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• W3023622052 abstract "Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth. Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth. Centromeres are cellular structures that are necessary for the propagation of hereditary information (1Cleveland D.W. Mao Y. Sullivan K.F. Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling.Cell. 2003; 112 (12600307): 407-42110.1016/S0092-8674(03)00115-6Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar, 2Hayden K.E. Human centromere genomics: now it's personal.Chromosome Res. 2012; 20 (22801774): 621-63310.1007/s10577-012-9295-yCrossref PubMed Scopus (25) Google Scholar). Located centric to the ends of each chromosome, centromeres provide the structural foundation for kinetochores, multimeric complexes that serve as molecular interfaces between microtubule spindle fibers and individual chromatids during mitosis (1Cleveland D.W. Mao Y. Sullivan K.F. Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling.Cell. 2003; 112 (12600307): 407-42110.1016/S0092-8674(03)00115-6Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar). The centromere–kinetochore–microtubule interaction facilitates separation of the sister chromatids as mitosis proceeds from metaphase to anaphase. Centromeres are thus essential to ensuring faithful segregation of chromosomes in actively dividing cells. Efforts to study human centromeres have focused on the epigenetics that drive centromere assembly (3Hayashi T. Fujita Y. Iwasaki O. Adachi Y. Takahashi K. Yanagida M. Mis16 and Mis18 are required for CENP-A loading and histone deacetylation at centromeres.Cell. 2004; 118 (15369671): 715-72910.1016/j.cell.2004.09.002Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 4Kim I.S. Lee M. Park K.C. Jeon Y. Park J.H. Hwang E.J. Jeon T.I. Ko S. Lee H. Baek S.H. Kim K.I. Roles of Mis18α in epigenetic regulation of centromeric chromatin and CENP-A loading.Mol. Cell. 2012; 46 (22516971): 260-27310.1016/j.molcel.2012.03.021Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). α-Satellite sequences that define centromere DNA are primarily occupied by a centromere-specific histone H3 variant known as CENPA (3Hayashi T. Fujita Y. Iwasaki O. Adachi Y. Takahashi K. Yanagida M. Mis16 and Mis18 are required for CENP-A loading and histone deacetylation at centromeres.Cell. 2004; 118 (15369671): 715-72910.1016/j.cell.2004.09.002Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 5Foltz D.R. Jansen L.E. Black B.E. Bailey A.O. Yates 3rd, J.R. Cleveland D.W. The human CENP-A centromeric nucleosome-associated complex.Nat. Cell Biol. 2006; 8 (16622419): 458-46910.1038/ncb1397Crossref PubMed Scopus (520) Google Scholar). CENPA is a functionally conserved ∼17-kDa molecule that forms a centromere-specific nucleosome with H2A, H2B, and H4 (6Hasson D. Panchenko T. Salimian K.J. Salman M.U. Sekulic N. Alonso A. Warburton P.E. Black B.E. The octamer is the major form of CENP-A nucleosomes at human centromeres.Nat. Struct. Mol. Biol. 2013; 20 (23644596): 687-69510.1038/nsmb.2562Crossref PubMed Scopus (141) Google Scholar). Proper CENPA localization is an ubiquitin E3 ligase–dependent process requiring ubiquitination of lysine 124 for engagement with the CENPA-specific chaperone HJURP (7Niikura Y. Kitagawa R. Ogi H. Abdulle R. Pagala V. Kitagawa K. CENP-A K124 ubiquitylation is required for CENP-A deposition at the centromere.Dev. Cell. 2015; 32 (25727006): 589-60310.1016/j.devcel.2015.01.024Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). HJURP subsequently facilitates the incorporation of newly synthesized CENPA into nucleosomes occupying replicated α-satellite DNA (8Barnhart M.C. Kuich P.H. Stellfox M.E. Ward J.A. Bassett E.A. Black B.E. Foltz D.R. HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore.J. Cell Biol. 2011; 194 (21768289): 229-24310.1083/jcb.201012017Crossref PubMed Scopus (232) Google Scholar, 9Foltz D.R. Jansen L.E. Bailey A.O. Yates 3rd, J.R. Bassett E.A. Wood S. Black B.E. Cleveland D.W. Centromere-specific assembly of CENP-A nucleosomes is mediated by HJURP.Cell. 2009; 137 (19410544): 472-48410.1016/j.cell.2009.02.039Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). CENPA nucleosomes have a unique set of binding partners that facilitate proper genomic localization, including CENPB, CENPC, and the constitutive centromere-associated network (CCAN) that comprises the inner kinetochore (10Falk S.J. Lee J. Sekulic N. Sennett M.A. Lee T.-H. Black B.E. CENP-C directs a structural transition of CENP-A nucleosomes mainly through sliding of DNA gyres.Nat. Struct. Mol. Biol. 2016; 23 (26878239): 204-20810.1038/nsmb.3175Crossref PubMed Scopus (50) Google Scholar, 11McKinley K.L. Sekulic N. Guo L.Y. Tsinman T. Black B.E. Cheeseman I.M. The CENP-L-N complex forms a critical node in an integrated meshwork of interactions at the centromere–kinetochore interface.Mol. Cell. 2015; 60 (26698661): 886-89810.1016/j.molcel.2015.10.027Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The CCAN serves as a multimeric interface between the DNA-enveloped CENPA nucleosomes and the KNL-1–Mis12–Ndc80 complex network that comprises the outer kinetochore and directly interacts with the microtubule spindle fibers (12Schleiffer A. Maier M. Litos G. Lampert F. Hornung P. Mechtler K. Westermann S. CENP-T proteins are conserved centromere receptors of the Ndc80 complex.Nat. Cell Biol. 2012; 14 (22561346): 604-61310.1038/ncb2493Crossref PubMed Scopus (129) Google Scholar). CENPA and its associated proteins therefore represent structural components that are essential to the integrity of cell division, and appropriate genomic localization of centromeric proteins is consequently a critical event in the cell cycle. Diseases of uncontrolled cell division, particularly cancer, are thus compelling to examine from the epigenetic perspective of centromere biology, primarily as it pertains to the key epigenetic mark CENPA. A number of studies have identified aberrant expression of centromeric/kinetochore proteins in cancers, where overexpression is predictive of survival and response to therapy, although their mechanistic contribution to cancer pathogenesis remains elusive (13Thiru P. Kern D.M. McKinley K.L. Monda J.K. Rago F. Su K.-C. Tsinman T. Yarar D. Bell G.W. Cheeseman I.M. Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program.Mol. Biol. Cell. 2014; 25 (24829384): 1983-199410.1091/mbc.e14-03-0837Crossref PubMed Google Scholar, 14Tomonaga T. Matsushita K. Ishibashi M. Nezu M. Shimada H. Ochiai T. Yoda K. Nomura F. Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer.Cancer Res. 2005; 65 (15930286): 4683-468910.1158/0008-5472.CAN-04-3613Crossref PubMed Scopus (103) Google Scholar, 15Valdivia M.M. Hamdouch K. Ortiz M. Astola A. CENPA a genomic marker for centromere activity and human diseases.Curr. Genomics. 2009; 10 (20119530): 326-33510.2174/138920209788920985Crossref PubMed Scopus (25) Google Scholar, 16Zhang W. Mao J.-H. Zhu W. Jain A.K. Liu K. Brown J.B. Karpen G.H. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy.Nat. Commun. 2016; 7 (27577169): 1261910.1038/ncomms12619Crossref PubMed Scopus (122) Google Scholar, 17Sun X. Clermont P.-L. Jiao W. Helgason C.D. Gout P.W. Wang Y. Qu S. Elevated expression of the centromere protein-A (CENP-A)–encoding gene as a prognostic and predictive biomarker in human cancers: elevated expression of the CENP-A–encoding gene in cancer.Int. J. Cancer. 2016; 139 (27062469): 899-90710.1002/ijc.30133Crossref PubMed Scopus (62) Google Scholar, 18Montes de Oca R. Gurard-Levin Z.A. Berger F. Rehman H. Martel E. Corpet A. de Koning L. Vassias I. Wilson L.O. Meseure D. Reyal F. Savignoni A. Asselain B. Sastre-Garau X. Almouzni G. The histone chaperone HJURP is a new independent prognostic marker for luminal A breast carcinoma.Mol. Oncol. 