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Plexin-B1 Mutations in Prostate Cancer

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Plexin-B1 Mutations in Prostate Cancer Plexin-B1 mutations in prostate cancer Oscar Gee-Wan Wong*, Tharani Nitkunan*, Izumi Oinuma†, Chun Zhou*, Veronique Blanc*, Richard S. D. Brown*, Simon R. J. Bott*, Joseph Nariculam*, Gary Box‡, Phillipa Munson§, Jason Constantinou*, Mark R. Feneley*, Helmut Klocker¶, Suzanne A. Eccles‡, Manabu Negishi†, Alex Freeman§, John R. Masters*, and Magali Williamson*ʈ *Prostate Cancer Research Centre, Institute of Urology, University College London, 67 Riding House Street, London W1W 7EJ, United Kingdom; †Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan; ‡Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom; §Department of Histopathology, University College London, London WC1E 6BT, United Kingdom; and ¶Department of Urology, University of Innsbruck, 6020 Innsbruck, Austria Edited by Stanley M. Gartler, University of Washington, Seattle, WA, and approved September 28, 2007 (received for review March 19, 2007) Semaphorins are a large class of secreted or membrane-associated The cytoplasmic domain and part of the extracellular domain proteins that act as chemotactic cues for cell movement via their (exons 1–5) of plexins A1, A3, and B1 were sequenced using cDNA transmembrane receptors, plexins. We hypothesized that the func- from the human prostate cancer cell lines PC3, DU145, and tion of the semaphorin signaling pathway in the control of cell LNCaP. A single nucleotide change (A5359G) was found in migration could be harnessed by cancer cells during invasion and Plexin-B1 in LNCaP, which potentially changes threonine 1697 to metastasis. We now report 13 somatic missense mutations in the an alanine. The A5359G mutation alters a BstZ171 site, and cytoplasmic domain of the Plexin-B1 gene. Mutations were found estimation of the relative intensities of the mutant and WT restric- in 89% (8 of 9) of prostate cancer bone metastases, in 41% (7 of 17) tion digest products (Fig. 1 a and b), as well as the genomic DNA of lymph node metastases, and in 46% (41 of 89) of primary sequence, suggests that the mutation is present in two of the three cancers. Forty percent of prostate cancers contained the same copies of chromosome 3p in LNCaP cells. LNCaP was derived from mutation. Overexpression of the Plexin-B1 protein was found in a patient of Caucasian origin, and the sequence change was absent the majority of primary tumors. The mutations hinder Rac and from the DNA of 60 control individuals of a similar genetic R-Ras binding and R-RasGAP activity, resulting in an increase in cell background. The rest of the Plexin-B1 cDNA was sequenced in motility, invasion, adhesion, and lamellipodia extension. These PC3, DU145, and LNCAP, and no further mutations were found. results identify a key role for Plexin-B1 and the semaphorin Quantitative RT-PCR showed high levels of Plexin-B1 in LNCaP signaling pathway it mediates in prostate cancer. (Fig. 1c) relative to the other prostate cancer cell lines lacking a mutation. adhesion ͉ migration ͉ R-Ras ͉ Rac ͉ semaphorin Mutation of Plexin-B1 in Clinical Samples of Prostate Cancer. SSCP emaphorins are a large class of secreted or membrane- analysis was used to screen exons 22–29 of the cytoplasmic domain Sassociated proteins that act as chemotactic cues for cell of the Plexin-B1 gene in DNA microdissected from tumor tissue. migration and axon guidance (1). Although best characterized in Aberrant bands were excised from the gel and sequenced (Fig. 2). the nervous system, they are widely expressed in other tissues Nine prostate cancer bone metastases were screened, and 17 where they have a role in angiogenesis, organogenesis, and further missense mutations were found in eight (89%) of the immune cell regulation (2, 3). Semaphorins inhibit integrin- samples. The mutations were absent from uninvolved tissue of the mediated adhesion (4) and act as chemorepellants, although same patient where available (seven cases) and are therefore some also act as chemoattractants. somatic [Table 1 and supporting information (SI) Fig. 7]. Seventeen Plexins are single-pass transmembrane receptors for semaphor- lymph node metastases contained nine missense mutations in 7 of ins (5, 6). Nine vertebrate plexins have been identified, grouped into 17 (41%) of the samples (Table 1 and SI Fig. 7). Eighty-nine four subfamilies, Plexin-A to Plexin-D, according to structure (5–7). primary prostate tumors treated by radical prostatectomy were Semaphorin 4D (Sema4D) is the ligand for Plexin-B1 and is screened, including DNA from 9 samples of fresh tissue, and expressed in both membrane-bound and secreted forms (6, 8). The mutations were found in 41 of 89 (46%) samples (Table 1 and SI cytoplasmic domain of plexins is