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Correlations of gene expression with IFN-gamma and IFN-gamma-inducible immune suppressive molecules in the tumor.

Kaplan-Meier survival curves in 97 ccRCC patients stratified according to the sex, age, Stage and Fuhrman grade

Tumors commonly harbor multiple genetic alterations, some of which initiate tumorigenesis. Among these, some tumor-specific somatic mutations resulting in mutated protein have the potential to induce antitumor immune responses. To examine the relevance of the latter to immune responses in the tumor and to patient outcomes, we used datasets of whole-exome and RNA sequencing from 97 clear cell renal cell carcinoma (ccRCC) patients to identify neoepitopes predicted to be presented by each patient's autologous HLA molecules. We found that the number of nonsilent or missense mutations did not correlate with patient prognosis. However, combining the number of HLA-restricted neoepitopes with the cell surface expression of HLA or β2-microglobulin(β2M) revealed that an A-neohi/HLA-Ahi or ABC-neohi/β2Mhi phenotype correlated with better clinical outcomes. Higher expression of immune-related genes from CD8 T cells and their effector molecules [CD8A, perforin (PRF1) and granzyme A (GZMA)], however, did not correlate with prognosis. This may have been due to the observed correlation of these genes with the expression of other genes that were associated with immunosuppression in the tumor microenvironment (CTLA-4, PD-1, LAG-3, PD-L1, PD-L2, IDO1, and IL10). This suggested that abundant neoepitopes associated with greater antitumor effector immune responses were counterbalanced by a strongly immunosuppressive microenvironment. Therefore, immunosuppressive molecules should be considered high-priority targets for modulating immune responses in patients with ccRCC. Blockade of these molecular pathways could be combined with immunotherapies targeting neoantigens to achieve synergistic antitumor activity. Cancer Immunol Res; 4(5); 463–71. ©2016 AACR.

HLA-restricted neoepitopes and expression of HLA/B2M

Mutations, HLA types neoepitopes, and predicted HLA binding

Non-synonymous mutations and prognosis of 97 ccRCC patients

PDF file, 152K, Elevated motility by 14-3-3ζ overexpression in LNCaP cells.

Purpose: Genes of androgen and estrogen signaling cells and stem cell–like cells play crucial roles in prostate cancer. This study aimed to predict clinical failure by identifying these prostate cancer-related genes.Experimental Design: We developed models to predict clinical failure using biopsy samples from a training set of 46 and an independent validation set of 30 patients with treatment-naïve prostate cancer with bone metastasis. Cancerous and stromal tissues were separately collected by laser-captured microdissection. We analyzed the association between clinical failure and mRNA expression of the following genes androgen receptor (AR) and its related genes (APP, FOX family, TRIM 36, Oct1, and ACSL 3), stem cell–like molecules (Klf4, c-Myc, Oct 3/4, and Sox2), estrogen receptor (ER), Her2, PSA, and CRP.Results: Logistic analyses to predict prostate-specific antigen (PSA) recurrence showed an area under the curve (AUC) of 1.0 in both sets for Sox2, Her2, and CRP expression in cancer cells, AR and ERα expression in stromal cells, and clinical parameters. We identified 10 prognostic factors for cancer-specific survival (CSS): Oct1, TRIM36, Sox2, and c-Myc expression in cancer cells; AR, Klf4, and ERα expression in stromal cells; and PSA, Gleason score, and extent of disease. On the basis of these factors, patients were divided into favorable-, intermediate-, and poor-risk groups according to the number of factors present. Five-year CSS rates for the 3 groups were 90%, 32%, and 12% in the training set and 75%, 48%, and 0% in the validation set, respectively.Conclusions: Expression levels of androgen- and estrogen signaling components and stem cell markers are powerful prognostic tools. Clin Cancer Res; 20(17); 4625–35. ©2014 AACR.