2015; 9 (25497280): 657-67410.1016/j.molonc.2014.11.002Crossref PubMed Scopus (52) Google Scholar, 19Li Y. Zhu Z. Zhang S. Yu D. Yu H. Liu L. Cao X. Wang L. Gao H. Zhu M. shRNA-targeted centromere protein A inhibits hepatocellular carcinoma growth.PLoS One. 2011; 6 (21423629): e1779410.1371/journal.pone.0017794Crossref PubMed Scopus (91) Google Scholar, 20McGovern S.L. Qi Y. Pusztai L. Symmans W.F. Buchholz T.A. Centromere protein-A, an essential centromere protein, is a prognostic marker for relapse in estrogen receptor–positive breast cancer.Breast Cancer Res. 2012; 14 (22559056): R7210.1186/bcr3181Crossref PubMed Scopus (82) Google Scholar, 21Ma X.-J. Salunga R. Tuggle J.T. Gaudet J. Enright E. McQuary P. Payette T. Pistone M. Stecker K. Zhang B.M. Zhou Y.-X. Varnholt H. Smith B. Gadd M. Chatfield E. et al.Gene expression profiles of human breast cancer progression.Proc. Natl. Acad. Sci. U.S.A. 2003; 100 (12714683): 5974-597910.1073/pnas.0931261100Crossref PubMed Scopus (723) Google Scholar, 22Wu Q. Qian Y.-M. Zhao X.-L. Wang S.-M. Feng X.-J. Chen X.-F. Zhang S.-H. Expression and prognostic significance of centromere protein A in human lung adenocarcinoma.Lung Cancer. 2012; 77 (22542705): 407-41410.1016/j.lungcan.2012.04.007Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 23Qiu J.-J. Guo J.-J. Lv T.-J. Jin H.-Y. Ding J.-X. Feng W.-W. Zhang Y. Hua K.-Q. Prognostic value of centromere protein-A expression in patients with epithelial ovarian cancer.Tumour Biol. 2013; 34 (23712606): 2971-297510.1007/s13277-013-0860-6Crossref PubMed Scopus (59) Google Scholar, 24Gu X.-M. Fu J. Feng X.-J. Huang X. Wang S.-M. Chen X.-F. Zhu M.-H. Zhang S.-H. Expression and prognostic relevance of centromere protein A in primary osteosarcoma.Pathol. Res. Pract. 2014; 210 (24440098): 228-23310.1016/j.prp.2013.12.007Crossref PubMed Scopus (35) Google Scholar). In the setting of ectopic constitutive overexpression, CENPA mislocalization in HeLa cells is independent of aberrant E3 ligase activity but rather demonstrates a reliance on the histone chaperone DAXX (25Lacoste N. Woolfe A. Tachiwana H. Garea A.V. Barth T. Cantaloube S. Kurumizaka H. Imhof A. Almouzni G. Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX.Mol. Cell. 2014; 53 (24530302): 631-64410.1016/j.molcel.2014.01.018Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Ectopic localization of endogenously overexpressed CENPA has also been shown in colon cancer cell lines (26Athwal R.K. Walkiewicz M.P. Baek S. Fu S. Bui M. Camps J. Ried T. Sung M.-H. Dalal Y. CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.Epigenetics Chromatin. 2015; 8 (25788983): 210.1186/1756-8935-8-2Crossref PubMed Scopus (86) Google Scholar). The phenotypic consequences of such mislocalization in malignancy have yet to be elucidated, although ectopic binding to sites marked by DNase hypersensitivity and CCCTC-binding factor (CTCF) transcription factor affinity has hinted at a potential role in regulating gene transcription (25Lacoste N. Woolfe A. Tachiwana H. Garea A.V. Barth T. Cantaloube S. Kurumizaka H. Imhof A. Almouzni G. Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX.Mol. Cell. 2014; 53 (24530302): 631-64410.1016/j.molcel.2014.01.018Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 26Athwal R.K. Walkiewicz M.P. Baek S. Fu S. Bui M. Camps J. Ried T. Sung M.-H. Dalal Y. CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.Epigenetics Chromatin. 