Cancers

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Open AccessReview

Oncogenic MicroRNAs: Key Players in Malignant Transformation

by Tania Frixa, Sara Donzelli and Giovanni Blandino

Cancers 2015, 7(4), 2466-2485; https://doi.org/10.3390/cancers7040904 - 18 Dec 2015

Cited by 126 | Viewed by 5764

MicroRNAs (miRNAs) represent a class of non-coding RNAs that exert pivotal roles in the regulation of gene expression at the post-transcriptional level. MiRNAs are involved in many biological processes and slight modulations in their expression have been correlated with the occurrence of different [...] Read more.

MicroRNAs (miRNAs) represent a class of non-coding RNAs that exert pivotal roles in the regulation of gene expression at the post-transcriptional level. MiRNAs are involved in many biological processes and slight modulations in their expression have been correlated with the occurrence of different diseases. In particular, alterations in the expression of miRNAs with oncogenic or tumor suppressor functions have been associated with carcinogenesis, malignant transformation, metastasis and response to anticancer treatments. This review will mainly focus on oncogenic miRNAs whose aberrant expression leads to malignancy. Full article

(This article belongs to the Special Issue Non-Coding RNAs in Cancers)

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Open AccessFeature PaperCase Report

Acute Myeloid Leukemia with Isolated Trisomy 19 Associated with Diffuse Myelofibrosis and Osteosclerosis

by Adam Stelling, Brian A. Jonas, Hooman H. Rashidi, Mehrdad Abedi and Mingyi Chen

Cancers 2015, 7(4), 2459-2465; https://doi.org/10.3390/cancers7040903 - 14 Dec 2015

Cited by 2 | Viewed by 5333

Abstract

Primary myelofibrosis (PMF), per WHO criteria, is a clonal myeloproliferative neoplasm that usually presents with a proliferation of granulocytic and megakaryocytic lineages with an associated fibrous deposition and extramedullary hematopoiesis. The bone marrow histologic findings of this disorder are typically characterized by the [...] Read more.

Primary myelofibrosis (PMF), per WHO criteria, is a clonal myeloproliferative neoplasm that usually presents with a proliferation of granulocytic and megakaryocytic lineages with an associated fibrous deposition and extramedullary hematopoiesis. The bone marrow histologic findings of this disorder are typically characterized by the presence of myeloid metaplasia with an associated reactive fibrosis, angiogenesis, and osteosclerosis. However, marked myelofibrosis is not solely confined to PMF and may also be associated with other conditions including but not limited to acute megakaryoblastic leukemias (FAB AML-M7). Here, we describe a rare case of a non-megakaryoblastic acute myeloid leukemia with marked myelofibrosis with osteosclerosis and an isolated trisomy 19. A 19-year-old male presented with severe bone pain of one week duration with a complete blood cell count and peripheral smear showing a mild anemia and occasional circulating blasts. A follow up computed tomography (CT) scan showed diffuse osteosclerosis with no evidence of hepatosplenomegaly or lymphadenopathy. Subsequently, the bone marrow biopsy showed markedly sclerotic bony trabeculae and a hypercellular marrow with marked fibrosis and intervening sheets of immature myeloid cells consistent with myeloblasts with monocytic differentiation. Importantly, these myeloblasts were negative for megakaryocytic markers (CD61 and vWF), erythroid markers (hemoglobin and E-cadherin), and lymphoid markers (CD3, CD19, and TdT). Metaphase cytogenetics showed an isolated triosomy 19 with no JAK2 V617F mutation. The patient was treated with induction chemotherapy followed by allogenic hematopoietic stem cell transplantation which subsequently resulted in a rapid resolution of bone marrow fibrosis, suggesting graft-anti-fibrosis effect. This is a rare case of a non-megakaryoblastic acute myeloid leukemia with myelofibrosis and osteosclerosis with trisomy 19 that may provide insights into the prognosis and therapeutic options of future cases. Full article

(This article belongs to the Special Issue Cancer-Associated Fibroblasts)

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Open AccessReview

Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth

by Kazuyoshi Shiga, Masayasu Hara, Takaya Nagasaki, Takafumi Sato, Hiroki Takahashi and Hiromitsu Takeyama

Cancers 2015, 7(4), 2443-2458; https://doi.org/10.3390/cancers7040902 - 11 Dec 2015

Cited by 613 | Viewed by 15388

Abstract

Cancer tissues are composed of cancer cells and the surrounding stromal cells (e.g., fibroblasts, vascular endothelial cells, and immune cells), in addition to the extracellular matrix. Most studies investigating carcinogenesis and the progression, invasion, metastasis, and angiogenesis of cancer have focused on alterations [...] Read more.

Cancer tissues are composed of cancer cells and the surrounding stromal cells (e.g., fibroblasts, vascular endothelial cells, and immune cells), in addition to the extracellular matrix. Most studies investigating carcinogenesis and the progression, invasion, metastasis, and angiogenesis of cancer have focused on alterations in cancer cells, including genetic and epigenetic changes. Recently, interactions between cancer cells and the stroma have attracted considerable attention, and increasing evidence has accumulated on this. Several researchers have gradually clarified the origins, features, and roles of cancer-associated fibroblasts (CAFs), a major component of the cancer stroma. CAFs function in a similar manner to myofibroblasts during wound healing. We previously reported the relationship between CAFs and angiogenesis. Interleukin-6 (IL-6), a multifunctional cytokine, plays a central role in regulating inflammatory and immune responses, and important roles in the progression, including proliferation, migration, and angiogenesis, of several cancers. We showed that CAFs are an important IL-6 source and that anti-IL-6 receptor antibody suppressed angiogenesis and inhibited tumor-stroma interactions. Furthermore, CAFs contribute to drug-resistance acquisition in cancer cells. The interaction between cancer cells and the stroma could be a potential target for anti-cancer therapy. Full article

(This article belongs to the Special Issue Cancer-Associated Fibroblasts)

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Open AccessFeature PaperArticle

Deficiency of iPLA₂β Primes Immune Cells for Proinflammation: Potential Involvement in Age-Related Mesenteric Lymph Node Lymphoma

by Johannes Inhoffen, Sabine Tuma-Kellner, Beate Straub, Wolfgang Stremmel and Walee Chamulitrat

Cancers 2015, 7(4), 2427-2442; https://doi.org/10.3390/cancers7040901 - 9 Dec 2015

Cited by 14 | Viewed by 4436

Abstract

Proinflammation can predispose the body to autoimmunity and cancer. We have reported that iPLA₂β^−/− mice are susceptible to autoimmune hepatitis and colitis. Here we determined whether cytokine release by immune cells could be affected by iPLA₂β deficiency alone [...] Read more.

Proinflammation can predispose the body to autoimmunity and cancer. We have reported that iPLA₂β^−/− mice are susceptible to autoimmune hepatitis and colitis. Here we determined whether cytokine release by immune cells could be affected by iPLA₂β deficiency alone or combined with CD95/FasL-antibody treatment in vivo. We also determined whether cancer risk could be increased in aged mutant mice. Immune cells were isolated from 3-month old male WT and iPLA₂β^−/− mice, and some were injected with anti-CD95/FasL antibody for 6 h. Kupffer cells (KC) or splenocytes and liver lymphocytes were stimulated in vitro by lipopolysaccharide or concanavalinA, respectively. Whole-body iPLA₂β deficiency caused increased apoptosis in liver, spleen, and mesenteric lymph node (MLN). KC from mutant mice showed suppressed release of TNFα and IL-6, while their splenocytes secreted increased levels of IFNγ and IL-17a. Upon CD95/FasL activation, the mutant KC in turn showed exaggerated cytokine release, this was accompanied by an increased release of IFNγ and IL-17a by liver lymphocytes. Aged iPLA₂β^−/− mice did not show follicular MLN lymphoma commonly seen in aged C57/BL6 mice. Thus, iPLA₂β deficiency renders M1- and Th1/Th17-proinflammation potentially leading to a reduction in age-related MLN lymphoma during aging. Full article

(This article belongs to the Special Issue Stress Responses in Tumors and The Tumor Microenvironment)

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Deficiency of iPLA2β increased apoptosis in spleen associated with the sensitized Th1/Th17 release by splenocytes. (A) Representative cleaved caspase 3 IHC-staining of a spleen of WT and KO (left panel) and its quantification of positive cells (right panel). Male mice at 19–24-months old were used (N = 8–14 per group); (B) ELISA determination of spontaneous and ConA-stimulated release of IFNγ, IL-17, and TNFα (pg/mL) by splenocytes isolated from 3-month old WT and mutant male mice (N = 4–6 per group). Saline or 10 µg/mL ConA was used to treat WT and KO splenocytes for 48 h, and a fold-increase of cytokine release by ConA treatment was calculated. * p < 0.05 vs. WT; ** p < 0.005 vs. WT. Full article ">Figure 2
iPLA2β deficiency leads to increased hepatic apoptosis associated with suppressed M1 cytokine release by KC either spontaneously or during LPS-stimulation. Male mice at 3 months old were used. Liver lymphocytes were treated with 10 µg/mL ConA for 48 h. KC were treated with 1 µg/mL LPS for 7 h. (A) iPLA2β IHC of human liver showed positive brown staining. IgG was used as (-) control; (B) In left panel, iPLA2β mRNA expression in livers of young mice was determined by qRT-PCR. In right panel, caspase 3/7 activity measured by luminescence normalized to the WT levels was obtained in liver homogenates of WT and KO mice (N = 3 per group for PCR and N = 4–5 per group for luminescence); (C) Spontaneous or ConA-stimulated release of IFNγ, IL-17 and IL-10 measured by ELISA was determined in liver lymphocytes of 3-month old WT and KO (N = 4–6 per group); Spontaneous or LPS-stimulated release of IL-6 and TNFα (D) as well as IL-10 and Il-4 (E) measured by ELISA was determined in KC isolated from WT and KO (left panel), and their fold increase by LPS was calculated (right panel) (N = 6 per group). * p < 0.05 vs. WT; ** p < 0.005 vs. WT. Full article ">Figure 3
Sublethal dose Jo2 treatment caused a very mild effect on liver injury and apoptosis but primes KC from mutant mice for a marked increase of IL-6 release either spontaneously or during LPS stimulation. Three-month old mice were treated with saline or 0.125 µg/g body weight Jo2 antibody for 6 h. KC were treated with saline or 1 µg/mL LPS for 7 h. (A) Activity of serum transaminases (AST and ALT) in U/L were determined in WT and KO mice (N = 3–7 per group); (B) Caspase 8 and caspase 3/7 activities measured by luminescence were determined in liver homogenates of WT and KO (N = 3–7 per group); Spontaneous (C) and LPS-stimulated (D) release of IL-6 and TNFα measured by ELISA was determined in KC isolated from WT and KO (N = 6 per group), and the fold increase by Jo2 was calculated (right-hand panel). * p < 0.05 vs. untreated; ** p < 0.005 vs. untreated. Full article ">Figure 4
Sublethal in vivo Jo2 treatment of KO mice led to a weak increase in Th1 cytokine release by liver lymphocytes and splenocytes. Three-month old mice were treated with saline or 0.125 µg/g body weight Jo2 antibody for 6 h. (A) The spontaneous release of IFNγ and IL-17a measured by ELISA was determined in liver lymphocytes isolated from WT and KO (N = 4–5 per group); (B) The release of IFNγ and IL-10 measured by ELISA was determined in splenocytes of WT and KO (N = 5–6 per group); (C) The ConA-stimulated release of IFNγ, IL-17aγ, and IL-10 was determined in liver lymphocytes from WT and KO (N = 6 per group); (D) The ConA-stimulated release of IFNγ, IL-17a, and IL-4 was determined in splenocytes of WT and KO (N = 4 per group). * p < 0.05 vs. control; ** p < 0.005 vs. control. Full article ">Figure 5
iPLA2β deficiency caused MLN apoptosis, loss of B cell cellularity, reduction in the incidence of follicular center cell lymphoma, and a rare MLN histiocytosis. Male mice at 19–24 months old were used (A) Representative H&E staining showed tingible body macrophages (TBM indicated by a white arrow) in the germinal centers of WT and KO (left panel), and TBM quantification (right panel) (N = 6–8 per group); (B) Representative cleaved caspase 3 IHC (indicated by a white arrow) showed apoptosis in WT and KO MLN (left panel), and apoptotic cell quantification (right panel) (N = 9 per group); (C) Representative CD45R IHC staining showed cellularity of cortex and medulla of WT and KO MLN (left panel), and CD45R (+) quantification (right panel) (N = 3–4 per group for medulla, and N = 5–9 per group for cortex); (D) Quantification of lymphoma incidence (N = 11–16 per group, upper panel). Bottom panel shows H&E of MLN follicular lymphoma from a WT mouse with centroblasts indicated by an arrow; (E) Representative CD3 (top) and CD45R (bottom) IHC staining of from left to right normal and lymphoma of MLN of WT mice. (F) Macroscopic picture, H&E and MLN lymphoma of KO#352 mouse. * p < 0.05 vs. control; ** p < 0.005 vs. control; ×40, ×100, ×200, ×400 magnification. Full article ">

