Inactivation Of Tumor Suppressor Genes Research Articles

Regulation of DNA methylation machinery by epi-miRNAs in human cancer: emerging new targets in cancer therapy.

Disruption in DNA methylation processes can lead to alteration in gene expression and function that would ultimately result in malignant transformation. In this way, studies have shown that, in cancers, methylation-associated silencing inactivates tumor suppressor genes, as effectively as mutations. DNA methylation machinery is composed of several genes, including those with DNA methyltransferases activity, proteins that bind to methylated cytosine in the promoter region, and enzymes with demethylase activity. Based on a prominent body of evidence, DNA methylation machinery could be regulated by microRNAs (miRNAs) called epi-miRNAs. Numerous studies demonstrated that dysregulation in DNA methylation regulators like upstream epi-miRNAs is indispensable for carcinogenesis; consequently, the malignant capacity of these cells could be reversed by restoring of this regulatory system in cancer. Conceivably, recognition of these epi-miRNAs in cancer cells could not only reveal novel molecular entities in carcinogenesis, but also render promising targets for cancer therapy. In this review, at first, we have an overview of the methylation alteration in cancers, and the effect of this phenomenon in miRNAs expression and after that, we conduct an in-depth discussion about the regulation of DNA methylation regulators by epi-miRNAs in cancer cells.

MicroRNAs in Lung Cancer Oncogenesis and Tumor Suppression: How it Can Improve the Clinical Practice?

Background Lung cancer (LC) development is a process that depends on genetic mutations. The DNA methylation, an important epigenetic modification, is associated with the expression of non-coding RNAs, such as microRNAs. MicroRNAs are particularly essential for cell physiology, since they play a critical role in tumor suppressor gene activity. Furthermore, epigenetic disruptions are the primary event in cell modification, being related to tumorigenesis. In this context, microRNAs can be a useful tool in the LC suppression, consequently improving prognosis and predicting treatment.Conclusion This manuscript reviews the main microRNAs involved in LC and its potential clinical applications to improve outcomes, such as survival and better quality of life.

Genomic Database Analysis of Uterine Leiomyosarcoma Mutational Profile.

Uterine Leiomyosarcoma (uLMS) is by far the most common type of uterine sarcoma, characterized by an aggressive clinical course, a heterogeneous genetic profile and a very scarce response to cytotoxic chemotherapy. The genetic make-up of uLMS is an area of active study that could provide essential cues for the development of new therapeutic approaches. A total of 216 patients with uLMS from cBioPortal and AACR-GENIE databases were included in the study. The vast majority of patients (81%) carried at least one mutation in either TP53, RB1, ATRX or PTEN. The most frequently mutated gene was TP53, with 61% of the patients harboring at least one mutation, followed by RB1 at 48%. PTEN alteration was more frequent in metastases than in primary lesions, consistent with a later acquisition during tumor progression. There was a significant trend for TP53 and RB1 mutations to occur together, while both TP53 and RB1 were mutually exclusive with respect to CDKN2A/B inactivation. Overall survival did not show significant correlation with the mutational status, even if RB1 mutation emerged as a favorable prognostic factor in the TP53-mutant subgroup. This comprehensive analysis shows that uLMS is driven almost exclusively by the inactivation of tumor suppressor genes and suggests that future therapeutic strategies should be directed at targeting the main genetic drivers of uLMS oncogenesis.

Epigenetic Silencing of LMX1A Contributes to Cancer Progression in Lung Cancer Cells.

