Sebastiano Battaglia

Transcription factor co-repressors in cancer biology: roles and targeting

Normal transcription displays a high degree of flexibility over the choice, timing and magnitude of mRNA expression levels that tend to oscillate and cycle. These processes allow for combinatorial actions, feedback control and fine‐tuning. A central role has emerged for the transcriptional co‐repressor proteins such as NCOR1, NCOR2/SMRT, CoREST and CTBPs, to control the actions of many transcriptional factors, in large part, by recruitment and activation of a range of chromatin remodeling enzymes. Thus, co‐repressors and chromatin remodeling factors are recruited to transcription factors at specific promoter/enhancer regions and execute changes in the chromatin structure. The specificity of this recruitment is controlled in a spatial‐temporal manner. By playing a central role in transcriptional control, as they move and target transcription factors, co‐repressors act as a key driver in the epigenetic economy of the nucleus. Co‐repressor functions are selectively distorted in malignancy, by both loss and gain of function and contribute to the generation of transcriptional rigidity. Features of transcriptional rigidity apparent in cancer cells include the distorted signaling of nuclear receptors and the WNTs/β‐catenin axis. Understanding and predicting the consequences of altered co‐repressor expression patterns in cancer cells has diagnostic and prognostic significance, and also have the capacity to be targeted through selective epigenetic therapies.

VDR regulation of microRNA differs across prostate cell models suggesting extremely flexible control of transcription

The Vitamin D Receptor (VDR) is a member of the nuclear receptor superfamily and is of therapeutic interest in cancer and other settings. Regulation of microRNA (miRNA) by the VDR appears to be important to mediate its actions, for example, to control cell growth. To identify if and to what extent VDR-regulated miRNA patterns change in prostate cancer progression, we undertook miRNA microarray analyses in 7 cell models representing non-malignant and malignant prostate cells (RWPE-1, RWPE-2, HPr1, HPr1AR, LNCaP, LNCaP-C4–2, and PC-3). To focus on primary VDR regulatory events, we undertook expression analyses after 30 minutes treatment with 1α,25(OH)2D3. Across all models, 111 miRNAs were significantly modulated by 1α,25(OH)2D3 treatment. Of these, only 5 miRNAs were modulated in more than one cell model, and of these, only 3 miRNAs were modulated in the same direction. The patterns of miRNA regulation, and the networks they targeted, significantly distinguished the different cell types. Integration of 1α,25(OH)2D3-regulated miRNAs with published VDR ChIP-seq data showed significant enrichment of VDR peaks in flanking regions of miRNAs. Furthermore, mRNA and miRNA expression analyses in non-malignant RWPE-1 cells revealed patterns of miRNA and mRNA co-regulation; specifically, 13 significant reciprocal patterns were identified and these patterns were also observed in TCGA prostate cancer data. Lastly, motif search analysis revealed differential motif enrichment within VDR peaks flanking mRNA compared to miRNA genes. Together, this study revealed that miRNAs are rapidly regulated in a highly cell-type specific manner, and are significantly co-integrated with mRNA regulation.

Integrative genomic analysis in K562 chronic myelogenous leukemia cells reveals that proximal NCOR1 binding positively regulates genes that govern erythroid differentiation and Imatinib sensitivity

To define the functions of NCOR1 we developed an integrative analysis that combined ENCODE and NCI-60 data, followed by in vitro validation. NCOR1 and H3K9me3 ChIP-Seq, FAIRE-seq and DNA CpG methylation interactions were related to gene expression using bootstrapping approaches. Most NCOR1 combinations (24/44) were associated with significantly elevated level expression of protein coding genes and only very few combinations related to gene repression. DAVIDs biological process annotation revealed that elevated gene expression was uniquely associated with acetylation and ETS binding. A matrix of gene and drug interactions built on NCI-60 data identified that Imatinib significantly targeted the NCOR1 governed transcriptome. Stable knockdown of NCOR1 in K562 cells slowed growth and significantly repressed genes associated with NCOR1 cistrome, again, with the GO terms acetylation and ETS binding, and significantly dampened sensitivity to Imatinib-induced erythroid differentiation. Mining public microarray data revealed that NCOR1-targeted genes were significantly enriched in Imatinib response gene signatures in cell lines and chronic myelogenous leukemia (CML) patients. These approaches integrated cistrome, transcriptome and drug sensitivity relationships to reveal that NCOR1 function is surprisingly most associated with elevated gene expression, and that these targets, both in CML cell lines and patients, associate with sensitivity to Imatinib.

