Gene Cell Cycle Progression Research Articles

Abstract 871: Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL)

Abstract CDC25A is a member of the CDC25 family of phosphatases that plays a major role in cell cycle progression. Here, we present evidence that expression of CDC25a in T-ALL is regulated at the transcriptional level by oncogenic Casein Kinase II (CK2) via direct phosphorylation of Ikaros, a transcription factor and tumor suppressor protein. Global chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) studies in both primary human acute lymphoblastic leukemia cells and cell lines, demonstrated that Ikaros binds to the promoter of the CDC25a gene. Ikaros functions as a tumor suppressor protein and deletion of which is associated with development of T-ALL. Ikaros binding to CDC25a promoter was confirmed by quantitative chromatin immunoprecipitation (qChIP) in primary T-ALL cells. Ikaros knock-down with shRNA results in increased transcription of CDC25a in T-ALL. In mice, T-ALL cells that were derived from Ikaros-knockout mice express high levels of CDC25a. Transduction of these cells with Ikaros-containing retrovirus results in sharp reduction of CDC25a expression. Overexpression of CK2 via retroviral transduction resulted in increased transcription of the CDC25a gene, as measured by qRT-PCR, as well as increased overall expression of CDC25a, as measured by Western blot. Increased expression of CK2 was associated with a loss of Ikaros binding to the CDC25a gene promoter. Molecular inhibition of CK2 using shRNA, as well as pharmacological inhibition with a specific CK2 inhibitor resulted in reduced expression of CDC25a in primary human T-ALL. CK2 inhibition was also associated with strong reduction in AKT phosphorylation, emphasizing that CK2 inhibition downregulates CDC25a and other cell cycle progression genes. Inhibition of CK2 was associated with increased Ikaros binding at the promoter of CDC25a. Ikaros knock-down restored high expression of CDC25a in T-ALL cells that were treated with CK2 inhibitors. These data showed that CK2 and Ikaros are major transcriptional regulators of CDC25a transcription in T-ALL and that CK2 inhibition represses CDC25a transcription via Ikaros-mediated repression. In conclusion, these results indicate that expression of the CDC25a oncogene in T-ALL is regulated by the CK2 which modulates Ikaros activity. Presented data revealed a novel mechanism of therapeutic action of CK2 inhibitors - repression of CDC25a expression via Ikaros. Results provide a rationale for the use of novel CK2 inhibitors in T-ALL. Citation Format: Soumya C. Iyer, Shriya Kane, Chandrika Gowda, Chunhua Song, Yali Ding, Jon Payne, Pavan Kumar Dhanyam Raju, Bihua Tan, Mary McGrath, Yevgeniya Bamme, Mario Solimon, Nathalia Moreno Cury, Dhimant Desai, Arati Sharma, Kimberly J. Payne, Sinisa Dovat. Regulation of CDC25a expression by the ikaros and casein kinase II (CK2) in T-cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 871.

Integrated Analysis of TP53 Gene and Pathway Alterations in The Cancer Genome Atlas

The TP53 tumor suppressor gene is frequently mutated in human cancers. An analysis of five data platforms in 10,225 patient samples from 32 cancers reported by The Cancer Genome Atlas (TCGA) enables comprehensive assessment of p53 pathway involvement in these cancers. More than 91% of TP53-mutant cancers exhibit second allele loss by mutation, chromosomal deletion, or copy-neutral loss of heterozygosity. TP53 mutations are associated with enhanced chromosomal instability, including increased amplification of oncogenes and deep deletion of tumor suppressor genes. Tumors with TP53 mutations differ from their non-mutated counterparts in RNA, miRNA, and protein expression patterns, with mutant TP53 tumors displaying enhanced expression of cell cycle progression genes and proteins. A mutant TP53 RNA expression signature shows significant correlation with reduced survival in 11 cancer types. Thus, TP53 mutation has profound effects on tumor cell genomic structure, expression, and clinical outlook.

Genome-wide association study of multisite chronic pain in UK Biobank.