Epigenetic modification is considered a major mechanism of the inactivation of tumor suppressor genes that finally contributes to carcinogenesis. LIM homeobox transcription factor 1α (LMX1A) is one of the LIM-homeobox-containing genes that is a critical regulator of growth and differentiation. Recently, LMX1A was shown to be hypermethylated and functioned as a tumor suppressor in cervical cancer, ovarian cancer, and gastric cancer. However, its role in lung cancer has not yet been clarified. In this study, we used public databases, methylation-specific PCR (MSP), reverse transcription PCR (RT-PCR), and bisulfite genomic sequencing to show that LMX1A was downregulated or silenced due to promoter hypermethylation in lung cancers. Treatment of lung cancer cells with the demethylating agent 5-aza-2’-deoxycytidine restored LMX1A expression. In the lung cancer cell lines H23 and H1299, overexpression of LMX1A did not affect cell proliferation but suppressed colony formation and invasion. These suppressive effects were reversed after inhibition of LMX1A expression in an inducible expression system in H23 cells. The quantitative RT-PCR (qRT-PCR) data showed that LMX1A could modulate epithelial mesenchymal transition (EMT) through E-cadherin (CDH1) and fibronectin (FN1). NanoString gene expression analysis revealed that all aberrantly expressed genes were associated with processes related to cancer progression, including angiogenesis, extracellular matrix (ECM) remodeling, EMT, cancer metastasis, and hypoxia-related gene expression. Taken together, these data demonstrated that LMX1A is inactivated through promoter hypermethylation and functions as a tumor suppressor. Furthermore, LMX1A inhibits non-small cell lung cancer (NSCLC) cell invasion partly through modulation of EMT, angiogenesis, and ECM remodeling.

S-adenosylmethionine in combination with decitabine shows enhanced anti-cancer effects in repressing breast cancer growth and metastasis.

Abnormal DNA methylation orchestrates many of the cancer‐related gene expression irregularities such as the inactivation of tumour suppressor genes through hypermethylation as well as activation of prometastatic genes through hypomethylation. The fact that DNA methylation abnormalities can be chemically reversed positions the DNA methylation machinery as an attractive target for anti‐cancer drug development. However, although in vitro studies suggested that targeting concordantly hypo‐ and hypermethylation is of benefit in suppressing both oncogenic and prometastatic functions of breast cancer cells, this has never been tested in a therapeutic setting in vivo. In this context, we investigated the combined therapeutic effects of an approved nutraceutical agent S‐adenosylmethionine (SAM) and FDA‐approved hypomethylating agent decitabine using the MDA‐MB‐231 xenograft model of breast cancer and found a pronounced reduction in mammary tumour volume and lung metastasis compared to the animals in the control and monotherapy treatment arms. Immunohistochemical assessment of the primary breast tumours showed a significantly reduced expression of proliferation (Ki‐67) and angiogenesis (CD31) markers following combination therapy as compared to the control group. Global transcriptome and methylome analyses have revealed that the combination therapy regulates genes from several key cancer‐related pathways that are abnormally expressed in breast tumours. To our knowledge, this is the first preclinical study demonstrating the anti‐cancer therapeutic potential of using a combination of methylating (SAM) and demethylating agent (decitabine) in vivo. Results from this study provide a molecularly founded rationale for clinically testing a combination of agents targeting the epigenome to reduce the morbidity and mortality from breast cancer.

Prostate Carcinogenesis: Insights in Relation to Epigenetics and Inflammation.

Prostate cancer is a multifactorial disease that mainly occurs due to the accumulation of somatic, genetic, and epigenetic changes, resulting in the inactivation of tumor-suppressor genes and activation of oncogenes. Mutations in genes, specifically those that control cell growth and division or the repair of damaged DNA, make the cells grow and divide uncontrollably to form a tumor. The risk of developing prostate cancer depends upon the gene that has undergone the mutation. Identifying such genetic risk factors for prostate cancer poses a challenge for the researchers. Besides genetic mutations, many epigenetic alterations, including DNA methylation, histone modifications (methylation, acetylation, ubiquitylation, sumoylation, and phosphorylation) nucleosomal remodeling, and chromosomal looping, have significantly contributed to the onset of prostate cancer as well as the prognosis, diagnosis, and treatment of prostate cancer. Chronic inflammation also plays a major role in the onset and progression of human cancer, via modifications in the tumor microenvironment by initiating epithelialmesenchymal transition and remodeling the extracellular matrix. In this article, the authors present a brief history of the mechanisms and potential links between the genetic aberrations, epigenetic changes, inflammation, and inflammasomes that are known to contribute to the prognosis of prostate cancer. Furthermore, the authors examine and discuss the clinical potential of prostate carcinogenesis in relation to epigenetics and inflammation for its diagnosis and treatment..

Contribution of DNA methylation and EZH2 in SRBC down-regulation in gastric cancer.

