The α6β4 integrin maintains the survival of human breast carcinoma cells in vivo

The alpha 6 beta 4 integrin has been widely implicated in carcinoma function in vitro; however, in vivo data are scarce. To determine the importance of alpha 6 beta 4 in tumor progression, a SUM-159 breast carcinoma cell line that is essentially devoid of alpha 6 beta 4 expression was generated using an RNA interference strategy. Loss of alpha 6 beta 4 expression inhibits colony formation in soft agar assays, suggesting a vital role for alpha 6 beta 4 in survival signaling and anchorage-independent growth. Orthotopic injection of the beta 4-deficient cell line into the mammary fat pad of immunocompromised mice yielded significantly fewer and smaller tumors than the control cell line, revealing a role for the alpha 6 beta 4 integrin in tumor formation. Under conditions that mimicked the in vivo environment, decreased expression of the alpha 6 beta 4 integrin led to enhanced apoptosis as determined by the percentage of Annexin V-FITC+, PI- cells and the presence of caspase-3 cleavage products. Recombinant vascular endothelial growth factor (VEGF) significantly inhibited the cell death observed in the beta 4-deficient cell line, demonstrating the importance of VEGF expression in this survival pathway. Furthermore, loss of alpha 6 beta 4 expression leads to enhanced apoptosis and reduced expression of VEGF in breast carcinoma cells in vivo. Importantly, the specificity of alpha 6 beta 4 in both the in vitro and in vivo assays showed that reexpression of the beta 4 subunit into the beta 4-deficient cell line could rescue the functional phenotype. Taken together, these data implicate the a,604 integrin in tumor formation by regulating tumor cell survival in a VEGF-dependent manner.