Chronic pain is highly prevalent worldwide and represents a significant socioeconomic and public health burden. Several aspects of chronic pain, for example back pain and a severity-related phenotype ‘chronic pain grade’, have been shown previously to be complex heritable traits with a polygenic component. Additional pain-related phenotypes capturing aspects of an individual’s overall sensitivity to experiencing and reporting chronic pain have also been suggested as a focus for investigation. We made use of a measure of the number of sites of chronic pain in individuals within the UK general population. This measure, termed Multisite Chronic Pain (MCP), is a complex trait and its genetic architecture has not previously been investigated. To address this, we carried out a large-scale genome-wide association study (GWAS) of MCP in ~380,000 UK Biobank participants. Our findings were consistent with MCP having a significant polygenic component, with a Single Nucleotide Polymorphism (SNP) heritability of 10.2%. In total 76 independent lead SNPs at 39 risk loci were associated with MCP. Additional gene-level association analyses identified neurogenesis, synaptic plasticity, nervous system development, cell-cycle progression and apoptosis genes as enriched for genetic association with MCP. Genetic correlations were observed between MCP and a range of psychiatric, autoimmune and anthropometric traits, including major depressive disorder (MDD), asthma and Body Mass Index (BMI). Furthermore, in Mendelian randomisation (MR) analyses a causal effect of MCP on MDD was observed. Additionally, a polygenic risk score (PRS) for MCP was found to significantly predict chronic widespread pain (pain all over the body), indicating the existence of genetic variants contributing to both of these pain phenotypes. Overall, our findings support the proposition that chronic pain involves a strong nervous system component with implications for our understanding of the physiology of chronic pain. These discoveries may also inform the future development of novel treatment approaches.

Flavin Oxidase-Induced ROS Generation Modulates PKC Biphasic Effect of Resveratrol on Endothelial Cell Survival.

Background: Dietary intake of natural antioxidants is thought to impart protection against oxidative-associated cardiovascular diseases. Despite many in vivo studies and clinical trials, this issue has not been conclusively resolved. Resveratrol (RES) is one of the most extensively studied dietary polyphenolic antioxidants. Paradoxically, we have previously demonstrated that high RES concentrations exert a pro-oxidant effect eventually elevating ROS levels leading to cell death. Here, we further elucidate the molecular determinants underpinning RES-induced oxidative cell death. Methods: Using human umbilical vein endothelial cells (HUVECs), the effect of increasing concentrations of RES on DNA synthesis and apoptosis was studied. In addition, mRNA and protein levels of cell survival or apoptosis genes, as well as protein kinase C (PKC) activity were determined. Results: While high concentrations of RES reduce PKC activity, inhibit DNA synthesis and induce apoptosis, low RES concentrations elicit an opposite effect. This biphasic concentration-dependent effect (BCDE) of RES on PKC activity is mirrored at the molecular level. Indeed, high RES concentrations upregulate the proapoptotic Bax, while downregulating the antiapoptotic Bcl-2, at both mRNA and protein levels. Similarly, high RES concentrations downregulate the cell cycle progression genes, c-myc, ornithine decarboxylase (ODC) and cyclin D1 protein levels, while low RES concentrations display an increasing trend. The BCDE of RES on PKC activity is abrogated by the ROS scavenger Tempol, indicating that this enzyme acts downstream of the RES-elicited ROS signaling. The RES-induced BCDE on HUVEC cell cycle machinery was also blunted by the flavin inhibitor diphenyleneiodonium (DPI), implicating flavin oxidase-generated ROS as the mechanistic link in the cellular response to different RES concentrations. Finally, PKC inhibition abrogates the BCDE elicited by RES on both cell cycle progression and pro-apoptotic gene expression in HUVECs, mechanistically implicating PKC in the cellular response to different RES concentrations. Conclusions: Our results provide new molecular insight into the impact of RES on endothelial function/dysfunction, further confirming that obtaining an optimal benefit of RES is concentration-dependent. Importantly, the BCDE of RES could explain why other studies failed to establish the cardio-protective effects mediated by natural antioxidants, thus providing a guide for future investigation looking at cardio-protection by natural antioxidants.

Cell cycle progression score differentiates indolent from aggressive prostate cancer in men diagnosed by TURP.

e16560 Background: Improved selection of men with localized prostate cancer for conservative management remains a clinical need. We previously showed that the Cell Cycle Progression (CCP) score provides a significant amount of independent prognostic information in men with prostate cancer who opt for conservative management. Here, we extend those results by evaluating the performance of the CCP score in a newly analyzed cohort of conservatively managed men and in a large pooled cohort of men who deferred initial curative therapy. Methods: The CCP score (based on 31 CCP genes and 15 housekeepers) was derived from FFPE tumor sections as previously described. For this study, the CCP score was evaluated in several independent cohorts: 1) a newly studied retrospective cohort from the U.K. diagnosed by a transurethral resection of the prostate (TURP1B, N = 192); 2) a previously studied cohort from the U.K. diagnosed by TURP (TURP1A, N = 200); and 3) two previously studied cohorts from the U.K. diagnosed by needle biopsy (TAPG1, N = 180 and TAPG2, N = 585). The combined cohort included 1,157 conservatively managed men with complete clinical data. Median follow-up for all cohorts was 9.8 years. Hazard ratios (HR) are for a one-unit change in score, and the endpoint for this study was disease specific mortality (DSM). Results: The CCP score was a highly significant predictor of DSM in TURP1B. The univariate HR was 2.96 (95% CI 2.32, 4.04, p = 3.6x10-9), and the multivariate HR after adjusting for CAPRA was 2.27 (95% CI 1.59, 3.23, p = 1.3x10-5). These results are very similar to the HRs obtained in TURP1A cohort, and the test for HR heterogeneity was not significant after adjusting for CAPRA (p = 0.30). In the pooled analyses, the interaction between CCP and cohort was not significant after adjusting for clinical variables. Interestingly, sample type (TURP vs. Needle biopsy) was prognostic even after adjusting for clinical variables. Summaries of the pooled analyses are given in Table. Conclusions: The CCP score adds a significant amount of independent prognostic information for outcome in conservatively managed men. As such, it can be used to help select appropriate patients for deferred treatment regimens like AS or watchful waiting. [Table: see text]

