Putative Tumor Suppressor Gene Research Articles

Abstract 5346: Recurrent genomic copy alterations deregulate the transcriptome and key signaling networks in head and neck cancer cell lines and tumors with worse prognosis

Abstract Human cancer cell lines have been important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations and subtypes found in tumors could further accelerate functional and therapeutic discoveries. We conducted an integrated analysis of the genomic and transcriptomic profiles of 15 human papillomavirus negative (HPV−) and 11 HPV(+) head and neck squamous cell carcinoma (HNSCC) lines, to identify models of important molecular subtypes uncovered among 279 HNSCC tumors from The Cancer Genome Atlas (TCGA). We developed analytic approaches to detect copy number alterations (CNAs) driving altered expression and predict deleterious mutations in HNSCC lines for comparison with TCGA. Strikingly, HNSCC lines displayed recurrent amplifications on chromosomes 3q22-29, 5p15, 11q13/22, and 8p11 that drive increased expression of a cluster of ~100 mRNAs encoding multiple known and understudied candidate oncogenes. These CNAs, together with loss and under-expression or mutations of putative tumor suppressor genes, recapitulate genomic alterations found in more aggressive HNSCC tumor subtypes. Among these, concurrent 3q26.3 amplification and TP53 mutation in HPV(−) HNSCCs is associated with worse overall survival. Common and distinctive CNAs, transcriptome, and mutations converge on biologically and therapeutically important signal pathways and functions in HNSCCs differing in HPV status, including PI3K, Hippo, TGF-β, Wnt/β-catenin, TP63, TP53/death, cell cycle/mitosis, stemness/differentiation, chromatin remodelling/DNA replication, post-transcriptional regulation, and mitochondrial biosynthesis. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with historic and recently derived HNSCC lines, and provide valuable models for future preclinical and therapeutic studies in subsets of HNSCCs with worse prognosis. Supported by NIDCD/NIH intramural projects ZIA-DC-000073, ZIA-DC-000074. This work utilized the computational resources of the NIH HPC Biowulf cluster. (http://hpc.nih.gov) Citation Format: Hui Cheng, Xinping Yang, Han Si, Anthony Saleh, Wenming Xiao, Jamie Coupar, Susanne M. Gollin, Robert L. Ferris, Natalia Issaeva, Wendell G. Yarbrough, Mark E. Prince, Thomas E. Carey, Carter Van Waes, Zhong Chen. Recurrent genomic copy alterations deregulate the transcriptome and key signaling networks in head and neck cancer cell lines and tumors with worse prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5346.

CHL1 gene acts as a tumor suppressor in human neuroblastoma.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

E-cadherin breast tumor expression, risk factors and survival: Pooled analysis of 5,933 cases from 12 studies in the Breast Cancer Association Consortium

E-cadherin (CDH1) is a putative tumor suppressor gene implicated in breast carcinogenesis. Yet, whether risk factors or survival differ by E-cadherin tumor expression is unclear. We evaluated E-cadherin tumor immunohistochemistry expression using tissue microarrays of 5,933 female invasive breast cancers from 12 studies from the Breast Cancer Consortium. H-scores were calculated and case-case odds ratios (OR) and 95% confidence intervals (CIs) were estimated using logistic regression. Survival analyses were performed using Cox regression models. All analyses were stratified by estrogen receptor (ER) status and histologic subtype. E-cadherin low cases (N = 1191, 20%) were more frequently of lobular histology, low grade, >2 cm, and HER2-negative. Loss of E-cadherin expression (score < 100) was associated with menopausal hormone use among ER-positive tumors (ever compared to never users, OR = 1.24, 95% CI = 0.97–1.59), which was stronger when we evaluated complete loss of E-cadherin (i.e. H-score = 0), OR = 1.57, 95% CI = 1.06–2.33. Breast cancer specific mortality was unrelated to E-cadherin expression in multivariable models. E-cadherin low expression is associated with lobular histology, tumor characteristics and menopausal hormone use, with no evidence of an association with breast cancer specific survival. These data support loss of E-cadherin expression as an important marker of tumor subtypes.

