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Mol Med. 1998 December; 4(12): 807–822.
PMCID: PMC2230392

Alterations of p16-pRb pathway and chromosome locus 9p21-22 in sporadic invasive breast carcinomas.

Abstract

The p16-pRb pathway represents a vital cell-cycle checkpoint. In the present study we investigated the alterations of this G1-phase protein pathway using immunohistochemical and molecular methods in a series of 55 breast carcinomas and correlated the findings with clinicopathological features of the patients. Furthermore, we examined its relationship with the status of the chromosomal region 9p21-22 performing a deletion map analysis because there are indications that, in addition to CDKN2 and MTS2/p15(INK4B) tumor suppressor genes (TSGs), this area harbors other TSG(s). Aberrant expression (Ab) of p16 and pRb was observed in 26 (47%) and 16 (29%) of the carcinomas, respectively. A statistical trend pointing out an inverse relationship between p16 and pRb expression was found (p = 0.079). Analysis of the region that encodes for p16 by deletion mapping, a PCR-based methylation assay and PCR-SSCP, revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16(INK4A) inactivation in breast carcinomas. The results of deletion mapping also suggest that another TSG(s) may reside at the 9p21-22 area particularly at the D9S162 loci and that co-deletion of this putative gene with CDKN2/p16(INK4A) may play a role in breast carcinogenesis. In addition, microsatellite instability (MI), a marker of replication error phenotype (RER+), was observed with a frequency of 16% in the area examined and was inversely related with loss of heterozygosity (LOH). Interestingly, most cases with MI at the region encoding for p16 were aggregated in a subgroup of breast carcinomas with no other obvious genetic and/or epigenetic CDKN2/p16(INK4A) alterations. We speculate that there is an additional mechanism of CDKN2/p16(INK4A) inactivation. The relationship of p16 protein level pRb, status, the p16-pRb combined immunoprofiles, and the microsatellite alterations detected at the 9p21-22 locus with the patients' clinicopathological parameters revealed two significant correlations: one between normal pRb expression and lymph node involvement (p = 0.0263), and the other between microsatellite alterations (LOH and or MI) and tumor size (p = 9.2 x 10(-3)). In view of the heterogenous nature of breast cancer, we suggest that in a significant proportion of breast carcinomas, deregulation of the p16-pRb pathway in association with another, as-yet unidentified, TSG(s) of the 9p21-22 region may play a role in initiating or progressing the oncogenic procedure, while in other subgroups, alternative molecules may play this role.

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  • Devilee P, Cornelisse CJ. Somatic genetic changes in human breast cancer. Biochim Biophys Acta. 1994 Dec 30;1198(2-3):113–130. [PubMed]
  • Cordon-Cardo C. Mutations of cell cycle regulators. Biological and clinical implications for human neoplasia. Am J Pathol. 1995 Sep;147(3):545–560. [PubMed]
  • Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993 Dec 16;366(6456):704–707. [PubMed]
  • Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS, 3rd, Johnson BE, Skolnick MH. A cell cycle regulator potentially involved in genesis of many tumor types. Science. 1994 Apr 15;264(5157):436–440. [PubMed]
  • Knudson AG. Antioncogenes and human cancer. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10914–10921. [PubMed]
  • Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature. 1994 Apr 21;368(6473):753–756. [PubMed]
