Signal Transduction Colorectal Carcinogenesis: Connecting K-RAS–Induced Transformation and CREB Activity In Vitro and In Vivo

Oncogenic transformation is often associatedwith an increased expression of the cAMP response element binding (CREB) transcription factor controlling the expressionof genes involved in cell proliferation, cell cycle, apoptosis, and tumor development, but a link between K-RAS-induced transformation and CREB has not yet been determined. Therefore, the constitutive and/or inhibitor-regulated mRNA and protein expression of CREB and signal transduction components and growth properties of parental fibroblasts, K-RAS-transformed counterparts, shCREB KRAS transfectants and human colon carcinoma cells were determined. Increased CREB transcript and protein levels accompanied by an enhanced CREB activity was detected in K-RAStransformed murine fibroblasts and K-RAS-mutated human tumor cells, which is dependent on theMAPK/MEK, PI3K, and/or PKC signal transduction. Immunohistochemical (IHC) staining of colorectal carcinoma lesions and murine tumors, with known KRAS gene mutation status, using antibodies specific for CREB and phospho-CREB, revealed a mechanistic link between CREB expression and K-RAS-mutated colorectal carcinoma lesions when comparedwith control tissues. Silencing of CREB by shRNA and/or treatment with a CREB inhibitor (KG-501) reverted the neoplastic phenotype of K-RAS transformants as demonstrated by a more fibroblast-like morphology, enhanced apoptosis sensitivity, increased doubling time, decreased migration, invasion and anchorage-independent growth, reduced tumorigenesis, and enhanced immunogenicity in vivo. The impaired shCREB-mediated invasion of K-RAS transformants was accompanied by a transcriptional downregulation of different matrix metalloproteinases (MMP) coupled with their reduced enzymatic activity. Implications: CREB plays a key role in the K-RAS-mediated neoplastic phenotype and represents a suitable therapeutic target for murine and human K-RAS-induced tumors. Mol Cancer Res; 13(8); 1248–62. 2015 AACR.