The cAMP/PKA/CREB and TGFβ/SMAD4 Pathways Regulate Stemness and Metastatic Potential in Colorectal Cancer Cells

Metastasis is responsible for the majority of deaths of patients with cancer. However, mechanisms governing metastasis in colorectal cancer remain largely unknown. Here we investigated how colorectal cancer cells acquire metastatic potential using a novel mouse model of colorectal cancer that spontaneously develops liver metastasis, gen-erated by introducing sporadic mutations of Ctnnb1, Kras, Trp53, and Smad4 (CKPS) genes. Proteomic analyses revealed elevated expres-sion of colorectal cancer stem cell markers ALCAM (CD166) and PROM1 (CD133) in colorectal cancer cells from the metastatic model compared with those from a nonmetastatic model. Spleen-to-liver metastasis assays using colorectal cancer cells derived from the CKPS model (CKPS cells) demonstrated the functional importance of ALCAM and PROM1 in initiating metastasis. Genetic and pharma-cologic analyses using CKPS cells in 2D and spheroid culture revealed that expression of ALCAM and PROM1 is regulated positively and negatively by the cAMP/PKA/CREB and TGFI3/SMAD4 pathways, respectively. Consistently, phospho-CREB was expressed in both primary and metastatic lesions of CKPS mice and patients with colorectal cancer, and knockout of CREB in CKPS cells reduced their spheroid-forming and metastasis-initiating abilities. Treatment with a CREB inhibitor potentiated the effect of irinotecan in suppres-sing liver metastasis by CKPS cells. These results reveal the essential roles of ALCAM and PROM1, as well as their upstream regulators, the cAMP/PKA/CREB and TGFI3/SMAD4 pathways, in maintaining the stemness and metastatic potential of colorectal cancer cells and indicate that CREB inhibition may be a potential therapeutic strategy against metastatic colorectal cancer. Significance: This study identifies signaling pathways essential for maintaining the stemness and metastatic potential of colorectal cancer cells and proposes CREB as a therapeutic target in metastatic colorectal cancer.