Gastric cancer (GC), a high mortality malignancy, is induced by genetic and epigenetic factors. DNA and histone methylation play critical roles in tumor suppressor genes inactivation. SRBC (serum deprivation response factor-related gene product that binds to the c-kinase), suggested as a tumor suppressor gene, participates in apoptosis, tumor chemoresistance and DNA damage response and is repressed in various cancers. Inspecting the mechanisms underlying SRBC suppression is important for cancer treatments. We investigated SRBC promoter DNA methylation status and expression of SRBC and EZH2 histone methyltrasferase in gastric cancer. Also, we surveyed SRBC expression after 5-azacitidine and UNC1999 treatments of AGS cell line. In current work, we used gastric adenocarcinoma tissues, marginal samples and normal gastric biopsies. DNA methylation was detected by Methylation- Specific PCR and mRNA expression was measured by Real-Time PCR. SRBC promoter methylation analysis, showed fully and partial methylated versions that were associated with patient's age (p = 0.001). SRBC expression significantly decreased in GC compare with marginal and normal samples (p-value < 0.001). EZH2 showed remarkable up-regulation in GC than controls and demonstrated a strong inverse correlation with SRBC expression (r = - 0.69). Restoration of SRBC expression was observed after 5-azacitidine and UNC1999 applications with a remarkable increase by combinational treatment. We showed that EZH2 plays role in SRBC silencing in addition to DNA methylation. Our study, suggests that DNA methylation and EZH2 are involved in SRBC silencing and their inhibitors can be considered in cancer treatment investigations to overcome chemoresistance induced by SRBC inactivation.

Abstract A64: Functional genomics of metastatic Ewing sarcoma

Abstract Ewing sarcoma (ES) is a poorly differentiated bone and soft tissue tumor of high metastatic potential. ES mainly affects children, adolescents, and young adults (AYAs) at a frequency of ~1.5 cases per million globally. Ewing neoplasms strikingly converge on a single recurrent initiating event, which is a chromosomal translocation that generates a fusion transcript between the EWSR1 gene and a gene of the ETS family of transcription factors, most commonly FLi1 (85%). After 20 years since the discovery of the EWS-FLi1 fusion, ES remains a clinical challenge with unacceptably low survival rates, primarily due to metastasis. The bulk of research conducted to date (&amp;gt;95%) being focused on the primary tumor has resulted in a critical knowledge gap. To address this issue and unravel the dysregulated pathways in ES tumor evolution and metastatic dissemination, we harnessed the genome-wide insertional mechanism of transposons and the transduction efficiency of lentiviruses to engineer ES cell models. Human mesenchymal stem cells (hMSC), the putative cells of origin of ES, were engineered to constitutively express EWS-Fli1 accompanied by the inducible expression of a highly active transposase. Upon activation of the former, transposon-mediated mutagenesis will activate oncogenes and inactivate tumor suppressor genes, to mediate transformation of the transduced that can now engraft when implanted in recipient mice. Xenografted tumors are resected and the mice observed for development of distant metastases. Matching primary and metastatic tumors are sequenced to uncover the genes commonly affected by transposition in the two compartments. Pathway-oriented bioinformatic analysis will further reveal candidate metastatic driver genes. Matching of the targeted pathways with drugs will be used to validate the candidates by in vitro and in vivo metastasis assays. Citation Format: Wajih Jawhar, Paul Waterhouse, Rama Khokha, Takeaki Ishii, Robert Turcotte, Nada Jabado, Livia Garzia. Functional genomics of metastatic Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A64.