  • Cairns P, Mao L, Merlo A, Lee DJ, Schwab D, Eby Y, Tokino K, van der Riet P, Blaugrund JE, Sidransky D. Rates of p16 (MTS1) mutations in primary tumors with 9p loss. Science. 1994 Jul 15;265(5170):415–417. [PubMed]
  • Okamoto A, Demetrick DJ, Spillare EA, Hagiwara K, Hussain SP, Bennett WP, Forrester K, Gerwin B, Serrano M, Beach DH, et al. Mutations and altered expression of p16INK4 in human cancer. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11045–11049. [PubMed]
  • Spruck CH, 3rd, Gonzalez-Zulueta M, Shibata A, Simoneau AR, Lin MF, Gonzales F, Tsai YC, Jones PA. p16 gene in uncultured tumours. Nature. 1994 Jul 21;370(6486):183–184. [PubMed]
  • Zhang SY, Klein-Szanto AJ, Sauter ER, Shafarenko M, Mitsunaga S, Nobori T, Carson DA, Ridge JA, Goodrow TL. Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck. Cancer Res. 1994 Oct 1;54(19):5050–5053. [PubMed]
  • Reissmann PT, Koga H, Takahashi R, Figlin RA, Holmes EC, Piantadosi S, Cordon-Cardo C, Slamon DJ. Inactivation of the retinoblastoma susceptibility gene in non-small-cell lung cancer. The Lung Cancer Study Group. Oncogene. 1993 Jul;8(7):1913–1919. [PubMed]
  • Dublin EA, Patel NK, Gillett CE, Smith P, Peters G, Barnes DM. Retinoblastoma and p16 proteins in mammary carcinoma: their relationship to cyclin D1 and histopathological parameters. Int J Cancer. 1998 Feb 20;79(1):71–75. [PubMed]
  • Ito Y, Kobayashi T, Takeda T, Nakano Y, Tamaki Y, Komoike Y, Wakasugi E, Shin E, Takatsuka Y, Kikkawa N, et al. Expression of p16 and cyclin-dependent kinase 4 proteins in primary breast carcinomas. Oncology. 1997 Nov-Dec;54(6):508–515. [PubMed]
  • Geradts J, Wilson PA. High frequency of aberrant p16(INK4A) expression in human breast cancer. Am J Pathol. 1996 Jul;149(1):15–20. [PubMed]
  • Merlo A, Mabry M, Gabrielson E, Vollmer R, Baylin SB, Sidransky D. Frequent microsatellite instability in primary small cell lung cancer. Cancer Res. 1994 Apr 15;54(8):2098–2101. [PubMed]
  • Rush EB, Abouezzi Z, Borgen PI, Anelli A. Analysis of MTS1/CDK4 in female breast carcinomas. Cancer Lett. 1995 Mar 2;89(2):223–226. [PubMed]
  • Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, Sidransky D, Baylin SB. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 1995 Oct 15;55(20):4525–4530. [PubMed]
  • Quesnel B, Fenaux P, Philippe N, Fournier J, Bonneterre J, Preudhomme C, Peyrat JP. Analysis of p16 gene deletion and point mutation in breast carcinoma. Br J Cancer. 1995 Aug;72(2):351–353. [PMC free article] [PubMed]
  • Berns EM, Klijn JG, Smid M, van Staveren IL, Gruis NA, Foekens JA. Infrequent CDKN2 (MTS1/p16) gene alterations in human primary breast cancer. Br J Cancer. 1995 Oct;72(4):964–967. [PMC free article] [PubMed]
  • Brenner AJ, Aldaz CM. Chromosome 9p allelic loss and p16/CDKN2 in breast cancer and evidence of p16 inactivation in immortal breast epithelial cells. Cancer Res. 1995 Jul 1;55(13):2892–2895. [PubMed]
  • Xu L, Sgroi D, Sterner CJ, Beauchamp RL, Pinney DM, Keel S, Ueki K, Rutter JL, Buckler AJ, Louis DN, et al. Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas. Cancer Res. 1994 Oct 15;54(20):5262–5264. [PubMed]
  • Marsh KL, Varley JM. Frequent alterations of cell cycle regulators in early-stage breast lesions as detected by immunohistochemistry. Br J Cancer. 1998 May;77(9):1460–1468. [PMC free article] [PubMed]
  • Musgrove EA, Lilischkis R, Cornish AL, Lee CS, Setlur V, Seshadri R, Sutherland RL. Expression of the cyclin-dependent kinase inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer. Int J Cancer. 1995 Nov 15;63(4):584–591. [PubMed]
  • Marsh KL, Varley JM. Loss of heterozygosity at chromosome 9p in ductal carcinoma in situ and invasive carcinoma of the breast. Br J Cancer. 1998 May;77(9):1439–1447. [PMC free article] [PubMed]
