Alterations in RD(INK4/ARF) -mediated en bloc regulation of the INK4-ARF locus in human squamous cell carcinoma of the head and neck.
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ABSTRACT:
SUBMITTER: Poi MJ
PROVIDER: S-EPMC4102663 | biostudies-literature | 2015 Jul
REPOSITORIES: biostudies-literature
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Project description:Recent identification of an enhancer element, RD(INK4/ARF) (RD), in the prominent INK4/ARF locus provides a novel mechanism to simultaneously regulate the transcription of p15(INK4B) (p15), p14(ARF) , and p16(INK4A) (p16) tumor suppressor genes. While genetic inactivation of p15, p14(ARF) , and p16 in human tumors has been extensively studied, little is known about genetic alterations of RD and its impact on p15, p14(ARF) , and p16 in human cancer. The purpose of this study was to investigate the potential existence of genetic alterations of RD in human cancer cells. DNAs extracted from 17 different cancer cell lines and 31 primary pheochromocytoma tumors were analyzed for deletion and mutation of RD using real-time PCR and direct DNA sequencing. We found that RD was deleted in human cancer cell lines and pheochromocytoma tumors at frequencies of 41.2% (7/17) and 13.0% (4/31), respectively. While some of these RD deletion events occurred along with deletions of the entire INK4/ARF locus, other RD deletion events were independent of genetic alterations in p15, p14(ARF) , and p16. Furthermore, the status of RD was poorly associated with the expression of p15, p14(ARF) , and p16 in tested cancer cell lines and tumors. This study demonstrates for the first time that deletion of the RD enhancer is a prevalent event in human cancer cells. Its implication in carcinogenesis remains to be further explored.
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Project description:The mechanisms involved in the reprogramming of differentiated cells into induced pluripotent stem (iPS) cells by the three transcription factors Oct4 (also known as Pou5f1), Klf4 and Sox2 remain poorly understood. The Ink4/Arf locus comprises the Cdkn2a-Cdkn2b genes encoding three potent tumour suppressors, namely p16(Ink4a), p19(Arf) and p15(Ink4b), which are basally expressed in differentiated cells and upregulated by aberrant mitogenic signals. Here we show that the locus is completely silenced in iPS cells, as well as in embryonic stem (ES) cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, the three factors together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of 'stemness'. This downregulation also occurs in cells carrying the oncoprotein large-T, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. All together, we conclude that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.
| S-EPMC3578184 | biostudies-literature
Project description:9p21 locus is one of the most reproducible regions in genome-wide association studies (GWAS). The region harbors CDKN2A/B genes that code for p16INK4a, p15INK4b, and p14ARF proteins, and it also harbors a long gene desert adjacent to these genes. The polymorphisms that are associated with several diseases and cancers are present in these genes and the gene desert region. These proteins are critical cell cycle regulators whose transcriptional dysregulation is strongly linked with cellular regeneration, stemness, aging, and cancers. Given the importance of this locus, intense scientific efforts on understanding the regulation of these genes via promoter-driven mechanisms and recently, via the distal regulatory mechanism have provided major insights. In this review, we describe these mechanisms and propose the ways by which this locus can be targeted in pathologies and aging.
| S-EPMC9500187 | biostudies-literature
Project description:MicroRNAs (mirs) are small noncoding RNA molecules (~22 nucleotides) that regulate posttranscriptional gene expression. Currently, there has not been a comprehensive study of their role in primary head and neck squamous cell carcinoma (HNSCC). To determine the role of mirs in HNSCC, we screened for altered microRNA expression in HNSCC primary tissue and cell lines. We then further tested the functional impact of alterations of specific mirs. An initial screening of 4 primary HNSCC, 4 normal mucosal controls and 4 HNSCC cell lines was analyzed for mature microRNA expression by microarray. Significance was determined using significance analysis of microarrays (SAM). Nine microRNAs were found by SAM to be upregulated or downregulated in tumor tissue including mir-21, let-7, 18, 29c, 142-3p, 155, 146b (overexpressed) and 494 (underexpressed). Mir-21 was validated by qRT-PCR. Functional validation by growth assays was performed, further validating mir-21. Transfection of mir-21 into JHU-011 and JHU-012 cell lines showed a 39% increase in cell growth at 72 hr relative to controls (p < 0.05). Transfection of the inhibitor into JHU-O12 cell lines showed a 92% decrease in cell growth relative to controls at 72 hr (p < 0.05). In addition, flow cytometry analysis of JHU-012 cells 48 hr after mir-21 inhibitor transfection showed a statistically significant increase in cytochrome c release and increased apoptosis. These differentially expressed microRNAs may be of interest as potential novel oncogenes and tumor suppressor genes in HNSCC. Mir-21 is a putative oncogenic microRNA in head and neck cancer.
| S-EPMC3184846 | biostudies-literature
Project description:Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of β-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.
| S-EPMC5386305 | biostudies-literature
Project description:Head and neck cancer collectively describes malignant tumors originating from the mucosal surface of the upper aerodigestive tract. These tumors pose a great threat to public health because of their high incidence and mortality. Traditional risk factors are tobacco and alcohol abuse. More recently, infection by high-risk types of human papilloma virus (HPV) has been identified as an additional risk factor, especially for oropharyngeal squamous cell carcinoma (OPSCC). Moreover, HPV-positive OPSCC is considered a distinct tumor entity with an improved clinical outcome compared to HPV-negative OPSCC. Epigenetic alterations act as key events in the pathogenesis of cancer and are of special interest for basic and translational oncology because of their reversible nature. This review provides a comprehensive summary of alterations of the epigenome in head and neck squamous cell carcinoma (HNSCC) with a focus on the methylome (hypomethylation and hypermethylation) and its predictive value in the evaluation of pathologic states and clinical outcome, or monitoring response rates to certain therapies.