Purpose: Androgen receptor is an essential transcriptional factor that contributes to the development and progression of prostate cancer. In this study, we investigated the androgen regulation and functional analysis of 14-3-3ζ in prostate cancer.Experimental Design: Using chromatin immunoprecipitation (ChIP) combined with DNA microarray (ChIP-chip) analysis in LNCaP cells, we identified a functional androgen receptor–binding site in the downstream region of the 14-3-3ζ gene. Androgen regulation was examined by quantitative reverse transcription PCR and Western blot analysis. Prostate cancer cells stably expressing 14-3-3ζ and siRNA knockdown were used for functional analyses. We further examined 14-3-3ζ expression in clinical samples of prostate cancer by immunohistochemistry and quantitative reverse transcription PCR.Results: Androgen-dependent upregulation of 14-3-3ζ was validated at the mRNA and protein levels. The 14-3-3ζ gene is favorable for cancer-cell survival, as its ectopic expression in LNCaP cells contributes to cell proliferation and the acquired resistance to etoposide-induced apoptosis. 14-3-3ζ expression was associated with androgen receptor transcriptional activity and prostate-specific antigen (PSA) mRNA expression. Immunoprecipitation indicated that 14-3-3ζ was associated with androgen receptor in the nucleus. Clinicopathologic studies further support the relevance of 14-3-3ζ in prostate cancers, as its higher expression is associated with malignancy and lymph node metastasis.Conclusions: 14-3-3ζ is a novel androgen-responsive gene that activates proliferation, cell survival, and androgen receptor transcriptional activity. 14-3-3ζ may facilitate the progression of prostate cancer. Clin Cancer Res; 18(20); 5617–27. ©2012 AACR.

Supplementary Figure S1. Correlation between mRNA expression and immunoreactivity of AR and Klf4.

Supplementary Figure S10. Association of CtBP2 binding sites with CtBP2 regulated genes.

ACI/Seg (ACI) rats develop prostate cancers spontaneously with aging, similar to humans. Here, to identify genes involved in prostate cancer susceptibility, we did linkage analysis and oligonucleotide microarray analysis. Linkage analysis was done using 118 effective rats, and prostate cancer susceptibility 1 (Pcs1), whose ACI allele dominantly induced prostate cancers, was mapped on chromosome 19 [logarithm of odds (LOD) score of 5.0]. PC resistance 1 (Pcr1), whose ACI allele dominantly and paradoxically suppressed the size of prostate cancers, was mapped on chromosome 2 (LOD score of 5.0). When linkage analysis was done in 51 rats with single or no macroscopic testicular tumors, which had larger prostates and higher testosterone levels than those with bilateral testicular tumors, Pcs2 and Pcr2 were mapped on chromosomes 20 and 1, respectively. By oligonucleotide microarray analysis with 8,800 probe sets and confirmation by quantitative reverse transcription-PCR, only two genes within these four loci were found to be differentially expressed >1.8-fold. Membrane metalloendopeptidase (Mme), known to inhibit androgen-independent growth of prostate cancers, on Pcr1 was expressed 2.0- to 5.5-fold higher in the ACI prostate, in accordance with its paradoxical effect. Cdkn1a on Pcs2 was expressed 1.5- to 4.5-fold lower in the ACI prostate. Additionally, genes responsible for testicular tumors and unilateral renal agenesis were mapped on chromosomes 11 and 14, respectively. These results showed that prostate cancer susceptibility of ACI rats involves at least four loci, and suggested Mme and Cdkn1a as candidates for Pcr1 and Pcs2.

Supplementary Figure S5. Validation of CtBP2 ChIP-seq study by conventional ChIP assay.

Supplementary Table S1. Primer sequences. Supplementary Table S2. Association between CtBP2 immunohistochemical status and clinicopathological parameters in 105 human prostate carcinomas.

Supplementary Figure S7. Analysis of CtBP2 function for PTEN promoter repression by androgen.

Supplementary Figure S1. CtBP2 is induced by androgen treatment.