  • Van Zee KJ, Calvano JE, Bisogna M. Hypomethylation and increased gene expression of p16INK4a in primary and metastatic breast carcinoma as compared to normal breast tissue. Oncogene. 1998 May 28;16(21):2723–2727. [PubMed]
  • T'Ang A, Varley JM, Chakraborty S, Murphree AL, Fung YK. Structural rearrangement of the retinoblastoma gene in human breast carcinoma. Science. 1988 Oct 14;242(4876):263–266. [PubMed]
  • Barbareschi M, Pelosio P, Caffo O, Buttitta F, Pellegrini S, Barbazza R, Dalla Palma P, Bevilacqua G, Marchetti A. Cyclin-D1-gene amplification and expression in breast carcinoma: relation with clinicopathologic characteristics and with retinoblastoma gene product, p53 and p21WAF1 immunohistochemical expression. Int J Cancer. 1997 Apr 22;74(2):171–174. [PubMed]
  • Geradts J, Hu SX, Lincoln CE, Benedict WF, Xu HJ. Aberrant RB gene expression in routinely processed, archival tumor tissues determined by three different anti-RB antibodies. Int J Cancer. 1994 Jul 15;58(2):161–167. [PubMed]
  • Nielsen NH, Emdin SO, Cajander J, Landberg G. Deregulation of cyclin E and D1 in breast cancer is associated with inactivation of the retinoblastoma protein. Oncogene. 1997 Jan 23;14(3):295–304. [PubMed]
  • Borg A, Zhang QX, Alm P, Olsson H, Sellberg G. The retinoblastoma gene in breast cancer: allele loss is not correlated with loss of gene protein expression. Cancer Res. 1992 May 15;52(10):2991–2994. [PubMed]
  • Anderson JJ, Tiniakos DG, McIntosh GG, Autzen P, Henry JA, Thomas MD, Reed J, Horne GM, Lennard TW, Angus B, et al. Retinoblastoma protein in human breast carcinoma: immunohistochemical study using a new monoclonal antibody effective on routinely processed tissues. J Pathol. 1996 Sep;180(1):65–70. [PubMed]
  • Berns EM, de Klein A, van Putten WL, van Staveren IL, Bootsma A, Klijn JG, Foekens JA. Association between RB-1 gene alterations and factors of favourable prognosis in human breast cancer, without effect on survival. Int J Cancer. 1995 Apr 21;64(2):140–145. [PubMed]
  • Jares P, Rey MJ, Fernández PL, Campo E, Nadal A, Muñoz M, Mallofré C, Muntané J, Nayach I, Estapé J, et al. Cyclin D1 and retinoblastoma gene expression in human breast carcinoma: correlation with tumour proliferation and oestrogen receptor status. J Pathol. 1997 Jun;182(2):160–166. [PubMed]
  • Sawan A, Randall B, Angus B, Wright C, Henry JA, Ostrowski J, Hennessy C, Lennard TW, Corbett I, Horne CH. Retinoblastoma and p53 gene expression related to relapse and survival in human breast cancer: an immunohistochemical study. J Pathol. 1992 Sep;168(1):23–28. [PubMed]
  • Varley JM, Armour J, Swallow JE, Jeffreys AJ, Ponder BA, T'Ang A, Fung YK, Brammar WJ, Walker RA. The retinoblastoma gene is frequently altered leading to loss of expression in primary breast tumours. Oncogene. 1989 Jun;4(6):725–729. [PubMed]
  • Trudel M, Mulligan L, Cavenee W, Margolese R, Côté J, Gariépy G. Retinoblastoma and p53 gene product expression in breast carcinoma: immunohistochemical analysis and clinicopathologic correlation. Hum Pathol. 1992 Dec;23(12):1388–1394. [PubMed]
  • Koh J, Enders GH, Dynlacht BD, Harlow E. Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition. Nature. 1995 Jun 8;375(6531):506–510. [PubMed]
  • Li Y, Nichols MA, Shay JW, Xiong Y. Transcriptional repression of the D-type cyclin-dependent kinase inhibitor p16 by the retinoblastoma susceptibility gene product pRb. Cancer Res. 1994 Dec 1;54(23):6078–6082. [PubMed]
  • Olopade OI, Pomykala HM, Hagos F, Sveen LW, Espinosa R, 3rd, Dreyling MH, Gursky S, Stadler WM, Le Beau MM, Bohlander SK. Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6489–6493. [PubMed]