Abstract B65: Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma

Abstract Clinical efficacy of immune checkpoint therapy in high-grade serous carcinoma (HGSC) has been disappointing to date and highlights the need to better understand the relationship between HGSC and its associated immune microenvironment. Tractable model systems for interrogating the tumor microenvironment in HGSC could be used to address this unmet need. We have generated genetically engineered mouse models (GEMMs) of HGSC utilizing oviduct-restricted tamoxifen-regulated Cre recombinase to drive conditional inactivation of key tumor suppressor genes (Brca1, Trp53, Rb1, Nf1) observed to be recurrently altered in human HGSC samples. These autochthonous tumors arise from the murine fallopian tube (oviduct) in the context of an intact immune system. We employed RNA sequencing (RNA-seq) of bulk tumors from 19 mouse HGSCs to compare their gene expression profiles to those of normal oviduct and ovary tissues (4 each), and 6 mouse endometrioid-like oviductal carcinomas based on conditional inactivation of Apc and Pten. We employed immunogenomic analysis of the RNA-seq data to characterize the intratumoral infiltrate of 28 immune cell types in these samples in silico. The analysis revealed a prevalence of gene expression signatures associated with immunosuppressive cell types, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the mouse HGSCs, not observed in the other sample groups. Human HGSCs with available RNA-seq data (from TCGA samples) showed similar enrichment of MDSC and Treg gene signatures in the subset of human HGSCs classified as mesenchymal or immunoreactive expression subtypes. To further validate the findings in the mouse HGSCs, we used immunohistochemistry to detect subsets of tumor-infiltrating lymphocytes (TILs) in the tumor tissues. Specifically, CD8+ TIL infiltrates recapitulated epithelial (E-TIL), stromal (S-TIL) and combined epithelial/stromal (ES-TIL) patterns described in human HGSCs. Moreover, FoxP3+ Tregs were frequently observed to be co-infiltrated with CD8+ TILs as expected in an immunosuppressive tumor microenvironment. Gene set enrichment analysis showed that NF-kappa B signaling is among the top five pathways upregulated in HGSCs versus normal oviducts, suggesting a plausible mechanism by which tumor cells can alter and respond to the microenvironment during tumor development. These studies provide an experimental paradigm for improving our understanding of the role of the tumor microenvironment in HGSC pathogenesis and may aid in efforts to enhance the efficacy of immunotherapies in this lethal disease. Citation Format: Kevin W. McCool, Rork Kuick, Yali Zhai, Zach Freeman, Rong Wu, Eric Fearon, Kathleen R. Cho. Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B65.

Abstract B50: Direct interrogation of the incessant ovulation hypothesis in a high-fidelity mouse model of high-grade serous cancer

Abstract The biologic basis for factors that modify risk of developing high-grade serous carcinoma remains understudied. In particular, the “Incessant Ovulation” hypothesis remains a plausible model to unify the observed risks associated with more ovulations (increasing age, early menarche/late menopause) as well as protection conferred by oral contraceptive use, multiparity, and breastfeeding. We used genetically engineered mouse models (GEMMs) of HGSC to directly interrogate the role of aging and multiparity as deleterious and protective risk factors, respectively, for HGSC development. Specifically, we utilized oviductal-restricted tamoxifen-inducible Cre recombinase to drive conditional bi-allelic inactivation of key tumor-suppressor genes (Brca1, Trp53, Rb1, Nf1) in the murine fallopian tube (oviduct). In our genetically engineered mice, autochthonous tumors arise with high penetrance in response to tamoxifen treatment, albeit with prolonged latency. As a consequence, the models allow us to evaluate effects of multiparity and aging on tumor development and progression. To test effects of multiparity, two cohorts of mice were tested, one with two floxed alleles of all 4 TSGs (BPRN), and another in which one allele of Nf1 was wild-type (BPRNfl/+). Mice were treated with tamoxifen and then continuously caged with a breeding male for a total of up to 9 pregnancies. Control mice were treated with tamoxifen and then remained nulliparous until sacrifice. The total number of pregnancies ranged from 5 to 9 for each mouse in the multiparous groups. Mice were euthanized 62 weeks post-TAM or earlier, if humane endpoints were reached. No apparent differences in diagnosis at study endpoint was observed in the multiparous BPRN versus nulliparous group (p=0.34, Mantel-Haenszel Chi-square test of association). Interestingly, the control mice showed a trend for worse diagnosis compared to the BPRNfl/+ mice at study endpoint (p=0.02). In the aging cohort, control BPRN mice were exposed to tamoxifen at 8 weeks of age and followed to humane endpoints. The experimental group was aged to 9 months, exposed to tamoxifen and then followed to humane endpoints. Survival post-TAM was observed to be shortened in the older animals (median 45.5 weeks versus 57 weeks in control mice, log rank test p=0.0033). Using Cox proportional hazard models, aging was associated with a hazard ratio of 2.8 with 95% CI of 1.4-5.9. Collectively these data suggest that multiple pregnancies alone delays HGSC development and progression in certain genetic contexts, while in contrast, tumor induction in aged mice is associated with significantly shorter survival. Together, these results help credential these GEMMs as useful tools with which to explore the biologic basis for risk factors presumed to underly the development of HGSC. Citation Format: Yali Zhai, Kevin McCool, Rork Kuick, Lixing Chen, Eric R. Fearon, Kathleen R. Cho. Direct interrogation of the incessant ovulation hypothesis in a high-fidelity mouse model of high-grade serous cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B50.

