141. Akt2 Is a Critical Regulator of Colorectal Cancer Metastasis Establishment

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; approximately 35-55% of patients with CRC will develop liver metastases during the course of their disease. Previously, we demonstrated the importance of the phosphatidylinositol 3-kinase (PI3K) pathway, acting through its downstream effector protein, Akt, in the proliferation and metastasis of CRC using novel preclinical models of liver metastasis. The purpose of our present study was to delineate the roles of the Akt isoforms (Akt1 and Akt2) in the systemic invasion and metastasis of CRC. Methods: (i) First, we evaluated the relative expression of Akt1 and Akt2 in different histotypes of human CRC and representative normal tissues (n = 45) using tissue microarray analysis and, in experimental colon neoplasms, by immunohistochemistry. To develop additional supportive evidence for a role for Akt2 in CRC metastasis, we compared expression between primary CRC and matched metastases in the same patients (n = 36) by tissue microarray analysis. The relative levels of Akt2 mRNA expression were examined in different stages of CRC and normal tissue (n=48) using real time PCR. (ii) To further analyze the contribution of the Akt isoforms to CRC metastasis, we transiently expressed Akt1 or Akt2 in non-metastatic human CRC cells (ie, SW480GFP and Caco-2GFP); the cells were injected into the spleens of athymic nude mice and mice were examined periodically by whole-body, real-time fluorescence imaging for in vivo visualization of metastasis. Conversely, we transfected a metastatic CRC cell line (ie, KM20GFP) with siRNA to either Akt1 or Akt2 or non-targeting control (NTC); cells were injected in the spleens of athymic nude mice and metastasis assessed as described above. Results: (i) Marked immunoreactivity was detected in premalignant colonic mucosa, 70% of experimental murine colon neoplasms and in different histotypes of human CRC (well- or moderately-differentiated); Akt1 expression was lower than Akt2 expression for all histotypes. In 32 of 36 cases, Akt2 expression was greater in the liver metastases than in primary colon tumors. In contrast to the higher expression of Akt2 in many of the liver metastases, there was no differential pattern of Akt1 expression between primary tumor and liver metastasis. (ii) Suppression of Akt2 expression in the highly metastatic KM20 CRC cells specifically inhibited their ability to metastasize in an experimental liver metastasis model (see figure comparing Akt2 siRNA vs. control). Overexpression of wild-type Akt1 did not restore metastatic potential in cells with downregulated Akt2. In contrast, ectopic expression of wild-type or constitutively active Akt2 resulted in metastasis by the non-metastatic CRC cell lines.