  • BLOOM HJ, RICHARDSON WW. Histological grading and prognosis in breast cancer; a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer. 1957 Sep;11(3):359–377. [PMC free article] [PubMed]
  • Kinne DW. Staging and follow-up of breast cancer patients. Cancer. 1991 Feb 15;67(4 Suppl):1196–1198. [PubMed]
  • Mouridsen HT, Palshof T, Mattheiem W, Sylvester RJ, Rotmensz N, Paridaens RJ. Currently active protocols in the EORTC Breast Cancer Cooperative Group. Recent Results Cancer Res. 1984;91:263–267. [PubMed]
  • Gorgoulis VG, Rassidakis GZ, Karameris AM, Papastamatiou H, Trigidou R, Veslemes M, Rassidakis AN, Kittas C. Immunohistochemical and molecular evaluation of the mdm-2 gene product in bronchogenic carcinoma. Mod Pathol. 1996 May;9(5):544–554. [PubMed]
  • Gorgoulis VG, Zoumpourlis V, Rassidakis GZ, Karameris A, Rassidakis AN, Spandidos DA, Kittas C. A molecular and immunohistochemical study of the MDM2 protein isoforms and p53 gene product in bronchogenic carcinoma. J Pathol. 1996 Oct;180(2):129–137. [PubMed]
  • Fong KM, Zimmerman PV, Smith PJ. Microsatellite instability and other molecular abnormalities in non-small cell lung cancer. Cancer Res. 1995 Jan 1;55(1):28–30. [PubMed]
  • Chaubert P, Guillou L, Kurt AM, Bertholet MM, Metthez G, Leisinger HJ, Bosman F, Shaw P. Frequent p16INK4 (MTS1) gene inactivation in testicular germ cell tumors. Am J Pathol. 1997 Sep;151(3):859–865. [PubMed]
  • Gorgoulis V, Zoumpourlis V, Rassidakis G, Karameris A, Barbatis C, Spandidos DA, Kittas C. Molecular analysis of p53 gene in laryngeal premalignant and malignant lesions. p53 protein immunohistochemical expression is positively related to proliferating cell nuclear antigen labelling index. Virchows Arch. 1995;426(4):339–344. [PubMed]
  • Merlo A, Mabry M, Gabrielson E, Vollmer R, Baylin SB, Sidransky D. Frequent microsatellite instability in primary small cell lung cancer. Cancer Res. 1994 Apr 15;54(8):2098–2101. [PubMed]
  • Otterson GA, Khleif SN, Chen W, Coxon AB, Kaye FJ. CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2'deoxycytidine. Oncogene. 1995 Sep 21;11(6):1211–1216. [PubMed]
  • Tam SW, Shay JW, Pagano M. Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16Ink4. Cancer Res. 1994 Nov 15;54(22):5816–5820. [PubMed]
  • Xu HJ, Hu SX, Benedict WF. Lack of nuclear RB protein staining in G0/middle G1 cells: correlation to changes in total RB protein level. Oncogene. 1991 Jul;6(7):1139–1146. [PubMed]
  • Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, Baylin SB, Sidransky D. 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med. 1995 Jul;1(7):686–692. [PubMed]
  • Gonzalez-Zulueta M, Bender CM, Yang AS, Nguyen T, Beart RW, Van Tornout JM, Jones PA. Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res. 1995 Oct 15;55(20):4531–4535. [PubMed]
  • de Vos S, Miller CW, Takeuchi S, Gombart AF, Cho SK, Koeffler HP. Alterations of CDKN2 (p16) in non-small cell lung cancer. Genes Chromosomes Cancer. 1995 Nov;14(3):164–170. [PubMed]
  • Neville EM, Stewart M, Myskow M, Donnelly RJ, Field JK. Loss of heterozygosity at 9p23 defines a novel locus in non-small cell lung cancer. Oncogene. 1995 Aug 3;11(3):581–585. [PubMed]
  • Gilmore AP, Ohanian V, Spurr NK, Critchley DR. Localisation of the human gene encoding the cytoskeletal protein talin to chromosome 9p. Hum Genet. 1995 Aug;96(2):221–224. [PubMed]
  • Albigès-Rizo C, Frachet P, Block MR. Down regulation of talin alters cell adhesion and the processing of the alpha 5 beta 1 integrin. J Cell Sci. 1995 Oct;108(Pt 10):3317–3329. [PubMed]