SERS-Based Assessment of MRD in Acute Promyelocytic Leukemia?

Acute promyelocytic leukemia (APL) is characterized by a unique chromosome translocation t(15;17)(q24;q21), which leads to the PML/RARA gene fusion formation. However, it is acknowledged that this rearrangement alone is not able to induce the whole leukemic phenotype. In addition, epigenetic processes, such as DNA methylation, may play a crucial role in leukemia pathogenesis. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), involves the covalent transfer of a methyl group (-CH3) to the fifth carbon of the cytosine ring in the CpG dinucleotide and results in the formation of 5-methylcytosine (5-mC). The aberrant gene promoter methylation can be an alternative mechanism of tumor suppressor gene inactivation. Understanding cancer epigenetics and its pivotal role in oncogenesis, can offer us not only attractive targets for epigenetic treatment but can also provide powerful tools in monitoring the disease and estimating the prognosis. Several genes of interest, such as RARA, RARB, p15, p16, have been studied in APL and their methylation status was correlated with potential diagnostic and prognostic significance. In the present manuscript we comprehensively examine the current knowledge regarding DNA methylation in APL pathogenesis. We also discuss the perspectives of using the DNA methylation patterns as reliable biomarkers for measurable residual disease (MRD) monitoring and as a predictor of relapse. This work also highlights the possibility of detecting aberrant methylation profiles of circulating tumor DNA (ctDNA) through liquid biopsies, using the conventional methods, such as methylation-specific polymerase chain reaction (MS-PCR), sequencing methods, but also revolutionary methods, such as surface-enhanced Raman spectroscopy (SERS).

Clinical significance of expression of fibrous sheath interacting protein 1 in colon cancer.

BACKGROUNDThe occurrence and development of colon cancer are complex, involving a variety of genetic changes, such as mutation and activation of oncogenes, inactivation of tumour suppressor genes, and aberrant proliferation and apoptosis regulation mechanisms. Fibrous sheath interacting protein 1 (FSIP1) is a newly discovered oncogene that is frequently activated in a variety of tumours such as breast cancer and bladder cancer. However, the clinical significance of FSIP1 in colon cancer is unclear. In this study, we analysed the clinical significance of expression of FSIP1 in human colon cancer, aimed to clarify the biological role of FSIP1 in the development and progression of colon cancer.AIMTo investigate the clinical significance of expression of FSIP1 in colon cancer.METHODSFrom March 2011 to March 2014, 302 specimens of tumour tissues and paracancerous tissues were obtained from patients pathologically diagnosed with colon cancer at Shengjing Hospital of China Medical University. Immunohistochemistry was used to detect FSIP1 expression in colon cancer tissues and adjacent normal tissues. Spearman correlation coefficient and Cox regression analyses were used to determine the relationship between FSIP1 expression and clinicopathological factors and prognosis, as well as the impact on survival.RESULTSCompared with its expression in adjacent normal tissues, FSIP1 was expressed at higher levels in colon cancer tissues. Spearman correlation analysis showed that high expression of FSIP1 was positively correlated with clinicopathological stage, lymph node metastasis, and poor prognosis in colon cancer; it was negatively correlated with the degree of tumour differentiation. Cox regression analysis showed that high FSIP1 expression was an independent risk factor for the prognosis of colon cancer patients.CONCLUSIONHigh expression of FSIP1 may be one of the important factors affecting the clinical outcome of colon cancer patients and leading to poor prognosis.

Progress in the correlation between PTPN12 gene expression and human tumors.