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Open AccessReview

RASSF6; the Putative Tumor Suppressor of the RASSF Family

by Hiroaki Iwasa, Xinliang Jiang and Yutaka Hata

Cancers 2015, 7(4), 2415-2426; https://doi.org/10.3390/cancers7040899 - 9 Dec 2015

Cited by 18 | Viewed by 5188

Abstract

Humans have 10 genes that belong to the Ras association (RA) domain family (RASSF). Among them, RASSF7 to RASSF10 have the RA domain in the N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred [...] Read more.

Humans have 10 genes that belong to the Ras association (RA) domain family (RASSF). Among them, RASSF7 to RASSF10 have the RA domain in the N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred to as the C-RASSF proteins. The C-RASSF proteins have the RA domain in the middle region and the Salvador/RASSF/Hippo domain in the C-terminal region. RASSF6 additionally harbors the PSD-95/Discs large/ZO-1 (PDZ)-binding motif. Expression of RASSF6 is epigenetically suppressed in human cancers and is generally regarded as a tumor suppressor. RASSF6 induces caspase-dependent and -independent apoptosis. RASSF6 interacts with mammalian Ste20-like kinases (homologs of Drosophila Hippo) and cross-talks with the Hippo pathway. RASSF6 binds MDM2 and regulates p53 expression. The interactions with Ras and Modulator of apoptosis 1 (MOAP1) are also suggested by heterologous protein-protein interaction experiments. RASSF6 regulates apoptosis and cell cycle through these protein-protein interactions, and is implicated in the NF-κB and JNK signaling pathways. We summarize our current knowledge about RASSF6 and discuss what common and different properties RASSF6 and the other C-RASSF proteins have. Full article

(This article belongs to the Special Issue RASSF Signalling in Cancer)

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Structures of Caernorhabditis elegans RSF-1, Drosophila melanogaster dRASSF. Homo sapiens RASSF1A, and Homo sapiens RASSF6. C1, phorbol esters/diacylglycerol-binding domain. RA, Ras association domain. SARAH, Salvador/RASSF/Hippo domain. LIM, Zinc-binding domain present in Lin-11, Isl-1, Mec-3. The PDZ-binding motif of RASSF6 is depicted by a red star. The amino acid number of each protein is shown on the right. The RASSF6-interacting proteins are shown on the bottom. The interactions with MST1/2. MAGI1, and MDM2 are demonstrated at the endogenous level (red letters). Ras binds to the RA domain. MST1/2 (mammalian Ste20-like kinase 1/2) interacts with the SARAH domain. MAGI1 (membrane-associated guanylate kinase inverted 1) binds to the PDZ-binding motif. The interacting regions of MDM2 and MOAP1 (modulator apoptosis 1) are not precisely determined. Full article ">Figure 2
(A) Interaction of RASSF6 and DLG1. Endogenous RASSF6 was immunoprecipitated from rat liver with anti-RASSF6 antibody. DLG1 was co-immunoprecipitated; (B) FLAG-RASSF6 was expressed in HEK293 cells and immunoprecipitated with anti-FLAG antibody. Endogenous Lin-7 was co-immunoprecipitated; (C) FLAG-RASSF6 was co-expressed with Myc-RASSF1A, RASSF2, RASSF3, RASSF4, Nore1 (for simplicity, described as RASSF5 in this figure), and RASSF6. The immunoprecipitation was performed with anti-Myc antibody. The lower panel was the immunoblotting of the inputs. The upper panel was the immunoblotting of the immunoprecipitates. All Myc-C-RASSF proteins were co-immunoprecipitated with FLAG-RASSF6. Full article ">Figure 3
The core structure of the Drosophila Hippo pathway. Hippo and Warts form the kinase cassette. Mats and Salvador function as an activator and a linker to promote the Hippo-mediated activation of Warts. Salvador harbors two WW domains and the SARAH domain. Unphosphorylated Yorkie co-operates with Scalloped to regulate the transcription of cell cycle-promoting and anti-apoptotic genes. When Yorkie is phosphorylated by Warts, Yorkie is recruited from the nucleus to the cytoplasm and undergoes degradation (small entities symbolize degradation). dRASSF blocks the interaction between Hippo and Salvador. Inset: dRASSF suppresses the cell overproliferation phenotype caused by the hippo mutant lacking the SARAH domain but cannot rescue the phenotype caused by the kinase-dead mutant. Full article ">Figure 4
RASSF1A functions as an upstream activator for the Hippo pathway. For the precise mechanism of how RASSF1A activates MST2, the readers should refer to the other chapter in this issue. In contrast, RASSF6 works as a partner of MST kinases. Under the normal condition, RASSF6 and MST1/2 form a complex and inhibit each other. Under the condition that the Hippo pathway is activated, RASSF6 and MST1/2 are dissociated. MST1/2 are autophosphorylated (P). RASSF6-mediated apoptosis is concomitantly triggered. Note that, in the case that the machinery underlying RASSF6-mediated apoptosis is impaired, RASSF6 overexpression could lead to oncogenesis through the inhibition of the Hippo pathway. Full article ">Figure 5
Under the normal condition, MDM2 degrades RASSF6 and p53. When cells are exposed to stress such as ultraviolet exposure, MDM2 self-ubiquitination is enhanced and p53 is stabilized. RASSF6 also induces apoptosis independently of p53, but the mechanism remains to be clarified. Small entities symbolize degradation of each ubiquitinated protein (Ub-RASSF6, Ub-p53, and Ub-MDM2). Full article ">

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Open AccessReview

Anti-Tumor Immunity in Head and Neck Cancer: Understanding the Evidence, How Tumors Escape and Immunotherapeutic Approaches

by Clint T. Allen, Paul E. Clavijo, Carter Van Waes and Zhong Chen

Cancers 2015, 7(4), 2397-2414; https://doi.org/10.3390/cancers7040900 - 9 Dec 2015

Cited by 59 | Viewed by 7040

Abstract

Many carcinogen- and human papilloma virus (HPV)-associated head and neck cancers (HNSCC) display a hematopoietic cell infiltrate indicative of a T-cell inflamed phenotype and an underlying anti-tumor immune response. However, by definition, these tumors have escaped immune elimination and formed a clinically significant [...] Read more.

Many carcinogen- and human papilloma virus (HPV)-associated head and neck cancers (HNSCC) display a hematopoietic cell infiltrate indicative of a T-cell inflamed phenotype and an underlying anti-tumor immune response. However, by definition, these tumors have escaped immune elimination and formed a clinically significant malignancy. A number of both genetic and environmental mechanisms may allow such immune escape, including selection of poorly antigenic cancer cell subsets, tumor produced proinflammatory and immunosuppressive cytokines, recruitment of immunosuppressive immune cell subsets into the tumor and expression of checkpoint pathway components that limit T-cell responses. Here, we explore concepts of antigenicity and immunogenicity in solid tumors, summarize the scientific and clinical data that supports the use of immunotherapeutic approaches in patients with head and neck cancer, and discuss immune-based treatment approaches currently in clinical trials. Full article

(This article belongs to the Special Issue Head and Neck Cancer)

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Open AccessFeature PaperArticle

Association Studies of HFE C282Y and H63D Variants with Oral Cancer Risk and Iron Homeostasis Among Whites and Blacks

by Nathan R. Jones, Joseph H. Ashmore, Sang Y. Lee, John P. Richie, Philip Lazarus and Joshua E. Muscat

Cancers 2015, 7(4), 2386-2396; https://doi.org/10.3390/cancers7040898 - 9 Dec 2015

Cited by 4 | Viewed by 3935

Abstract

Background: Polymorphisms in the hemochromatosis (HFE) gene are associated with excessive iron absorption from the diet, and pro-oxidant effects of iron accumulation are thought to be a risk factor for several types of cancer. Methods: The C282Y (rs1800562) and H63D (rs1799945) [...] Read more.

Background: Polymorphisms in the hemochromatosis (HFE) gene are associated with excessive iron absorption from the diet, and pro-oxidant effects of iron accumulation are thought to be a risk factor for several types of cancer. Methods: The C282Y (rs1800562) and H63D (rs1799945) polymorphisms were genotyped in 301 oral cancer cases and 437 controls and analyzed in relation to oral cancer risk, and serum iron biomarker levels from a subset of 130 subjects. Results: Individuals with the C282Y allele had lower total iron binding capacity (TIBC) (321.2 ± 37.2 µg/dL vs. 397.7 ± 89.0 µg/dL, p = 0.007) and higher percent transferrin saturation (22.0 ± 8.7 vs. 35.6 ± 22.9, p = 0.023) than wild type individuals. Iron and ferritin levels approached significantly higher levels for the C282Y allele (p = 0.0632 and p = 0.0588, respectively). Conclusions: Iron biomarker levels were elevated by the C282Y allele, but neither (rs1800562) nor (rs1799945) was associated with oral cancer risk in blacks and whites. Full article

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Open AccessArticle

Loss of E2F1 Extends Survival and Accelerates Oral Tumor Growth in HPV-Positive Mice

by Rong Zhong, John Bechill and Michael T. Spiotto

Cancers 2015, 7(4), 2372-2385; https://doi.org/10.3390/cancers7040895 - 8 Dec 2015

Cited by 6 | Viewed by 4551

Abstract

The Human Papillomavirus (HPV) is associated with several human cancers, including head and neck squamous cell carcinomas (HNSCCs). HPV expresses the viral oncogene E7 that binds to the retinoblastoma protein (RB1) in order to activate the E2F pathway. RB1 can mediate contradictory pathways—cell [...] Read more.