Unmasking targets of antitumor immunity via high-throughput antigen profiling

More than three decades of evidence has established that antitumor immune responses, initially shown with IL-2 treatment, can result in complete, durable eradication of malignant disease in metastatic patients. Recent studies have demonstrated that immune checkpoint blockade as well as cellular therapies, including dendritic cell activation of T cells and adoptive T cell transfer, can induce long-lasting responses. To elicit cytolysis of tumor cells, effector T cells rely on tumor expression of target antigens. However, the antigens targeted during antitumor responses are largely unknown. Technological advancements and availability of sequencing data have paved the way for more efficient screening and validation of tumor-associated antigens and neoantigens derived from non-synonymous mutations targeted by T cells under baseline conditions and in the context of immunotherapy.

Replication and validation of genetic polymorphisms associated with survival after allogeneic blood or marrow transplant

Multiple candidate gene-association studies of non-HLA single-nucleotide polymorphisms (SNPs) and outcomes after blood or marrow transplant (BMT) have been conducted. We identified 70 publications reporting 45 SNPs in 36 genes significantly associated with disease-related mortality, progression-free survival, transplant-related mortality, and/or overall survival after BMT. Replication and validation of these SNP associations were performed using DISCOVeRY-BMT (Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT), a well-powered genome-wide association study consisting of 2 cohorts, totaling 2888 BMT recipients with acute myeloid leukemia, acute lymphoblastic leukemia, or myelodysplastic syndrome, and their HLA-matched unrelated donors, reported to the Center for International Blood and Marrow Transplant Research. Gene-based tests were used to assess the aggregate effect of SNPs on outcome. None of the previously reported significant SNPs replicated at P < .05 in DISCOVeRY-BMT. Validation analyses showed association with one previously reported donor SNP at P < .05 and survival; more associations would be anticipated by chance alone. No gene-based tests were significant at P < .05. Functional annotation with publicly available data shows these candidate SNPs most likely do not have biochemical function; only 13% of candidate SNPs correlate with gene expression or are predicted to impact transcription factor binding. Of these, half do not impact the candidate gene of interest; the other half correlate with expression of multiple genes. These findings emphasize the peril of pursing candidate approaches and the importance of adequately powered tests of unbiased genome-wide associations with BMT clinical outcomes given the ultimate goal of improving patient outcomes.

Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells.

BACKGROUND The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. METHODS In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. RESULTS These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. CONCLUSIONS These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer.

Paternal lineage early onset hereditary ovarian cancers: A Familial Ovarian Cancer Registry study

Given prior evidence that an affected woman conveys a higher risk of ovarian cancer to her sister than to her mother, we hypothesized that there exists an X-linked variant evidenced by transmission to a woman from her paternal grandmother via her father. We ascertained 3,499 grandmother/granddaughter pairs from the Familial Ovarian Cancer Registry at the Roswell Park Cancer Institute observing 892 informative pairs with 157 affected granddaughters. We performed germline X-chromosome exome sequencing on 186 women with ovarian cancer from the registry. The rate of cancers was 28.4% in paternal grandmother/granddaughter pairs and 13.9% in maternal pairs consistent with an X-linked dominant model (Chi-square test X2 = 0.02, p = 0.89) and inconsistent with an autosomal dominant model (X2 = 20.4, p<0.001). Paternal grandmother cases had an earlier age-of-onset versus maternal cases (hazard ratio HR = 1.59, 95%CI: 1.12–2.25) independent of BRCA1/2 status. Reinforcing the X-linked hypothesis, we observed an association between prostate cancer in men and ovarian cancer in his mother and daughters (odds ratio, OR = 2.34, p = 0.034). Unaffected mothers with affected daughters produced significantly more daughters than sons (ratio = 1.96, p<0.005). We performed exome sequencing in reported BRCA negative cases from the registry. Considering age-of-onset, one missense variant (rs176026 in MAGEC3) reached chromosome-wide significance (Hazard ratio HR = 2.85, 95%CI: 1.75–4.65) advancing the age of onset by 6.7 years. In addition to the well-known contribution of BRCA, we demonstrate that a genetic locus on the X-chromosome contributes to ovarian cancer risk. An X-linked pattern of inheritance has implications for genetic risk stratification. Women with an affected paternal grandmother and sisters of affected women are at increased risk for ovarian cancer. Further work is required to validate this variant and to characterize carrier families.