Background:The global morbidity of cancer is rising rapidly. Despite advances in molecular biology, immunology, and cytotoxic and immune-anticancer therapies, cancer remains a major cause of death worldwide. Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a new member of the cytoplasmic protein tyrosine phosphatase family, isolated from a cDNA library of adult colon tissue. Thus far, no studies have reviewed the correlation between PTPN12 gene expression and human tumors.Methods:This article summarizes the latest domestic and international research developments on how the expression of PTPN12 relates to human tumors. The extensive search in Web of Science and PubMed with the keywords including PTPN12, tumor, renal cell carcinoma, proto-oncogenes, tumor suppressor genes was undertaken.Results:More and more studies have shown that a tumor is essentially a genetic disease, arising from a broken antagonistic function between proto-oncogenes and tumor suppressor genes. When their antagonistic effect is out of balance, it may cause uncontrolled growth of cells and lead to the occurrence of tumors. PTPN12 is a tumor suppressor gene, so inhibiting its activity will lead directly or indirectly to the occurrence of tumors.Conclusion:The etiology, prevention, and treatment of tumors have become the focus of research around the world. PTPN12 is a tumor suppressor gene. In the future, PTPN12 might serve as a novel molecular marker to benefit patients, and even the development of tumor suppressor gene activation agents can form a practical research direction.

Chlorogenic Acid Decreases Malignant Characteristics of Hepatocellular Carcinoma Cells by Inhibiting DNMT1 Expression.

BackgroundHepatocellular carcinoma (HCC) is the most common malignant tumor of the adult liver, exhibiting rapid progression and poor prognosis. Chlorogenic acid (CGA), a polyphenol, has several biological activities, including the suppression of liver cancer cell invasion and metastasis. Increased levels or alterations in the function of DNMT1 are associated with the inactivation of tumor suppressor genes. However, the CGA-affected DNMT1 expression mediated mechanism is still unclear.MethodsThe human hepatocellular carcinoma (HCC) HepG2 cells were treated with a positive control drug (5-AZA) or varying doses of CGA. DNA methyltransferase 1 (DNMT1) protein levels and other relevant proteins were evaluated using Western blotting and immunocytochemistry. Cell-cycle analysis was performed by flow cytometry-based PI staining, and cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The transwell invasion and wound healing assays were used to evaluate cell migration and invasion. In vivo proliferation of the HCC cells was detected. We investigated the expression of DNMT1, p53, p21, p-ERK, MMP-2, and MMP-9 in tumors using immunohistochemical analysis.ResultsOur results showed that CGA inhibited the proliferation, colony formation, invasion, and metastasis of HepG2 cells both in vitro and in vivo by down-regulating DNMT1 protein expression, which enhanced p53 and p21 activity, and resulting in a significant reduction in cell proliferation and metastasis. Moreover, CGA inactivated ERK1/2 and reduced MMP-2 and MMP-9 expression in HepG2 cells.ConclusionsCGA can suppress liver cancer cell proliferation, invasion, and metastasis through several pathways. CGA could serve as a candidate chemopreventive agent for HCC.

Variability in retinoblastoma genome stability is driven by age and not heritability.

Retinoblastoma (RB) is a childhood intraocular cancer initiated by biallelic inactivation of the RB tumor suppressor gene (RB1-/- ). RB can be hereditary (germline RB1 pathogenic allele is present) or non-hereditary. Somatic copy number alterations (SCNAs) contribute to subsequent tumorigenesis. Previous studies of only enucleated RB eyes have reported associations between heritability status and the prevalence of SCNAs. Herein, we use an aqueous humor (AH) liquid biopsy to investigate RB genomic profiles in the context of germline RB1 status, age, and International Intraocular Retinoblastoma Classification (IIRC) clinical grouping for both enucleated and salvaged eyes. Between 2014 and 2019, AH was sampled from a total of 54 eyes of 50 patients. Germline RB1 status was determined from clinical blood testing, and cell-free DNA from AH was analyzed for SCNAs. Of the 50 patients, 23 (46.0%; 27 eyes) had hereditary RB, and 27 (54.0%, 27 eyes) had non-hereditary RB. Median age at diagnosis was comparable between hereditary (13 ± 10 months) and non-hereditary (13 ± 8 months) eyes (P = 0.818). There was no significant difference in the prevalence or number of SCNAs based on (1) hereditary status (P > 0.56) or (2) IIRC grouping (P > 0.47). There was, however, a significant correlation between patient age at diagnosis, and (1) number of total SCNAs (r[52] = 0.672, P < 0.00001) and (2) number of highly-recurrent RB SCNAs (r[52] = 0.616, P < 0.00001). This evidence does not support the theory that specific molecular or genomic subtypes exist between hereditary and non-hereditary RB; rather, the prevalence of genomic alterations in RB eyes is strongly related to patient age at diagnosis.