Abstract QS43: Blepharophimosis Ptosis Epicanthus Inversus Syndrome Caused by a ZC3H13 Gene Mutation

PURPOSE: Blepharophimosis Ptosis Epicanthus Inversus Syndrome (BPES) is a rare craniofacial autosomal dominant disorder characterized by severe bilateral ptosis, telecanthus, epicanthus inverses, belpharophimosis and orbital dysmorphology. BPES type 1 is accompanied by ovarian failure and attendant infertility, while type 2 manifests only eyelid and orbital involvement. While BPES is often associated with a dominantly inherited mutation in the forkhead transcription factor FOXL2 (3q23), we sought to capitalize on next generation whole exome sequencing to decipher the cause of BPES in a pedigree lacking a FOXL2 mutation. METHODS: A male patient with BPES was identified at the Yale-New Haven Hospital. Following informed consent, clinical data and blood samples from both affected and unaffected family members were obtained. Genomic DNA was extracted from whole blood samples and subjected to exome capture followed by next generation sequencing. Sequence reads were mapped to the human reference genome. Variants with minor allele frequencies in reference databases were selected and annotated for impact on the encoded protein and for conservation of the reference base and amino acid residue among orthologs across phylogeny. Sanger sequencing was employed to verify candidate mutations. In addition, all FOXL2 exons were subjected to Sanger sequencing to verify sequence status. RESULTS: A four-generation BPES pedigree was identified demonstrating autosomal dominant inheritance. In affected individuals, next generation whole exome sequencing identified a common non-sense mutation in ZC3H13 (13q14.13), which was confirmed by Sanger sequencing. A cytosine to thymine (C>T) alteration resulted in the conversion of arginine 1513 to a premature stop codon (R1513X). The variant 13:46538114 C / T was not found in dbSNP with an ExAC allele frequency of 1.651e-05. The amino acid residue arginine 1513 is evolutionarily conserved from Neurospora crassa (red bread mold) to Homo sapiens. All FOXL2 exons lacked disease-causing alterations. Conclusions: ZC3H13 is a putative tumor suppressor gene encoding a zinc finger CCCH-type containing protein. It localizes to nuclear speckles and is predicted to play a role in mRNA splicing and cell cycle progression. Our data suggest that ZC3H13 mutations represent an additional dominant subtype of BPES and point to a novel role for ZC3H13 in craniofacial development and disease pathogenesis. D.M. Balkin: None. C. Nelson-Williams: None. B.J. Sumpio: None. D.A. Scott: None. P. Le Pabic: None. T.F. Schilling: None. R.P. Lifton: None. D. Narayan: None.

Aging Mouse Models Reveal Complex Tumor-Microenvironment Interactions in Cancer Progression.

Mouse models and genetically engineered mouse models (GEMM) are essential experimental tools for the understanding molecular mechanisms within complex biological systems. GEMM are especially useful for inferencing phenocopy information to genetic human diseases such as breast cancer. Human breast cancer modeling in mice most commonly employs mammary epithelial-specific promoters to investigate gene function(s) and, in particular, putative oncogenes. Models are specifically useful in the mammary epithelial cell in the context of the complete mammary gland environment. Gene targeted knockout mice including conditional targeting to specific mammary cells can reveal developmental defects in mammary organogenesis and demonstrate the importance of putative tumor suppressor genes. Some of these models demonstrate a non-traditional type of tumor suppression which involves interplay between the tumor susceptible cell and its host/environment. These GEMM help to reveal the processes of cancer progression beyond those intrinsic to cancer cells. Furthermore, the, analysis of mouse models requires appropriate consideration of mouse strain, background, and environmental factors. In this review, we compare aging-related factors in mouse models for breast cancer. We introduce databases of GEMM attributes and colony functional variations.

