Protein Kinase C-β Gene Variants, Pathway Activation, and Enzastaurin Activity in Lung Cancer

Sang-Haak Lee; Tingan Chen; Jun Zhou; Jennifer Hofmann; Gerold Bepler

Disclosures

Clin Lung Cancer. 2010;11(3) 

In This Article

Discussion

The discovery of tumor-promoting signal-transduction molecules and pathways invigorated the development of targeted agents, with hopes of reducing cancer-related morbidity and mortality. Because it is the leading cause of cancer deaths in the United States,[10] lung cancer is one of the leading disease sites for this development. This is exemplified by the approval and implementation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors as active therapeutic agents. The successful implementation of these agents into clinical practice was substantially facilitated by the identification of subgroups of patients with favorable efficacy characteristics. Most notable among these characteristics was the discovery of mutations in the EGFR gene that render tumors exquisitely sensitive to blockades of EGFR-mediated signal transduction.[11–14] Many other signal-transduction pathways have been targeted for drug development, and novel inhibitors are undergoing clinical testing. Among these is enzastaurin, a selective and potent (IC50 of 6 nM) ATP-binding inhibitor of the serine/threonine kinase PKCβ2.[15] The preclinical activity of this agent was predominantly reported in lymphoma, glioblastoma, and colorectal cancer.[6,16] However, in patients with advanced NSCLC whose previous therapy had failed, the efficacy of this agent was marginal, with only 7 of 55 (13%) patients showing no disease progression 6 months after initiation of treatment.[9] Moreover, two studies presented at the 2009 meeting of the American Society of Clinical Oncology did not show additional efficacy when enzastaurin was added to a combination of carboplatin and pemetrexed or carboplatin, pemetrexed, and bevacizumab as first-line therapy in patients with NSCLC.[17,18]

We sequenced the complete coding region of the PRKCB1 gene in 28 lung cancer cell lines and found only one sequence variation that resulted in an amino acid substitution (T40I) in exon 1. This variation was evident in two SCLC cell lines, in the conserved region 1, which is a cysteine-rich zinc-binding domain. However, position 40 has not been implicated in zinc, diacylglycerol, or phorbol ester binding (NP_002729.2). The T40I substitution did not result in an obvious alteration of enzastaurin on PKCβ2 pathway activation or proliferation inhibition. This lack of effect on efficacy is not surprising, because the catalytic domain of PKCβ2 encompasses amino acids 341–663, including the ATP-binding site. We found no evidence for PKCβ2 deletions, insertions, truncations, or other putatively activating mutations in the cell lines examined. Because we limited our sequencing efforts to 28 cell lines, we cannot rule out the existence of PKC mutations with a potentially meaningful biologic effect in a small subset of lung cancers. Examples of a lowfrequency gene mutation with biologic and potentially clinical effects are found in the recently described EML4-Alk fusions, which have an estimated frequency of < 5% in NSCLC.[19] However, the clinical experience with enzastaurin in lung cancer to date does not support the existence of a hypersensitive population of patients, a finding consistent with our PRKCB1 gene sequencing data.

Our results demonstrated the concentration-dependent proliferation inhibition and suppression of PKC®2 kinase and pathway activity in NSCLC and SCLC cell lines with IC50 values in the 0.05–0.2-μM range, which are lower than the previously reported IC50 values in the 1.0-μM range in glioblastoma (U87MG), colorectal (HCT116), and prostate cancer (PC3) cell lines.[6] These concentrations are also lower than those reported for lung cancer cell lines with IC50 values in the 1–10-μM range (A549; approximately 2–3 μM).[20,21] This is most likely a result of differences in the reagents, cell densities, and exposure durations used for assessments of proliferation. In our studies, the drug concentrations required for the phosphorylation inhibition of PKCβ2 and the downstream molecules GSK3β, S6RP, Akt, and FKHR and proliferation inhibition are comparable (Table 3), which is consistent with previous data in colorectal cancer.[6] Our IC50 concentrations were below the steadystate plasma enzastaurin concentrations reported in clinical trials.[20] Our results suggest that a PRKCB1 gene variant (T40I) exists, and it does not affect enzastaurin's antiproliferative or phosphorylation- inhibitory activity. In addition, enzastaurin appears to be equally active in NSCLC and SCLC cell lines, with IC50 values in the range of 0.05–0.2 μM. The inhibition of phosphorylation of PKCβ2 and the downstream molecules GSK3β, S6RP, Akt, and FKHR is evident in the same concentration range, which suggests that the determination of phosphorylation levels of these molecules in human tissue specimens may be a useful pharmacodynamic parameter for in vivo target inhibition by enzastaurin. In a limited number of pretreatment tumor samples from pancreatic cancer patients treated with enzastaurin and gemcitabine, no statistically significant association between drug efficacy and biomarker levels was evident.[20] To our knowledge, a reduction in PKCβ2 phosphorylation has not been described in patients' specimens during therapy. However, in xenograft-bearing mice receiving enzastaurin, a reduction in GSK3βphosphorylation was described.[21] It is thus important to determine prospectively if a reduction in target phosphorylation by enzastaurin is a clinically useful predictive marker of therapeutic efficacy.

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