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Beta Human Papillomavirus and Merkel Cell Polyomavirus in Skin Neoplasms

Abstract B085: Low-sensitivity and drug-resistance to DNA-demethylating epi-drug decitabine in solid tumors: A challenge yet to overcome

Abstract Based on the current understanding of the epigenetic landscape, cancer methylome is highly disrupted, which makes DNA methylation an excellent target for anti-cancer cures. To date, azacytidine and its congener, decitabine (DAC) are the most successful DNA demethylating epigenetic drugs. However, scientists are yet to find the reversal of clinical resistance to these drugs. Yet another challenge remains to be the decreased efficacy of these promising therapies in the cure of solid tumors. We used a soild tumor, HCT116 colorectal cancer cell line and developed resistance against DAC. DAC-resistant HCT116 cells were used to study the epigenetic cross-talk between DNA methylation and chromatin modifications. The screening of parental and DAC-resistant HCT116 cells against inhibitors of epigenetic writers-erasers-readers unveiled increased sensitivity of resistant cells to BET inhibitor (inhibitor of reader enzymes), (+)-JQ1. BET inhibitor further mediated augmented response on cell cycle phases of resistant cells, increased anti-proliferative effects in xenograft models of resistant cells, and synergystic effects in combination with DAC in HCT116 parental cells, both in vitro and. We then sequenced the transcriptome of DAC-sensitive and -resistant HCT116 cells, and (+)-JQ1 treated-(DAC-resistant) cells, using RNA-seq. The RNA-seq data revealed the overexpression of critical oncogenes, and their binding inactivation of key tumor suppressor genes (TSGs) in resistant cells. The most significant and biologically relevant transcriptional changes were further validated by qRT-PCR, and in addition their methylation status was determined by bisulphite sequencing. We discovered that the expressions of down-regulated TSGs were driven by promoter methylation, exposing these tumor-suppressive signatures as biomarkers which might differentiate between DAC-resistance and sensitivity, whereas, overexpression of oncogenes was independent of promoter methylation. Interestingly, the expressions of up-regulated oncogenes which define cell identity, mainly those involved in signaling of inflammatory pathways (including some bromodomain-specific genes) were reversed on treatment with (+)-JQ1. Further, siRNA-mediated genetic inhibition of bromodomains in resistant cells phenocopied therapeutic inhibition by (+)-JQ1. These data unveil the chromatin “reader proteins”, as regulators of dysregulated oncogenic expressions in DAC-resistant cells. The present study provides novel insights into the epigenomic landscape of DAC-resistant colorectal cancer cells, and put forward, the alternative therapeutic regimen for DAC-resistant patients. This study was supported by Czech Ministry of Education, Youth and Sports (LO1304, LM2015091, LM2015064), Technology Agency of the Czech Republic (TE01020028) and Internal Grant Agency of Palacky University (IGA_LF_2016_019). Citation Format: Khushboo Agrawal, Petr Vojta, Rastislav Slavkosky, Ivo Frydrych, Petr Dzubak, Marian Hajduch. Low-sensitivity and drug-resistance to DNA-demethylating epi-drug decitabine in solid tumors: A challenge yet to overcome [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B085.

