US007807373B2 (12) United States Patent (10) Patent No.: US 7,807,373 B2 Srivastava et al. (45) Date of Patent: Oct. 5, 2010 (54) PROSTATE SPECIFIC GENE, PCGEM1, AND GenBank. Accession No. ACO 13401, GI:8569780, 169700 bp DNA, METHODS OF USING PCEGM1 TO DETECT, Jul. 7, 2000. TREAT, AND PREVENT PROSTATE CANCER “AC003046.” EBI Database XPO02143197, Dec. 1, 2001. (75) Inventors: Shiv Srivastava, Potomac, MD (US); Srikantanet al., “Structure and Expression of a Novel Prostate Spe Judd W. Moul, Bethesda, MD (US); cific Gene: PCGEM1. Proc. American Assoc. Cancer Research Vasantha Srikantan, Rockville, MD Annual, XP000929230, vol. 40, p. 37 (Mar. 1999). (US); Zhiqiang Zou, Gaithersburg, MD Bussemakers et al., “A New Prostate-Specific Arker, Strongly (US) Overexpressed in Prostatic Tumors.” Urological Research, XP0020743.05, vol. 25, No. 1, p. 76 (Feb. 1997). (73) Assignee: Henry M. Jackson Foundation for the Wang et al., “Genes Regulated by Androgen in the Rat Ventral Pros Advancement of Military Medicine, tate.” Proc. Nat.1 Acad. Sci., U.S.A., vol. 94, pp. 12999-13004 (Nov. Rockville, MD (US) 1997). “ACO 13401, EBI Database XP002143200, Apr. 16, 2005. (*) Notice: Subject to any disclaimer, the term of this Srikantan et al., “PCGEM1, a Prostate-Specific Gene, Is Overex patent is extended or adjusted under 35 posed in Prostate Cancer.” Proc. Natl. Acad. Sci., vol.97, No. 22, pp. U.S.C. 154(b) by 285 days. 12216-12221 (Oct. 24, 2000). (21) Appl. No.: 12/166,723 Srikantan et al., Identification of Prostate Cancer Associated Novel Gene Expression Alterations, Proc. American Assoc. Cancer (22) Filed: Jul. 2, 2008 Research, vol.39, p. 362 (Mar. 1998). Srikantan et al., Identification of Prostate Cancer Associated Novel (65) Prior Publication Data Gene Expression Alterations, USUHS Research Day-Graduate Stu US 2010/O184O23 A1 Jul. 22, 2010 dent Colloquium, p. 164 (Apr. 1-2, 1998). Srikantan et al., Identification of Prostate Cancer Associated Novel Related U.S. Application Data Gene Expression Alterations, J. Urology, AUA 93rd Annual Meeting, (60) Division of application No. 10/802,823, filed on Mar. p. 40 (May 30-Jun. 4, 1998). 