Supplementary Figure S6. CtBP2 negatively regulates FOXO1 promoter activation

Supplementary Table 2 from Linkage and Microarray Analyses of Susceptibility Genes in ACI/Seg Rats: A Model for Prostate Cancers in the Aged

Supplementary Figure S8. Analysis of CtBP2 bindings in AR co-repressors by ChIP-seq.

Supplementary Table 1 from Linkage and Microarray Analyses of Susceptibility Genes in ACI/Seg Rats: A Model for Prostate Cancers in the Aged

Supplementary Figure S4. ChIP-seq views of representative AR binding genes.

Supplementary Figure S3. CtBP2 promotes androgen-dependent prostate cancer cell proliferation.

Supplementary Figure S9. CtBP2 regulates androgen receptor co-repressors.

Supplementary Table S1. Primer sequences. Supplementary Table S2. Association between CtBP2 immunohistochemical status and clinicopathological parameters in 105 human prostate carcinomas.

Supplementary Figure S11. A model for the functions of CtBP2 in modulating AR signaling in prostate cancer.

Purpose: Androgen receptor is an essential transcriptional factor that contributes to the development and progression of prostate cancer. In this study, we investigated the androgen regulation and functional analysis of 14-3-3ζ in prostate cancer. Experimental Design: Using chromatin immunoprecipitation (ChIP) combined with DNA microarray (ChIP-chip) analysis in LNCaP cells, we identified a functional androgen receptor–binding site in the downstream region of the 14-3-3ζ gene. Androgen regulation was examined by quantitative reverse transcription PCR and Western blot analysis. Prostate cancer cells stably expressing 14-3-3ζ and siRNA knockdown were used for functional analyses. We further examined 14-3-3ζ expression in clinical samples of prostate cancer by immunohistochemistry and quantitative reverse transcription PCR. Results: Androgen-dependent upregulation of 14-3-3ζ was validated at the mRNA and protein levels. The 14-3-3ζ gene is favorable for cancer-cell survival, as its ectopic expression in LNCaP cells contributes to cell proliferation and the acquired resistance to etoposide-induced apoptosis. 14-3-3ζ expression was associated with androgen receptor transcriptional activity and prostate-specific antigen (PSA) mRNA expression. Immunoprecipitation indicated that 14-3-3ζ was associated with androgen receptor in the nucleus. Clinicopathologic studies further support the relevance of 14-3-3ζ in prostate cancers, as its higher expression is associated with malignancy and lymph node metastasis. Conclusions: 14-3-3ζ is a novel androgen-responsive gene that activates proliferation, cell survival, and androgen receptor transcriptional activity. 14-3-3ζ may facilitate the progression of prostate cancer. Clin Cancer Res; 18(20); 5617–27. ©2012 AACR.

Prostate cancer is one of the most common malignancies and ranks the second most common cause of cancer-related deaths in men in the United States. Catechol-O-methyltransferase (COMT) is a phase II enzyme that detoxifies various catechol compounds that are reactive toward DNA and damaging to the cell. Studies have shown COMT to play a protective role against cancers such as renal and breast, but their effect on prostate is not well understood. In this study, the biological properties and function of COMT in prostate cancer were studied. Expression of COMT was initially measured in normal/benign and cancerous prostate tissues by immunohistochemistry, and cell lines by real-time PCR and western blotting. Cancerous cells displaying the lowest levels was then transfected with COMT. Gene effect on various cellular properties such as cell proliferation, migration, invasion and apoptosis, as well as growth in athymic nude mice were determined. COMT protein expression was lower in cancer regions compared to benign and normal regions of prostate tissues. Cancerous DU145 and DuPro cells also had reduced mRNA levels of COMT but with undetectable protein levels. Interestingly, re-expressing COMT in DU145 and DuPro cells led to decreased cell proliferation, migration, wound healing ability and invasion, and increased apoptosis compared to vector control. COMT also inhibited cell tumor formation in animal models. As a possible target, the TNFRSF11B gene was upregulated due to COMT. These results demonstrate COMT to protect against prostate cancer progression and to have a functional role by affecting apoptosis. COMT may thus be a potential biomarker or therapeutic target for prostate cancer. Citation Format: Shigekatsu Maekawa, Taku Kato, Yutaka Hashimoto, Marisa Shiina, Ryan K. Wong, Varahram Shahryari, Soichiro Yamamura, Sahana Majid, Sharanjot Saini, Laura Z. Tabatabai, Yukio Homma, Rajvir Dahiya, Yuichiro Tanaka. Role of catechol-O-methyltransferase gene in prostate cancer [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 1404.