A novel germline ARMC5 mutation in a patient with bilateral macronodular adrenal hyperplasia: a case report

BackgroundBilateral macronodular adrenal hyperplasia (BMAH) is a rare cause of Cushing’s syndrome (CS). BMAH is predominantly believed to be caused by two mutations, a germline and somatic one, respectively, as described in the two-hit hypothesis. In many familial cases of BMAH, mutations in armadillo repeat containing 5 (ARMC5), a putative tumor suppressor gene, are thought to induce the disorder. The objective of this study was to report a case in which the patient presented with BMAH induced by a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) alone rather than a two-hit mutation.Case presentationA 51-year-old woman was identified with masses in the bilateral adrenals. Serum cortisol levels were increased significantly both in the morning (08:00 AM) and late at night (24:00 AM), while plasma adrenocorticotropic hormone was normal. The patient underwent a left adrenalectomy and histopathology substantiated the BMAH diagnosis. WES of the germline DNA discovered a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) and in silico analysis predicted that the mutation significantly impaired protein function, resulting in inactivated ARMC5. Subsequently, WES of the tumor specimen identified 79 somatic single nucleotide polymorphisms (SNPs)/insertion-deletion (indel) mutations, including 32 missense/nonsense/splice/stop-loss mutations. None of these mutations were CS-related.ConclusionsA novel germline ARMC5 mutation (c. 517C > T, p. Arg173*) was identified that induced BMAH alone without a second mutation. ARMC5 sequencing may improve the identification of clinical forms of BMAH and allow earlier diagnosis of this disease.

Downregulation of glycine decarboxylase enhanced cofilin-mediated migration in hepatocellular carcinoma cells.

Metabolic reprogramming is a hallmark of cancer. Glycine decarboxylase (GLDC), an oxidoreductase, plays an important role in amino acid metabolism. While GLDC promotes tumor initiation and proliferation in non-small cell lung cancer and glioma and it was reported as a putative tumor suppressor gene in gastric cancer, the role of GLDC in hepatocellular carcinoma (HCC) is unknown. In the current study, microarray-based analysis suggested that GLDC expression was low in highly malignant HCC cell lines, and clinicopathological analysis revealed a decrease in GLDC in HCC tumor samples. While the knockdown of GLDC enhanced cancer cell migration and invasion, GLDC overexpression inhibited them. Mechanistic studies revealed that GLDC knockdown increased the levels of reactive oxygen species (ROS) and decreased the ratio of glutathione/oxidized glutathione (GSH/GSSG), which in turn dampened the ubiquitination of cofilin, a key regulator of actin polymerization. Consequently, the protein level of cofilin was elevated, which accounted for the increase in cell migration. The overexpression of GLDC reversed the phenotype. Treatment with N-acetyl-L-cysteine decreased the protein level of cofilin while treatment with H2O2 increased it, further confirming the role of ROS in regulating cofilin degradation. In a tumor xenographic transplant nude mouse model, the knockdown of GLDC promoted intrahepatic metastasis of HCC while GLDC overexpression inhibited it. Our data indicate that GLDC downregulation decreases ROS-mediated ubiquitination of cofilin to enhance HCC progression and intrahepatic metastasis.

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients

ABSTRACTSecreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.

Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR-mediated direct in vivo screening.

Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas some mutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct high-throughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adeno-associated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and died within 4 months. We used molecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis in immunocompetent mice. AAV-mediated autochthonous CRISPR screens provide a powerful means for mapping a provisional functional cancer genome atlas of tumor suppressors in vivo.

Abberent expression of NOR1 protein in tumor associated macrophages contributes to the development of DEN-induced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and the sixth most common lethal cancer worldwide. Recent evidences demonstrated that oxidored nitro domain containing protein 1 (NOR1), a putative tumor suppressor gene, is overexpressed in human HCC tissues. However, the role of NOR1 in HCC development remains unclear. Here, we described that NOR1 protein level is elevated in HCC and is associated with poorer clinical outcome. However, ecotopic overexpression of NOR1 protein in human HCC cell line HepG2 cells had no effect on cells proliferation, migration, and clonality. Immunofluoresence assay indicates NOR1 protein is mainly expressed at CD163 positive M2 tumor associated macrophages (TAMs). To explore the role of NOR1 in the development of HCC, we interrogated the susceptibility of mice lacking the NOR1 gene to DEN-induced hepatocarcinogenesis. NOR1 deficient mice displayed resistance to DEN-induced HCC. We also demonstrate that mNOR1 protein is enriched in F4/80 positive Kupffer cells (KCs) infiltrated in DEN induced murine HCC tissues. Loss of NOR1 led to increase of iNOS whereas decrease of Arg1, Ym1 expression in KCs. Overexpression of NOR1 in THP-1 macrophages led to decrease of iNOS but increase of Arg1. Mechanistic investigations showed that inflammatory cytokines IL-6, TNF-α production, and NF-κB activation were also decreased in NOR1 knockout mice exposed to DEN treatment. Our data suggested that NOR1 is overexpressed in HCC associated TAMs and promotes M2 alternative polarization. Genetic deletion of NOR1 in mice leads to impairment of IL-6 production and NF-κB activation, which in turn attenuates DEN-induced HCC development.