2015; 8 (25788983): 210.1186/1756-8935-8-2Crossref PubMed Scopus (86) Google Scholar). Here we report that CENPA is highly overexpressed in prostate cancer (PCA) and that disease progression correlates with CENPA expression within a large patient cohort. CENPA knockdown markedly decreases proliferation of prostate cancer cells but not that of benign prostate cells and increased expression of CENPA causes benign prostate epithelial cells to proliferate more rapidly. Interestingly, CENPA appears to affect proliferation of prostate cancer cells by acting as a transcriptional regulator that modulates expression of genes critical to proliferation, cell cycle progression, and centromere/kinetochore integrity in addition to its role in the centromere. The significance of centromeres to cell division suggests that centromeric components may play important roles in development and in diseases involving cell division gone awry, particularly in cancer. Previous work identified a centromere-kinetochore (CEN/KT) signature that was associated with aggressive, treatment-refractory malignancy (16Zhang W. Mao J.-H. Zhu W. Jain A.K. Liu K. Brown J.B. Karpen G.H. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy.Nat. Commun. 2016; 7 (27577169): 1261910.1038/ncomms12619Crossref PubMed Scopus (122) Google Scholar). We therefore profiled the transcriptomes of different types of malignancies across a compiled catalogue of publicly available RNA-sequencing (RNA-seq) databases (n = 10,848) (27Iyer M.K. Niknafs Y.S. Malik R. Singhal U. Sahu A. Hosono Y. Barrette T.R. Prensner J.R. Evans J.R. Zhao S. Poliakov A. Cao X. Dhanasekaran S.M. Wu Y.-M. Robinson D.R. et al.The landscape of long noncoding RNAs in the human transcriptome.Nat. Genet. 2015; 47 (25599403): 199-20810.1038/ng.3192Crossref PubMed Scopus (1875) Google Scholar). We found that CENPA is ubiquitously overexpressed in malignant tissue relative to respective normal counterparts (Fig. S1A and Table S1). These observations, combined with the well-characterized contributions of centromeric components like CENPA to cell division, suggested conducting a more focused interrogation of these components in cancers that display poor prognosis in the context of high proliferation indices. Prostate cancer is one such disease, where a high proliferation index is predictive of poor outcomes (28Fisher G. Yang Z.H. Kudahetti S. Møller H. Scardino P. Cuzick J. Berney D.M. Transatlantic Prostate Group Prognostic value of Ki-67 for prostate cancer death in a conservatively managed cohort.Br. J. Cancer. 2013; 108 (23329234): 271-27710.1038/bjc.2012.598Crossref PubMed Scopus (84) Google Scholar, 29Li R. Heydon K. Hammond M.E. Grignon D.J. Roach 3rd, M. Wolkov H.B. Sandler H.M. Shipley W.U. Pollack A. Ki-67 staining index predicts distant metastasis and survival in locally advanced prostate cancer treated with radiotherapy: an analysis of patients in Radiation Therapy Oncology Group protocol 86-10.Clin Cancer Res. 2004; 10 (15217948): 4118-412410.1158/1078-0432.CCR-1052-03Crossref PubMed Scopus (92) Google Scholar). New treatment strategies are much needed for prostate cancer, which remains the most diagnosed malignancy in men and the second leading cause of cancer-related death in men (30American Cancer Society Cancer Facts & Figures (2018) https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2018.html (accessed June 4, 2018).Google Scholar). Although hormonal therapy and chemotherapeutic options are available, resistant metastatic disease and life-altering side effects, such as urinary incontinence and erectile dysfunction, are everlasting concerns (31Cooperberg M.R. Prostate cancer: a new look at prostate cancer treatment complications.Nat. Rev. Clin. Oncol. 2014; 11 (24687033): 304-30510.1038/nrclinonc.2014.58Crossref PubMed Scopus (8) Google Scholar). In view of the above considerations, we performed sample set enrichment analysis (SSEA) in the prostate tissue type cohort containing RNA-seq data from 685 tissue samples (27Iyer M.K. Niknafs Y.S. Malik R. Singhal U. Sahu A. Hosono Y. Barrette T.R. Prensner J.R. Evans J.R. Zhao S. Poliakov A. Cao X. Dhanasekaran S.M. Wu Y.