The Human Papillomavirus (HPV) is associated with several human cancers, including head and neck squamous cell carcinomas (HNSCCs). HPV expresses the viral oncogene E7 that binds to the retinoblastoma protein (RB1) in order to activate the E2F pathway. RB1 can mediate contradictory pathways—cell growth and cell death via E2F family members. Here, we assessed the extent to which E2F1 mediates lethality of HPV oncogenes. Ubiquitous expression of the HPV oncogenes E6 and E7 caused lethality in mice that was associated with focal necrosis in hepatocytes and pancreatic tissues. Furthermore, all organs expressing HPV oncogenes displayed up-regulation of several E2F1 target genes. The E2F1 pathway mediated lethality in HPV-positive mice because deletion of E2F1 increased survival of mice ubiquitously expressing HPV oncogenes. E2F1 similarly functioned as a tumor suppressor in HPV-positive oral tumors as tumors grew faster with homozygous loss of E2F1 compared to tumors with heterozygous loss of E2F1. Re-expression of E2F1 caused decreased clonogenicity in HPV-positive cancer cells. Our results indicate that HPV oncogenes activated the E2F1 pathway to cause lethality in normal mice and to suppress oral tumor growth. These results suggest that selective modulation of the E2F1 pathway, which is activated in HPV tumors, may facilitate tumor regression. Full article

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Ubiquitous induction of HPV oncogenes caused lethality in adult mice. (A) TAM-induced recombination of the iHPV transgene in the skin, spleen, liver, brain and pancreas of RosaHPV mice. PCR identification of LSL-E6E7 transgene recombination (upper panel) or the control Cre transgene; (B) TAM-induced expression of E6E7 and luciferase transgenes in RosaHPV mice treated with TAM or vehicle for 5d. qRT-PCR for E6E7 and luciferase transgenes in RosaHPV mice treated with or without TAM. Data represent from organs isolated from five individual mice per group. Error bars represent standard deviation (st. dev.) p values determined by Student’s t-test; (C) TAM treatment caused lethality in RosaHPV mice but not in control mice. RosaHPV mice or KH mice were treated with TAM or vehicle. Mice were monitored for survival and depicted in a Kaplan-Meier survival plot. p value determined by Log-rank test; (D) TAM treatment caused death associated with or without paralysis. Of 17 RosaHPV mice that died, five displayed evidence of paralysis while 12 had normal limb movement. Paralysis was scored as positive if mice had impaired gait or loss of lower limb function. p value determined by Likelihood Ratio; (E) paralyzed RosaHPV mice that died had significant weight loss. Weights of RosaHPV mice treated with vehicle or TAM were measured at the start of the experiment and twice weekly. Change in body weight was calculated using body weight at the time of death or end of the experiment compared to body weight at the start of the experiment. Data displayed in quantile box-and-whisker plots where upper and lower whiskers represent the first and fourth quartiles, respectively, and the upper and lower boxes represent the second and third quartiles, respectively. p value determined by t-test with appropriate Bonferroni corrections; and (F) liver and pancreas demonstrated necrosis. Histology of indicated organs Rosa-HPV mice treated with TAM. Upper panels scale bar = 500 μM; Lower panels scale bar = 100 μM. Box in lower magnification field area depicted in the higher magnification field. * denotes p < 0.01. Full article ">Figure 2
TAM treatment of RosaHPV mice did not cause lethality due to immune responses against induced HPV oncogenes. (A) TAM treatment caused lethality in immunodeficient Rosa-HPV-Rag1−/− mice. RosaHPV mice also containing the Rag1−/−, Rag1+/− or Rag1+/+ genotypes were treated with TAM. In addition, control RosaHPV-Rag1+/+ mice were treated with vehicle (-TAM); (B) TAM caused lethality in immunosuppressed RosaHPV mice also transgenic for the 2C T cell receptor. RosaHPV mice containing the 2C TCR transgene contain >95% of T cells specific for the 2C T cell receptor making mice immunosuppressed and unable to reject immunogenic tissues. p value determined by log-rank test. Full article ">Figure 3
E2F target genes are induced in different organs of RosaHPV mice treated with TAM. The skin, spleen, liver, and brain from RosaHPV mice treated with TAM (filled bars) or vehicle (open bars) subjected to qRT-PCR for the E2F target genes CCNE1, RRM1, MCM6, TTK, CCNE2, MCM2, MCM5, and LIG1. Data from tissues isolated from five mice per group and assayed in duplicate. * denotes p < 0.01. Error bars represent SD. p values determined by Student’s t-test. Full article ">Figure 4
Loss of E2F1 rescued HPV oncogene lethality and paralysis in adult mice. (A) E2F1 loss rescued HPV oncogene lethality in adult RosaHPV mice. RosaHPV-E2F1+/+ (long dashed line), RosaHPV-E2F1+/− (short dashed line), and RosaHPV-E2F1−/− (solid line) mice were treated with TAM and monitored for survival; (B) Rosa-HPV-E2F1+/+, Rosa-HPV-E2F1+/− and Rosa-HPV-E2F1−/− mice were treated with TAM and monitored for paralysis. p value determined by Log-rank test; (C) E2F1 expression increased in the skin, spleen, and brain after TAM. RosaHPV mice were treated with vehicle (open bars) or TAM (filled bars) and, 5d later, E2F1 mRNA expression was detected by qRT-PCR. Full article ">Figure 5
E2F1 suppressed HPV oral tumor growth. (A) HPV oral tumors grew faster with loss of E2F1. KHR-E2F1+/+(n = 3), KHR-E2F1+/− (n = 4) and KHR-E2F1−/− (n = 5) mice were treated with TAM to induce primary oral tumors and tumor growth was monitored every 3 d. Representative of three independent experiments; (B) KHR-E2F1−/− mice expressed low levels of E2F1 transcripts but similar or higher levels of E2F2 and E2F3 transcripts. qRT-PCR analysis of E2F1, E2F2, and E2F3 was performed on RNA isolated from KHR-E2F1+/+, KHR-E2F1+/−, and KHR-E2F1−/− oral tumors; (C) loss of E2F1 caused similar or higher levels of E2F1 target genes. RNA isolated from KHR-E2F1+/+, KHR-E2F1+/− and KHR-E2F1−/− oral tumors was assessed for expression of the indicated genes using qRT-PCR. Average of three tumors performed in duplicate in <a href="#cancers-07-00895-f005" class="html-fig">Figure 5</a>B,C; (D) loss of E2F1 increased tumor cell proliferation. KHR-E2F1+/− and KHR-E2F1−/− oral tumors were assessed for PCNA by immunohistochemistry; and (E) quantitation of PCNA in KHR tumors with intact or deficient E2F1. Eight random fields from four individual KHR-E2F1+/− and KHR-E2F1−/− oral tumors were assessed for PCNA nuclear staining as previously described [<a href="#B27-cancers-07-00895" class="html-bibr">27</a>]. * denotes p < 0.01. Error bars represent SD. p values determined by Student’s t-test. Full article ">Figure 6
E2F1 overexpression suppressed clonogenicity in HPV-positive cells. (A) Compared to control transfected cells, HeLa cells transfected with E2F1 vector expressed elevated levels of E2F1 but similar levels of E2F3a. Total RNA was extracted from HeLa cells transfected with E2F1 or control vectors and subjected to qRT-PCR for the indicated genes; and (B) overexpression of E2F1 causes loss of clonogenicity in HPV-positive HeLa cells. HeLa cells were transfected with vectors expressing E2F1 or control enhanced green fluorescence protein and assessed for clonogenic survival. * denotes p < 0.01. Error bars represent SD. p values determined by Student’s t-test. Full article ">

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Open AccessFeature PaperReview

Paclitaxel Through the Ages of Anticancer Therapy: Exploring Its Role in Chemoresistance and Radiation Therapy

by Anna Maria Barbuti and Zhe-Sheng Chen

Cancers 2015, 7(4), 2360-2371; https://doi.org/10.3390/cancers7040897 - 3 Dec 2015

Cited by 200 | Viewed by 11273

Abstract

Paclitaxel (Taxol^®) is a member of the taxane class of anticancer drugs and one of the most common chemotherapeutic agents used against many forms of cancer. Paclitaxel is a microtubule-stabilizer that selectively arrests cells in the G2/M phase of the cell [...] Read more.

Paclitaxel (Taxol^®) is a member of the taxane class of anticancer drugs and one of the most common chemotherapeutic agents used against many forms of cancer. Paclitaxel is a microtubule-stabilizer that selectively arrests cells in the G2/M phase of the cell cycle, and found to induce cytotoxicity in a time and concentration-dependent manner. Paclitaxel has been embedded in novel drug formulations, including albumin and polymeric micelle nanoparticles, and applied to many anticancer treatment regimens due to its mechanism of action and radiation sensitizing effects. Though paclitaxel is a major anticancer drug which has been used for many years in clinical treatments, its therapeutic efficacy can be limited by common encumbrances faced by anticancer drugs. These encumbrances include toxicities, de novo refraction, and acquired multidrug resistance (MDR). This article will give a current and comprehensive review of paclitaxel, beginning with its unique history and pharmacology, explore its mechanisms of drug resistance and influence in combination with radiation therapy, while highlighting current treatment regimens, formulations, and new discoveries. Full article

(This article belongs to the Special Issue Drug/Radiation Resistance in Cancer Therapy)

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Open AccessCommentary

The Microenvironment in Gliomas: Phenotypic Expressions

by Davide Schiffer, Laura Annovazzi, Marta Mazzucco and Marta Mellai

Cancers 2015, 7(4), 2352-2359; https://doi.org/10.3390/cancers7040896 - 3 Dec 2015

Cited by 16 | Viewed by 4249

Abstract

The microenvironment of malignant gliomas is described according to its definition in the literature. Beside tumor cells, a series of stromal cells (microglia/macrophages, pericytes, fibroblasts, endothelial cells, normal and reactive astrocytes) represents the cell component, whereas a complex network of molecular signaling represents [...] Read more.

The microenvironment of malignant gliomas is described according to its definition in the literature. Beside tumor cells, a series of stromal cells (microglia/macrophages, pericytes, fibroblasts, endothelial cells, normal and reactive astrocytes) represents the cell component, whereas a complex network of molecular signaling represents the functional component. Its most evident expressions are perivascular and perinecrotic niches that are believed to be the site of tumor stem cells or progenitors in the tumor. Phenotypically, both niches are not easily recognizable; here, they are described together with a critical revision of their concept. As for perinecrotic niches, an alternative interpretation is given about their origin that regards the tumor stem cells as the residue of those that populated hyperproliferating areas in which necroses develop. This is based on the concept that the stem-like is a status and not a cell type, depending on the microenvironment that regulates a conversion of tumor non-stem cells and tumor stem cells through a cell reprogramming. Full article

(This article belongs to the Special Issue Cancer Stem Cells and Tumor Microenvironment)

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Graphical abstract

Graphical abstract
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Glioblastoma. Close relationship of Nestin+ cells with small vessels (A), but not with GFAP cells (B); DAB, ×400; Ring of tumor cells around a vessel: the inner cells are Nestin+ (C) and GFAP+ cells are external (D), DAB, ×200. Full article ">Figure 2
Relationship between a stem cell/progenitor and an endothelial cell. Full article ">Figure 3
Development of necrosis in a hyperproliferating area from ischemia due to the imbalance between proliferation rate of tumor and endothelial cells. Full article ">Figure 4
Glioblastoma. (A) Hyperproliferating area with scarce GFAP-positive cells, DAB, ×200; (B) Id. with abundant Nestin+ cells, DAB, ×200; (C) Circumscribed necrosis developing in a Nestin-rich hyperproliferating area, DAB, ×200; (D) Circumscribed necrosis developed in a SOX2-rich hyperproliferating area, DAB, ×200; (E) Most cells are Nestin+ in a perinecrotic palisade, immunofluorescence, ×400; (F) Nestin+ cells around a circumscribed necrosis, immunofluorescence, ×400. Full article ">Figure 5
Hypotheses on the origin of glioblastoma stem cells (GSCs)/glioma initiating cells (GICs). Full article ">Figure 6
Possible dynamics of stemness and differentiation. Full article ">

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Open AccessReview

Aberrant GLI1 Activation in DNA Damage Response, Carcinogenesis and Chemoresistance

by Komaraiah Palle, Chinnadurai Mani, Kaushlendra Tripathi and Mohammad Athar

Cancers 2015, 7(4), 2330-2351; https://doi.org/10.3390/cancers7040894 - 27 Nov 2015

Cited by 69 | Viewed by 7811

Abstract

The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of [...] Read more.