Nature of tumour rejection antigens in ovarian cancer

Major progress in the analysis of human immune responses to cancer has been made through the molecular characterization of human tumour antigens. The development of therapeutic strategies for eliciting immune‐mediated rejection of tumours has accelerated due to the elucidation of the molecular basis for tumour cell recognition and destruction by immune cells. Of the various human tumour antigens defined to date in ovarian cancer, the cancer‐testis (CT) family of antigens have been studied extensively preclinically and clinically because of their testis‐restricted expression in normal tissues and ability to elicit robust immune responses. Recent developments in cancer sequencing technologies offer a unique opportunity to identify tumour mutations with the highest likelihood of being expressed and recognized by the immune system. Such mutations, or neoantigens, could potentially serve as specific immune targets for T‐cell‐mediated destruction of cancer cells. This review will highlight current work in selecting tumour rejection antigens in ovarian cancer for improving the efficacy of immunotherapy.

LSD1 dual function in mediating epigenetic corruption of the vitamin D signaling in prostate cancer

BackgroundLysine-specific demethylase 1A (LSD1) is a key regulator of the androgen (AR) and estrogen receptors (ER), and LSD1 levels correlate with tumor aggressiveness. Here, we demonstrate that LSD1 regulates vitamin D receptor (VDR) activity and is a mediator of 1,25(OH)2-D3 (vitamin D) action in prostate cancer (PCa).MethodsAthymic nude mice were xenografted with CWR22 cells and monitored weekly after testosterone pellet removal. Expression of LSD1 and VDR (IHC) were correlated with tumor growth using log-rank test. TRAMP tumors and prostates from wild-type (WT) mice were used to evaluate VDR and LSD1 expression via IHC and western blotting. The presence of VDR and LSD1 in the same transcriptional complex was evaluated via immunoprecipitation (IP) using nuclear cell lysate. The effect of LSD1 and 1,25(OH)2-D3 on cell viability was evaluated in C4-2 and BC1A cells via trypan blue exclusion. The role of LSD1 in VDR-mediated gene transcription was evaluated for Cdkn1a, E2f1, Cyp24a1, and S100g via qRT-PCR-TaqMan and via chromatin immunoprecipitation assay. Methylation of Cdkn1a TSS was measured via bisulfite sequencing, and methylation of a panel of cancer-related genes was quantified using methyl arrays. The Cancer Genome Atlas data were retrieved to identify genes whose status correlates with LSD1 and DNA methyltransferase 1 (DNMT1). Results were correlated with patients’ survival data from two separate cohorts of primary and metastatic PCa.ResultsLSD1 and VDR protein levels are elevated in PCa tumors and correlate with faster tumor growth in xenograft mouse models. Knockdown of LSD1 reduces PCa cell viability, and gene expression data suggest a dual coregulatory role of LSD1 for VDR, acting as a coactivator and corepressor in a locus-specific manner. LSD1 modulates VDR-dependent transcription by mediating the recruitment of VDR and DNMT1 at the TSS of VDR-targeted genes and modulates the epigenetic status of transcribed genes by altering H3K4me2 and H3K9Ac and DNA methylation. Lastly, LSD1 and DNMT1 belong to a genome-wide signature whose expression correlates with shorter progression-free survival and overall survival in primary and metastatic patients’ samples, respectively.ConclusionsResults demonstrate that LSD1 has a dual coregulatory role as corepressor and coactivator for VDR and defines a genomic signature whose targeting might have clinical relevance for PCa patients.

No evidence that genetic variation in the myeloid-derived suppressor cell pathway influences ovarian cancer survival

Background: The precise mechanism by which the immune system is adversely affected in cancer patients remains poorly understood, but the accumulation of immunosuppressive/protumorigenic myeloid-derived suppressor cells (MDSCs) is thought to be a prominent mechanism contributing to immunologic tolerance of malignant cells in epithelial ovarian cancer (EOC). To this end, we hypothesized genetic variation in MDSC pathway genes would be associated with survival after EOC diagnoses. Methods: We measured the hazard of death due to EOC within 10 years of diagnosis, overall and by invasive subtype, attributable to SNPs in 24 genes relevant in the MDSC pathway in 10,751 women diagnosed with invasive EOC. Versatile Gene-based Association Study and the admixture likelihood method were used to test gene and pathway associations with survival. Results: We did not identify individual SNPs that were significantly associated with survival after correction for multiple testing (P < 3.5 × 10−5), nor did we identify significant associations between the MDSC pathway overall, or the 24 individual genes and EOC survival. Conclusions: In this well-powered analysis, we observed no evidence that inherited variations in MDSC-associated SNPs, individual genes, or the collective genetic pathway contributed to EOC survival outcomes. Impact: Common inherited variation in genes relevant to MDSCs was not associated with survival in women diagnosed with invasive EOC. Cancer Epidemiol Biomarkers Prev; 26(3); 420–4. ©2016 AACR.