-M. Robinson D.R. et al.The landscape of long noncoding RNAs in the human transcriptome.Nat. Genet. 2015; 47 (25599403): 199-20810.1038/ng.3192Crossref PubMed Scopus (1875) Google Scholar). Gene expression of numerous centromeric components exhibited strong enrichments in prostate cancer tissue relative to their normal counterparts (Fig. 1A and Table S2). Our analysis corroborates on a significantly greater scale previous reports that characterize some of these components as part of the centromere-kinetochore (CEN/KT) signature that is strongly associated with poor disease outcomes (16Zhang W. Mao J.-H. Zhu W. Jain A.K. Liu K. Brown J.B. Karpen G.H. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy.Nat. Commun. 2016; 7 (27577169): 1261910.1038/ncomms12619Crossref PubMed Scopus (122) Google Scholar). We selected CENPA from this panel of genes for further assessment, given its central role in centromere biology, importance for development, and highly conserved function, and found a significant increase in expression with disease progression (Fig. 1B) (32Smith C.L. Blake J.A. Kadin J.A. Richardson J.E. Bult C.J. Mouse Genome Database Group Mouse Genome Database (MGD)-2018: knowledgebase for the laboratory mouse.Nucleic Acids Res. 2018; 46 (29092072): D836-D84210.1093/nar/gkx1006Crossref PubMed Scopus (145) Google Scholar). This in silico finding was validated at the protein level through prostate tissue microarrays stained for CENPA, notably demonstrating marked overexpression of CENPA that increased with disease severity (n = 58 total tissues, n = 174 cores) (Fig. 1C). Importantly, receiver operator characteristic analysis of the CENPA-stained prostate tissue microarray produced an area under the curve of 0.89, orthogonally demonstrating a strong association between elevated CENPA expression and metastatic prostate cancer (Fig. S1B). Assessment of CENPA expression was also examined in cancer cell line models to determine feasibility for more focused molecular inquiry. We verified robust overexpression of CENPA in prostate cancer cell lines, as compared with benign prostatic epithelial lines (Fig. 1D). The PNT2 benign cell line was a notable exception, likely because of its rapid proliferation rate relative to other cell lines we tested (Fig. S1C). Taken together, CENPA is a functionally conserved, developmentally important factor abundant in prostate cancer tissue, as seen in a large number of patients and in prostate cancer cell lines, and an increase in its expression at the RNA and protein levels is highly correlated with more aggressive disease. The abundance of CENPA in prostate cancer raised the question of whether overexpression plays a functional role in disease pathogenesis and progression. We thus first conducted a comparative analysis of CENPA expression relative to the remaining transcriptome in prostate cancer to identify associations with biological concepts that could computationally guide functional assessments. Our efforts to profile transcriptomes in human cancer and normal tissue facilitates performing transcriptome-wide correlations against nominated genes of interest in a tissue-specific manner within a large catalogue of samples (n = 685). We thus correlated CENPA mRNA levels to the expression levels of all other protein coding elements (Data Set S1) to deconvolute its relative contribution to prostate cancer progression. CENPA expression tracks tightly with a number of previously identified prostate cancer pathogenesis factors including CENPF, UBE2C, and EZH2 (Fig. 2A; Fig. S2, C and D; and Data Set S1). MKI67 (gene encoding proliferation marker Ki67) also performed well in our analysis, further suggesting a role for CENPA in cellular proliferation (Fig. 