Clear cell renal cell carcinomas (ccRCC) show a broad range of clinical behavior, and prognostic biomarkers are needed to stratify patients for appropriate management. We sought to determine whether long intergenic non-coding RNAs (lincRNAs) might predict patient survival. Candidate prognostic lincRNAs were identified by mining The Cancer Genome Atlas (TCGA) transcriptome (RNA-seq) data on 466 ccRCC cases (randomized into discovery and validation sets) annotated for ~21,000 lncRNAs. A previously uncharacterized lincRNA, SLINKY (Survival-predictive LINcRNA in KidneY cancer), was the top-ranked prognostic lincRNA, and validated in an independent University of Tokyo cohort (P=0.004). In multivariable analysis, SLINKY expression predicted overall survival independent of tumor stage and grade [TCGA HR=3.4 (CI, 2.1-5.4), P < 0.001; Tokyo HR=9.2 (CI, 2.2-43), P = 0.003], and by decision tree, ROC and decision curve analysis, added independent prognostic value. In ccRCC cell lines, SLINKY knockdown reduced cancer cell proliferation (with cell-cycle G1 arrest) and induced transcriptome changes enriched for cell proliferation and survival processes. Notably, the genes affected by SLINKY knockdown in cell lines were themselves prognostic and correlated with SLINKY expression in the ccRCC patient samples. From a screen for binding partners, we identified direct binding of SLINKY to Heterogeneous Nuclear Ribonucleoprotein K (HNRNPK), whose knockdown recapitulated SLINKY knockdown phenotypes. Thus, SLINKY is a robust prognostic biomarker in ccRCC, where it functions possibly together with HNRNPK in cancer cell proliferation. Citation Format: Xue Gong, Zurab Siprashvili, Okyaz Eminaga, Zhewei Shen, Yusuke Sato, Haruki Kume, Yukio Homma, Seishi Ogawa, Paul A. Khavari, Jonathan R. Pollack, James D. Brooks. Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4721. doi:10.1158/1538-7445.AM2017-4721

Backgrounds: Upper urinary tract urothelial carcinoma (UTUC) is relatively rare, accounting for 5-10% of urothelial malignancies with frequent multifocal development. To clarify distinct characteristics of UTUC, we comprehensively investigated the genetic alterations of this disease. Materials & methods: Surgical specimens of UTUC and matched normal samples were obtained from 99 patients with various stages who underwent nephroureterectomy, and subjected to whole exome and RNA sequencing. We compared our results in UTUC with datasets previously reported in bladder urothelial carcinoma (BUC). Mutations in apparently normal urothelial epithelia in 5 cases were also interrogated. Results: Genetic alterations were most frequently observed in TERT promoter (51% of cases), followed by KMT2D (48%), FGFR3 (44%), CDKN2A (42%), TP53 (31%), and RAS pathway (HRAS/KRAS/NRAS, 21%). More than 95% of cases harbored either TP53/MDM2, FGFR3, or RAS pathway mutations in an almost mutually exclusive manner, based on which UTUCs are classified into 3 distinct subgroups with unique molecular and clinical features; FGFR3-mutated tumors showed a significantly better postoperative overall survival than those with TP53/MDM2 (p Mutation patterns were compared between different urothelial cancers with regard to their location. Despite common genes affected, their mutation frequencies were substantially different; KMT2D mutations were more frequent in UTUC, while RB1 alterations were more prevalent in BUC. In addition, KMT2D mutations were significantly more common in UTUCs in the ureter than those in renal pelvis (85% vs. 35%, p In the analysis of normal ureter tissues (N=25 from 5 patients), driver mutations were identified in 6 samples from 2 patients. In one case, tumor and normal samples shared 10-42 mutations, indicating that the cancer evolved within a background of clonal precancerous proliferation in apparently normal epithelia. By contrast, in the other case, none of the mutations were shared between tumors and normal epithelia, suggesting the presence of a field effect on urothelial carcinogenesis. Conclusions: UTUC tumors are classified into 3 molecularly and clinically distinct subtypes based on the status of mutations in TP53/MDM2, FGFR3, and RAS pathway. Depending on their location, urothlial cancers have different genetic backgrounds, where a field effect unique to urothelial epithelia might contribute to multifocal occurrence of UTUC. Citation Format: Yoichi Fujii, Yusuke Sato, Hiromichi Suzuki, Tetsuichi Yoshizato, Yusuke Shiozawa, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Tohru Nakagawa, Haruki Kume, Hiroaki Nishimatsu, Toshikazu Okaneya, Masashi Sanada, Hideki Makishima, Satoru Miyano, Yukio Homma, Seishi Ogawa. Distinct genomic landscape of upper urinary tract urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2017-2456