The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor β (TGFβ), wingless-type MMTV integration site family (WNT) and β-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for aberrant GLI1 expression and discuss GLI1-mediated HH signaling on DNA damage responses, carcinogenesis and chemoresistance. Full article

(This article belongs to the Special Issue Hedgehog Signaling Pathway in Cancer: Smoothened and GLI Take Center Stage)

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Canonical and non-canonical hedgehog signaling (HH) through glioma-associated oncogene homolog 1 (GLI1). Canonical HH signaling is activated by the binding of HH ligand to protein patched homolog 1(PTCH1), preventing its association with smoothened (SMO). This activates SMO leading to the dissociation of GLI and its translocation into the nucleus, where it serves as a transcription factor. In non-canonical activation of GLI1, various oncogenic signaling molecules, such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor (TGF)β, wingless-type MMTV integration site family (WNT), and β-catenin directly activate GLI1 in a Hedgehog-independent manner. Full article ">Figure 2
Role of glioma-associated oncogene homolog 1 (GLI1) in DNA damage response and repair. (A) Regulation of GLI1 by DNA damage. DNA damage increases expression of p53, which in turn activates the ubiquitin-mediated degradation of GLI1, suppressing cellular proliferation. In contrast, after DNA damage, cells lacking functional p53 are unable to suppress GLI1 in this manner and, therefore, are more proliferative; (B) Regulation of DNA repair by GLI1. GLI1 inhibits mismatch repair (MMR) and double strand break (DSB) repair through its regulation of MutL homolog 1 (MLH1) and ataxia telangiectasia-mutated protein kinase (ATR)/checkpoint kinase 1 (CHK1) signaling, respectively. At the same time, GLI1 activates nucleotide excision repair (NER) and DSB repair by regulating c-JUN and BH3 domain-only death agonist protein (BID)-ATR/CHK1 pathway, respectively. The magnitude of DNA damage and cell line individuality may decide the fate of GLI1 following DNA damage and its subsequent repair. Full article ">Figure 3
Role of glioma-associated oncogene homolog 1 (GLI1) in carcinogenesis. GLI1 regulates processes involved in all six of the traditional hallmarks of cancer. GLI1 protects against apoptosis by inducing anti-apoptotic proteins, such as B-cell lymphoma 2 (BCL2). GLI1 promotes cell invasion and metastasis through induction of epithelial-mesenchymal transition (EMT) markers such as SNAIL1, C-terminal binding protein 2 (ctBP2), transforming growth factor β (TGFβ), rat sarcoma viral oncogene homolog (RAS) and wingless-type MMTV integration site family (WNT). Replicative immortality can be achieved through GLI1-mediated regulation of human telomerase reverse transcriptase (hTERT) protein expression. GLI1 can aid in evasion of growth suppressors by regulating p53 and promotes proliferation by inducing expression of Ki67, proliferating cell nuclear antigen (PCNA) and mitotic spindle assembly checkpoint protein L1 (MAD2L1). Finally, GLI1 stimulates new blood vessel formation by enhancing expression of the potent pro-angiogenic protein cysteine-rich protein 61 (CYR61). Full article ">Figure 4
Role of glioma-associated oncogene homolog 1 (GLI1) in chemoresistance. GLI1 regulates the ATP-binding cassette (ABC) transporter family of proteins inducing drug efflux. Apart from this, GLI1 also induces the expression of proteins involved in cell cycle arrest and DNA damage repair. It is well known that these proteins repair the DNA damage induced by therapeutic agents while treating cancer cells and thus promotes cancer cell survival and facilitates chemoresistance. Full article ">

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Open AccessReview

Optimizing Management of Patients with Adult T Cell Leukemia-Lymphoma

by Jean A. Yared and Amy S. Kimball

Cancers 2015, 7(4), 2318-2329; https://doi.org/10.3390/cancers7040893 - 25 Nov 2015

Cited by 17 | Viewed by 4659

Abstract

Adult T cell leukemia-lymphoma is a rare disease with a high mortality rate, and is challenging for the clinician. Early allogeneic stem cell transplant can confer durable remission. As novel therapeutic agents become available to treat T cell malignancies, it is increasingly important [...] Read more.

Adult T cell leukemia-lymphoma is a rare disease with a high mortality rate, and is challenging for the clinician. Early allogeneic stem cell transplant can confer durable remission. As novel therapeutic agents become available to treat T cell malignancies, it is increasingly important that medical oncologists, hematologists, and hematopathologists recognize and accurately diagnose adult T cell leukemia-lymphoma. There is no uniform standard of treatment of adult T cell leukemia-lymphoma, and clinical trials remain critical to improving outcomes. Here we present one management approach based on the recent advances in treatment for adult T cell leukemia-lymphoma patients. Full article

(This article belongs to the Special Issue Lymphoma)

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Open AccessFeature PaperReview

Diet and Pancreatic Cancer Prevention

by Ilaria Casari and Marco Falasca

Cancers 2015, 7(4), 2309-2317; https://doi.org/10.3390/cancers7040892 - 23 Nov 2015

Cited by 39 | Viewed by 6639

Abstract

Pancreatic cancer is without any doubt the malignancy with the poorest prognosis and the lowest survival rate. This highly aggressive disease is rarely diagnosed at an early stage and difficult to treat due to its resistance to radiotherapy and chemotherapy. Therefore, there is [...] Read more.

Pancreatic cancer is without any doubt the malignancy with the poorest prognosis and the lowest survival rate. This highly aggressive disease is rarely diagnosed at an early stage and difficult to treat due to its resistance to radiotherapy and chemotherapy. Therefore, there is an urgent need to clarify the causes responsible for pancreatic cancer and to identify preventive strategies to reduce its incidence in the population. Some circumstances, such as smoking habits, being overweight and diabetes, have been identified as potentially predisposing factors to pancreatic cancer, suggesting that diet might play a role. A diet low in fat and sugars, together with a healthy lifestyle, regular exercise, weight reduction and not smoking, may contribute to prevent pancreatic cancer and many other cancer types. In addition, increasing evidence suggests that some food may have chemo preventive properties. Indeed, a high dietary intake of fresh fruit and vegetables has been shown to reduce the risk of developing pancreatic cancer, and recent epidemiological studies have associated nut consumption with a protective effect against it. Therefore, diet could have an impact on the development of pancreatic cancer and further investigations are needed to assess the potential chemo preventive role of specific foods against this disease. This review summarizes the key evidence for the role of dietary habits and their effect on pancreatic cancer and focuses on possible mechanisms for the association between diet and risk of pancreatic cancer. Full article

(This article belongs to the Special Issue Targeted Intervention for Pancreatic Cancer Associated with Smoking, Alcohol Abuse and Psychological Stress)

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Open AccessReview

Prostate Cancer Stem-like Cells Contribute to the Development of Castration-Resistant Prostate Cancer

by Diane Ojo, Xiaozeng Lin, Nicholas Wong, Yan Gu and Damu Tang

Cancers 2015, 7(4), 2290-2308; https://doi.org/10.3390/cancers7040890 - 18 Nov 2015

Cited by 53 | Viewed by 5696

Abstract

Androgen deprivation therapy (ADT) has been the standard care for patients with advanced prostate cancer (PC) since the 1940s. Although ADT shows clear benefits for many patients, castration-resistant prostate cancer (CRPC) inevitably occurs. In fact, with the two recent FDA-approved second-generation anti-androgens abiraterone [...] Read more.

Androgen deprivation therapy (ADT) has been the standard care for patients with advanced prostate cancer (PC) since the 1940s. Although ADT shows clear benefits for many patients, castration-resistant prostate cancer (CRPC) inevitably occurs. In fact, with the two recent FDA-approved second-generation anti-androgens abiraterone and enzalutamide, resistance develops rapidly in patients with CRPC, despite their initial effectiveness. The lack of effective therapeutic solutions towards CRPC largely reflects our limited understanding of the underlying mechanisms responsible for CRPC development. While persistent androgen receptor (AR) signaling under castration levels of serum testosterone (<50 ng/mL) contributes to resistance to ADT, it is also clear that CRPC evolves via complex mechanisms. Nevertheless, the physiological impact of individual mechanisms and whether these mechanisms function in a cohesive manner in promoting CRPC are elusive. In spite of these uncertainties, emerging evidence supports a critical role of prostate cancer stem-like cells (PCSLCs) in stimulating CRPC evolution and resistance to abiraterone and enzalutamide. In this review, we will discuss the recent evidence supporting the involvement of PCSLC in CRPC acquisition as well as the pathways and factors contributing to PCSLC expansion in response to ADT. Full article

(This article belongs to the Special Issue Cancer Stem Cells and Tumor Microenvironment)

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Open AccessArticle

Non-Surgical Breast-Conserving Treatment (KORTUC-BCT) Using a New Radiosensitization Method (KORTUC II) for Patients with Stage I or II Breast Cancer

by Yasuhiro Ogawa, Kei Kubota, Nobutaka Aoyama, Tomoaki Yamanishi, Shinji Kariya, Norihiko Hamada, Munenobu Nogami, Akihito Nishioka, Masahide Onogawa and Mitsuhiko Miyamura

Cancers 2015, 7(4), 2277-2289; https://doi.org/10.3390/cancers7040891 - 17 Nov 2015

Cited by 18 | Viewed by 5710

Abstract

The purpose of the present study was to establish a non-surgical breast-conserving treatment (BCT) using KORTUC II radiosensitization treatment. A new radiosensitizing agent containing 0.5% hydrogen peroxide and 0.83% sodium hyaluronate (a CD44 ligand) has been developed for intra-tumoral injection into various tumors. [...] Read more.