18, 2004, now abandoned, which is a continuation of Petrovics, et al. Oncogene, vol. 23, pp. 605-611 (2004). application No. 09/534,072, filed on Mar. 24, 2000, Genbank. Accession No. AC003046, GI:2583140, 22.9335bp DNA, now Pat. No. 6,828,429. Nov. 3, 1997. Moreno, J.G. et al., “Detection of Hematogenous Micrometastasis in (60) Provisional application No. 60/126,469, filed on Mar. Patients with Prostate Cancer, Cancer Research, 52.61 10-61 12 26, 1999. (Nov. 1992). (51) Int. Cl. Dixon et al., Cancer Chemother. Pharmacol., (1999)43(Suppl.):S78 CI2O I/68 (2006.01) S84. (52) U.S. Cl. ......................... 435/6: 435/91.2:536/23.1; Genbank. Accession No. AF099810, GI:38.00891, 193824 bp DNA, 536/24.33:436/64 Oct. 28, 1998. (58) Field of Classification Search ..................... 435/6, Genbank. Accession No. AC006925, GI:4417322, 176186 bp DNA, 435/91.2: 536/23.1, 24.33: 436/64 Mar. 16, 1999. See application file for complete search history. Office Action dated Dec. 20, 2005, from European Application No. (56) References Cited OO 9183732. Office Action dated Jul. 19, 2007, from European Application No. 00 U.S. PATENT DOCUMENTS 9183732. 6,828,429 B1 12/2004 Srivastava et al. 2001/005.3519 A1 12, 2001 Fodor et al. * cited by examiner Primary Examiner Stephen L Rawlings FOREIGN PATENT DOCUMENTS (74) Attorney, Agent, or Firm—MH2 Technology Law Group WO WO95/19434 7, 1995 LLP WO WO99.00498 1, 1999 WO PCT/USOO.O7906 5, 2000 (57) ABSTRACT OTHER PUBLICATIONS A nucleic acid sequence that exhibits prostate-specific Petrovics et al. (Oncogene. Jan. 15, 2004; 23 (2): 605-611).* Fu et al. (DNA Cell Biol. Mar. 2006; 25 (3): 135-141).* expression and over-expression in tumor cells is disclosed. Ifere et al. (Recent Pat. DNA Gene Seq. Nov. 2009; 3 (3): 151-163).* The sequence and fragments thereof are useful for detecting, GenBank. Accession No. ACO 13401, GI:6289254, 163807 bp DNA, diagnosing, preventing, and treating prostate cancer and other Nov. 9, 1999. prostate related diseases. The sequence is also useful for GenBank. Accession No. ACO 13401, GI:8050939, 170695 bp DNA, measuring hormone responsiveness of prostate cancer cells. May 24, 2000. GenBank. Accession No. ACO 13401, GI:8099084, 172760 bp DNA, May 26, 2000. 7 Claims, 21 Drawing Sheets U.S. Patent Oct. 5, 2010 Sheet 1 of 21 US 7,807,373 B2 STRATEGY FORTHEIDENTIFICATION OF GENE EXPRESSIONALTERATIONS IN PROSTATE CANCER OCTEMBEDDED FROZEN PROSTATE TUMOR/NORMAL TISSUE MAKE 6 am SERIAL SECTIONS HISTOLOGICALEXAMINATION OF H & ESLIDE RNA PREPARATION RT-PCRAMPLIFICATIONUSING ARBITRARY AND ANCHORED PRIMER CONTAINING 5 M13 ORT7 SEQUENCES HIGHRESOLUTIONGEL