OR02-4 YAP1 Overexpression Associates With Worse Prognosis In Patients With Adrenal Tumors And YAP1 Inhibition By Verteporfin Reduces Tumorigenicity And Reverses Epithelial-mesenchymal Transition In Adrenocortical Tumor Cells

Background: There is no effective adjuvant therapy for patients with advanced adrenocortical tumors (ACT). Abnormal YAP1, a HIPPO pathway effector, is involved in some types of cancers. Recently, we showed association of the overexpression of YAP1 in pediatric ACT (pACT) with worse prognosis, but its association with tumorigenicity and metastasis in ACT or its involvement in adult ACT (aACT) is unknown. Aims:To analyze the association of YAP1 expression in ACT with prognosis and the effect of YAP1 inhibition on tumor growth and epithelial-mesenchymal transition (EMT) in adrenocortical tumor cells.Methods: The YAP1 mRNA expression in pACT and aACT was analyzed using data available in the Gene Expression Omnibus database (GSE76019, ACT=34) and The Cancer Genome Atlas database (ACC, n=76), respectively. In vitro, we treated NCI-H295 adrenocortical tumor cells with Verteporfin (10uM), a FDA-approved drug that inhibits YAP1/TEAD complex. We evaluated mRNA expression of the HIPPO pathway genes (LATS2, STK3, STK4), its targets (CTGF), TEAD4, CTNNB1 and cycle cell progression markers (CCND1, CKD2 and CCNE1) by qPCR. In addition, we analyzed YAP1, phosphoYAP1 (Ser127), Beta-catenin and mesenchymal markers (Vimentin, N-cadherin and the transcription factor, Snail1) protein expression by Western blot (WB). Cell proliferation (MTS), cell invasion (transwell system), and tumorigenesis (soft agar colony formation assay) were assessed after VP treatment. Results:Higher YAP1 mRNA expression was associated with lower disease free survival (DFS) and lower total survival in adults with and with lower DFS in pediatric patients. Verteporfin considerably reduced cell proliferation (-53.2% after 48h and -67.7% after 72h), cell invasion (-85.6%) and the tumorigenicity, shown by reduction of anchorage-independent growth. Verteporfin antitumor effect on ACT cells was shown by reduced mRNA expression of the Beta-catenin encoding gene (CTNNB1; p<0.05), cell cycle progression genes (CCND1, CDK2 and CCNE1; p<0.005) and YAP1 target gene (CTGF) and TEAD4 (p<0.0001), besides increased mRNA expression of core kinase genes of the HIPPO pathway (LATS2, STK3 and STK4; p<0.05). Verteporfin reduced protein levels of YAP1 (-88.5%), phosphoYAP1 (-94.6%) (p<0.0005), Beta-catenin (-50.4%, p<0.001) and the mesenchymal markers Vimentin (-36%, p<0.01), N-cadherin (-66%, p<0.001) and Snail1 (-53%, p<0.05).Conclusions: YAP1 overexpression is a marker in worse prognosis in ACT. YAP1 inhibition by Verteporfin reduced tumor growth through activation of HIPPO pathway, inhibition of cell cycle progression, reduction of Beta-catenin expression, loss of capacity of anchorage-independent growth, reversed EMT by reduction of mesenchymal markers and reduced cell viability in adrenal cells. These results indicate to YAP1 inhibition by Verteporfin as a novel potential adjuvant therapy for patients with ACT.