Abstract IA26: Multiplexed functional cancer genomics

Abstract Extensive cancer genotyping has identified a large number of putative tumor-suppressor genes. However, genome sequencing data alone are insufficient to uncover the importance and role of these genes during carcinogenesis. Moreover, whether tumor suppressors restrict the rate of tumor growth, decrease the probability of incipient tumorigenesis, or limit the emergence of particularly fast-growing clones remains unknown for even the most well-studied tumor suppressors. I will discuss the development and use of multiplexed autochthonous mouse models of human lung adenocarcinoma that integrate tumor barcoding, CRISPR/Cas9-based genome editing, and high-throughput barcode sequencing (Tuba-seq). Using these methods, the impact of large panels of putative tumor suppressor genes can be quantified in parallel. We find that many previously understudied genes have strong effects on multiple aspects of tumor growth in vivo. While some functional tumor-suppressor genes have particularly strong effects only during some but not other phases of tumorigenesis, many genes impacted multiple facets of cancer development. Tuba-seq-based studies across oncogenic contexts and with combinatorial tumor suppressor gene inactivation further highlight the context dependency of gene function during carcinogenesis. These cause-and-effect analyses uncover an unexpectedly complex taxonomy of tumor suppression. We have further employed these multiplexed and quantitative in vivo models to assess the relationship between tumor genotype and therapeutic responses at scale. We coupled Tuba-seq with robust statistical methods to enable the generation of a pharmacogenomic map of lung cancer treatment responses in vivo. We uncover a surprisingly informative map of genotype-specific therapeutic responses, which highlights the importance of tumor-suppressor genotype in driving precision treatment approaches. These blueprints of the multifaceted nature of carcinogenesis have implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches for precision cancer treatment. Citation Format: Hongchen Cai, Chuan Li, Su Kit Chew, Maryam Yousefi, Giorgia Foggetti, Wen-Yang Lin, Zoë N. Rogers, Ian P. Winters, Christopher D. McFarland, Katherina Politi, Charlie Swanton, Dmitri A. Petrov, Monte M. Winslow. Multiplexed functional cancer genomics [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA26.

A case of von Hippel-Lindau disease with multi-organ involvement: a rare case report

Von Hippel-Lindau (VHL) disease is a rare autosomal dominant syndrome manifested by a spectrum of tumours in the central nervous system (CNS) and other visceral organs. We herein report a case of 35 years aged newly diagnosed diabetic female patient presented with headache, gait instability, loss of vision in both eyes, left sided hearing impairment and subsequently diagnosed to have VHL disease. The pathophysiology involves the inactivation of the VHL tumour suppressor gene. Early recognition and treatment remains the mainstay of management. Even many years after the complete tumour excision, newer neoplasms may develop. Increasing knowledge about the molecular enabled us to investigate the role of anti-angiogenic drugs. Continuous surveillance at regular interval must be conducted in patients with VHL disease.

Analysis of Promoter Hypermethylation of DAPK and BAX Apoptotic Genes in Iranian Gastric Cancer Patients Undergoing Chemotherapy

Background: The apoptotic route is mostly damaged in gastric cancer tumor cells. DNA methylation of promoter associated CpG islands inactivates tumor suppressor genes. The objective of the present study was to analyze the hypermethylation of death-associated protein kinase (DAPK) and Bcl-2-associated X protein (BAX) genes in individuals suffering from gastric cancer and undergoing chemotherapy. Methods: Genomic DNA was extracted from blood samples and the tissue fixed in the paraffin of 30 patients and normal individuals. Hypermethylation investigation of DAPK and BAX genes was conducted via methylation specific PCR technique, the outcomes of which were analyzed through electrophoresis and SPSS software version 20. Results: Methylation of both BAX and DAPK genes with a frequency of (28.3%, 21.7%) in blood and (23.3%, 23.3%) in tissue, respectively, had a significant relationship with gastric cancer (P˂0.01). A significant relationship was also observed between the methylation of BAX gene in tissue and tumor type (12, 35.3% and P˂0.01). No relationship was found between methylation and grade, stage, node, age, sex, and other pathologic and clinical data of the patients (P>0.05). There was a significant association between simultaneous methylation of DAPK and BAX genes in tumor and typical tissues with methylation a frequency of 40% and 95.83%, respectively (P˂ 0.01). Conclusion: Methylation of the BAX and DAPK genes can be used as a biomarker in bloodand an approach in the early detection of malignity and illness management. Methylation inhibitors with the potential for drug targeting of DAPK and BAX can further be employed in pharmacotherapy.