ELECTROPHORESIS (GENOMYX SYSTEM) AND EXCISION OF DIFFERENTIALLY EXPRESSEDBANDS REAMPLIFICATIONUSING M13 AND T7 PRIMERS PURIFICATION AND AUTOMATED DNA SEQUENCING ONAB1377 USING M13/T7 PRIMER DNA SEQUENCE DATABASE SEARCH (NCBI, CGAP) RT-PCR USING GENE SPECIFIC PRIMER FORTUMOR SPECIFIC ALTERATIONS TO SCREEN RNAFROMDEFINED GROUP OF PATIENTS AND CELL LINES ANALYZE EXPRESSION PATTERN STUDY BIOLOGICAL FUNCTION FOR CLINICAL CORRELATIONS OF SELECTED GENES FIG. 1 U.S. Patent Oct. 5, 2010 Sheet 2 of 21 US 7,807,373 B2 TNT NTNT NT T-TUMOR N-NORMAL - or t 1. 8 FIG 2 T-TUMOR St T N T N T N T N T N - No RT - N-NORMAL : : -- CYTOKERATIN T N T N T N T N T N - No RT : - PCGEM 1 FIG 3 U.S. Patent Oct. 5, 2010 Sheet 3 of 21 US 7,807,373 B2 123 CD >>H O >< LLI CY<C H= Z -- PCGEM 1 FIG 4 U.S. Patent Oct. 5, 2010 Sheet 4 of 21 US 7,807,373 B2 WHSOOd| SDENTO}}_LNOO O 1 nM 10 nM O 12 HR FIG 5A U.S. Patent Oct. 5, 2010 Sheet 5 of 21 US 7,807,373 B2 K b 2.37 - 18S 1.35 FIG 5B U.S. Patent Oct. 5, 2010 Sheet 6 of 21 US 7,807,373 B2 MO&&WWNO9 - "... ONWS) TWNBOW - WHOWAL - SOON HoWA is OOO TWNS OOAH HOWWOLS - rt : SWAONW - ANOX 98 SW TWTXS - WIT SDN?. - WLNOW NW 9 - LAWH - . 3 E3 : MOOX?. OOO8 WHA (SNINI IWSOOnW) NOOO SNILSSLN TWWS - MAWAO - SISL - WISOd SWAHL - NB9TS - 2 : ; ; ; U.S. Patent Oct. 5, 2010 Sheet 7 of 21 US 7,807,373 B2 appendix lung trachoa placent fetal fitn fatal fetal at it fit brin heart karey er open thymus ting yeast A a st an co "Poly (A) human 00 ng 100 ng 100 ng 100ng 100 gg isong a U.S. Patent Oct. 5, 2010 Sheet 8 of 21 US 7,807,373 B2 U.S. Patent Oct. 5, 2010 Sheet 9 of 21 US 7,807,373 B2 cDNA sequence of PCGEM Sed. ID No. 1 AAGGCACTCT GGCACCCAGTTTTGGAACTG CAGTTTTAAA AGTCATAAATTGAATGAAAA TGATAGCAAA 70 CCTGGAGGTTTTTAAAGAGCTATTTATAGGTCCCTGGACA GCATCTTTTTTCAATTAGGC AGCAACCTTT 140 TTGCCCTATG CCGTAACCTG TGTCTGCAAC TTCCTCTAATTGGGAAATAG TAAGCAGATTCATAGAGCT 210 CAATGATAAA ATTGAACTAC GAGATGCACT GGGACTCAAC GTGACCTTAT CAAGTGAGCA GGCTTGGTGC 280 ATTTGACAC, TCATGATATC AGCCAAAGTG GAACTAAAAA CAGCTCCTGG AAGAGGACTA TGACATCATC 350 AGGTTGGGAG TCTCCAGGGA CAGCGGACCC TTTGGAAAAG GACTAGAAAGTGTGAAATCT ATTAGTCTTC 420 CATATGAAATTCTCTGTCTC TGTAAAAGCA TTTCATATTT ACAAGACACA GGCCTACTCC TAGGGCAGCA 490 AAAAGTGGCA ACAGGCAAGC AGAGGGAAAA GAGATCATGA GGCATTTCAG AGTGCACTGT CTTTTCATAT 560 ATTTCCAAT GCCGTATGTTTGGTTTTATTTTGGCCAAGC ATAACAATCT GCTCAAGAAAAAAAAATCTG 630 (AGAAAACAA ACGTCCCTTT GCCAATGTTA TGTTTCTTTTTGACAAGCCC