Aberrant methylation of FAT4 and SOX11 in peripheral blood leukocytes and their association with gastric cancer risk.

Background: Aberrant DNA methylation, especially tumor suppressor gene hypermethylation, is a well-recognized biomarker of initial tumorogenesis stages. FAT4 and SOX11 are putative tumor suppressor genes and can be down-regulated by hypermethylation in various cancers tissues. However, in peripheral blood leukocytes, the association between these two genes methylation status, as well as the effects of gene-environment interactions, and gastric cancer (GC) risk remain unclear.Methods: A hospital-based case-control study including 375 cases and 394 controls was conducted. Peripheral blood leukocytes DNA methylation status were detected by methylation-sensitive high-resolution melting (MS-HRM) assay. Logistic regression was adopted to analyze the relationship of FAT4 and SOX11 methylation with GC susceptibility.Results: Positive methylation (Pm) and total positive methylation (Tpm) of FAT4 were significantly increased the risk of GC (OR = 2.204, 95% CI: 1.168-4.159, P = 0.015; OR = 1.583, 95% CI: 1.031-2.430, P = 0.036, respectively). Compared with controls, cases exhibited higher SOX11 Pm frequencies with OR of 2.530 (95% CI: 1.289-4.969, P = 0.007). Nonetheless, no statistically significant association between SOX11 Tpm and GC risk was observed. Additionally, interactions between FAT4 Tpm and increased consumption of freshwater fish (≥1 times/week) displayed an antagonistic effect on GC (OR = 0.328, 95% CI: 0.142-0.762, P = 0.009), and high salt intake interacted with SOX11 Tpm also showed statistically significant (OR = 0.490, 95% CI: 0.242-0.995, P = 0.048).Conclusions: FAT4 aberrant methylation in peripheral blood leukocytes and gene-environment interactions were associated with the risk of GC, while SOX11 was controversial and needed to be more investigated.

Decreased expression of chromodomain helicase DNA-binding protein 9 is a novel independent prognostic biomarker for colorectal cancer.

Previous studies suggested that chromodomain helicase DNA-binding proteins (CHDs), including CHD 1–8, were associated with several human diseases and cancers including lymphoma, liver cancer, colorectal cancer, stomach cancer, etc. To date, little research on CHD 9 in human cancers has been reported. In this study, we assessed the prognostic value of CHD 9 in patients with colorectal cancer (CRC). We screened for CHD 9 expression using immunohistochemical analysis in 87 surgical CRC specimens and found that the expression was upregulated in 81.5% of the cases, while 7.4% were decreased; in the remaining 11.1% of the cases, levels were not altered. Kaplan-Meier analysis showed that patients with high CHD 9 expression had better prognosis than those with low CHD 9 expression (54.5 vs 32.1%, P=0.034). Subsequently, Cox multi-factor survival regression analysis revealed that expression of CHD 9 protein was an independent predictor for CRC, with a hazard ratio of 0.503 (P=0.028). In addition, we found that CHD 9 expression was positively correlated with MSH2 (rs=0.232, P=0.036). We speculated that CHD9 might be a putative tumor suppressor gene, and could inhibit the development of CRC by participating in DNA repair processes. Our findings suggest that CHD 9 could be a novel prognostic biomarker and a therapeutic target for CRC. Further studies are needed to detect the effect of CHD 9 on cellular function and the expression of mismatch repair genes.

SLURP-1 is mutated in Mal de Meleda, a potential molecular signature for melanoma and a putative squamous lineage tumor suppressor gene.