The purpose of the present study was to establish a non-surgical breast-conserving treatment (BCT) using KORTUC II radiosensitization treatment. A new radiosensitizing agent containing 0.5% hydrogen peroxide and 0.83% sodium hyaluronate (a CD44 ligand) has been developed for intra-tumoral injection into various tumors. This new method, named KORTUC II, was approved by our local ethics committee for the treatment of breast cancer and metastatic lymph nodes. A total of 72 early-stage breast cancer patients (stage 0, 1 patient; stage I, 23; stage II, 48) were enrolled in the KORTUC II trial after providing fully informed consent. The mean age of the patients was 59.7 years. A maximum of 6 mL (usually 3 mL for tumors of less than approximately 3 cm in diameter) of the agent was injected into breast tumor tissue twice a week under ultrasonographic guidance. For radiotherapy, hypofraction radiotherapy was administered using a tangential fields approach including an ipsilateral axillary region and field-in-field method; the energy level was 4 MV, and the total radiation dose was 44 Gy administered as 2.75 Gy/fraction. An electron boost of 3 Gy was added three times. Treatment was well tolerated with minimal adverse effects in all 72 patients. No patients showed any significant complications other than mild dermatitis. A total of 24 patients under 75 years old with stage II breast cancer underwent induction chemotherapy (EC and/or taxane) prior to KORTUC II treatment, and 58 patients with estrogen receptor-positive tumors also received hormonal therapy following KORTUC II. The mean duration of follow-up as of the end of September 2014 was 51.1 months, at which time 68 patients were alive without any distant metastases. Only one patient had local recurrence and died of cardiac failure at 6.5 years. Another one patient had bone metastases. For two of the 72 patients, follow-up ended after several months following KORTUC II treatment. In conclusion, non-surgical BCT can be performed using KORTUC II, which has three major characteristics: imaging guidance; enzyme-targeting; and targeting of breast cancer stem cells via the CD44 receptor. Full article

(This article belongs to the Special Issue Drug/Radiation Resistance in Cancer Therapy)

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Open AccessArticle

Differences in Redox Regulatory Systems in Human Lung and Liver Tumors Suggest Different Avenues for Therapy

by Ryuta Tobe, Bradley A. Carlson, Petra A. Tsuji, Byeong Jae Lee, Vadim N. Gladyshev and Dolph L. Hatfield

Cancers 2015, 7(4), 2262-2276; https://doi.org/10.3390/cancers7040889 - 10 Nov 2015

Cited by 17 | Viewed by 4948

Abstract

A common characteristic of many cancer cells is that they suffer from oxidative stress. They, therefore, require effective redox regulatory systems to combat the higher levels of reactive oxygen species that accompany accelerated growth compared to the normal cells of origin. An elevated [...] Read more.

A common characteristic of many cancer cells is that they suffer from oxidative stress. They, therefore, require effective redox regulatory systems to combat the higher levels of reactive oxygen species that accompany accelerated growth compared to the normal cells of origin. An elevated dependence on these systems in cancers suggests that targeting these systems may provide an avenue for retarding the malignancy process. Herein, we examined the redox regulatory systems in human liver and lung cancers by comparing human lung adenocarcinoma and liver carcinoma to their respective surrounding normal tissues. Significant differences were found in the two major redox systems, the thioredoxin and glutathione systems. Thioredoxin reductase 1 levels were elevated in both malignancies, but thioredoxin was highly upregulated in lung tumor and only slightly upregulated in liver tumor, while peroxiredoxin 1 was highly elevated in lung tumor, but downregulated in liver tumor. There were also major differences within the glutathione system between the malignancies and their normal tissues. The data suggest a greater dependence of liver on either the thioredoxin or glutathione system to drive the malignancy, while lung cancer appeared to depend primarily on the thioredoxin system. Full article

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Figure 1
Expression of proteins of the TXN system. (A,B) Expression of TXNRD1, TXN and PRDX1 in normal and tumor lung and liver tissues, respectively, as analyzed by western blotting. N designates normal tissue and T, tumor tissue. Coomassie blue staining is shown in the bottom panels and used as a control for protein loading; (C) Quantification of band intensities on western blots. Relative band intensities of duplicate lanes were quantified as described in the Experimental Section and tumor sample (T) values were normalized to the control tissue (N). * Denotes statistical differences, p < 0.05; (D) TXNRD catalytic activities in normal and tumor tissues. Activities are expressed as µmol of TNB/min/mg of protein for lung (upper panel) and liver tissues (lower panel). Values are the means ± S.D. of three independent experiments. Experimental details are given in the Experimental Section. Full article ">Figure 2
Expression of proteins of the GSH system. (A,B) Expression of GPX1, GPX2, GPX4, GSR, GCLC, GSS, GLRX, GGT1, GSTA1, and GSTP1 in normal and tumor lung and liver tissues, respectively, as analyzed by western blotting. GSTA1 and GPX2 were only detected in liver samples and therefore not included with the lung samples. N designates normal tissue and T, tumor tissue. Coomassie blue staining is shown in the bottom panels and used as a control for protein loading; (C) Quantification of band intensities on western blots. Relative band intensities of duplicate lanes were quantified as described in the Experimental Section and tumor sample values were normalized to the control tissue (N). * Denotes statistical differences, p < 0.05; (D) Glutathione peroxidase activities in normal and tumor tissues. Activities are expressed as units/mg of protein for lung (upper panel) and liver tissues (lower panel). Values are the means ± S.D. of three independent experiments. Experimental details are given in Experimental Section; (E) Amounts of total GSH (GSH + GSSG) in lung (upper panel) and liver tissues (lower panel). Total GSH levels are expressed as nmol per mg of protein and are the means ± S.D. for three independent experiments. Details are given in the Experimental Section. Full article ">Figure 3
Expression of SOD1, CAT and G6PD. (A,B) Expression of SOD1, CAT and G6PD in normal and tumor lung and liver tissues as analyzed by western blotting. N designates normal tissue and T, tumor tissue. Coomassie blue staining is shown in the bottom panels and used as a control for protein loading; (C) Quantification of band intensities on western blots. Relative band intensities of duplicate lanes were quantified as described in the Experimental Section and tumor sample (T) values were normalized to the control tissue (N). * Denotes statistical differences, p < 0.05. Full article ">Figure 4
Concentrations of ascorbic acid and uric acid. (A,B) Amounts of ascorbic acid and uric acid, respectively, in lung (upper panel) and liver tissues (lower panel). The concentration of ascorbic acid and uric acid are expressed as nmol per mg of protein and are the means ± S.D. for three independent experiments. * Denotes statistical difference, p < 0.05. Experimental details are given in the Experimental Section. Full article ">

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Open AccessFeature PaperArticle

Tumor Volumes and Prognosis in Laryngeal Cancer

by Mohamad R. Issa, Stuart E. Samuels, Emily Bellile, Firas L. Shalabi, Avraham Eisbruch and Gregory Wolf

Cancers 2015, 7(4), 2236-2261; https://doi.org/10.3390/cancers7040888 - 10 Nov 2015

Cited by 30 | Viewed by 5960

Abstract

Tumor staging systems for laryngeal cancer (LC) have been developed to assist in estimating prognosis after treatment and comparing treatment results across institutions. While the laryngeal TNM system has been shown to have prognostic information, varying cure rates in the literature have suggested [...] Read more.

Tumor staging systems for laryngeal cancer (LC) have been developed to assist in estimating prognosis after treatment and comparing treatment results across institutions. While the laryngeal TNM system has been shown to have prognostic information, varying cure rates in the literature have suggested concern about the accuracy and effectiveness of the T-classification in particular. To test the hypothesis that tumor volumes are more useful than T classification, we conducted a retrospective review of 78 patients with laryngeal cancer treated with radiation therapy at our institution. Using multivariable analysis, we demonstrate the significant prognostic value of anatomic volumes in patients with previously untreated laryngeal cancer. In this cohort, primary tumor volume (GTV_P), composite nodal volumes (GTV_N) and composite total volume (GTV_P + GTV_N = GTV_C) had prognostic value in both univariate and multivariate cox model analysis. Interestingly, when anatomic volumes were measured from CT scans after a single cycle of induction chemotherapy, all significant prognosticating value for measured anatomic volumes was lost. Given the literature findings and the results of this study, the authors advocate the use of tumor anatomic volumes calculated from pretreatment scans to supplement the TNM staging system in subjects with untreated laryngeal cancer. The study found that tumor volume assessment after induction chemotherapy is not of prognostic significance. Full article

(This article belongs to the Special Issue Head and Neck Cancer)

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Open AccessArticle

Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

by Janet M. Doolittle-Hall, Danielle L. Cunningham Glasspoole, William T. Seaman and Jennifer Webster-Cyriaque

Cancers 2015, 7(4), 2217-2235; https://doi.org/10.3390/cancers7040887 - 10 Nov 2015

Cited by 23 | Viewed by 6128

Abstract

Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting [...] Read more.

Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures. Full article

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Location of viral integration sites in the human genome. Human chromosomes (1–22, X, Y) are arranged around the circle. The inner-most ring shows viral integration sites, stacking multiple events that occurred at the same location. (a) HPV integration sites; (b) HBV integration sites; (c) MCPyV integration sites; (d) HIV integration sites. Full article ">Figure 2
GO biological process term enrichment of genes near viral integration sites. GO terms that were significant after the Fisher exact test with Bonferroni multiple testing correction (p < 0.05) are shown. For HIV, only the terms with the 20 lowest p-values are shown. Full article ">Figure 3
Genomic features near integration sites. (a) Categories of genomic features in the context of chromatin; (b) windows of four sizes are defined around viral integration sites, and features present in the human genome within each window are scored. Integration sites may be precisely mapped or be broader regions. Full article ">Figure 4
Significant differences were detected between viral integration sites and random sites. (a) HPV; (b) HBV; (c) HIV. Significance was determined using a two-sided Mann–Whitney U-test with Bonferroni correction, α < 0.05. Comparisons using the gene constraint set are indicated with GC. No significant differences were found for MCPyV. Only the features from the most relevant cell lines were considered for each virus. Full article ">Figure 5
Predictive genomic features for each DNA tumor virus. Random forest models were developed for each virus and window size, using either the background set or the gene constraint set as the negative class. Starting from only the genomic features that were considered relevant to each virus, feature elimination was used to select the smallest set of features that gave an ROC within 2% of the best model using three-fold cross-validation repeated 10 times on the training set. The optimal model was then used to classify a held-out test set (75% of data for training, 25% for testing). The entire process was repeated 10 times, once for each of the randomly-selected background sets. The number of times each feature was selected for inclusion in the optimal model is shown (white: zero, black: 10). Only features selected at least five times for at least one window size are shown. (a) Features predictive of HPV integration. (b) ChIP-qPCR of two histone marks predictive of HPV integration, H3K36me3 and H3K4me3. The cartoon shows the locations of primers designed to tile across an approximately ±500-bp window around the two identical HPV-16 integrants at 13q22.1 in SiHa cells. The graph shows the mean and standard deviation of two replicates of qPCR, and a representative gel of the products is shown at the right. All primer pairs produced bands at the expected sizes, but 5′-300 showed additional bands (the arrow indicates the expected size). qPCR quantification showed high fold enrichment for 5′-300, some of which may be due to non-specific amplification. However, a band is clearly present at the expected size (arrow), suggesting the presence of H3K36me3 and H3K4me3 near the integration site. Satellite region 2 (SAT2) and total H3 were used as positive controls. (c) Features predictive of HPV integration. (d) Features predictive of HPV integration. Comparisons using the gene constraint set are indicated with GC. Full article ">Figure 6
Significant differences were detected between types of viral integration sites. (a) Certain features in 7/8 categories were significantly different near HPV-18 integrations compared to HPV-16 (HPV-16 n = 382, HPV-18 n = 133); and (b) integrations in cervical tissue compared to those in head and neck cancers (HNC) (cervical n = 431, HNC n = 59). Regardless of window size or whether or not the number of genes was controlled for, gene expression, repeats and certain transcription factors differed significantly between HPV types (a) and tissues (b). (c) Significant differences between cervical cancer (n = 419) and W12 cell line (n = 28) integration sites. (d) Significant differences between HBV integration sites in HCC (n = 628) and tumor-adjacent tissues (n = 618). Significance was determined using a two-sided Mann–Whitney U-test with Bonferroni correction, α < 0.05. Comparisons using the gene constraint set are indicated with GC. Full article ">

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Open AccessFeature PaperReview

Multiparametric Evaluation of Head and Neck Squamous Cell Carcinoma Using a Single-Source Dual-Energy CT with Fast kVp Switching: State of the Art

by Stephanie Lam, Rajiv Gupta, Hillary Kelly, Hugh D. Curtin and Reza Forghani

Cancers 2015, 7(4), 2201-2216; https://doi.org/10.3390/cancers7040886 - 6 Nov 2015

Cited by 46 | Viewed by 7517

Abstract

There is an increasing body of evidence establishing the advantages of dual-energy CT (DECT) for evaluation of head and neck squamous cell carcinoma (HNSCC). Focusing on a single-source DECT system with fast kVp switching, we will review the principles behind DECT and associated [...] Read more.

