Genomic analysis of benign prostatic hyperplasia implicates cellular re-landscaping in disease pathogenesis.

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.

[1]  R. Dhir,et al.  Symptomatic and asymptomatic benign prostatic hyperplasia: Molecular differentiation by using microarrays , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Mayer,et al.  Testosterone and 17β-estradiol induce glandular prostatic growth, bladder outlet obstruction, and voiding dysfunction in male mice. , 2012, Endocrinology.

[3]  C. Roehrborn,et al.  Lower urinary tract symptoms revisited: a broader clinical perspective. , 2008, European urology.

[4]  J. Zhang,et al.  Proliferation and differentiation of prostatic stromal cells , 2001, BJU international.

[5]  E. Noel,et al.  Differential gene expression in the peripheral zone compared to the transition zone of the human prostate gland , 2008, Prostate Cancer and Prostatic Diseases.

[6]  R. Dahiya,et al.  Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1 , 2007, In Vitro Cellular & Developmental Biology - Animal.

[7]  P. Walsh,et al.  The development of human benign prostatic hyperplasia with age. , 1984, The Journal of urology.

[8]  P. Walsh,et al.  The induction of prostatic hypertrophy in the dog with androstanediol. , 1976, The Journal of clinical investigation.

[9]  W. Christens-Barry,et al.  Prostatic growth rate determined from MRI data: age-related longitudinal changes. , 1999, Journal of andrology.

[10]  D. Williams,et al.  A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal-epithelial interactions in prostatic neoplasia. , 1999, Carcinogenesis.

[11]  D. Bostwick,et al.  The relationship between prostate inflammation and lower urinary tract symptoms: examination of baseline data from the REDUCE trial. , 2008, European urology.

[12]  R. West,et al.  Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ , 2017, Genome Research.

[13]  R. Shah,et al.  CXCL12 overexpression and secretion by aging fibroblasts enhance human prostate epithelial proliferation in vitro , 2005, Aging cell.

[14]  M. Sogayar,et al.  Bone Morphogenetic Proteins: structure, biological function and therapeutic applications. , 2014, Archives of biochemistry and biophysics.

[15]  T. Mueller,et al.  Structural insights into BMP receptors: Specificity, activation and inhibition. , 2016, Cytokine & growth factor reviews.

[16]  Christopher A. Miller,et al.  VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. , 2012, Genome research.

[17]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[18]  T. Hubbard,et al.  A census of human cancer genes , 2004, Nature Reviews Cancer.

[19]  M. Barry,et al.  THE AMERICAN UROLOGICAL ASSOCIATION SYMPTOM INDEX FOR BENIGN PROSTATIC HYPERPLASIA , 1992, The Journal of urology.

[20]  W. Butler,et al.  Dentin sialophosphoprotein in biomineralization , 2010, Connective tissue research.

[21]  Gabor T. Marth,et al.  Haplotype-based variant detection from short-read sequencing , 2012, 1207.3907.

[22]  P. di Sant'Agnese,et al.  Relationship of neuroendocrine cells of prostate and serotonin to benign prostatic hyperplasia. , 1993, Urology.

[23]  K. Miyazono,et al.  BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. , 2005, Cytokine & growth factor reviews.

[24]  Serotonin regulates prostate growth through androgen receptor modulation , 2017, Scientific Reports.

[25]  Ohad Parnes,et al.  Inflammation , 2008, The Lancet.

[26]  Arthur P. Grollman,et al.  Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing , 2016, Proceedings of the National Academy of Sciences.

[27]  J. Macoska,et al.  Prostatic fibrosis, lower urinary tract symptoms, and BPH , 2013, Nature Reviews Urology.

[28]  E. Giovannucci,et al.  Association between markers of the metabolic syndrome and lower urinary tract symptoms in the Third National Health and Nutrition Examination Survey (NHANES III) , 2005, International Journal of Obesity.

[29]  W. Mcdougal,et al.  Androgen responsiveness of stromal cells of the human prostate: regulation of cell proliferation and keratinocyte growth factor by androgen. , 1998, The Journal of urology.

[30]  M. Oberholzer,et al.  Light microscopic stereological analysis of the normal human prostate and of benign prostatic hyperplasia. , 1979, The Journal of urology.

[31]  P. di Sant'Agnese,et al.  Neuroendocrine cells in the human prostate gland. , 1993, Journal of andrology.

[32]  H. Hakonarson,et al.  ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data , 2010, Nucleic acids research.

[33]  M. Carini,et al.  Fat boosts, while androgen receptor activation counteracts, BPH‐associated prostate inflammation , 2013, The Prostate.

[34]  S. Simon,et al.  The genomic landscape of fibrolamellar hepatocellular carcinoma: whole genome sequencing of ten patients , 2015, Oncotarget.

