Targeting ErbB receptors in a three-dimensional cell culture model of K-Ras mutant colorectal cancer

Abstract

K-Ras is a key signaling molecule regulating central biological processes like proliferation, polarization and survival, and is essential in the control of tissue homeostasis. Constitutive active K-Ras mutations are found in 40% of all colorectal cancers (CRC) contributing to tumor development, progression and therapy resistance. Despite constant efforts there are no targeted therapies available for K-Ras mutated CRC thus far. To develop new, mechanism-based treatment strategies the contribution of oncogenic Ras to transformation and therapy resistance has to be understood in detail. For this purpose, the human epithelial colorectal adenocarcinoma cell line Caco-2 was used as a model in this thesis. Cultured Caco-2 cells represent an early stage of CRC. They express wild-type K-Ras and have no further mutations in downstream Ras signaling pathways. Seeded into a three-dimensional culture system, Caco-2 cells are able to polarize, partly recapitulating the morphological features of the normal colorectal epithelium. By introducing an oncogenic K-Ras variant (G12V) in this organotypic model system, using an inducible expression system, two new aspects of oncogenic Ras signaling could be described for the first time: Firstly, the acute expression of K-RasG12V disrupted polarized morphogenesis of Caco-2 grown in 3D culture. I was able to identify a novel autocrine signaling loop that mediated the hyperproliferation and loss of cell polarity induced by K-RasG12V expression, which involved the receptor tyrosine kinase ErbB3 and transcriptional upregulation of its ligand heregulin (HRG). Secondly, in Caco-2 3D cultures, K-RasG12V expression led to resistance against a targeted single-chain TRAIL molecule (Db-scTRAIL) comprising an ErbB1 blocking moiety derived from cetuximab and three TRAIL monomers. Here, I identified a resistance mechanism triggered by K-RasG12V involving the upregulation of the anti-apoptotic proteins cIAP1/2. The combination of Db-scTRAIL with a new Smac mimetic (SM83) was able to override this resistance not only in the Caco-2 model but also in additional Ras-mutated CRC cell lines. Taken together these findings provide the basis for a new rational approach: combining ErbB3 blockade in Ras mutant CRC with an apoptosis inducing TRAIL molecule plus a sensitizing Smac mimetic. This combination might efficiently block the autocrine Ras-HRG-ErbB3 loop and therefore suppress transformation whilst simultaneously inducing apoptosis.