There is an increasing body of evidence establishing the advantages of dual-energy CT (DECT) for evaluation of head and neck squamous cell carcinoma (HNSCC). Focusing on a single-source DECT system with fast kVp switching, we will review the principles behind DECT and associated post-processing steps that make this technology especially suitable for HNSCC evaluation and staging. The article will review current applications of DECT for evaluation of HNSCC including use of different reconstructions to improve tumor conspicuity, tumor-normal soft tissue interface, accuracy of invasion of critical structures such as thyroid cartilage, and reduce dental artifact. We will provide a practical approach for DECT implementation into routine clinical use and a multi-parametric approach for scan interpretation based on the experience at our institution. The article will conclude with a brief overview of potential future applications of the technique. Full article

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Different dual-energy CT (DECT) scanners currently in clinical use. (A) Illustration of a dual source DECT, consisting of two source x-ray tubes with corresponding detectors; (B) Illustration of a single source DECT with rapid kVp switching. With this type of scanner, the tube voltage follows a pulsed curve, and projection data are collected twice for every projection, one at high and one at low tube voltage, during rapid kVp switching; (C) Illustration of a dual layer DECT, consisting of a single source and single (but layered) detector. The detector is composed of two scintillation layers enabling separation of high and low energy spectra produced by a single source. Full article ">Figure 2
Increased tumor attenuation on 40 keV virtual monochromatic images (VMIs). (A) 70 keV single energy equivalent CT image of a right base of tongue tumor (large black arrow) and pathologic right level IIA lymph node (small white arrow) is shown. Note the similar density of both lesions compared to the normal right sternocleidomastoid muscle (M); (B) On the 40 keV image displayed using the same window-level settings, note the higher lesion density as well as higher relative contrast compared to muscle (M). Also note the increased image noise on the 40 keV VMI (B) compared to 70 keV VMI (A). Full article ">Figure 3
Virtual monochromatic images (VMIs) and iodine overlay map of a laryngeal tumor (T) invading the left thyroid cartilage. (A) 65 keV VMI; (B) 40 keV VMI; and (C) iodine-water (iodine overlay) material decomposition maps are shown. Note the increased tumor conspicuity on the 40 keV (B) compared to the 65 keV (A) VMIs. The iodine overlay map provides a quantitative estimate of iodine content of different tissues and demonstrates iodine containing tumor transgressing the left thyroid cartilage (arrow). It is noteworthy that the tumor edge of the extralaryngeal component (arrow) is more clearly seen on the 40 keV VMI (B) and iodine overlay map (C) than on the SECT equivalent 65 keV VMI (A). Full article ">Figure 4
Quantitative region of interest analysis comparing the spectral Hounsfield unit attenuation curves of HNSCC to non-ossified thyroid cartilage (NOTC) [<a href="#B16-cancers-07-00886" class="html-bibr">16</a>]. Pooled analysis of 30 tumors and NOTC from 30 normal patients is shown. At 65 or 70 keV, the attenuation of tumor can be very similar to normal NOTC, explaining the difficulties that may be encountered in differentiating between the 2 on conventional single energy CT scans. On the other hand, there is spectral separation at either end of the curve, with the best density separation achieved in the high energy range. Full article ">Figure 5
High energy virtual monochromatic images (VMIs) for evaluation of non-ossified thyroid cartilage (NOTC). 140 keV image from the same patient as in <a href="#cancers-07-00886-f003" class="html-fig">Figure 3</a> is shown. The laryngeal tumor invades the left thyroid cartilage, and the invaded portion appears as a relatively low density defect (double arrows) because of suppression of iodine density within the enhancing tumor on high keV images (compare to <a href="#cancers-07-00886-f003" class="html-fig">Figure 3</a>A,B). In this case, there is partial non-ossification of the thyroid cartilage on the left posteriorly. Note the preserved high attenuation of the NOTC (single arrow). There is clear attenuation difference between normal NOTC and tumor on the 140 keV image but the density on conventional single energy equivalent 65 keV image is nearly identical (compare to <a href="#cancers-07-00886-f003" class="html-fig">Figure 3</a>A). It is noteworthy that the tumor itself is not well seen on the 140 keV images, and these VMIs should be used in conjunction with the 65 and/or 40 keV VMIs and not in isolation. Full article ">Figure 6
Use of high energy DECT virtual monochromatic images (VMIs) for dental artifact reduction. (A) 65 keV and (B) 140 keV VMIs are shown from the same level in the neck. Note significant reduction of artifact such as in the region of retromolar trigone (black arrow) or oral tongue (white arrow) on the higher energy, 140 keV VMI compared to the 65 keV VMI. Full article ">Figure 7
Use of high energy DECT virtual monochromatic images (VMIs) for dental artifact reduction in a patient with a gingival-buccal tumor. (A) 65 keV (equivalent to single energy CT) and (B) 95 keV VMIs are shown from the same level in the neck. Note reduction of artifact and improved visualization of the enhancing tumor on the 95 keV VMI compared to the 65 keV VMI. Full article ">

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Open AccessReview

Revealing the Complexity of Breast Cancer by Next Generation Sequencing

by John Verigos and Angeliki Magklara

Cancers 2015, 7(4), 2183-2200; https://doi.org/10.3390/cancers7040885 - 6 Nov 2015

Cited by 28 | Viewed by 6240

Abstract

Over the last few years the increasing usage of “-omic” platforms, supported by next-generation sequencing, in the analysis of breast cancer samples has tremendously advanced our understanding of the disease. New driver and passenger mutations, rare chromosomal rearrangements and other genomic aberrations identified [...] Read more.

Over the last few years the increasing usage of “-omic” platforms, supported by next-generation sequencing, in the analysis of breast cancer samples has tremendously advanced our understanding of the disease. New driver and passenger mutations, rare chromosomal rearrangements and other genomic aberrations identified by whole genome and exome sequencing are providing missing pieces of the genomic architecture of breast cancer. High resolution maps of breast cancer methylomes and sequencing of the miRNA microworld are beginning to paint the epigenomic landscape of the disease. Transcriptomic profiling is giving us a glimpse into the gene regulatory networks that govern the fate of the breast cancer cell. At the same time, integrative analysis of sequencing data confirms an extensive intertumor and intratumor heterogeneity and plasticity in breast cancer arguing for a new approach to the problem. In this review, we report on the latest findings on the molecular characterization of breast cancer using NGS technologies, and we discuss their potential implications for the improvement of existing therapies. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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Open AccessReview

The Clinical Relevance of Long Non-Coding RNAs in Cancer

by Andreia Silva, Marc Bullock and George Calin

Cancers 2015, 7(4), 2169-2182; https://doi.org/10.3390/cancers7040884 - 27 Oct 2015

Cited by 129 | Viewed by 7490

Abstract

Non-coding RNAs have long been associated with cancer development and progression, and since their earliest discovery, their clinical potential in identifying and characterizing the disease has been pursued. Long non-coding (lncRNAs), a diverse class of RNA transcripts >200 nucleotides in length with limited [...] Read more.

Non-coding RNAs have long been associated with cancer development and progression, and since their earliest discovery, their clinical potential in identifying and characterizing the disease has been pursued. Long non-coding (lncRNAs), a diverse class of RNA transcripts >200 nucleotides in length with limited protein coding potential, has been only modestly studied relative to other categories of non-coding RNAs. However, recent data suggests they too may be important players in cancer. In this article, we consider the value of lncRNAs in the clinical setting, and in particular their potential roles as diagnostic and prognostic markers in cancer. Furthermore, we summarize the most significant studies linking lncRNA expression in human biological samples to cancer outcomes. The diagnostic sensitivity, specificity and validity of these non-coding RNA transcripts is compared in the various biological compartments in which they have been detected including tumor tissue, whole body fluids and exosomes. Full article

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Open AccessReview

The Interactions of Obesity, Inflammation and Insulin Resistance in Breast Cancer

by David P. Rose, Peter J. Gracheck and Linda Vona-Davis

Cancers 2015, 7(4), 2147-2168; https://doi.org/10.3390/cancers7040883 - 26 Oct 2015

Cited by 101 | Viewed by 10456

Abstract

Obese postmenopausal women have an increased breast cancer risk, the principal mechanism for which is elevated estrogen production by adipose tissue; also, regardless of menstrual status and tumor estrogen dependence, obesity is associated with biologically aggressive breast cancers. Type 2 diabetes has a [...] Read more.

Obese postmenopausal women have an increased breast cancer risk, the principal mechanism for which is elevated estrogen production by adipose tissue; also, regardless of menstrual status and tumor estrogen dependence, obesity is associated with biologically aggressive breast cancers. Type 2 diabetes has a complex relationship with breast cancer risk and outcome; coexisting obesity may be a major factor, but insulin itself induces adipose aromatase activity and estrogen production and also directly stimulates breast cancer cell growth and invasion. Adipose tissue inflammation occurs frequently in obesity and type 2 diabetes, and proinflammatory cytokines and prostaglandin E2 produced by cyclooxygenase-2 in the associated infiltrating macrophages also induce elevated aromatase expression. In animal models, the same proinflammatory mediators, and the chemokine monocyte chemoattractant protein-1, also stimulate tumor cell proliferation and invasion directly and promote tumor-related angiogenesis. We postulate that chronic adipose tissue inflammation, rather than body mass index-defined obesity per se, is associated with an increased risk of type 2 diabetes and postmenopausal estrogen-dependent breast cancer. Also, notably before the menopause, obesity and type 2 diabetes, or perhaps the associated inflammation, promote estrogen-independent, notably triple-negative, breast cancer development, invasion and metastasis by mechanisms that may involve macrophage-secreted cytokines, adipokines and insulin. Full article

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Open AccessReview

Roles of TRPM8 Ion Channels in Cancer: Proliferation, Survival, and Invasion

by Nelson S. Yee

Cancers 2015, 7(4), 2134-2146; https://doi.org/10.3390/cancers7040882 - 23 Oct 2015

Cited by 68 | Viewed by 7839

Abstract

The goal of this article is to provide a critical review of the transient receptor potential melastatin-subfamily member 8 (TRPM8) in cancers, with an emphasis on its roles in cellular proliferation, survival, and invasion. The TRPM8 ion channels regulate Ca²⁺ homeostasis and function [...] Read more.

