Selective chemical and genetic inhibition of PKCβII strongly sensitizes colon cancer cells to anoikis.

BACKGROUND

The protein kinase CβII (PKCβII) is a conventional PKC isoform that exerts both over-expression and downregulation in tumors. Roles of PKCβII in tumor formation and progression remains poorly understood. In the present study, we aimed to investigate the hypothesis that PKCβII overexpression promotes anoikis resistance in colon cancer cells.

METHODS AND RESULTS

Colon cancer cells were grown in suspension, and their susceptibility to anoikis was assessed using cell viability assay and number of apoptotic cells were measured with flow cytometry. We analyzed expression of PKC isoforms by western blot and qRT-PCR. Our findings demonstrated that PKCβII was overexpressed by ~ threefold in LoVo and T84 compared to CCD-18Co and SW480 cells. Anoikis resistant cells were treated with PKC inhibitors to assess requirement for PKCβII activity in the growth of these cells. To further elucidate the role of PKCβII in regulating growth and anoikis resistance, we used both Crispr/Cas9 to knock out PKCβII and shRNA approaches for partial PKCβII knockdown. Furthermore, we overexpressed PKCβII in anoikis-sensitive cells and assessed its impact on cell survival under suspension conditions. Our in vitro experiments revealed a correlation between PKCβII expression levels and anoikis resistance in T84 and LoVo cell lines. At the molecular level, reduced PKCβII expression or activity in suspended T84 and LoVo cells resulted in induce apoptosis. Treatment with the PKC inhibitor staurosporine promoted anoikis in LoVo and T84 cells, while PKC activator induced anoikis resistance in sensitive cells, suggesting that PKCβII might regulate anoikis in suspended cells. Furthermore, overexpression of PKCβII in CCD-18Co and SW480 cells restored cells resistance to anoikis.

CONCLUSION

Taken together, our findings demonstrate that PKCβII selectively promotes anoikis resistance of human colon cancer cells and targeting PKCβII may provide new strategies for colon cancer therapy.