therapeutic target to sensitize cancer cells to chemotherapy and radiation. Here, we report that ARID1A is regulated by human antigen R (HuR), an RNA-binding protein that is highly expressed in a wide range of cancers and enables resistance to chemotherapy and radiation. Our results indicate that HuR binds ARID1A mRNA, thereby increasing its stability in breast cancer cells. We further find that ARID1A expression suppresses the accumulation of DNA double-strand breaks (DSBs) caused by radiation and can rescue the loss of radioresistance triggered by HuR inhibition, suggesting that ARID1A plays an important role in HuR-driven resistance to radiation. Taken together, our work shows that HuR and ARID1A form an important regulatory axis in radiation resistance that can be targeted to improve radiotherapy in breast cancer patients. Full article

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ARID1A is a bona fide human antigen R (HuR) target. (A) Twenty-four hours after transfection with siHuR, MDA-MB-231 cells were treated with Actinomycin D and collected at the indicated time points to evaluate their ARID1A mRNA levels by RT-qPCR. (B) H2AX mRNA levels were also evaluated as a negative control. (C) MDA-MB-231 cell lysates were used to immunoprecipitate HuR using an antibody against HuR or IgG. HuR immunocomplexes were subjected to RNA extraction, and the levels of ARID1A mRNA were measured by qRT-PCR. The levels of HuR and H2AX mRNAs were also measured as positive and negative controls, respectively. Inset shows HuR immunoprecipitation by HuR antibody, but not by IgG control. (D) Eight hours after transfection with HuR siRNA, MDA-MB-231 cells were transfected with a vector encoding either luciferase alone (Lux-No-UTR) or luciferase fused to the 3′UTR of ARID1A (Lux-ARID1A 3′UTR); 16 h later, luciferase activity was evaluated. (E) Three segments of ARID1A 3′UTR were cloned into a T7 containing vector to generate biotinylated RNA probes. Asterisk indicates regions of high probability for HuR binding. (F) These probes were incubated with MDA-MB-231 lysates, recovered with neutravidin-coated beads, and subjected to immunoblot analysis to evaluate the levels of HuR bound to the probes. Data represent the average of three independent experiments. Error bars represent SEM (standard error of the mean), * p ≤ 0.01 and ** p ≤ 0.001. Full article ">Figure 2
Genetic or pharmacological inhibition of HuR reduces ARID1A levels. Twenty-four hours after transfection with HuR siRNA, MDA-MB-231 and Hs578t cells were treated with a radiation dose of 5 Gy; 2 h later, they were evaluated for ARID1A expression by (A,B) Western blot or (C,D) (q) RT-PCR analyses. MDA-MB-231 and SUM159PT cells treated with HuR inhibitor, CMLD-2, for 24 h were collected to evaluate ARID1A expression levels. (E,F) As a control for HuR inhibition, p27 expression was also evaluated in MDA-MB-231 and SUM159PT following CMLD-2 treatment. Data presented are the average of three independent experiments. Error bars represent SEM (standard error of the mean), * p ≤ 0.01 and ** p ≤ 0.001. Full article ">Figure 3
HuR overexpression elevates ARID1A abundance. MCF-7 and MDA-MB-231 cells transfected with an empty or a HuR-FLAG tagged vector were collected to evaluate HuR and ARID1A expression by Western blot (A,C) and RT-qPCR (B,D) analyses. Data represent the average of three independent experiments. Error bars represent SEM (standard error of the mean), * p ≤ 0.01 and *** p ≤ 0.0001. Full article ">Figure 4
HuR and ARID1A expression levels correlate in vivo. (A) The levels of HuR and ARID1A in breast cancer were examined by immunohistochemical (IHC) analysis of tissue arrays. Scale bar equals 100 µm (B) Analysis of correlation in the levels of HuR and ARID1A in breast cancer tissue arrays. (C) Patient-derived xenograft tissues were analyzed by Western blot to evaluate HuR and ARID1A expression levels. (D) Quantified and normalized band intensities of ARID1A and HuR were subjected to correlation analysis. Full article ">Figure 5
Targeting ARID1A sensitizes breast cancer cells to radiation. (A–C) MDA-MB-231, MDA-MB-468, and SUM159PT cells transfected with ARID1A siRNA were exposed to the indicated doses of gamma radiation and re-plated for colony formation. Around 10 days later, the colonies were stained and quantitated to evaluate surviving fractions. (D,E) MDA-MB-231 and SUM-159PT cells transfected with either an empty vector or an ARID1A overexpressing vector were irradiated with the indicated doses and re-plated for colony formation analysis. Around 10 days later, the colonies were stained and quantitated to evaluate the surviving fractions. (F) MDA-MB-231 cells previously treated with HuR siRNA were transfected with ARID1A or empty vector to test the ability of ARID1A to complement HuR-dependent radioresistance. Error bars represent SEM (standard error of the mean), * p ≤ 0.05. Full article ">Figure 6
Ablating ARID1A leads to accumulation of DNA double-strand breaks (DSBs). In MDA-MB-231 cells transfected with ARID1A siRNA, followed by irradiation, the levels of DNA damage were evaluated with two approaches: Measurement of ɤH2AX levels by (A) Western blot analysis and (B) neutral comet assay. After MDA-MB-231 cells transfected with an empty vector or one that overexpresses ARID1A were irradiated, the levels of DNA damage were evaluated with two approaches: Measurement of ɤH2AX levels by Western blot analysis (C) and neutral comet assay (D). (E,F) MDA-MB-231 cells previously treated with HuR siRNA were transfected with an empty vector or a vector that expressed ARID1A to test the ability of ARID1A to reduce DNA DSBs in cells where HuR was downregulated. Error bars represent SEM (standard error of the mean), ** p ≤ 0.05; *** p ≤ 0.0001. Full article ">

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Cancers, Volume 11, Issue 12 (December 2019) – 223 articles

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