conserved between plexin family Fig. 7), including 5 of 9 samples of fresh tissue (Fig. 2a). members but has low homology to other receptor proteins (5) and DNA was also extracted from eight samples of adjacent mor- lacks intrinsic tyrosine kinase activity. Plexins have a role in the phologically normal fresh tissue from the same patients as the fresh regulation of the actin cytoskeleton, controlling the activity of the primary tumor samples. Two of eight apparently normal tissue small GTPases, Rnd1, R-Ras, Rac, and Rho (9–12). Plexins also samples contained a mutation. In both cases, the same mutation interact with and activate the receptor tyrosine kinases, ErbB2 (13) was present in the tumor and the adjacent histopathologically and c-Met (14). Plexins activate many characteristics of the invasive defined normal tissue. These findings suggest that, although retain- phenotype seen in cancer progression, inducing changes in extra- ing a normal morphology, the tissue may contain early changes cellular matrix (ECM) adhesion, motility, scatter, and branching associated with the development of prostate cancer or that morphogenesis (15, 16). Prostate cancer is the second leading cause contaminating tumor cells are present, as observed in other stud- ies (17). of cancer-related death in men in the U.S. and U.K. Mutations were confirmed by direct sequencing [i.e., amplifica- We hypothesized that the function of the semaphorin-signaling pathway in control of cell migration could be harnessed by the cancer cell during invasion and metastasis. Our findings suggest that Author contributions: O.G.-W.W. and T.N. contributed equally to this work; O.G.-W.W., mutation or overexpression of the Plexin-B1 gene contributes to T.N., I.O., C.Z., V.B., J.N., G.B., P.M., J.C., and M.W. performed research; R.S.B., S.R.B., M.F., prostate cancer progression. and H.K. contributed new reagents/analytic tools; S.E., M.N., A.F., and J.R.M. analyzed data; J.R.M. and M.W. designed research; M.W. and J.R.M. wrote the paper. Results Conflict of interest statement: A patent has been filed by M.W. and J.R.M. for mutations. Mutation of Plexin-B1 in Prostate Cancer Cell Lines. We hypothesized This article is a PNAS Direct Submission. that mutations in the cytoplasmic domain of the semaphorin ʈTo whom correspondence should be addressed. E-mail: [email protected]. receptors, plexins, would be most likely to affect semaphorin This article contains supporting information online at www.pnas.org/cgi/content/full/ signaling. We therefore focused our screen for mutations on 0702544104/DC1. stretches of the receptor genes encoding the cytoplasmic domain. © 2007 by The National Academy of Sciences of the USA 19040–19045 ͉ PNAS ͉ November 27, 2007 ͉ vol. 104 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702544104 Downloaded by guest on September 24, 2021 a c f a b no noisserpxe evital noisserpxe 30 510 30 non is i ii iii b 341 25 neoplastic i s tumour 169 20 e 20 123 15 rpxe 10 10 5 evitaleR b 0 ii DU 0 175 123456 110 eR LNCaP 145 PC3 65 ii 12 3 iii d e i ii i 1 2 1 2 1 2 Fig. 2. Plexin-B1 mutations detected by SSCP analysis in prostate cancer. (a) (i) SSCP analysis of nonmalignant DNA (lane 1) and DNA from a primary prostate cancer from fresh-frozen tissue (lane 2). The band marked with an arrow contains the C5060T sequence change. (ii) SSCP analysis of nonmalig- nant DNA (lane 1) and DNA from a primary prostate cancer from fresh-frozen iii iv ii tissue (lane 2). The band marked with arrows contains the A5653G mutation. (iii) SSCP analysis of nonmalignant DNA (lane 1) and DNA from a prostate cancer lymph node metastasis (lane 2). The bands marked with arrows contain the A5653G mutation. (b) Mutations in the Plexin-B1 gene. (i) Normal DNA sequence. (ii) Sequence of a SSCP band showing the A5653G mutation. (iii) Direct sequence of DNA from a metastasis in the same patient as in bii, showing the A5653G mutation. Fig. 1. Plexin-B1 expression in prostate cancer. (a) RT-PCR and BstZ171 restriction enzyme digestion of RNA from PC3 (lane 1), LNCaP (lane 2), and DU145 (lane 3). The A5359G mutation in LNCaP destroys a BstZ171 site. Band Ͻ sizes in base pairs are shown. (b) PCR of genomic DNA and BstZ171 restriction tissues (P 0.001, Mann–Whitney tumor vs. nonneoplastic, for enzyme digestion. Shown are undigested DNA (lane 1), PC3 DNA digested both extent and intensity). Expression of Plexin-B1 was found in with BstZ171 (lane 2), and LNCaP DNA digested with BstZ171 (lane 3). The 77% (65 of 84) of prostate tumors and 6% (5 of 83) of nonneoplastic A5359G mutation in LNCaP destroys a BstZ171 site. (c) Quantitative RT-PCR of tissue from the same patients. All nonneoplastic cores were from RNA from LNCaP, DU145, and PC3 cells. Results are the average of three patients diagnosed with prostate cancer.
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