Receptor interacting protein kinase 3 (RIP3) regulates iPSCs generation through modulating cell cycle progression genes

The molecular mechanisms involved in induced pluripotent stem cells (iPSCs) generation are poorly understood. The cell death machinery of apoptosis-inducing caspases have been shown to facilitate the process of iPSCs reprogramming. However, the effect of other cell death processes, such as programmed necrosis (necroptosis), on iPSCs induction has not been studied. In this study, we investigated the role of receptor-interacting protein kinase 3 (RIP3), an essential regulator of necroptosis, in reprogramming mouse embryonic fibroblast cells (MEFs) into iPSCs. RIP3 was found to be upregulated in iPSCs compared to MEFs. Deletion of RIP3 dramatically suppressed the reprogramming of iPSCs (~82%). RNA-seq analysis and qRT-PCR showed that RIP3 KO MEFs expressed lower levels of genes that control cell cycle progression and cell division and higher levels of extracellular matrix-regulating genes. The growth rate of RIP3 KO MEFs was significantly slower than WT MEFs. These findings can partially explain the inhibitory effects of RIP3 deletion on iPSCs generation and show for the first time that the necroptosis kinase RIP3 plays an important role in iPSC reprogramming. In contrast to RIP3, the kinase and scaffolding functions of RIPK1 appeared to have distinct effects on reprogramming.

Moringa peregrina Leaves Extracts Induce Apoptosis and Cell Cycle Arrest of Hepatocellular Carcinoma.

Moringa grows in the tropical and subtropical regions of the world. The genus Moringa belongs to family Moringaceae. It is found to possess various medicinal uses including hypoglycemic, analgesic, anti-inflammatory, hypolipidemic, and antioxidant activities. In this study, we investigated the antimicrobial and the anticancer activity of the Moringa peregrina as well as Moringa oleifera leaves extracts grown locally in Egypt. Results indicated that most of the extracts were found to possess high antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and fungus. The survival rate of cancer cells was decreased in both hepatocellular carcinoma (HepG2) and breast carcinoma (MCF-7) cell lines when treated with Moringa leaves extracts. In addition, the cell cycle progression, apoptosis, and cancer-related genes confirmed its anticancer effect. The toxicity of each extract was also tested using the normal melanocytes cell line HFB4. The toxicity was low in both Moringa peregrina and Moringa oleifera leaves extracts. Furthermore, GC/MS analysis fractionized the phytochemicals content for each potential extract. In conclusion, results suggested that the Moringa peregrina and Moringa oleifera leaves extracts possess antimicrobial and anticancer properties which could be attributed to the bioactive phytochemical compounds present inside the extracts from this plant. These findings can be used to develop new drugs, especially for liver cancer chemotherapy.

Correction to: Gene overexpression screen for chromosome instability in yeast primarily identifies cell cycle progression genes.

In the original publication, 'Frumkin JP et al.' reference was missed to include in the reference list. The complete reference should read as below.

Fibroblast Growth Factor 15-Dependent and Bile Acid-Independent Promotion of Liver Regeneration in Mice.

The role of intestine-derived factors in promoting liver regeneration after partial hepatectomy (PHx) are not entirely known, but bile acids (BAs) and fibroblast growth factor 15 (Fgf15) that is highly expressed in the mouse ileum could promote hepatocyte proliferation. Fgf15 strongly suppresses the synthesis of BAs, and emerging evidence indicates that Fgf15 is important for liver regeneration. The mechanisms by which Fgf15 promotes liver regeneration are unclear, but Fgf15 may do so indirectly by reducing BA levels and/or directly by promoting cell proliferation. However, it remains undetermined whether these two mechanisms are independent or integrated. In this study, we aimed to clarify these relationships by generating Fgf15 Tet-Off, transgenic mice (Fgf15 Tg) that had very low BA levels as a result from overexpressed Fgf15-mediated suppression of BA synthesis. Compared with wild-type mice, the Fgf15 Tg mice showed increased hepatocyte proliferation even without surgery, and a further induction of the genes in cell-cycle progression after PHx. Moreover, overexpression of Fgf15 by adeno-associated virus (AAV)-Fgf15 transduction or treatment with the recombinant Fgf15 protein led to increased cell proliferation in vivo. Furthermore, Fgf15 Tg mice exhibited an earlier and greater activation of mitogen-activated protein kinase, signal transducer and activator of transcription 3, and NF-κB signaling pathways in the priming stage, and a disruption of the hippo signaling pathway in the termination stage of liver regeneration. Conclusion: Direct in vivo evidence demonstrates that Fgf15 is critical in stimulating the phases of priming and termination of liver regeneration that are critical for cell survival and liver-size determination, independent of BA levels. (Hepatology 2018; 00:000-000).

