Abstract
P21‐activated kinase‐1 (PAK‐1) is a serine/threonine kinase involved in multiple signaling pathways that mediate cellular functions such as cytoskeletal motility, cell proliferation, and survival. PAK‐1 expression is altered in various cancers, including prostate and breast. Our recent studies showed that prostate cancer cells expressing higher levels of PAK‐1 were resistant to the cytotoxic effects of the PAK‐1 inhibitor, inhibitor targeting PAK‐1 activation‐3 (IPA‐3), compared to those with lower expression. This study expanded these findings to other cancers (breast and melanoma) by testing the hypothesis that genetic and pharmacological inhibition of PAK‐1 alters cell growth, migration, and invasion in prostate, breast, and skin cancer cell lines. We also tested the specificity of IPA‐3 for PAK‐1 and the hypothesis that gene silencing of PAK‐1 altered the efficacy of sterically stabilized liposomes (SSL) containing IPA‐3 (SSL‐IPA‐3). PAK‐1 expression was identified in four different breast cancer cell lines, and in a melanoma cell line. The expression of PAK‐1 correlated to the IC50 of IPA‐3 as measured by MTT staining. PAK‐1 inhibition using shRNA correlated with decreased cell migration and invasion in prostate cancer DU‐145 and breast cancer MCF‐7 cells. Decreased migration and invasion also correlated to decreased expression of E‐cadherin and alterations in C‐X‐C Chemokine Receptor type 4 and Homing Cell Adhesion Molecule expression. PAK‐1 inhibition increased the cytotoxicity of IPA‐3, and the cytotoxicity of SSL‐IPA‐3 to levels comparable to that of free drug. These data demonstrate that both pharmacological and molecular inhibition of PAK‐1 decreased growth in prostate, breast, and melanoma cancer cell lines, and increased the toxicity of IPA‐3 and its liposomal formulation. These data also show the specificity of IPA‐3 for PAK‐1, are some of the first data suggesting that IPA‐3 is a therapeutic treatment for breast cancer and melanoma, and demonstrate the efficacy of liposome‐encapsulated IPA‐3 in breast cancer cells.
Highlights
This study used both pharmacological and molecular approaches to investigate the hypothesis that the efficacy of free inhibitor targeting P21‐activated kinase‐1 (PAK‐1) activation‐3 (IPA‐3) and sterically stabilized liposomes (SSL)‐IPA‐3 is dependent on the expression of P21‐activated kinases (PAKs)‐1 in diverse cancer cells, including prostate and breast cancer, as well as melanoma cells. These studies showed that cancer cells with high PAK‐1 expression were less responsive to the cytotoxicity of IPA‐3 and SSL‐IPA‐3 compared to cells with low PAK‐1 expression. This current study investigated the ability of PAK‐1 gene silencing to alter prostate and breast cancer cell growth and alter markers of epithelial‐mesenchymal transition (EMT)
Data from our study support that PAK‐1 mediated cell growth in several different cancer cell lines, including those derived from breast and melanoma
Our previous study suggested that prostate cancer cells with the highest expression of PAK‐1 (DU‐145) as well as the breast cancer cells (MCF‐7) were the least susceptible to IPA‐318, raising the concern that the toxicity of IPA‐3 may not be dependent on PAK‐1 expression
Summary
The human breast cancer cell lines BT‐474, MCF‐7, MDA‐231, MDA‐468, the melanoma‐derived cell line, MDA‐435, and the immortalized breast epithelial cell line, MCF‐10A, were purchased from ATCC. We further assessed the effect of PAK‐1 inhibition on DU‐145 and MCF‐7 cell growth by determining differences in the expression of E‐cadherin, N‐cadherin, CXCR‐4, and HCAM using immunoblot analysis (Figure 6). These proteins were chosen as many studies have shown their involvement in cancer proliferation and progression.[23,24,25,26] E‐ and N‐cadherin have been shown to play an important role in EMT while CXCR4 plays an important role in cancer cell metastasis through chemoattraction. Treatment of DU‐145 and MCF‐7 PAK‐1 KD cells with free IPA‐3 and SSL‐IPA‐3 resulted in concentration‐dependent increases in annexin V and PI staining at 48 hours. Treatment of DU‐145 PAK‐1 KD and MCF‐7 PAK‐1 KD cells with free IPA‐3 resulted in concentration‐dependent increases in cells staining
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