| S-EPMC4905189 | biostudies-other
Project description:BackgroundEpigenetic changes associated with human papillomavirus (HPV)-driven tumors have been described; however, HPV type-specific alterations are less well understood. We sought to compare HPV16-specific methylation changes with those in virus-unassociated head and neck squamous cell carcinomas (HNSCC).MethodsWithin The Cancer Genome Atlas, 59 HPV16+ HNSCC, 238 nonviral HNSCC (no detectable HPV or other viruses), and 50 normal head and neck tissues were evaluated. Significant differentially methylated regions (DMR) were selected, and key associated genes were identified. Partial least squares models were generated to predict HPV16 status in additional independent samples.ResultsHPV infection in HNSCC is associated with type-specific methylomic profiles. Multiple significant DMRs were identified between HPV16+, nonviral, and normal samples. The most significant differentially methylated genes, SYCP2, MSX2, HLTF, PITX2, and GRAMD4, demonstrated HPV16-associated methylation patterns with corresponding alterations in gene expression. Phylogenetically related HPV types (alpha-9 species; HPV31, HPV33, and HPV35) demonstrated a similar methylation profile to that of HPV16 but differed from those seen in other types, such as HPV18 and 45 (alpha-7).ConclusionsHNSCC linked to HPV16 and types from the same alpha species are associated with a distinct methylation profile. This HPV16-associated methylation pattern is also detected in cervical cancer and testicular germ cell tumors. We present insights into both shared and unique methylation alterations associated with HPV16+ tumors and may have implications for understanding the clinical behavior of HPV-associated HNSCC.ImpactHPV type-specific methylomic changes may contribute to understanding biologic mechanisms underlying differences in clinical behavior among different HPV+ and HPV- HNSCC.
| S-EPMC8983563 | biostudies-literature
Project description:The INK4/ARF (CDKN2A/B) locus has been identified as a hotspot for susceptibility to genetic changes associated with a vast variety of cancers and aging related diseases including coronary artery disease and type II diabetes. Using chromatin Capture-C technologies, we discovered a distal enhancer upstream INK4/ARF looped to CDKN2A, ARF and CDKN2B in human fibroblast cells IMR90 and cancer cell lines HCT116 and SEM, but absent in human fibroblast cell-derived induced pluripotent stem cells, which maintained an epigenetically silenced INK4/ARF locus. Bi-allelic deletion of the ~1.4 Kb core enhancer sequence by CRISPR/Cas9 technology in human HCT116 cells abrogated the long-range chromatin interaction, thereafter disrupting gene expression of these tumor suppressor genes. Moreover, cross-species conservation of the distal enhancer elements between human and mouse was observed.
2024-08-01 | GSE119947 | GEO
Project description:The INK4/ARF (CDKN2A/B) locus has been identified as a hotspot for susceptibility to genetic changes associated with a vast variety of cancers and aging related diseases including coronary artery disease and type II diabetes. Using chromatin Capture-C technologies, we discovered a distal enhancer upstream INK4/ARF looped to CDKN2A, ARF and CDKN2B in human fibroblast cells IMR90 and cancer cell lines HCT116 and SEM, but absent in human fibroblast cell-derived induced pluripotent stem cells, which maintained an epigenetically silenced INK4/ARF locus. Bi-allelic deletion of the ~1.4 Kb core enhancer sequence by CRISPR/Cas9 technology in human HCT116 cells abrogated the long-range chromatin interaction, thereafter disrupting gene expression of these tumor suppressor genes. Moreover, cross-species conservation of the distal enhancer elements between human and mouse was observed.
2024-08-01 | GSE119148 | GEO
Project description:BACKGROUND:Advanced stage head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with low survival rates. Loss-of-heterozygosity/allelic imbalance (LOH/AI) analysis has been widely used to identify genomic alterations in solid tumors and the tumor microenvironment (stroma). We hypothesize that these identified alterations can point to signaling networks functioning in HNSCC epithelial-tumor and surrounding stroma (tumor microenvironment). RESULTS:Under the assumption that genes in proximity to identified LOH/AI regions are correlated with the tumorigenic phenotype, we mined publicly available biological information to identify pathway segments (signaling proteins connected to each other in a network) and identify the role of tumor microenvironment in HNSCC. Across both neoplastic epithelial cells and the surrounding stromal cells, genetic alterations in HNSCC were successfully identified, and 75 markers were observed to have significantly different LOH/AI frequencies in these compartments (p < 0.026). We applied a network identification approach to the genes in proximity to these 75 markers in cancer epithelium and stroma in order to identify biological networks that can describe functional associations amongst these marker-associated genes. CONCLUSIONS:We verified the involvement of T-cell receptor signaling pathways in HNSCC as well as associated oncogenes such as LCK and PLCB1, and tumor suppressors such as STAT5A, PTPN6, PARK2. We identified expression levels of genes within significant LOH/AI regions specific to stroma networks that correlate with better outcome in radiation therapy. By integrating various levels of high-throughput data, we were able to precisely focus on specific proteins and genes that are germane to HNSCC.
| S-EPMC3170587 | biostudies-literature