Gene overexpression screen for chromosome instability in yeast primarily identifies cell cycle progression genes

Loss of heterozygosity (LOH) in a vegetatively growing diploid cell signals irregularity of mitosis. Therefore, assays of LOH serve to discover pathways critical for proper replication and segregation of chromosomes. We screened for enhanced LOH in a whole-genome collection of diploid yeast strains in which a single gene was strongly overexpressed. We found 39 overexpression strains with substantially increased LOH caused either by recombination or by chromosome instability. Most of them, 32 in total, belonged to the category of “cell division”, a broadly defined biological process. Of those, only one, TOP3, coded for an enzyme that uses DNA as a substrate. The rest related to establishment and maintenance of cell polarity, chromosome segregation, and cell cycle checkpoints. Former studies, in which gene deletions were used, showed that an absence of a protein participating in the DNA processing machinery is a potent stimulator of genome instability. As our results suggest, overexpression of such proteins is not comparably damaging as the absence of them. It may mean that the harmful effect of overexpression is more likely to occur in more complex and multistage processes, such as chromosome segregation. We also report a side finding, resulting from the fact that we worked with the yeast strains bearing a 2-micron plasmid. We noted that intense transcription from such a plasmid led to an enhanced rate of an entire chromosome loss (as opposed to LOH produced by recombination). This observation may support models linking segregation of 2-micron plasmids to segregation of chromosomes.

Role of Host GPR120 in Mediating Dietary Omega-3 Fatty Acid Inhibition of Prostate Cancer.

GPR120, a G protein-coupled receptor for long-chain polyunsaturated fatty acids (FAs), mediates the anti-inflammatory effects of omega-3 (ω-3) FAs. We investigated whether host or tumor GPR120 plays a role in the anti-prostate cancer effects of ω-3 FAs. MycCap prostate cancer allografts were grown in immunocompetent wild-type (WT) and GPR120 knockout (KO) mice fed ω-3 (fish oil) or ω-6 (corn oil) diets. Immune cell infiltration was quantified by flow cytometry, and gene expression of immune cell markers in isolated tumor-associated macrophages (TAMs) was quantified by quantitative real-time polymerase chain reaction. Archived tissue from a fish oil intervention trial was used to correlate gene expression of GPR120 with cell cycle progression (CCP) genes and Ki67 index (n = 11-15 per group). All statistical tests were two-sided. In WT mice (n = 7 per group), dietary ω-3 FAs decreased MycCap allograft tumor growth (mean [SD] final tumor volume ω-6 = 491 [437] mm3 vs ω-3 = 127 [77] mm3, P = .04), whereas in global GPR120KO mice (n = 7 per group) ω-3 FAs had no anticancer effects. Dietary ω-3 FAs inhibited GPR120KO-MycCaP allografts grown in WT mice (n = 8 per group; mean [SD] final tumor volume ω-6 = 776 [767] mm3 vs ω-3 = 36 [34] mm3, P = .02). Omega-3 FA treatment decreased the number of M2-like TAMs in tumor tissue and gene expression of M2 markers in isolated TAMs compared with ω-6 controls in WT (n = 7 per group) but not in GPR120KO mice (n = 7 per group). In human tissue, higher expression of stromal GPR120 correlated with greater reduction in expression of CCP genes in men with prostate cancer on a high-ω-3 diet (r = -.57, P = .04). Host GPR120 plays a central role in the anti-prostate cancer effects of dietary ω-3 FAs. Future studies are required to determine if the anticancer effects of ω-3 FAs are mediated through inhibition of M2-like macrophages and if host GPR120 status predicts anticancer effects of dietary ω-3 FAs in men with prostate cancer.

CCPG1, an unconventional cargo receptor for ER-phagy, maintains pancreatic acinar cell health

ER stress-mediated induction of a new vertebrate-specific autophagy cargo receptor, CCPG1 (cell-cycle progression gene 1), drives degradation of endoplasmic reticulum. CCPG1 acts via ATG8-family interaction and, non-canonically, via discrete interactions with FIP200. CCPG1 ameliorates ER stress in the exocrine pancreas. This has potential implications for inflammation and cancer, discussed here.

APRIL signaling via TACI mediates immunosuppression by T regulatory cells in multiple myeloma: therapeutic implications.