The goal of this article is to provide a critical review of the transient receptor potential melastatin-subfamily member 8 (TRPM8) in cancers, with an emphasis on its roles in cellular proliferation, survival, and invasion. The TRPM8 ion channels regulate Ca²⁺ homeostasis and function as a cellular sensor and transducer of cold temperature. Accumulating evidence has demonstrated that TRPM8 is aberrantly expressed in a variety of malignant solid tumors. Clinicopathological analysis has shown that over-expression of TRPM8 correlates with tumor progression. Experimental data have revealed important roles of TRPM8 channels in cancer cells proliferation, survival, and invasion, which appear to be dependent on the cancer type. Recent reports have begun to reveal the signaling mechanisms that mediate the biological roles of TRPM8 in tumor growth and metastasis. Determining the mechanistic roles of TRPM8 in cancer is expected to elucidate the impact of thermal and chemical stimuli on the formation and progression of neoplasms. Translational research and clinical investigation of TRPM8 in malignant diseases will help exploit these ion channels as molecular biomarkers and therapeutic targets for developing precision cancer medicine. Full article

(This article belongs to the Special Issue Cancer Cell Proliferation)

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Open AccessArticle

Hypermethylation of MAPK13 Promoter in Oesophageal Squamous Cell Carcinoma Is Associated with Loss of p38δ MAPK Expression

by Carol O' Callaghan, Liam J. Fanning and Orla P. Barry

Cancers 2015, 7(4), 2124-2133; https://doi.org/10.3390/cancers7040881 - 23 Oct 2015

Cited by 4 | Viewed by 4547

Abstract

The loss of tumour suppressor gene function is a hallmark of malignant transformation and can occur by a variety of genetic and/or epigenetic alterations. We have previously characterised p38δ mitogen-activated protein kinase (MAPK) as a tumour suppressor in oesophageal squamous cell carcinoma (OESCC) [...] Read more.

The loss of tumour suppressor gene function is a hallmark of malignant transformation and can occur by a variety of genetic and/or epigenetic alterations. We have previously characterised p38δ mitogen-activated protein kinase (MAPK) as a tumour suppressor in oesophageal squamous cell carcinoma (OESCC) and outlined how loss of p38δ MAPK expression promotes increased proliferation and migration, as well as reduced chemosensitivity. Our aim was to investigate the underlying molecular causes of loss of p38δ MAPK expression in OESCC. Sequence analysis of DNA from p38δ MAPK positive and p38δ MAPK negative OESCC cell lines was used to investigate potential loss of function causing mutations. Epigenetic control of p38δ expression in OESCC was examined using methylation-specific PCR and sequencing of bisulfite-converted DNA. We did not identify any mutations in the MAPK13 sequence in OESCC cell lines which lack p38δ MAPK expression. However, we identified a differential pattern of methylation between p38δ MAPK positive and p38δ MAPK negative cell lines. We outline here for the first time differential MAPK13 promoter methylation in OESCC. Our results suggest that epigenetic alterations are responsible, in part, for the suppression of p38δ MAPK expression and promotion of tumourigenesis in OESCC. Full article

(This article belongs to the Special Issue Cancer Cell Proliferation)

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Figure 1
Effect of p38δ MAPK status on oesophageal cancer cell growth and migration. Oesophageal p38δ MAPK negative (KE-3 and -8, KYSE-70, OE-21, and OC-1) (A) and positive (KE-4, -5, -6, and -10, KYSE-450, OC-3, OE-19, -21 and -33) (B) cell lines were examined for their cell proliferation rate. Cells were seeded (3 × 104) in six-well plates and counted for 24–120 h (A,B); (C) Effect of p38δ on cell migration using a Boyden Chamber as described in the Experimental Section. The results shown are mean ± S.E. of three independent experiments. Full article ">Figure 1 Cont.
Effect of p38δ MAPK status on oesophageal cancer cell growth and migration. Oesophageal p38δ MAPK negative (KE-3 and -8, KYSE-70, OE-21, and OC-1) (A) and positive (KE-4, -5, -6, and -10, KYSE-450, OC-3, OE-19, -21 and -33) (B) cell lines were examined for their cell proliferation rate. Cells were seeded (3 × 104) in six-well plates and counted for 24–120 h (A,B); (C) Effect of p38δ on cell migration using a Boyden Chamber as described in the Experimental Section. The results shown are mean ± S.E. of three independent experiments. Full article ">Figure 2
p38δ MAPK mRNA expression in oesophageal cancer. (A,B) Agarose gel electrophoresis analysis of DNA fragments produced by PCR amplification of p38δ MAPK mRNA from p38δ MAPK protein positive (KE-4, KE-5, KE-6, KE-10, KYSE-450, OC-3, OE-19, OE-33) and p38δ MAPK protein negative (KE-3, KE-8, KYSE-70, OE-21, OC-1) oesophageal carcinoma cells with (A) P002F and P001R primers and (B) P003F and P001R primers; and (C) relative p38δ MAPK mRNA levels in p38δ MAPK protein negative OESCC cell lines KE-3, KE-8, KYSE-70, OE-21 and OC-1 were compared with p38δ MAPK protein positive KE-6 and KYSE-450 cells. GAPDH was measured as an internal control. Relative values were calculated by normalizing 2−ΔΔCT. Results shown are the mean ± S.E. of three independent qrt-PCR experiments. *** p < 0.001, ** p < 0.01, significant changes in p38δ MAPK expression from (black) KE-6 and (grey) KYSE-450 cells were determined by application of Student′s t-test. Full article ">Figure 3
MAPK13 promoter methylation analysis. (A) Agarose gel electrophoresis analysis of MAPK13 (Methylation-Specific PCR) MSP products (p38δ MAPK protein positive cell lines, KE-6, KYSE-450 and p38δ MAPK protein negative cell lines, KE-3, KE-8, KYSE-70, OE-21, OC-1; (B) DNA sequence analysis of MAPK13 CpG island Bisulfite Sequencing PCR (BSP) products. Residues identical to the reference sequence (MAPK13 NC_000006.12) residue at the same position are represented by a point (•), residues which differ are in uppercase; and (C) representative BSP analysis of CpG methylation. An unmethylated cytosine is depicted as a white circle, a methylated cytosine as a black circle. Full article ">

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Open AccessReview

Targeting the Hedgehog Pathway in Pediatric Medulloblastoma

by Sherri Y. Huang and Jer-Yen Yang

Cancers 2015, 7(4), 2110-2123; https://doi.org/10.3390/cancers7040880 - 23 Oct 2015

Cited by 38 | Viewed by 5658

Abstract

Medulloblastoma (MB), a primitive neuroectomal tumor of the cerebellum, is the most common malignant pediatric brain tumor. The cause of MB is largely unknown, but aberrant activation of Hedgehog (Hh) pathway is responsible for ~30% of MB. Despite aggressive treatment with surgical resection, [...] Read more.

Medulloblastoma (MB), a primitive neuroectomal tumor of the cerebellum, is the most common malignant pediatric brain tumor. The cause of MB is largely unknown, but aberrant activation of Hedgehog (Hh) pathway is responsible for ~30% of MB. Despite aggressive treatment with surgical resection, radiation and chemotherapy, 70%–80% of pediatric medulloblastoma cases can be controlled, but most treated patients suffer devastating side effects. Therefore, developing a new effective treatment strategy is urgently needed. Hh signaling controls transcription of target genes by regulating activities of the three Glioma-associated oncogene (Gli1-3) transcription factors. In this review, we will focus on current clinical treatment options of MB and discuss mechanisms of drug resistance. In addition, we will describe current known molecular pathways which crosstalk with the Hedgehog pathway both in the context of medulloblastoma and non-medulloblastoma cancer development. Finally, we will introduce post-translational modifications that modulate Gli1 activity and summarize the positive and negative regulations of the Hh/Gli1 pathway. Towards developing novel combination therapies for medulloblastoma treatment, current information on interacting pathways and direct regulation of Hh signaling should prove critical Full article

(This article belongs to the Special Issue Hedgehog Signaling Pathway in Cancer: Smoothened and GLI Take Center Stage)

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Open AccessReview

The Tip of the Iceberg: Clinical Implications of Genomic Sequencing Projects in Head and Neck Cancer

by Andrew C. Birkeland, Megan L. Ludwig, Taha S. Meraj, J. Chad Brenner and Mark E. Prince

Cancers 2015, 7(4), 2094-2109; https://doi.org/10.3390/cancers7040879 - 21 Oct 2015

Cited by 17 | Viewed by 6222

Abstract

Recent genomic sequencing studies have provided valuable insight into genetic aberrations in head and neck squamous cell carcinoma. Despite these great advances, certain hurdles exist in translating genomic findings to clinical care. Further correlation of genetic findings to clinical outcomes, additional analyses of [...] Read more.

Recent genomic sequencing studies have provided valuable insight into genetic aberrations in head and neck squamous cell carcinoma. Despite these great advances, certain hurdles exist in translating genomic findings to clinical care. Further correlation of genetic findings to clinical outcomes, additional analyses of subgroups of head and neck cancers and follow-up investigation into genetic heterogeneity are needed. While the development of targeted therapy trials is of key importance, numerous challenges exist in establishing and optimizing such programs. This review discusses potential upcoming steps for further genetic evaluation of head and neck cancers and implementation of genetic findings into precision medicine trials. Full article

(This article belongs to the Special Issue Head and Neck Cancer)

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Figure 1
Five year overall survival (x-axis in months) and genetic status from TCGA. PIK3CA-activating mutations and amplifications do not correlate with worse overall survival (p = 0.292) (A) in the TCGA cohort, while EGFR amplifications do correlate with worse overall survival (p = 0.016) (B). Survival trends are consistent when analyzing all stages and when analyzing advanced-staged (III/IV) tumors specifically (data not shown). Full article ">Figure 1 Cont.
Five year overall survival (x-axis in months) and genetic status from TCGA. PIK3CA-activating mutations and amplifications do not correlate with worse overall survival (p = 0.292) (A) in the TCGA cohort, while EGFR amplifications do correlate with worse overall survival (p = 0.016) (B). Survival trends are consistent when analyzing all stages and when analyzing advanced-staged (III/IV) tumors specifically (data not shown). Full article ">Figure 2
Copy number variation and mRNA level in TCGA. The mRNA level does not necessarily correlate with DNA copy number. As an example, mRNA levels of several commonly amplified (EGFR, CCND1, FGF19) (A–C) and deleted (CDKN2A) (D) genes show poor correlation with copy number in the TCGA dataset. Data generated from cBioPortal [<a href="#B7-cancers-07-00879" class="html-bibr">7</a>,<a href="#B8-cancers-07-00879" class="html-bibr">8</a>]. Of note, the copy number caller on cBioPortal has upper and lower limits. Full article ">Figure 3
Survival based on tumor subsite, EGFR amplification status. Five-year overall survival (x-axis in months) from 522 TCGA patients with CNV data on EGFR amplification in oral cavity (OC) and laryngeal (L) SCCs. A trend to worse overall survival based on EGFR amplification is seen with laryngeal SCCs (log-rank p = 0.08), but not oral cavity SCCs (log-rank p = 0.263). Full article ">

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Open AccessArticle

Aberrant MUC1-TRIM46-KRTCAP2 Chimeric RNAs in High-Grade Serous Ovarian Carcinoma

by Kalpana Kannan, Gona Karimi Kordestani, Anika Galagoda, Cristian Coarfa and Laising Yen

Cancers 2015, 7(4), 2083-2093; https://doi.org/10.3390/cancers7040878 - 19 Oct 2015

Cited by 10 | Viewed by 5370

Abstract

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. By analyzing the mRNA-seq reads from The Cancer Genome Atlas (TCGA), we uncovered a novel cancer-enriched chimeric RNA as the result of splicing between MUC1, a highly glycosylated [...] Read more.

