ORIGINAL PAPERS Incomplete inhibition of the Rb tumor suppressor pathway in the context of inactivated p53 is sufficient for pancreatic islet tumorigenesis Oriol Casanovas 1,2 , Jeffrey H Hager 1,2,3 , Matthew Gee Hong Chun 1 and Douglas Hanahan* ,1 1 Department of Biochemistry and Biophysics, Diabetes Center and Comprehensive Cancer Center, University of California-San Francisco, 513 Parnassus Avenue HSW-1090, UCSF, San Francisco, CA-94143, USA Here, we describe the surprising residual capability of the Rb pathway to negatively regulate proliferation and tumorigenesis in a SV40 large T antigen (Tag)-driven mouse model of pancreatic islet carcinogenesis. Hetero- geneous Tag expression during all progression stages suggested that a threshold level of the T antigen oncoprotein might be deterministic for b-cell hyperproli- feration and led us to hypothesize that Tag might not be fully inhibiting the tumor suppressor activity of Rb. Moreover, genomic profiling of these tumors by array CGH pointed to regions of loss on chromosomes 6 and 14, where the Rb pathway inhibitor p27 and Rb itself, respectively, reside. Indeed, genetic ablation of the p27 Kip1 or Rb genes accentuated Tag-induced tumorigen- esis, with loss of Rb in particular broadly enhancing multiple parameters of tumorigenesis including the frequency and growth rates of premalignant lesions, of nascent solid tumors, and of invasive carcinomas. The data indicate that attenuation rather than complete inactivation of Rb tumor suppressor gene function, in the context of p53 inhibition, is sufficient to initiate tumorigenesis in this model of islet cell cancer, with the demonstrable possibility that subsequent losses of Rb or its regulators can enhance malignant progression. The results may be relevant to human papillomavirus (HPV)- induced cervical neoplasias where E7 oncogene expression levels or activity (in the case of intermediate/low-risk HPV subtypes) incompletely inhibits Rb tumor suppressor functions, as well as to other neoplasias where initiating oncogenic or tumor suppressor events reduce but do not abrogate Rb function. Oncogene (2005) 24, 6597–6604. doi:10.1038/sj.onc.1208823; published online 27 June 2005 Keywords: oncogenesis; retinoblastoma protein (Rb); tumor suppressor genes; transgenic mice; RIP-Tag2 Introduction The viral oncogenes human papillomavirus (HPV) 16 E6/E7 and SV40 large T antigen (Tag) are well- established promoters of tumorigenesis in humans and animal models. These viral oncoproteins inhibit Rb and p53 tumor suppressor genes as their primary mechanism of tumorigenesis. In contrast to the potent effects of the E6/7 oncogenes carried in the high-risk HPV genotypes such as HPV16, the low/intermediate-risk HPV virus subtypes carry variants of the E6 and E7 oncogenes that are not able to fully inhibit Rb and p53, rather only partially attenuating their tumor suppressor functions, although they are also able to trigger cervical carcinoma after a long latency period (Sang and Barbosa 1992; Giannoudis and Herrington, 2000). The RIP-Tag2 mouse model of multistage islet cell carcinogenesis shows an intriguing latency in the initiation of aberrant proliferation: although SV40 T antigen expression begins during embryonic develop- ment (day E8.5) (Hanahan, 1985), focal hyperproliferation is not evident until 4 or 5 weeks of age. During this dichotomous period of oncogene expression with proliferative latency, the development, morphological appearance and functional characteristics of the islets of transgenic animals are indistinguishable from wild-type littermates. However, beginning at 4–5 weeks of age, hyperproliferation is activated in a subset of the oncogene-expressing islets, transforming them into lesions describable as hyperplasias and dysplasias (Hanahan, 1985). The existence of this proliferative latency raises questions about the sufficiency of Tag for the full functional inactivation of the Rb proliferation and/or the p53 antiapoptotic signals, suggestive of possible similarity to the low/intermediate-risk subclasses of HPV viral oncogenes in cervical carcino- genesis. Previous studies have assessed SV40 large T antigen’s (Tag’s) actions in abrogating p53 tumor suppressor function by crossing RIP-Tag2 mice to p53-null mice. The characteristic rates of apoptosis in the distinctive stages of the tumorigenesis pathway were unaltered in the RIP-Tag2;p53 / mice, indicating that Tag was suppressing p53’s proapoptotic actions. Nevertheless, and counterintuitively, tumor size was reduced, as was the characteristic rate of tumor cell proliferation (Herzig et al., 1999). These effects were attributed to the mutual Received 14 March 2005; revised 22 April 2005; accepted 23 April 2005; published online 27 June 2005 *Correspondence: D Hanahan; E-mail: dh@biochem.ucsf.edu 2 These authors contributed equally to this work 3 Current address: Kalypsys Inc., 10420 Wateridge Circle, San Diego, CA-92121, USA Oncogene (2005) 24, 6597–6604 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc