Janknecht, Ralf

Abstract The pivotal roles of the K-RAS and TGFβ signaling pathways have been well characterized in a number of normal and pathological conditions including human cancer. Hyperactivation of the ERBB/KRAS pathway is known to drive neoplastic transformation, tumorigenesis and metastasis in a large percentage of human cancers while the growth inhibitory actions of TGFβ are critical in preventing cancer. A better understanding of the antagonistic interaction and cross-talk between the K-RAS and TGFβ signaling pathways that control tumorigenesis and metastasis may give rise to novel therapeutic interventions. Our previous work has highlight the importance of SIAH2 in K-RAS-dependent tumorigenesis and metastasis in human cancer. In this study, we showed that SMAD2/3/4 – the essential downstream mediators of TGFβ signaling, are new SIAH2 substrates. SIAH2 physically interacts with, ubiquitinates and degrades SMAD2/3/4 (but not SMAD1 – the downstream mediator of the BMP signaling pathway). The identification of the SIAH2-mediated SMAD2/3/4 modulation/degradation may provide an additional mechanism to explain the well documented, but less understood ERBB/K-RAS and TGFβ antagonism described in normal development, tumorigenesis and metastasis. We propose that the tumor promoting activity of RAS/SIAH2 may be mediated through the degradation of SMAD2/3/4 proteins and thus the relief of TGFβ-dependent growth inhibition in response to ERBB/K-RAS activation. The discovery of the SIAH2-SMAD2/3/4 antagonism downstream of K-RAS and TGFβ signaling pathways may provide a novel mode of antagonistic cross-talk between these two important signaling pathways to explain the growth inhibitory action of the TGFβ pathway and the growth-promoting activity of the K-RAS pathway in the genesis, progression and metastasis of human cancer. Since the substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligase, our results provide new insights into the role of altered proteolysis in mediating the context-dependent ERBB/RAS-TGFβ antagonism and cross-talk in human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1980. doi:10.1158/1538-7445.AM2011-1980

Abstract ETS variant 1 (ETV1), also known as ETS-related protein 81, is overexpressed in prostate tumors, but whether and how this transcription factor affects tumorigenesis has remained elusive. Here, we show that ETV1 is primarily overexpressed in the most aggressive human prostate tumors. Transgenic ETV1 mice developed prostatic intraepithelial neoplasia as well as hyperplasia/neoplasia in seminal vesicles. Moreover, ETV1 cooperated with the androgen receptor (AR) to bind to the prostate-specific antigen enhancer and stimulate gene transcription. Consistent with its ability to physically interact with AR, ETV1 rendered an ETV1 binding site–driven reporter androgen inducible, and, on the other hand, ETV1 superinduced transcription from an AR binding site on androgen stimulation. In conclusion, our study substantiates that ETV1 overexpression is an underlying cause in the development of prostate and possibly also seminal vesicle cancer. Its interaction with and activation of AR provides a molecular mechanism on how ETV1 exerts its deleterious function. Thus, inhibiting ETV1 or blocking its interaction with AR may represent novel strategies in prostate cancer therapy. [Cancer Res 2009;69(20):8102–10]

Abstract The homologous proteins p68 and p72 are members of the DEAD box family of RNA helicases. Here, we show that expression of both of these helicases strongly increases during the polyp→adenoma→adenocarcinoma transition in the colon. Furthermore, p68 and p72 form complexes with β-catenin and promote the ability of β-catenin to activate gene transcription. Conversely, simultaneous knockdown of p68 and p72 leads to reduced expression of the β-catenin–regulated genes, c-Myc, cyclin D1, c-jun, and fra-1, all of which are proto-oncogenes. Moreover, transcription of the cell cycle inhibitor p21WAF1/CIP1, whose expression is suppressed by c-Myc, is enhanced on p68/p72 knockdown. Thus, p68/p72 may contribute to colon cancer formation by directly up-regulating proto-oncogenes and indirectly by down-regulating the growth suppressor p21WAF1/CIP1. Accordingly, knockdown of p68 and p72 in colon cancer cells inhibits their proliferation and diminishes their ability to form tumors in vivo. Altogether, these results suggest that p68/p72 overexpression is not only a potential marker of colon cancer but is also causally linked to this disease. Therefore, p68 and p72 may be novel targets in the combat against colon cancer. [Cancer Res 2007;67(16):7572–8]

Abstract Purpose: Tumor markers ideally allow monitoring and prediction of disease progression. In Ewing’s sarcoma, a devastating childhood cancer, only a few reliable prognostic markers have been identified. To this end, we analyzed the expression of four tumor-promoting proteins, cyclin D1, HER2/Neu, Mdm2, and vascular endothelial growth factor (VEGF), in Ewing’s sarcoma. Experimental Design and Results: Thirty-one tissue samples from patients with Ewing’s sarcoma were stained with antibodies against cyclin D1, HER2/Neu, Mdm2, or VEGF. Whereas no significant expression of HER2/Neu and Mdm2 was detected, positive cyclin D1 and VEGF staining was observed in 42% and 55% of all tumors, respectively. Importantly, VEGF expression was found to be an independent negative predictor of survival in Ewing’s sarcoma patients, whereas cyclin D1 expression did not correlate with survival in these patients. Consistently, the Ewing’s sarcoma-specific EWS-ETS oncoproteins were capable of activating both the cyclin D1 and VEGF promoters in transient transfections of tissue culture cells. Furthermore, this activation was enhanced by coexpression of the Sp1 transcription factor. Using a mammalian two-hybrid system, some evidence was obtained that this may involve a physical interaction between EWS-ETS and Sp1 proteins. Conclusions: Our data reveal that VEGF may serve as a prognostic marker in Ewing’s sarcoma patients and provide a molecular mechanism by which VEGF and cyclin D1 expression is up-regulated in approximately half of all Ewing’s sarcomas.

BRCA1 is a breast and ovarian cancer-specific tumor suppressor, with properties of a transcription factor involved in DNA repair. We previously have shown the transactivation of heterologous promoters by the carboxyl terminus of BRCA1. We now describe that BRCA1-mediated transactivation is enhanced by p300/CBP (CREB binding protein) and that this effect was suppressed by the adenovirus E1A oncoprotein. We show a physical association of BRCA1 with the transcriptional coactivators/acetyltransferases p300 and CBP. Endogenous as well as overexpressed BRCA1 and p300 were found to associate in a phosphorylation-independent manner. BRCA1 interacts with the cAMP response element binding protein (CREB) domain of p300/CBP via both its amino and carboxyl termini. Finally, full-length BRCA1 is shown to transcriptionally activate the Rous sarcoma virus-long terminal repeat promoter, which was further stimulated by p300. Immunocolocalization analyses suggest that BRCA1 and p300 associate in a cell cycle-dependent manner. Our results support a role for BRCA1 in transcription.