Abstract #236: CAIX expression increases the aggressive behavior of a luminal breast cancer cell line without medium acidification Free

Carbonic anhydrase IX (CAIX) is a membrane-bound enzyme whose catalytic domain faces the interstitial space and catalyzes the reversible dehydration of CO₂. As such, CAIX is proposed to regulate extracellular pH in the few normal tissues in which it is expressed. However, CAIX is overexpressed in many solid tumors, including breast cancer, and is associated with hypoxia and poor prognosis. The specific function of CAIX in cancer cells is still not well understood which is important for the development of new interventional tools for cancer therapy. In this in vitro study, we demonstrate that CAIX expression increases the aggressive behavior of T47D breast cancer cells without causing acidification of medium. Methods: The estrogen-receptor positive, ductal adenocarcinoma cell line T47D, was plated in McCoy\#8217;s medium. pcDNA3-CAIX was transfected into T47D cells followed by G418 selection. T47D-CAIX clones were selected for CAIX expression using the M75 monoclonal antibody by western blot. CAIX activity was verified by previously published methods. The wound healing assay and matrigel insert assay were used to evaluate cell migration and invasion. The effect of pH on cell behavior was determined by reducing medium pH to 6.8. Results: We have shown previously that T47D cells do not express CAIX. CAIX expression and carbonic anhydrase activity in T47D-CAIX cells were confirmed. T47D-CAIX cells showed a 15% increase in cell proliferation. CAIX overexpression also increased cell migration over 24 h in serum free medium. In addition, CAIX overexpression doubled the number of invading cells through matrigel-coated inserts using 10% serum as the chemotaxic agent. There was no morphological change between T47D-CAIX and control, T47D cells. Low pH increased control cell migration over 24h. There was also a significant increase in the number of invading cells. Low pH did not affect proliferation in contrast to the increase induced by CAIX overexpression. Low pH also caused a morphologic change from an epithelial-like to a mensenchymal-like cell. Western blotting did not find changes of p-Erk, GRP78, vimentin (a mesenchymal cell marker), or CAIX levels. While low pH and CAIX overexpression caused similar changes in cell behavior, we did not see significant changes in medium acidification between the control and T47D-CAIX cells. Further, there was no difference in Glut 1, vimentin, p-ERK, or GRP78 expression. Conclusion: Low extracellular pH increased migration and invasion capacity of T47D breast cancer cells. CAIX overexpression caused similar changes in aggressive behavior, as well as increased cell proliferation. Morphological changes were only observed under low pH. Ecotopic expression of CAIX did not cause a reduction in medium pH. These data suggest that CAIX-induced cellular changes in vitro may not be directly related to acidification.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 236.