We here investigate how APRIL impacts immune regulatory T cells and directly contributes to the immunosuppressive multiple myeloma (MM) bone marrow (BM) microenvironment. First, APRIL receptor TACI expression is significantly higher in regulatory T cells (Tregs) than conventional T cells (Tcons) from the same patient, confirmed by upregulated Treg markers, i.e., Foxp3, CTLA-4. APRIL significantly stimulates proliferation and survival of Tregs, whereas neutralizing anti-APRIL monoclonal antibodies (mAbs) inhibit theses effects. Besides TACI-dependent induction of cell cycle progression and anti-apoptosis genes, APRIL specifically augments Foxp3, IL-10, TGFβ1, and PD-L1 in Tregs to further enhance Treg-inhibited Tcon proliferation. APRIL further increases MM cell-driven Treg (iTreg) via TACI-dependent proliferation associated with upregulated IL-10, TGFβ1, and CD15s in iTreg, which further inhibits Tcons. Osteoclasts producing APRIL and PD-L1 significantly block Tcon expansion by iTreg generation, which is overcome by combined treatment with anti-APRIL and -PD1/PD-L1 mAbs. Finally, APRIL increases IL-10-producing B regulatory cells (Bregs) via TACI on BM Bregs of MM patients. Taken together, these results define novel APRIL actions via TACI on Tregs and Bregs to promote MM cell survival, providing the rationale for targeting APRIL/TACI system to alleviate the immunosuppressive BM milieu and improve patient outcome in MM.

Abstract 5900: Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells

Abstract Background and objective: Androgen deprivation therapy (ADT) is among the most effective of all cancer therapies for prostate cancer; however, resistance to therapy leading to castrate resistant prostate cancer (CRPC) is inevitable in most patients. Recently we demonstrated that CYP3A5 expressed in prostate cancer cells facilitates the nuclear translocation of AR, directly impacting the transcriptional regulation of AR-dependent genes promoting prostate cancer cell growth. Our preliminary data show that CYP3A5 is a part of the HSP90-AR complex, known to regulate AR nuclear localization and activation. Additionally, we observed that CYP3A5 inhibition also blocks growth of prostate cancer cells lacking AR ligand binding site (22RV1). This observation suggested presence of an alternative mechanism of regulation of prostate cell growth by CYP3A5, independent of its AR activation. We hypothesize that CYP3A5 inhibition can also sensitize prostate cancer cells to therapy by downregulating expression of drug resistance genes independent of its AR regulation. Our aim is to decipher the role of CYP3A5 in therapeutic resistance, by screening for CYP3A5 regulated cancer drug resistance genes and to further understand the underlying mechanism of this regulation. Method: We used cancer drug resistance PCR array after CYP3A5 siRNA knockdown to identify CYP3A5 regulated genes involved in therapeutic resistance. Further we evaluated the role of CYP3A5 in inducing synthetic lethality in combination with DNA damage repair inhibiting drugs by imagining the γ-H2AX foci. Results: CYP3A5 siRNA knockdown downregulates 11 genes involved in cancer drug resistance: TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1 with a p≤0.005 value. Of the list of downregulated genes, the three genes TOP2A, BRCA2 and BRCA1 have the most fold change and their loss is known to impair homologous recombination (HR), leading to synthetic lethality. HR defects in CRPC, sensitizes these tumors to PARP and topoisomerase inhibitors. We tested the effect of CYP3A5 inhibition in inducing HR defects by analyzing γ-H2AX foci counts in combination with topoisomerase inhibitor etoposide. Increased number of γ-H2AX foci suggest that CYP3A5 siRNA may induce synthetic lethality in prostate cancer cells when used in combination with DNA damage repair inhibiting drugs by impairing HR and downregulating TOP2A and BRCA1/2 expression. Conclusions: CYP3A5 inhibition downregulates DNA damage repair genes (TOP2A and BRCA1/2) and cell cycle progression genes (CCNE1, CDK 2/4) and impairs HR. CYP3A5 inhibition can sensitize CRPC to PARP and topoisomerase inhibitors as it induces synthetic lethality by downregulating TOP2A and BRCA1/2 expression. CYP3A5 inhibition can be potentially exploited therapeutically in combination with PARP inhibitors as it functionally impairs HR and causes synthetic lethality. Citation Format: Priyatham Gorjala, Ranjana Mitra, Oscar Goodman. Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells [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 5900.

PO-374 LINC00152 long non-coding RNA promotes the proliferation of SW480 colon carcinoma cells through regulation of cell cycle and WNT signalling pathway