Mal de Meleda (MDM) is a rare inherited autosomal recessive genodermatosis characterized by palmoplantar keratoderma (PPK) with transgrediens and caused by mutations in the SLURP1 gene. Uncommonly, cutaneous tumors have been found at PPK sites in MDM patients. To study a Middle Eastern family with MDM with both PPK and skin tumors. We studied a Middle Eastern (Palestinian) family with clinical features of MDM and cutaneous tumors. Histopathological analysis was performed on biopsies from skin lesions found in the affected individuals. Direct sequencing of SLURP1 was performed in MDM affected members. In silico analysis of publicly available datasets was used to survey SLURP1 mRNA levels in normal and malignant tissues. Statistical analysis was performed in the R statistical language. Affected members from the Middle Eastern family displayed severe forms of PPK consistent with MDM. Histopathological analysis of the skin lesions revealed that the examined affected members exhibited skin squamous cell carcinomas (SCCs) and melanoma. Sequence analysis revealed homozygous SLURP1 mutations (c.82delT) in the affected members. Following analysis of various publicly available expression datasets, SLURP1 mRNA levels were found to be markedly elevated in tissues of epithelial lineage, relative to tissues of other lineages, and significantly suppressed in malignant tumors of epithelial lineage relative to normal or their premalignant counterparts. There was significant decrease in SLURP-1 expression in melanomas versus melanocytic nevi as well as a highly significant decrease in SLURP-1 expression in metastatic melanomas as compared to primary melanoma. Our study underscores cases of Middle Eastern MDM with SLURP1 mutations and skin malignancies at PPK sites. Our findings also highlight a plausible epithelial lineage-specific tumor suppressor role for the SLURP1 gene, as well as a role in the development and metastasis of melanoma and thus a potential molecular signature for melanoma.

Somatic PRDM2 c.4467delA mutations in colorectal cancers control histone methylation and tumor growth.

The chromatin modifier PRDM2/RIZ1 is inactivated by mutation in several forms of cancer and is a putative tumor suppressor gene. Frameshift mutations in the C-terminal region of PRDM2, affecting (A)8 or (A)9 repeats within exon 8, are found in one third of colorectal cancers with microsatellite instability, but the contribution of these mutations to colorectal tumorigenesis is unknown. To model somatic mutations in microsatellite unstable tumors, we devised a general approach to perform genome editing while stabilizing the mutated nucleotide repeat. We then engineered isogenic cell systems where the PRDM2 c.4467delA mutation in human HCT116 colorectal cancer cells was corrected to wild-type by genome editing. Restored PRDM2 increased global histone 3 lysine 9 dimethylation and reduced migration, anchorage-independent growth and tumor growth in vivo. Gene set enrichment analysis revealed regulation of several hallmark cancer pathways, particularly of epithelial-to-mesenchymal transition (EMT), with VIM being the most significantly regulated gene. These observations provide direct evidence that PRDM2 c.4467delA is a driver mutation in colorectal cancer and confirms PRDM2 as a cancer gene, pointing to regulation of EMT as a central aspect of its tumor suppressive action.

ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma.

Pheochromocytoma (PCC) is related to germline mutations in 12 susceptibility genes. Although comparative genomic hybridization array has revealed some putative tumor suppressor genes on the short arm of chromosome 1 that are likely to be involved in PCC tumorigenesis, the molecules involved, except for those encoded by known susceptibility genes, have not been found in the generation of sporadic tumors. In the present work, we first identified that the unmethylated allele of Aplasia Ras homolog member I (ARHI) was deleted in most PCC tumors which retained a hypermethylated copy, while its mRNA level was significantly correlated with the unmethylated copy. De-methylation experiments confirmed that expression of ARHI was also regulated by the methylation level of the remaining allele. Furthermore, ARHI overexpression inhibited cell proliferation, with cell cycle arrest and induction of apoptosis, in ARHI-negative primary human PCC cells, whereas knockdown of ARHI demonstrated the opposite effect in ARHI-positive primary human PCC cells. Finally, we demonstrated that ARHI has the ability to suppress pAKT and pErK1/2, to promote the expression of p21Waf1/Cip1 and p27Kip1, and also to increase p27Kip1 protein stability. In summary, ARHI was silenced or downregulated in PCC tissues harboring only one hypermethylated allele. ARHI contributes to tumor suppression through inhibition of PI3K/AKT and MAKP/ERK pathways, to upregulate cell cycle inhibitors such as p27Kip1. We therefore reasoned that ARHI is a novel epigenetic silenced tumor suppressor gene on chromosome 1p that is involved in sporadic PCC tumorigenesis.