  • Gorgoulis VG, Zacharatos P, Kotsinas A, Liloglou T, Kyroudi A, Veslemes M, Rassidakis A, Halazonetis TD, Field JK, Kittas C. Alterations of the p16-pRb pathway and the chromosome locus 9p21-22 in non-small-cell lung carcinomas: relationship with p53 and MDM2 protein expression. Am J Pathol. 1998 Dec;153(6):1749–1765. [PubMed]
  • Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science. 1993 May 7;260(5109):816–819. [PubMed]
  • Jiricny J. Colon cancer and DNA repair: have mismatches met their match? Trends Genet. 1994 May;10(5):164–168. [PubMed]
  • Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, Vogelstein B, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science. 1995 Jun 2;268(5215):1336–1338. [PubMed]
  • Shaw JA, Walsh T, Chappell SA, Carey N, Johnson K, Walker RA. Microsatellite instability in early sporadic breast cancer. Br J Cancer. 1996 Jun;73(11):1393–1397. [PMC free article] [PubMed]
  • Wooster R, Cleton-Jansen AM, Collins N, Mangion J, Cornelis RS, Cooper CS, Gusterson BA, Ponder BA, von Deimling A, Wiestler OD, et al. Instability of short tandem repeats (microsatellites) in human cancers. Nat Genet. 1994 Feb;6(2):152–156. [PubMed]
  • Yee CJ, Roodi N, Verrier CS, Parl FF. Microsatellite instability and loss of heterozygosity in breast cancer. Cancer Res. 1994 Apr 1;54(7):1641–1644. [PubMed]
  • Paulson TG, Wright FA, Parker BA, Russack V, Wahl GM. Microsatellite instability correlates with reduced survival and poor disease prognosis in breast cancer. Cancer Res. 1996 Sep 1;56(17):4021–4026. [PubMed]
  • Patel U, Grundfest-Broniatowski S, Gupta M, Banerjee S. Microsatellite instabilities at five chromosomes in primary breast tumors. Oncogene. 1994 Dec;9(12):3695–3700. [PubMed]
  • Glebov OK, McKenzie KE, White CA, Sukumar S. Frequent p53 gene mutations and novel alleles in familial breast cancer. Cancer Res. 1994 Jul 15;54(14):3703–3709. [PubMed]
  • Sakaguchi M, Fujii Y, Hirabayashi H, Yoon HE, Komoto Y, Oue T, Kusafuka T, Okada A, Matsuda H. Inversely correlated expression of p16 and Rb protein in non-small cell lung cancers: an immunohistochemical study. Int J Cancer. 1996 Feb 8;65(4):442–445. [PubMed]
  • Kinoshita I, Dosaka-Akita H, Mishina T, Akie K, Nishi M, Hiroumi H, Hommura F, Kawakami Y. Altered p16INK4 and retinoblastoma protein status in non-small cell lung cancer: potential synergistic effect with altered p53 protein on proliferative activity. Cancer Res. 1996 Dec 15;56(24):5557–5562. [PubMed]
  • Yeager T, Stadler W, Belair C, Puthenveettil J, Olopade O, Reznikoff C. Increased p16 levels correlate with pRb alterations in human urothelial cells. Cancer Res. 1995 Feb 1;55(3):493–497. [PubMed]
  • Ueki K, Ono Y, Henson JW, Efird JT, von Deimling A, Louis DN. CDKN2/p16 or RB alterations occur in the majority of glioblastomas and are inversely correlated. Cancer Res. 1996 Jan 1;56(1):150–153. [PubMed]
  • Wang Y, Becker D. Differential expression of the cyclin-dependent kinase inhibitors p16 and p21 in the human melanocytic system. Oncogene. 1996 Mar 7;12(5):1069–1075. [PubMed]
  • Hangaishi A, Ogawa S, Imamura N, Miyawaki S, Miura Y, Uike N, Shimazaki C, Emi N, Takeyama K, Hirosawa S, et al. Inactivation of multiple tumor-suppressor genes involved in negative regulation of the cell cycle, MTS1/p16INK4A/CDKN2, MTS2/p15INK4B, p53, and Rb genes in primary lymphoid malignancies. Blood. 1996 Jun 15;87(12):4949–4958. [PubMed]
  • Medema RH, Herrera RE, Lam F, Weinberg RA. Growth suppression by p16ink4 requires functional retinoblastoma protein. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6289–6293. [PubMed]
  • Thorlacius S, Jonasdottir O, Eyfjord JE. Loss of heterozygosity at selective sites on chromosomes 13 and 17 in human breast carcinoma. Anticancer Res. 1991 Jul-Aug;11(4):1501–1507. [PubMed]

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