IntroductionLong non-coding RNAs (lncRNAs) contribute to different cancers including colorectal cancer (CRC). Previous studies have shown altered LINC00152 expression in CRC, but the detailed mechanism of its effects during CRC development and progression is not well studied. We aimed to study the effects of LINC00152 silencing on the cell cycle regulation and whole transcriptome in colon carcinoma cells. We also analysed the DNA methylation alterations caused by LINC00152 knockdown.Material and methodsLINC00152 were silenced in SW480 colon carcinoma cells using Stealth siRNAs. Flow cytometric cell cycle analysis was performed using propidium-iodide DNA staining. Cyclin D1 protein expression was detected using flow cytometry after labelling with anti-cyclin D1 antibody. The effect of LINC00152 silencing to genome-wide gene expression was studied on Human Transcriptome Array 2.0 microarrays. DNA methylation alterations after LINC00152 knockdown were evaluated using Reduced Representation Bisulfite Sequencing (RRBS) method using NextSeq500 device.Results and discussionsSilencing of LINC00152 significantly suppressed cell growth compared to negative control cells (p<0.05). LINC00152 knockdown caused approximately two-fold increase in apoptosis (48 hour: si-NEG: 4%, si-LINC00152: 8%; 72 hour: si-NEG: 11%, si-LINC00152: 23%) (p<0.05). In parallel with the growth inhibitory effect, silencing of LINC00152 could reduce cyclin D1 expression already after 48 hours, however, significant decrease was detected after 72 hours in the LINC00152 siRNA-treated cells (si-NEG MFI=0.70 and si-LINC00152 MFI=0.56) without attenuation of phospho-S6 protein. Whole transcriptome microarray analysis of LINC00152 silenced cells revealed significant under-expression of genes with oncogenic and/or metastasis promoting function (e.g. STC1, YES1, HES1, KLK6, PORCN) and up-regulation of tumour suppressor genes (e.g. DKK1, PERP) (FDR p<0.05, absolute value of logFC >1). Using RRBS, DNA methylation alterations after LINC00152 silencing could be detected genome-widely including hypomethylation in SFRP2 and ALDH1A3 gene promoters.ConclusionOur results suggest that LINC00152 lncRNA can contribute to CRC pathogenesis by facilitating cell proliferation through up-regulation of several oncogenes/metastatic genes in WNT, Notch and TP53 pathways and of cyclin D1 cell cycle progression gene, furthermore, by affecting the promoter methylation status of certain CRC associated genes.

A BTB-ZF protein, ZNF131, is required for early B cell development

Members of the BTB-ZF transcription factor family play important roles in lymphocyte development. During T cell development, ZNF131, a BTB-ZF protein, is critical for the double-negative (DN) to double-positive (DP) transition and is also involved in cell proliferation. Here, we report that knockout of Znf131 at the pre-pro-B cell stage in mb1-Cre knock-in mouse resulted in defect of pro-B to pre-B cell transition. ZNF131 was shown to be required for efficient pro-B cell proliferation as well as for immunoglobulin heavy chain gene rearrangement that occurs in the proliferating pro-B cells. We speculate that inefficient gene rearrangement may be due to loss of cell proliferation, since cell cycle progression and immunoglobulin gene rearrangement, which would occur in a mutually exclusive manner, may be interconnected or coupled to avoid occurrence of genomic instability. ZNF131 suppresses expression of Cdk inhibitor, p21cip1, and that of pro-apoptotic factors, Bax and Puma, targets of p53, to facilitate cell cycle progression and suppress unnecessary apoptosis, respectively, of pro-B cells. There results demonstrate the essential roles of ZNF131 in coordinating the B cell differentiation and proliferation.

Evaluating the impact of African American ancestry among men with localized prostate cancer treated with radical prostatectomy.

41 Background: Prostate cancer (PC) is the most common male malignancy. Prior data has suggested that African American (AA) men present with more aggressive disease relative to men of other ancestries. Here, we examined the effects of ancestry on clinical and molecular measures of disease aggressiveness as well as pathologic outcomes in men treated with radical prostatectomy (RP) for localized PC. Methods: Data was collected from patients undergoing RP at the Ochsner Clinic from 2006 to 2011. Formalin−fixed paraffin embedded biopsy tissue was analyzed for the RNA expression of 31 cell cycle progression (CCP) genes and 15 housekeeping genes to obtain a CCP score (a validated molecular measure of PC aggressiveness). Cancer of the Prostate Risk Assessment (CAPRA) scores were also determined based on clinicopathologic features at the time of diagnosis. Clinical (Gleason score, tumor stage, CAPRA score) and molecular (CCP score) measures of disease aggressiveness were compared based on ancestry (AA versus non−AA). Cox proportional hazards models were used to test association of ancestry to biochemical recurrence (BCR) and progression to metastatic disease. Fisher’s exact and Wilcoxon rank sum tests were used to compare ancestries. Results: A total of 384 patients were treated with RP, including 133 (34.8%) AA men. At the time of diagnosis, the median age was 62 years (interquartile range (IQR) 56, 66) and PSA was 5.4 ng/mL (IQR 4.2, 7.6). When compared by ancestry, there were no significant differences in biopsy Gleason score (p = 0.26), clinical stage (p = 0.27), CAPRA score (p = 0.58), or CCP score (p = 0.87). In addition, there was no significant difference in the risk of BCR between ancestries (p = 0.55). Only non−AA men progressed to metastatic disease within the ten years of follow−up. Conclusions: Contrary to prior reports, these data appears to indicate that men of AA ancestry do not necessarily present with or develop a more biologically aggressive form of PC. Although these data represents only one institution’s experience, it contains a highly robust AA population compared to prior reports. Further research is required to account for the discrepancy in the previously published literature.

