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Protein kinase C? is a therapeutic target in malignant melanoma with NRAS mutation.

ABSTRACT: NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase C? (PKC?). Here, we report that PKC? inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKC? were designed as chimeric hybrids of two naturally occurring PKC? inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated and validated is that combining two domains of two naturally occurring PKC? inhibitors into a chimeric or hybrid structure retains biochemical and biological activity and improves PKC? isozyme selectivity. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKC?, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKC? inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKC? inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKC? inhibition effectively induced cytotoxicity in BRAF mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKC? in patients who have relapsed following treatment with BRAF inhibitors. Taken together, the present work demonstrates that inhibition of PKC? by novel small molecule inhibitors causes caspase-dependent apoptosis mediated via the JNK-H2AX pathway in melanomas with NRAS mutations or BRAF inhibitor resistance.

SUBMITTER: Takashima A

PROVIDER: S-EPMC4160068 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Protein kinase Cδ is a therapeutic target in malignant melanoma with NRAS mutation.

Takashima Asami A English Brandon B Chen Zhihong Z Cao Juxiang J Cui Rutao R Williams Robert M RM Faller Douglas V DV

ACS chemical biology 20140218 4

NRAS is the second most frequently mutated gene in melanoma. Previous reports have demonstrated the sensitivity of cancer cell lines carrying KRAS mutations to apoptosis initiated by inhibition of protein kinase Cδ (PKCδ). Here, we report that PKCδ inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKCδ were designed as chimeric hybrids of two naturally occurring PKCδ inhibitors, staurosporine and rottlerin. The specific hypothesis interrogated a ...[more]

PMID: 24506253

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Project description:Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival; however, relapses are common. A number of receptor tyrosine kinases (RTKs) including EGFR and MET have been reported to be involved in CMM metastasis and in the development of resistance to therapy, targeting the mitogen-activated protein kinase (MAPK pathway). IHC analysis showed that patients with higher MET protein expression had a significantly shorter overall survival. In addition, silencing of MET caused an upregulation of EGFR and p-AKT, which was abrogated by concomitant silencing of MET and EGFR in CMM cells resistant to MAPK-targeting drugs. We therefore explored novel treatment strategies using clinically approved drugs afatinib (ERBB family inhibitor) and crizotinib (MET inhibitor), to simultaneously block MET and ERBB family RTKs. The effects of the combination were assessed in cell culture and spheroid models using established CMM and patient-derived short-term cell lines, and an in vivo xenograft mouse model. The combination had a synergistic effect, promoting cell death, concomitant with a potent downregulation of migratory and invasive capacity independent of their BRAF/NRAS mutational status. Furthermore, the combination attenuated tumor growth rate, as ascertained by the significant reduction of Ki67 expression and induced DNA damage in vivo. Importantly, this combination therapy had minimal therapy-related toxicity in mice. Lastly, the cell cycle G2 checkpoint kinase WEE1 and the RTK IGF1R, non-canonical targets, were altered upon exposure to the combination. Knockdown of WEE1 abrogated the combination-mediated effects on cell migration and proliferation in BRAF mutant BRAF inhibitor-sensitive cells, whereas WEE1 silencing alone inhibited cell migration in NRAS mutant cells. In summary, our results show that afatinib and crizotinib in combination is a promising alternative targeted therapy option for CMM patients, irrespective of BRAF/NRAS mutational status, as well as for cases where resistance has developed towards BRAF inhibitors.

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Protein kinase C? is a therapeutic target in malignant melanoma with NRAS mutation.

Publications

Protein kinase Cδ is a therapeutic target in malignant melanoma with NRAS mutation.

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