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. By analyzing the mRNA-seq reads from The Cancer Genome Atlas (TCGA), we uncovered a novel cancer-enriched chimeric RNA as the result of splicing between MUC1, a highly glycosylated transmembrane mucin, TRIM46, a tripartite motif containing protein, and KRTCAP2, a keratinocyte associated protein. Experimental analyses by RT-PCR (reverse transcription PCR) and Sanger sequencing using an in-house cohort of 59 HGSC patient tumors revealed a total of six MUC1-TRIM46-KRTCAP2 isoforms joined by different annotated splice sites between these genes. These chimeric isoforms are not detected in non-cancerous ovaries, yet are present in three out of every four HGSC patient tumors, a significant frequency given the exceedingly heterogeneous nature of this disease. Transfection of the cDNA of MUC1-TRIM46-KRTCAP2 isoforms in mammalian cells led to the translation of mutant MUC1 fusion proteins that are unglycosylated and cytoplasmically localized as opposed to the cell membrane, a feature resembling the tumor-associated MUC1. Because the parental MUC1 is overexpressed in 90% of HGSC tumors and has been proposed as a clinical biomarker and therapeutic target, the chimeric MUC1-TRIM46-KRTCAP2 isoforms identified in this report could represent significantly better MUC1 variants for the same clinical utilities. Full article

(This article belongs to the Special Issue Next Generation Sequencing Approaches in Cancer)

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MUC1 chimeric RNAs identified in TCGA database of HGSC patient samples. (A) Schematic showing the position of 25 paired chimeric reads aligning to both MUC1 and TRIM46 genes identified from nine patients in the 130 TCGA cohort. Arrows indicate PCR primer targeting locations. (B) An example of RT-PCR validation for MUC1-TRIM46 chimeric RNA using one of the in-house HGSC patient samples. The bands corresponding to the six isoforms are labeled as shown. Full article ">Figure 2
MUC1-TRIM46-KRTCAP2 chimeric RNA isoforms and predicted protein consequences. (A) Schematic of the parental MUC1 and the six isoforms of MUC1-TRIM46-KRTCAP2 chimeric RNAs are shown with MUC1 (red), TRIM46 (blue) and KRTCAP2 (green) regions. The indicated coding and non-coding regions of MUC1 are based on the annotation of specific transcripts in the UCSC genome browser. (B) The expected protein products of these chimeric RNAs are shown with the domains indicated. Full article ">Figure 3
MUC1-TRIM46-KRTCAP2 is a highly recurrent chimeric RNA in HGSC patient tumor samples and cell lines. (A) The results of RT-PCR for MUC1-TRIM46-KRTCAP2 in 59 HGSC tumor samples (denoted by “S”). (B) The results of 24 non-cancerous ovary samples (“OV”) are shown. NTC refers to “no cDNA control”. The different isoforms are indicated on samples S61 and S63. (C) The results of RT-PCR for MUC1-TRIM46-KRTCAP2 in three HGSC cell lines (ES2, OV90 and OVCAR8) are shown. Full article ">Figure 4
MUC1-TRIM46-KRTCAP2 chimeric RNAs give rise to fusion proteins. (A) MUC1-TRIM46-KRTCAP2 isoforms were cloned with a C-terminal FLAG tag and expressed in OVCAR8 cells. Western blot of protein extracts using FLAG antibody shows that most of the isoforms are translated with the expected sizes lacking glycosylation. Isoform 5 appears to form a homodimer. (B) Immunocytochemistry of OVCAR8 cells transfected with different MUC1-TRIM46-KRTCAP2-FLAG expression constructs. The fusion protein isoforms are seen mainly in the cytoplasm as visualized by FLAG antibody. Images were taken using deconvolution microscopy. Full article ">

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Open AccessReview

Development, Maintenance, and Reversal of Multiple Drug Resistance: At the Crossroads of TFPI1, ABC Transporters, and HIF1

by Terra Arnason and Troy Harkness

Cancers 2015, 7(4), 2063-2082; https://doi.org/10.3390/cancers7040877 - 16 Oct 2015

Cited by 31 | Viewed by 5961

Abstract

Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have [...] Read more.

Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize these tumor cells to therapy. In this review we discuss the development, maintenance, and reversal of MDR as three distinct phases of cancer biology. Possible means to exploit these stages to reverse MDR will be explored. Early molecular detection of MDRcancers before clinical failure has the potential to offer new approaches to fighting MDRcancer. Full article

(This article belongs to the Special Issue Drug/Radiation Resistance in Cancer Therapy)

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Open AccessReview

Direct Interaction between Carcinoma Cells and Cancer Associated Fibroblasts for the Regulation of Cancer Invasion

by Hideki Yamaguchi and Ryuichi Sakai

Cancers 2015, 7(4), 2054-2062; https://doi.org/10.3390/cancers7040876 - 14 Oct 2015

Cited by 109 | Viewed by 6852

Abstract

The tumor stroma acts as an essential microenvironment of the cancer cells, which includes many different types of non-cancerous cells and the extracellular matrix (ECM). Stromal fibroblasts (SFs) are the major cellular constituents of the tumor stroma and are often called cancer-associated fibroblasts [...] Read more.

The tumor stroma acts as an essential microenvironment of the cancer cells, which includes many different types of non-cancerous cells and the extracellular matrix (ECM). Stromal fibroblasts (SFs) are the major cellular constituents of the tumor stroma and are often called cancer-associated fibroblasts (CAFs). They are often characterized by α-smooth muscle actin (αSMA) expression, which is indicative of the myofibroblast phenotype and strong contractility. These characteristics contribute to the remodeling and stiffening of the stromal ECM, thereby offering an appropriate field for cancer cell invasion. Importance of the tumor stroma in cancer progression has recently been highlighted. Moreover, several reports suggest that stromal fibroblasts interact with adjacent cancer cells through soluble factors, exosomes, or direct cell-cell adhesion to promote cancer cell invasion. In this review, current models of the regulation of cancer cell invasion by surrounding fibroblasts are summarized, including our recent work on the interaction between stromal fibroblasts and scirrhous gastric carcinoma (SGC) cells by using a three-dimensional (3D) culture system. Further mechanistic insights into the roles of the interaction between cancer cells and stromal fibroblasts in cancer invasion will be required to identify novel molecular targets for preventing cancer cell invasion. Full article

(This article belongs to the Special Issue Cancer-Associated Fibroblasts)

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Direct interaction between cancer-associated fibroblasts (CAFs) and cancer cells promotes cancer cell invasion. CAFs and SGC cells indirectly interact via paracrine signaling mediated by soluble factors and exosomes. This interaction induces phenotypic changes in both the cell types, which in turn trigger cancer cell invasion. Through paracrine signaling, the two cell types attract each other, leading to a direct physical interaction that may be mediated by cell-surface adhesion molecules. This direct interaction may cause further changes in both cell types, resulting in a more efficient CAF-led cancer cell invasion. Full article ">Figure 2
Direct interaction between cancer-associated fibroblasts (CAFs) and scirrhous gastric carcinoma (SGC) cells. (A) CAFs and SGC cells cultured at the top of the 3D Matrigel form invasive foci. SGC cells come in contact with CAFs and co-invade the 3D Matrigel; (B) F-actin staining of the invasive foci consisting of CAFs and SGC cells showed that the foci invade the Matrigel and are associated with cleaved signals for collagen type IV; (C) CAFs expressing fibronectin (white arrowheads) physically associate with SGC cells by extending their lamellipodia and filopodia toward SGC cells (yellow arrowheads). The lower panels show magnified images of the boxed regions; (D) Treatment with an Src inhibitor dasatinib blocks the interaction between CAFs and SGC cells, resulting in the suppression of invasive foci formation and invasion. (A and D) Images are reproduced from Yamaguchi et al. [<a href="#B46-cancers-07-00876" class="html-bibr">46</a>]. Full article ">

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Open AccessReview

Hedgehog Cholesterolysis: Specialized Gatekeeper to Oncogenic Signaling

by Brian P. Callahan and Chunyu Wang

Cancers 2015, 7(4), 2037-2053; https://doi.org/10.3390/cancers7040875 - 14 Oct 2015

Cited by 16 | Viewed by 5601

Abstract

Discussions of therapeutic suppression of hedgehog (Hh) signaling almost exclusively focus on receptor antagonism; however, hedgehog’s biosynthesis represents a unique and potentially targetable aspect of this oncogenic signaling pathway. Here, we review a key biosynthetic step called cholesterolysis from the perspectives of structure/function [...] Read more.

Discussions of therapeutic suppression of hedgehog (Hh) signaling almost exclusively focus on receptor antagonism; however, hedgehog’s biosynthesis represents a unique and potentially targetable aspect of this oncogenic signaling pathway. Here, we review a key biosynthetic step called cholesterolysis from the perspectives of structure/function and small molecule inhibition. Cholesterolysis, also called cholesteroylation, generates cholesterol-modified Hh ligand via autoprocessing of a hedgehog precursor protein. Post-translational modification by cholesterol appears to be restricted to proteins in the hedgehog family. The transformation is essential for Hh biological activity and upstream of signaling events. Despite its decisive role in generating ligand, cholesterolysis remains conspicuously unexplored as a therapeutic target. Full article

(This article belongs to the Special Issue Hedgehog Signaling Pathway in Cancer: Smoothened and GLI Take Center Stage)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Components and selected antagonists of the Hh cell-cell signaling pathway. Hh precursor cholesterolysis occurs in the endoplasmic reticulum of the Hh expressing cell (top). Full article ">Figure 2
(A) Block diagram of Hh precursor protein, with signal peptide (grey), signaling ligand (blue), and autocatalytic segment (green). (B) Conserved residues in the autocatalytic segment displayed by Logoplot (<a href="http://weblogo.berkeley.edu/logo.cgi" target="_blank">http://weblogo.berkeley.edu/logo.cgi</a>). Solid line delineates the HINT domain; hashed line, the SRR region. Residues marked with red asterisks are required for cholesterolysis. Full article ">Figure 3
Proposed mechanism of Hh precursor cholesterolysis as a self-catalyzed event. Inset depicts the two chemical steps: an N-S acyl shift (Step 1) followed by transesterification (Step 2). Signaling ligand, HhN (blue); autocatalytic segment, HhC (green). Full article ">Figure 4
(A) HINT domain of Drosophila melanogaster Hh precursor (PDB#, 1AT0); (B) conserved catalytic residues of the HINT domain; an (C) alignment of Hh HINT domain with self-splicing intein (PDB#, 2IN0). Figures rendered using PyMol (DeLano Scientific LLC, Palo Alto, CA, USA). Full article ">Figure 5
(A) Cholesterol targets soluble Hh ligand to cell membranes; (B) Proteins associate with Hh ligand via cholesterol; and (C) Micellization of the Hh ligand stabilized by hydrophobic interactions of the attached lipids. Full article ">Figure 6
(A) Cholesterolysis assay using HhC (green) fused to the FLaSH, peptide-dye complex (red). (B) Cholesterolysis assay using HhC fused to cyan and yellow fluorescent proteins. Full article ">Figure 7
Proposed balancing of Hedgehog biosynthesis by cellular zinc concentrations and disruption in cancer. Full article ">

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Cancers, Volume 7, Issue 4 (December 2015) – 35 articles

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