TGAGATTTCT GAGGGGAATT 700 CACATAAATG GGATCAGGTC ATTCATTTAC CTTGTGTGCA AATATGATTT AAAGATACAA CCTTTGCAGA 770 CAGCAIGCTTTCCTAAGGGT AGCCACGTGG ACGACTAAGG GTAAAGCATT CTTCAAGATC ACTTAATCAA 840 CAAAGGTGCT CITTGCATTC TGAAATGCCCTTGTTGCAAA. TATTGGTTATATTGATTAAA TTTACACTTA 910 ATGGAAACAA CCTTTAACTT ACAGATGAAC AAACCCACAA AAGCAAAAAATCAAAAGCCC TACCTATGAT 980 TTCATATTTT CTGTGTAACT GGATTAAAGG ATTCCTGCTT GCTTTTGGGC ATAAATGATA ATGGAATATT 1050 TCCAGGTATT GTTTAAAATC AGGGCCCATC TACAAATTCT TAGCAATACT TTGGATAATT CTAAAATTCA 1120 CCTGGACATT GTCTAATTGTTTTTTATATA CATCTTGCT AGAATTTCAA ATTTTAAGTA TCTGAATTTA 1190 CTTAATTAGCTCTCCTGATC AATTCAAAAA CATTACTTTC CTAAATTTTAGACTATGAAG GTCATAAATT 1260 CAACAAATAT, ATCTACACAT ACAATTATAG ATTGTTTTTC ATTATAATGT CTTCATCTTA ACACAATTGT 1330 CTTTGTGATT GTTTTTAGAA AACTGAGAGTTTTAATTCAT AATTACTTCA TCAAAAAATT GTGGGAACAA 1400 TCCACCATTA, ATTCTATCTG ATTGTTTTTA TCTACATAAG GACTCTTAAG CTTCGTCCCTTGAACTCTTT 1470 TCTACTTAGT CCCATGTTTA AAATTACTAC TTTATATCTA AACCATTTAT GTTTTTCAATTCAATTTACA 1540 TGATGCTAAT TATGGCAATT ATAACAAATA TTAAAGATTT CGAAATAGAAAAAAAAAAAA AAA 1603 FIG. 8 U.S. Patent Oct. 5, 2010 Sheet 10 of 21 US 7,807,373 B2 cDNA sequence of PCGEM Seg. ID No.2 GCGGCCGCGT CGACGCAACTTCCTCTAATT GGGAAATAGT TAAGCAGATT CATAGAGCTG AATGATAAAA 70 TTGTACTTCG AGATGCACTG GGACTCAACG TGACCTTATC AAGTGAGATG GAGTCTTGCC CTGTCTCCAA 140 GGCEGGAGCC CAATGGTGTG ATCTTGGCTC ACTGCAACCT CCACCTCCCA GGTTCAAACGTTTCTCCTGC 210 CTCAGCCTCC CAAGTAACTG GGATTACAGC AGGCTTGGTG CATTTGACAC TTCATGATAT CAGCCAAAGT 280 GGAACTAAAA ACAGCTCCTG GAAGAGGACT ATGACATCAT CAGGTTGGGA GTCTCCAGGG ACAGCGGACC 350 CTTTGGAAAA GGACTAGAAA GTGTGAAATC TATTAGTCTT CGATATGAAA TTCTCTGTCT CCGTAAAAGC 420 ATTTCATATT TACAAGACAC AGGCCTACTC CTAGGGCAGC AAAAAGTGGC AACAGGCAAG CAGAGGGAAA 490 ACACATCATGAGCCATTTCA GAGTGCACTG TCTTTTCATA TATTTCCAA TGCCGTATGTTTGGTTTTAT 560 TTTGGCCAAG CATAACAATC TGCTCAAAAAAAAAAAATCT GGAGAAAACA AAGGTGCCTT TGCCAATGTT 630 ATGTTTCTTTTTGACAAGCC CTGAGATTTC TGAGGGGAATTCACATAAAT GGGATCAGGT CATTCATTTA 700 CCTTGTGTGC AAATATGATT TAAAGATACA ACCTTTGCAG AGAGCATGCTTTCCTAAGGG TAGGCACGTG 770 GACGACTAAG GGTAAAGCAT TCTTCAAGAT CAGTTAATCA AGAAAGGTGC TCTTGCATT CTGAAATGCC 840 CTTGTTCCAA ATATTGGTTA TATTGATTAA ATTTACACTTAATGGAAACA ACCTTTAACTTACAGATGAA 910 CAAACCCCAC AAAACCAAAA AATCAAAAGC CCTACCTATG ATTTCATATTTTCCTGTAA CTCCATTAAA 980 GGATTCCTCC TTCCTTTTGG GCATAAATGA TAATGGAATA TTTCCAGGTATTGTTTAAAA TCAGGGCCCA 1050 TCTACAAATT CTTAGCAATA