[35]  C. Roehrborn,et al.  The long-term effect of doxazosin, finasteride, and combination therapy on the clinical progression of benign prostatic hyperplasia. , 2003, The New England journal of medicine.

[36]  Biaoyang Lin,et al.  The program of androgen-responsive genes in neoplastic prostate epithelium , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[37]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[38]  J. McNeal Origin and evolution of benign prostatic enlargement. , 1978, Investigative urology.

[39]  S Miyano,et al.  Open source clustering software. , 2004, Bioinformatics.

[40]  O. Hofmann,et al.  VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research , 2016, Nucleic acids research.

[41]  J. Gustafsson,et al.  A role for epithelial-mesenchymal transition in the etiology of benign prostatic hyperplasia , 2009, Proceedings of the National Academy of Sciences.

[42]  G. Joyce,et al.  Economic costs of benign prostatic hyperplasia in the private sector. , 2005, The Journal of urology.

[43]  S. J. Higgins,et al.  The endocrinology and developmental biology of the prostate. , 1987, Endocrine reviews.

[44]  Steven A. Roberts,et al.  Mutational heterogeneity in cancer and the search for new cancer-associated genes , 2013 .

[45]  G. Bartsch,et al.  Human benign prostatic hyperplasia: a stromal disease? New perspectives by quantitative morphology. , 1980, Urology.

[46]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Macoska,et al.  The inflammatory microenvironment of the aging prostate facilitates cellular proliferation and hypertrophy. , 2008, Cytokine.

[48]  D. Peehl,et al.  Molecular and cellular pathogenesis of benign prostatic hyperplasia. , 2004, The Journal of urology.

[49]  Rohit Mehra,et al.  CXC-Type Chemokines Promote Myofibroblast Phenoconversion and Prostatic Fibrosis , 2012, PloS one.

[50]  John Trinick,et al.  Titin: properties and family relationships , 2003, Nature Reviews Molecular Cell Biology.

[51]  C. Roehrborn,et al.  1277: The Impact of Acute or Chronic Inflammation in Baseline Biopsy on the Risk of Clinical Progression of BPH: Results from the MTOPS Study , 2005 .

[52]  C. Roehrborn Prostate size: does it matter? , 2000, Reviews in urology.

[53]  R. Tibshirani Regression Shrinkage and Selection via the Lasso , 1996 .

[54]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[55]  G. Kramer,et al.  Phenotypic characterization of infiltrating leukocytes in benign prostatic hyperplasia. , 1992, Laboratory investigation; a journal of technical methods and pathology.

[56]  R C Bruskewitz,et al.  The effect of finasteride in men with benign prostatic hyperplasia. The Finasteride Study Group. , 2002, The New England journal of medicine.

[57]  J. Cyster,et al.  CXCL13 is required for B1 cell homing, natural antibody production, and body cavity immunity. , 2002, Immunity.

[58]  H. Klocker,et al.  Increased growth factor production in a human prostatic stromal cell culture model caused by hypoxia , 2003, The Prostate.

[59]  Alok J. Saldanha,et al.  Java Treeview - extensible visualization of microarray data , 2004, Bioinform..

[60]  M. Webber,et al.  Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18. , 1997, Carcinogenesis.

[61]  Andrew E. Jaffe,et al.  Bioinformatics Applications Note Gene Expression the Sva Package for Removing Batch Effects and Other Unwanted Variation in High-throughput Experiments , 2022 .

[62]  S. Monti,et al.  Prevalent decrease of the EGF content in the periurethral zone of BPH tissue induced by treatment with finasteride or flutamide. , 1997, Journal of andrology.

[63]  J. Trent,et al.  Gene expression signature of benign prostatic hyperplasia revealed by cDNA microarray analysis , 2002, The Prostate.

[64]  H. Ahn,et al.  Stromal cells of the human prostate: Initial isolation and characterization , 1996, The Prostate.

[65]  K. McFann,et al.  DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells. , 2006, American journal of physiology. Endocrinology and metabolism.

[66]  A. Sivachenko,et al.  Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples , 2013, Nature Biotechnology.

[67]  Steven A. Roberts,et al.  Mutational heterogeneity in cancer and the search for new cancer genes , 2014 .

[68]  G. Andriole,et al.  The effect of finasteride in men with benign prostatic hyperplasia. , 1992, The Journal of urology.

[69]  M. Carini,et al.  Metabolic syndrome and benign prostatic enlargement: a systematic review and meta‐analysis , 2015, BJU international.

[70]  N. Masumori,et al.  Distribution of Neuroendocrine Cells in the Transition Zone of the Prostate , 2017, Advances in urology.

[71]  F. Watzinger,et al.  Bone morphogenetic proteins 5 and 6 stimulate osteoclast generation , 2006 .

[72]  Satoru Miyano,et al.  Open source clustering software , 2004 .