Backgrounds Upper urinary tract urothelial carcinoma (UUTUC) is a relatively rare and poor prognostic cancer which accounts for 5-10% of all urothelial malignancies. Despite the histological similarity, there are epidemiological and clinical differences between UUTUC and bladder urothelial carcinoma (BUC). Compared to BUC, the molecular pathogenesis of UUTUC is poorly understood. Urothelial carcinoma is also characterized by frequent multifocal lesions, suggesting field effects from mutagenic agents in urine and/or a dissemination from the primary tumor. To reveal the origin of the multifocality as well as the molecular pathogenesis of UUTUC, we performed comprehensive molecular analysis of UUTUC with regional multiple sampling. Materials & methods Surgical specimens of UUTUC and matched normal samples were obtained from 57 patients with various stages who underwent nephroureterectomy. Morphologically normal urothelial epithelia were also obtained in 5 cases, all of which were confirmed to be pathologically intact by an expert pathologist. Genomic DNA was extracted from each specimen and was subjected to whole exome sequencing using Hiseq 2500 for the detection of both somatic mutations and copy number lesions. Results In copy number analysis, recurrent deletions in 8p, 9p and 9q were identified, where homo deletions of 9p21.3 (CDKN2A) were most frequent (40.7%) Focal amplifications in 11q13.3 (CCND1) and 12q15 (MDM2) were also found. On average, 230 somatic nonsynonymous mutations per sample were detected. The mutation signature was biased to age-related and APOBEC patterns. Mutations were most frequently observed in KMT2D (42% of cases), followed by TP53 (32%), FGFR3 (24%), KDM6A (24%), EP300 (21%) and CREBBP (13%), which were also reported to be significantly mutated in BUC. However, the frequencies of these genetic lesions were substantially different between UUTUC and BUC. For example, abnormalities in RB1, CDKN1A and PIK3CA were not detected or rare in UUTUC but have been reported to be common targets of genetic lesions in BUC. In the analysis of normal epithelia, an average of 12.7 somatic mutations per sample were found but with low variant allele frequencies between 0.05−0.1. Somatic mutations in adjacent epithelia and preoperative urine were frequently shared by primary tumor, supporting a possibility of tumor disseminations from the original sites through urinary flow. In contrast, in other cases, distant epithelia harbored driver gene mutations that were not shared by the primary tumors, suggesting the presence of a mutagenic field effect on urothelial carcinogenesis. Conclusions Despite the similarity in their mutation spectrum, UUTUC and BUC seemed to have distinct pathogenesis in terms of the difference in mutation frequencies. Our data suggested that both dissemination and field effect might contribute to multifocal occurrence of UUTUC. These findings will provide a new insight into the pathophysiology of UUTUC. Citation Format: Yoichi Fujii, Yusuke Sato, Shigekatsu Maekawa, Hiromichi Suzuki, Tetsuichi Yoshizato, Kenichi Yoshida, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Tohru Nakagawa, Haruki Kume, Hiroaki Nishimatsu, Yoshikazu Hirano, Masashi Sanada, Hideki Makishima, Satoru Miyano, Yukio Homma, Seishi Ogawa. Genomic landscape and clonal expansions of upper urinary tract urothelial carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 94.