What is New in Gastrointestinal Stromal Tumor?

The classification "gastrointestinal stromal tumor" (GIST) became commonplace in the 1990s and since that time various advances have characterized the GIST lineage of origin, tyrosine kinase mutations, and mechanisms of response and resistance to targeted therapies. In addition to tyrosine kinase mutations and their constitutive activation of downstream signaling pathways, GISTs acquire a sequence of chromosomal aberrations. These include deletions of chromosomes 14q, 22q, 1p, and 15q, which harbor putative tumor suppressor genes required for stepwise progression from microscopic, preclinical forms of GIST (microGIST) to clinically relevant tumors with malignant potential. Recent advances extend our understanding of GIST biology beyond that of the oncogenic KIT/PDGFRA tyrosine kinases and beyond mechanisms of KIT/PDGFRA-inhibitor treatment response and resistance. These advances have characterized ETV1 as an essential interstitial cell of Cajal-GIST transcription factor in oncogenic KIT signaling pathways, and have characterized the biologically distinct subgroup of succinate dehydrogenase deficient GIST, which are particularly common in young adults. Also, recent discoveries of MAX and dystrophin genomic inactivation have expanded our understanding of GIST development and progression, showing that MAX inactivation is an early event fostering cell cycle activity, whereas dystrophin inactivation promotes invasion and metastasis.

Recurrent alterations of the WW domain containing oxidoreductase gene spanning the common fragile site FRA16D in multiple myeloma and monoclonal gammopathy of undetermined significance.

The putative tumor suppressor gene WW domain containing oxidoreductase (WWOX) spans a common fragile site (CFS) on chromosome 16q23.3. CFSs are regions of profound genomic instability and sites for genomic deletions in cancer cells. Therefore, WWOX is structurally altered in diverse nonhematological cancer types. However, the function of WWOX in hematological tumor types, including multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) remains unclear. WWOX expression and methylation in patients with MM, MGUS, or noninvasive lymphoma (control) were analyzed using reverse transcription- and methylation specific-polymerase chain reaction analysis. Variant WWOX transcripts were detected in 65 and 50% of patients with MM and MGUS, respectively, compared with 10% of controls. WWOX expression was higher in patients with MM, and WWOX promoter methylation was detected in 35% of patients with MM compared with 5% of patients with MGUS and 4% of controls. WWOX promoter methylation was significantly associated with shorter overall survival time of patients, in particular those with MM who were never treated with novel agents. Genomic alterations, including deletions and promoter methylation that affect WWOX expression occur early and may be involved in the pathogenesis, progression, and prognosis of MM.

Multiple roles of RARRES1 in prostate cancer: Autophagy induction and angiogenesis inhibition.

BackgroundProstate cancer (PCa) poses a major health concern in men worldwide. Retinoic Acid Receptor Responder (RARRES1)/ Tazarotene-induced gene-1 (TIG-1) is a putative tumor suppressor gene that exerts its tumor suppressor function via unknown mechanisms. Epigenetic silencing of RARRES1 leads to its loss in several types of cancer, including PCa. Determining the molecular mechanisms that mediate the tumor suppressor role of RARRES1 in PCa is the focus of our study.FindingsOur data indicates that RARRES1 over expression in PCa cell lines represses mitogen-activated protein kinase (MAPK) activation. RARRES1 expression induces the levels of autophagy-related genes, beclin, ATG3 and increases LC3B-II conversion. A significant induction of SIRT1 along with mTOR inhibition is noted on RARRES1 expression. Furthermore, RARRES1 over expression elevates the levels of the antioxidant enzyme, catalase. Our results also indicate that RARRES1 expression inhibits angiogenesis in endothelial cells.ConclusionsIn summary, the data presented here indicate that forced expression of RARRES1 in PCa cells (a) induces ER stress and autophagic response; (b) increases SIRT1 levels; and (c) higher levels of anti-oxidant enzymes. Our study also implicates the role of RARRES1 as a novel anti-angiogenic molecule. Overall this study reports the molecular players that RARRES1 modulates to serve as a tumor suppressor molecule. Future studies will help determine the in vivo mechanisms by which RARRES1 may serve as a target for therapeutic intervention both in cancer and in angiogenesis-related disorders.