Evaluating cell cycle progression score as a prognostic marker for non-muscle invasive bladder cancer (NMIBC).

476 Background: Accurate grading and staging from transurethral resection of bladder tumors (TURBT) is vital for appropriate clinical management. Non-muscle-invasive bladder cancer (NMIBC) can recur progress with higher grade and or stage progression to MIBC, requiring radical intervention with poorer prognosis. Further, grade and stage may change in 20-50% of TURBTs following re-review by expert GU pathologists Objective measures of stage and grade might offer additional and/or improved risk stratification; therefore we evaluated a molecular RNA signature as a prognostic marker for NMIBC. Methods: Patients were diagnosed with NMIBC at the University of Minnesota (UM)or Spectrum Health System(SHS) from 2005-2012. A Cell Cycle Progression (CCP) score was determined from the average expression of 36 CCP genes for patients with available FFPE diagnostic TURBT. The combined cohort consisted of 293 patients (UM n = 152, SHS n = 141). Study outcome was time from NMIBC diagnosis to progression to MIBC. Median follow-up for patients who did not experience an event was 4.4 years for UM and 6.9 for SHS. Association with outcomes was evaluated by Cox proportional hazards survival analysis and likelihood ratio tests. All analyses were stratified by cohort. Results: CCP score was associated with progression to MIBC in univariable analysis [hazard ratio 1.42 (95% CI 1.19, 1.68), p = 4.3x10-5]. Tumor grade and stage were also highly prognostic. CCP score was strongly associated with stage (T1 vs Ta, p &lt; 10-6) and grade (high vs low, p &lt; 10-14) in both cohorts. As a result, CCP score did not provide independent prognostic information in multivariable analysis after adjusting for stage and grade (p = 0.32). There was a significant interaction between stage and CCP score (p = 0.0017), justifying an exploratory analysis of CCP score in Ta disease. In this subset, CCP score trended toward (p = 0.056) after adjusting for grade. Conclusions: In NMIBC, CCP score was highly correlated with tumor stage and grade and could serve as a quantitative measure of clinical parameters. The score may also provide prognostic information regarding risk of progression to MIBC, particularly in patients with Ta disease, but requires additional validation.

Nc886 is epigenetically repressed in prostate cancer and acts as a tumor suppressor through the inhibition of cell growth

BackgroundNc886 is a 102 bp non-coding RNA transcript initially classified as a microRNA precursor (Pre-miR-886), later as a divergent homologue of the vault RNAs (vtRNA 2–1) and more recently as a novel type of RNA (nc886). Although nc886/vtRNA2–1/Pre-miR-886 identity is still controversial, it was shown to be epigenetically controlled, presenting both tumor suppressor and oncogenic function in different cancers. Here, we study for the first time the role of nc886 in prostate cancer.MethodsNc886 promoter methylation status and its correlation with patient clinical parameters or DNMTs levels were evaluated in TCGA and specific GEO prostate tissue datasets. Nc886 level was measured by RT-qPCR to compare normal/neoplastic prostate cells from radical prostatectomies and cell lines, and to assess nc886 response to demethylating agents. The effect of nc886 recovery in cell proliferation (in vitro and in vivo) and invasion (in vitro) was evaluated using lentiviral transduced DU145 and LNCaP cell lines. The association between the expression of nc886 and selected genes was analyzed in the TCGA-PRAD cohort.ResultsNc886 promoter methylation increases in tumor vs. normal prostate tissue, as well as in metastatic vs. normal prostate tissue. Additionally, nc886 promoter methylation correlates with prostate cancer clinical staging, including biochemical recurrence, Clinical T-value and Gleason score. Nc886 transcript is downregulated in tumor vs. normal tissue -in agreement with its promoter methylation status- and increases upon demethylating treatment. In functional studies, the overexpression of nc886 in the LNCaP and DU145 cell line leads to a decreased in vitro cell proliferation and invasion, as well as a reduced in vivo cell growth in NUDE-mice tumor xenografts. Finally, nc886 expression associates with the prostate cancer cell cycle progression gene signature in TCGA-PRAD.ConclusionsOur data suggest a tumor suppressor role for nc886 in the prostate, whose expression is epigenetically silenced in cancer leading to an increase in cell proliferation and invasion. Nc886 might hold clinical value in prostate cancer due to its association with clinical parameters and with a clinically validated gene signature.