Introduction and Objective Testicular germ cell cancer (TGCC) is the most common solid malignancy among young adult males. Cure could be obtained by intensive chemotherapy combined with retroperitoneal lymph node dissection, but often with substantially compromised quality of life. As for the common genetic lesions, somatic mutations and amplifications of KIT are found in about 20%-30% of cases with TGCCs. However, the molecular pathogenesis of TGCC is still poorly understood. In this study, to obtain a better understanding of the genetic basis of TGCC and to identify druggable molecular targets, we performed whole exome sequencing as well as SNP array-based copy number analysis in TGCC. Methods All cases underwent high orchiectomy and were histologically diagnosed as TGCC. Genomic DNA was extracted from fresh frozen specimens of TGCC. Whole exome sequencing was performed for paired tumor/normal DNA from 10 TGCC patients, in which target exomes were captured using SureSelect Human All Exon V5 (Agilent Technologies) and subjected to massively parallel sequencing on the Illumina platform (HiSeq2000). Copy number variants were also interrogated in 40 TGCCs (25 seminomas and 15 non-seminomas) using Affymetrix 250K NspI array. Results TGCC genome was triploid in most cases with high level amplification in12p. Loss of heterozygosity (LOH) of chromosome 4 was frequently observed in both seminoma and non-seminoma, whereas 11q deletion was found predominantly in cases with seminoma. In addition, we identified recurrent focal amplifications involving 4q12 and 22q11, from which KIT and MAPK1 were identified, respectively. In whole exome sequencing, 15 somatic mutations were detected per sample on average, which was relatively lower than other solid malignancies. When combined the results of copy number analysis with those of whole-exome sequencing, genes involved in RAS signaling pathway and chromatin modification were frequently altered in TGCC. Conclusions Our comprehensive analyses revealed genomic aberrations of TGCC in terms of copy number alterations and gene mutations. Mutated/amplified KIT and MAPK1 and other RAS pathway gene could be potential targets of small molecule inhibitors for therapeutics. Citation Format: Yusuke Sato, Aiko Sato-Otsubo, Yasunobu Nagata, Kenichi Yoshida, Yuichi Shiraishi, Hiromichi Suzuki, Masashi Sanada, Haruki Kume, Satoru Miyano, Yukio Homma, Seishi Ogawa. Genome-wide analysis of copy number alterations and gene mutations in testicular germ cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2014-2223

Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer. The most frequent genetic event in the evolution of ccRCC is inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Recent studies have revealed frequent mutations of PBRM1 as well as other epigenetic regulators including BAP1, SETD2 and KDM5C, but their impact on therapeutics remains unclear. In this study, to obtain a better understanding of molecular pathogenesis of ccRCC, we performed an integrated genetic study of ccRCC including whole exome sequencing, copy number analysis as well as transcriptome and methylome analysis. A total of 106 paired specimens were analyzed by massively parallel sequencing of whole exomes (Agilent SureSelect, Illumina HiSeq2000) and SNP array-based copy number analysis (Affymetrix 250K array) as well as gene expression (Agilent Human Gene Expression 4x44k v2) and methylation (Illumina Infinium 450K) profiling and RNA sequencing (Illumina HiSeq2000). On average, 48.8 somatic mutations per sample were identified in whole exome analysis, in which VHL mutations were most frequent. PBRM1, BAP1 and SETD2 were also recurrently mutated and were further analyzed in 240 cases together with an additional 80 genes involved in chromatin regulation. PBRM1 mutations were found in 42% of the cases, while BAP1 and SETD2 were mutated in 12% and 10%, respectively. BAP1 mutations correlated with poor prognosis and SETD2 mutation was associated with the risk for metastatic diseases. Pathway analysis revealed frequent mutations of genes involved in mRNA processing. Among them, mutations of genes involved in 3’ splice site recognition, which were frequently mutated in MDS, were rare. Most of them were involved in release of intron, 3’-end processing or export to cytoplasm. In expression analysis, tumors were clustered into two clusters known as ccA and ccB, in which the ccA type was characterized by overexpressed genes involved in angiogenesis, whereas expression of genes in cell cycle regulators were a prominent feature in the ccB type tumors. In methylome analysis, 15 samples were clustered into hypermethylated subtype where all cases were included in the ccB type and were associated with poor prognosis. Homeobox genes were differently methylated in hypermethylated subtype which indicate deregulation of polycomb mediated gene silencing induce high-grade ccRCC. RUNX1 and SRPX2 were differently methylated between ccA type and ccB type, which may affect the differences of expression profile between two subtypes. In RNA sequencing, known fusion gene involving TFE3 and NONO was detected in single case. In total, 34 fusion genes were detected in 23 samples, although no recurrent fusion genes have been identified. Our results indicate that genomic analyses are useful for classification, prognostic prediction and development of treatment strategy in ccRCC. Citation Format: Yusuke Sato, Shigekatsu Maekawa, Yusuke Okuno, Yuichi Shiraishi, Aiko Sato, Genta Nagae, Teppei Shimamura, Yasunobu Nagata, Kenichi Yoshida, Masashi Sanada, Haruki Kume, Hiroyuki Aburatani, Satoru Miyano, Yukio Homma, Seishi Ogawa. Integrative analysis of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3184. doi:10.1158/1538-7445.AM2013-3184

Renal cell carcinoma (RCC) is the most common form of adult kidney cancer and accounts for 2-3% of all adult malignancies, in which clear cell carcinoma accounts for more than 80% of the cases. As for the pathogenesis of clear cell RCC, inactivation of the VHL gene has been reported in 80% of clear cell RCC and more recently, frequent mutations of epigenetic regulators, including PBRM1, SETD2, KDM5C and UTX, have been demonstrated through high-throughput mutation studies, including PBRM1 has been demonstrated in ∼40% of the cases. Nevertheless, probably, our knowledge of the full spectrum of gene mutations in RCC is still incomplete. In this study, to obtain a better understanding of molecular pathogenesis of clear cell RCC, we performed an integrated genetic study of clear cell RCC, where a total of 93 paired specimens were analyzed by massively parallel sequencing of SureSelect (Agilent)-enriched whole exomes, SNP array-based copy number analysis (Affymetrix), as well as gene expression profiling (Agilent). In whole exome sequencing, 42 somatic mutations per sample were identified on average, which involved not only previously reported genes, but also a number of novel gene targets. Among these, mutations of genes involved in chromatin regulation or histone modification were preferentially found in advanced cases. To understand whole picture of gene mutations of epigenetic mechanism in clear cell RCC, mutation analysis of 85 genes involved in chromatin regulation or histone modification were performed in 180 cases using multiplexed barcode sequencing. A total of 201 somatic mutations were validated and 74% cases had at least one somatic mutation. PBRM1 mutations were found in 43% cases and SETD2 were mutated in 10% of cases. When comparing clinical picture with mutation status, SETD2 mutation was associated with the risk of metastasis, while PBRM1 mutations had no impact on prognosis. Our results showed that in clear cell RCC, multiple component of complexes involved in epigenetic regulation undergo gene mutations, confirming that deregulated epigenetic apparatus play important roles in pathogenesis of clear cell RCC. In this meeting, we will present the result of our integrated genetic analysis of RCC and discuss the genetic basis of RCC in terms of copy number alterations, gene mutations, as well as gene expression profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2092. doi:1538-7445.AM2012-2092

