Function and Inhibition of Oncogenic Transcription Factors

The principal goal of our research is to elucidate the molecular mechanisms of cellular proliferation control and carcinogenesis. Specifically, we focus on the analysis of oncogenes and the definition of possible antagonists of oncogene function. The c-myc proto-oncogene encodes a transcription factor (Myc) with oncogenic potential. Myc and its dimerization partner Max are bHLH-Zip DNA binding proteins controlling fundamental cellular processes. Deregulation of c-myc leads to tumorigenesis and is a hallmark of many human cancers. We have recently identified ancestral forms of myc (myc1, myc2) and max genes from the early diploblastic cnidarian Hydra, the most basal metazoan organism employed so far for the analysis of these genes [Hartl et al., PNAS 107:4051-4056 (2010)]. Hydra myc1 is specifically activated in all rapidly proliferating cell types of the interstitial stem cell system. The ancestral Hydra Myc1 and Max proteins share the principal design and biochemical properties with their vertebrate derivatives, and Hydra Myc1 even displays basic oncogenic potential. Our results suggest that the principal functions of the Myc master regulator arose very early in metazoan evolution, allowing their dissection in a simple model organism. We will now also characterize in detail the structure and specific expression of the myc2 gene. Notably, we will analyze the role of these genes in Hydra development and stem cell biology by gene silencing procedures. Furthermore, we will identify and analyze effector genes downstream of Hydra Myc and compare these pathways with those proposed for the role of Myc in growth control or carcinogenesis in higher organisms. We will also extend our analyses to the putative non-transcriptional functions of Myc, e.g. control of DNA replication. Myc-induced cell transformation involves transcriptional activation or suppression of specific target genes. We have recently identified the BASP1 gene as a novel target suppressed by Myc [Hartl et al., PNAS 106:5604-5609 (2009)]. The acidic 25 kDa BASP1 protein was originally isolated as a cytoskeleton-associated protein from rat and chicken brain, but has also been found in other tissues and subcellular locations. BASP1 mRNA and protein expression is specifically suppressed in avian fibroblasts transformed by the v myc oncogene. Ectopic expression of BASP1 renders fibroblasts resistant to subsequent cell transformation by v myc. Mutational analysis showed that the basic N-terminal domain containing a myristoylation site, a calmodulin binding domain, and a putative nuclear localization signal is essential for the inhibitory function of the BASP1 protein. Our results suggest that downregulation of the BASP1 gene is a necessary event in myc-induced oncogenesis and define the BASP1 protein as a potential tumor suppressor. We will now reassess the apparent antagonism of Myc and BASP1 in mammalian systems, including human tumor cells. Furthermore, we will explore the possibility to use BASP1-derived small peptides as putative inhibitors of Myc function and myc-induced tumorigenesis.

Our research is focused on the molecular mechanisms of cellular proliferation control and carcinogenesis. Specifically, we analyze the function of oncogenes and search for possible antagonists and mechanisms for the inhibition of oncogene function. Cell transformation and tumor induction by activated oncogenes (mutated proto-oncogenes) or inactivated tumor suppressor genes involve changes in transcriptional control with subsequent alterations in gene expression patterns. One of the critical transcription factors (Myc) is encoded by a prominent proto-oncogene, c-MYC. Myc and its dimerization partner Max are bHLH-LZ DNA binding proteins controlling fundamental cellular processes. Deregulation of the master regulator c-MYC leads to tumorigenesis and is a hallmark of many human cancers. The specific aims of the project funded by this grant were focused on (i) the definition of basic functions of Myc homologs in very simple metazoan organisms, (ii) the identification of novel Myc transcriptional targets and protein interaction partners, and (iii) the identification of molecules and mechanisms to inhibit Myc function as a strategy towards therapeutic intervention in carcinogenesis. (i) We identified and fully characterized novel homologs of human c-MYC in the simple metazoan Hydra (myc1 and myc2), with different developmental expression patterns and different control by Wnt signaling. Studies of Myc function in very basic organisms may provide further insights into relevant Myc functions in more complex organisms, including man. (ii) We identified genes encoding components of the DNA replication machinery as direct transcriptional targets of Myc, implicating Myc in the transcriptional control of DNA replication. This is of particular interest since previous studies had reported that DNA replication is also under non-transcriptional control by Myc. We also identified the calcium sensor calmodulin as a novel protein-protein interaction partner of Myc, pointing to a possible synergism of Ca2+ and Myc signaling. (iii) In collaborative studies with the Scripps Research Institute in California, we identified and fully characterized small- molecule inhibitors of Myc:Max dimerization that work in the nanomolar range and are currently developed into potential therapeutic agents. In addition to these major project areas, we continued structural analyses of cancer-relevant proteins encoded by Myc transcriptional targets in cooperation with the University of Vienna, and established new methods of transcriptional silencing of cancer-relevant genes in cooperation with colleagues from the University of Innsbruck.