Mismatch Repair Proteins Initiate Epigenetic Alterations during Inflammation-Driven Tumorigenesis.

Aberrant silencing of genes by DNA methylation contributes to cancer, yet how this process is initiated remains unclear. Using a murine model of inflammation-induced tumorigenesis, we tested the hypothesis that inflammation promotes recruitment of epigenetic proteins to chromatin, initiating methylation and gene silencing in tumors. Compared with normal epithelium and noninflammation-induced tumors, inflammation-induced tumors gained DNA methylation at CpG islands, some of which are associated with putative tumor suppressor genes. Hypermethylated genes exhibited enrichment of repressive chromatin marks and reduced expression prior to tumorigenesis, at a time point coinciding with peak levels of inflammation-associated DNA damage. Loss of MutS homolog 2 (MSH2), a mismatch repair (MMR) protein, abrogated early inflammation-induced epigenetic alterations and DNA hypermethylation alterations observed in inflammation-induced tumors. These results indicate that early epigenetic alterations initiated by inflammation and MMR proteins lead to gene silencing during tumorigenesis, revealing a novel mechanism of epigenetic alterations in inflammation-driven cancer. Understanding such mechanisms will inform development of pharmacotherapies to reduce carcinogenesis. Cancer Res; 77(13); 3467-78. ©2017 AACR.

Abstract 2157: P73 isoforms regulate cellular survival and response to treatment in diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL) and is curable in more than 60% of the cases. Despite long-term remission achieved with current therapies, relapse occurs in almost one-third of the cases. Rearrangements of chromosome locus 1p36 with ensuing deletion or disruption of TP73, one of the most distally located putative tumor suppressor genes, is frequent in many NHL subtypes, including DLBCL, and confers inferior prognosis. Recent reports in normal B-cells and lymphoma animal models suggest an important role of p73 in B-cell lymphomas; however, the biological significance of p73 isoforms in DLBCL is not clear. TP73 is a member of the TP53 family that shares structural homology and hence is capable of transactivating p53 target genes. We investigated the relationship between rearrangements of chromosome locus 1p36 and p73 expression and examined whether differential expression of p73 isoforms (TAp73 and ΔNp73) correlate with proliferation and apoptosis in DLBCL. We utilized molecular techniques in conjunction with fluorescence in situ hybridization (FISH) in untreated histologically confirmed DLBCL patient specimens. In our data set, the 1p36 chromosomal locus was disrupted (mainly heterozygous deletion) in 35% of the cases and was significantly associated with ΔNp73 expression. Subsequent immunohistochemical analysis showed a significant positive correlation between TAp73 and cleaved caspase-3 expression and between ΔNp73 and Ki-67 expression. Additionally, we observed a negative correlation between TAp73 and the anti-apoptotic molecules Bcl-2 and Bcl-6. We then performed in vitro studies in DLBCL cells and modulated the expression of TAp73 and ΔNp73 using expression vectors and siRNA. TAp73-transfected cells were more susceptible to serum deprivation and doxorubicin treatment, conversely the ΔNp73-transfected cells were more resistant to serum deprivation and doxorubicin treatment compared to control cells. Thus, rearrangements of 1p36 consequently deregulates p73 isoform expression in DLBCL. Our data emphasizes the importance of delineating the functional aspect of genetic alterations and underscores the significant role of p73 isoforms in DLBCL pathogenesis and treatment. Citation Format: Hesham M. Hassan, Michelle L. Varney, Pamela A. Althof, Gabriel C. Caponetti, Kai Fu, Dennis D. Weisenburger, Rakesh K. Singh, Bhavana J. Dave. P73 isoforms regulate cellular survival and response to treatment in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2157. doi:10.1158/1538-7445.AM2017-2157