Renal cell carcinoma (RCC) is the most common form of adult kidney cancer and accounts for 2-3% of all adult malignancies. The majority of RCC are classified as clear cell RCC. The most frequent genetic event in the evolution of clear cell RCC is inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Advances in our understanding of the VHL pathway of clear cell RCC led to the development of several molecular targeted agents such as tyrosine kinase inhibitors (TKI) and mTOR inhibitors. Nevertheless, patients with metastatic disease still have an extremely short life expectancy. Other molecular pathways may contribute to tumor progression or poor prognosis, but such pathways are still unclear. Known cancer genes frequently mutated in other adult cancers, such as RAS, BRAF, TP53, RB, PIK3CA and EGFR, make only a small contribution to clear cell RCC. To improve clinical outcome, a better understanding of critical genes associated with the pathogenesis of RCC is required.In this study, to obtain a comprehensive registry of critical genetic lesions in RCC, we performed SNP array-based genome-wide copy number analysis for >200 paired RCC specimens as well as whole exome analysis for nucleotide alterations using high-throughout resequencing technologies for selected cases. Copy number alterations were quite common in RCC and 96% of 200 RCC specimens showed one or more copy number changes. These lesions typically involve whole chromosomal arms, whereas focal gains and losses were rare and not recurrent. Recurrent copy number alterations included 3p- (68%) and acquired uniparental disomy (aUPD) of 3p (23%), 5q+ (63%), 7q+ (41%), 9p- (17%), 14q- (27%), which were used for clustering RCC cases into several subgroups with discrete genomic profiles and clinical pictures. Approximately one fourth of RCC cases were hyperploid, which predicted frequent metastatic diseases and poor prognosis. Interestingly, the hyperploid cases showed genomic profiles very similar to those of near diploid cases except for their ploidity, indicating that the hyperploid tumors were derived from the near diploid components. Multivariate analysis revealed several independent predictors of prognosis, where hyperploid and 14q LOH consistently predict poor clinical outcomes. Whole exome analysis was performed for 10 RCC paired specimens by combining SureSelect technologies (Agilent) with high-throughput resequencing using Genome Analizer (Illumina), where tumor specific genetic changes were intensively explored. A large number of tumor-specific nucleotide mutations and insertions/deletions were identified involving PTPRG, ERG, and other genes, which were thought to be involved in RCC pathogenesis and thus to be possible targets for novel therapeutics. In this meeting, we will present the result of our comprehensive genetic analysis of RCC and discuss the genetic basis of RCC in terms of copy number alternations and gene mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2011-4702

The 14-3-3 proteins are a family of regulatory molecules that modulate the functions of their binding partners. They are involved in many processes including protein synthesis/degradation, cell cycle regulation, apoptosis, and tumor development. We have identified one of 14-3-3 protein isoforms 14-3-3zeta as a novel androgen target gene by chromatin immunoprecipitation (ChIP) coupled with microarray analysis. The expression of 14-3-3zeta was upregulated in LNCaP prostate cancer cells at both mRNA and protein levels by androgen stimulation. We generated LNCaP cells stably expressing 14-3-3zeta or control empty vector, and found that less etoposide-induced cell death was observed in 14-3-3zeta-expressing cells compared with control cells. Cell proliferation and motility were increased in 14-3-3zeta-expressing cells. Imunoprecipitation study showed that 14-3-3zeta binds to androgen receptor (AR) in these cells. Luciferase assay showed that androgen-dependent PSA promoter activity was significantly increased in 14-3-3ζ-expressing cells. Androgen-dependent PSA expression was also increased in 14-3-3ζ-expressing cells. siRNA-dependent 14-3-3zeta knockdown revealed that androgen-dependent AR transactivation was reduced in LNCaP cells. Taken together, the present study indicates that 14-3-3ζ is a bona-fide androgen-regulated gene that plays an important role in activating AR-dependent signaling, thereby promoting cell growth, motility and acquirement of apoptosis resistance in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1705.