2B). Of note, CENPA does not tightly correlate with ACTB (housekeeping gene), AMACR (prostate cancer biomarker), or AR (Fig. S2, A and B, and Data Set S1), suggesting a pathogenic process that is independent of androgen signaling, a pharmacologically relevant molecular pathway that is frequently targeted in prostate cancer treatment. Strong associations with cellular proliferation genes and select pathogenesis factors independent of AR implicate CENPA as a contributor to a biological process that is involved in androgen refractory prostate cancer progression. In fact, we found that AR signaling actually represses CENPA expression in cell culture (Fig. S3A). We additionally used the Database for Annotation, Visualization, and Integrated Discovery (DAVID) to conduct ontology assessments on the highest-performing genes from our transcriptome-wide correlation against CENPA expression in prostate cancer (r > 0.8) (33Huang D.W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4 (19131956): 44-5710.1038/nprot.2008.211Crossref PubMed Scopus (25346) Google Scholar). Our analysis revealed a correlation between CENPA gene expression and biological concept clusters that highlight centromeres, kinetochores, mitosis, and cell division (Fig. 2C and Fig. S3B). Concepts that include genes encoding components of the CCAN were additionally captured by our analysis. Of note, CDC25C, CDCA5, TOP2A, and CENPU, genes known to play roles in cellular proliferation, cell cycle progression, and centromere/kinetochore integrity, were included in these biological concepts. Preranked gene set enrichment analysis (GSEA) independently confirmed enrichments in gene signatures important for cell cycle, cell division, and mitosis (Fig. 2D and Fig. S3C). Taken together, CENPA expression is strongly linked to gene signatures that underlie processes that govern proliferation, cell cycle progression, and centromere/kinetochore integrity in prostate cancer. Significant association between CENPA and proliferation signatures is expected given the role CENPA plays in the structural integrity of the centromere. There is limited evidence, however, concerning CENPA function in human malignancy. We therefore performed loss-of- and gain-of-function experiments in cell lines stably expressing either doxycycline-inducible short hairpin RNAs against CENPA or EF1A promoter–driven full-length CENPA. Doxycycline administration at 2 μg/ml was sufficient to produce robust knockdown of CENPA after 72 h (Fig. 3A and Fig. S4, A and B). CENPA depletion led to a profound growth-inhibitory effect on 22Rv1, LnCaP, and DU145 prostate cancer cells (Fig. 3, B–D, and Fig. S4, C and D). CENPA depletion in prostate cancer cells results in an accumulation of cells in G1 that seem to be unable to progress through the cell cycle (Fig. 3E and Fig. S4, E and F). Conversely, overexpression of CENPA in the 957E-hTERT benign prostate epithelial cell line leads to a profound growth-promoting effect (Fig. 3, F and G). Interestingly, benign 957E-hTERT cells depleted of CENPA do not demonstrate significant proliferative changes, consistent with previous reports that nonmalignant cells can proliferate with low levels of CENPA (Fig. 3G) (34Black B.E. Jansen L.E. Maddox P.S. Foltz D.R. Desai A.B. Shah J.V. Cleveland D.W. Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain.Mol. Cell. 2007; 25 (17244537): 309-32210.1016/j.molcel.2006.12.018Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Taken together, our data show that CENPA is an essential factor for progression through the cell cycle and that overexpression drives proliferation of prostate cancer cells. Given prior reports of ectopic deposition in the setting of CENPA overexpression and the marked overexpression of CENPA in prostate cancer, we performed native ChIP followed by sequencing to identify noncentromeric and potentially regulatory binding sites for CENPA in prostate cancer. As expected, four α-satellites were enriched relative to the IgG control antibodies using a PCR assay we previously devised that can distinguish chromosome-specific α-satellite DNA from any given centromere, verifying the validity of the CENPA ChIP (Fig. S5A) (35Contreras-Galindo R. Fischer S. Saha A.K. Lundy J.D. Cervantes P.W. Mourad M. Wang C. Qian B. Dai M. Meng F. Chinnaiyan A. Omenn G.S. Kaplan M.H. Markovitz D.M. Rapid molecular assays to study human centromere genomics.Genome Res. 2017; 27 (29141960): 2040-204910.1101/gr.219709.116Crossref PubMed Scopus (13) Google Scholar). CENPA-directed ChIP-seq identified 569 noncentromeric binding sites in the VCaP prostate cancer cell line within three experimental replicates (Fig. 4A; Fig. S5, C and D; and Data Set S2). One example of such a CENPA-binding site is present in the promoter region of CDC25C (Fig. 4B), a cell cycle phosphatase that is critical for progression through anaphase that was also identified in our comparative gene expression analysis described above (Fig. 2A). Intriguingly, the promoter of CENPA itself was also bound by CENPA, consistent with previously reported results, hinting that CENPA might regulate its own transcription (26Athwal R.K. Walkiewicz M.P. Baek S. Fu S. Bui M. Camps J. Ried T. Sung M.-H. Dalal Y. CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells.Epigenetics Chromatin. 2015; 8 (25788983): 210.1186/1756-8935-8-2Crossref PubMed Scopus (86) Google Scholar). CENPA-directed ChIP was additionally conducted in the benign prostatic epithelial cell line 957E-hTERT to determine whether ectopic CENPA binding is a cancer-specific observation (Fig. S5B). CENPA enrichment over the four previously assessed α-satellites was significantly lower than that observed in the VCaP cell line, consistent with each cell line's respective CENPA abundance observed above (Fig. 1D). CENPA-directed ChIP-seq for the 957E-hTERT cell line was thus deferred. We next conducted a global assessment of CENPA-binding sites to obtain a functional taxonomy of CENPA-bound genes. Ontologic assessment of genes whose transcriptional start sites were in close proximity to CENPA-binding sites revealed enrichments in biological concepts that are involved with maintenance of nuclear architecture and organization, such as protein–DNA complex assembly (p = 6.44 × 10−18) and chromosome organization (p = 8.70 × 10−13) (Fig. 4C). Furthermore, binning CENPA-binding sites into categories corresponding to discreet locations within the human genome demonstrates a predilection toward binding regulatory elements such as promoters and CpG islands (Fig. 4D). Comparing the number of peaks present within any two genomic regions reveals significant overlap between loci considered to be regulatory areas (Fig. S5E). Taken together, we show that CENPA localizes to noncanonical genomic loci, with a predilection toward the regulatory elements of genes that control cellular proliferation. Histone variants have been well-characterized as modulators of aberrant gene expression in cancer. H2A.Z.2, macroH2A, and H3.3 are well-documented as key contributors to malignant phenotypes in a number of cancer types (36Sporn J.C. Kustatscher G. Hothorn T. Collado M. Serrano M. Muley T. Schnabel P. Ladurner A.G. Histone macroH2A isoforms predict the risk of lung cancer recurrence.Oncogene. 2009; 28 (19648962): 3423-342810.1038/onc.2009.26Crossref PubMed Scopus (146) Google Scholar, 37Vardabasso C. Hasson D. Ratnakumar K. Chung C.-Y. Duarte L.F. Bernstein E. Histone variants: emerging players in cancer biology.Cell. Mol. Life Sci. 2014; 71 (23652611): 379-40410.1007/s00018-013-1343-zCrossref PubMed Scopus (115) Google Sc" @default.
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