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


Clin Lung Cancer. 2010;11(3) 

In This Article

Abstract and Introduction


Background: Protein kinase C-β2 (PKCβ2) is a splice-variant of the PRKCB1 gene and belongs to a family of serine/threonine-specific kinases that are predominantly activated by diacylglycerol, calcium, and phorbol ester. Cellular functions associated with PKCβ2 activation include transformation, proliferation, and inhibition of apoptosis. Enzastaurin (LY317615) is an oral, selective, potent inhibitor of the PKCβ2 kinase. Preclinical activity for this agent was predominantly reported in lymphoma, glioblastoma, and colorectal cancer. In patients with advanced non–small-cell lung cancer (NSCLC) whose previous therapy had failed, 13% of patients had disease control for 6 months with single-agent therapy.
Patients and Methods: We investigated whether biologically relevant variants of PRKCB1 exist in lung cancer cell lines in the context of enzastaurin-induced proliferation and kinase inhibition, using exon sequencing, immunoblotting, and cytotoxicity assays in NSCLC and small-cell lung cancer (SCLC) cell lines.
Results: We discovered a total of 6 single-nucleotide variants, but only 1 resulted in an amino acid substitution (T40I). This substitution was not located in the kinase domain of PKCβ2 and did not affect enzastaurin's antiproliferative or phosphorylation-inhibitory activity. We found enzastaurin to be equally active in NSCLC and SCLC cell lines, with values of the 50% inhibitory concentration in a range of 0.05–0.2 μM.
Conclusion: The inhibition of phosphorylation of PKCβ2 and the downstream molecules glycogen synthase kinase-3β, S6RP, Akt, and forkhead transcription factor was evident in the same concentration range, which suggests the premise 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.


The protein kinase C (PKC) family of proteins is characterized by serine/threonine-specific kinase activity, triggered predominantly by diacylglycerol and calcium and by the tumor-promoting agent phorbol ester. They are encoded by 9 different genes (α,βγ,δ,ε,ζ η, θ, and ι) located on distinct chromosomal segments, and isoforms have been described as a result of alternative splicing. Their function is the phosphorylation of proteins involved in a variety of signal-transduction pathways, and several different family members display tissue-specific expression profiles.[1]

The PRKCB1 gene, located on chromosome segment 16p11.2, comprises 18 exons and encodes PKCβ, which occurs in 2 isoforms, β1 and β2. The isoforms are a result of the differential use of exons 17 and 18 through alternative splicing of the mRNA. Isoform β1 (NP_997700), which uses exon 18, has a shorter C-terminus than isoform β2 (NP_002729), which uses exon 17 (NCBI Gene Database, identification number 5579). Cellular functions associated with PKCβ activation include transformation, proliferation, inhibition of apoptosis,[2–4] and activation of Akt (protein kinase B, PKB) pathways.[5,6] The activation of Akt, which can be triggered by a loss of phosphatase and tensin homologue (PTEN), is associated with poor prognoses in non–small-cell lung cancer (NSCLC).[7,8]

Enzastaurin (LY317615) is an oral serine/threonine kinase inhibitor that targets the PKC and Akt pathways, and inhibits the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and S6 ribosomal protein (S6RP).[6] The antitumor activity of enzastaurin was indicated in experimental models of several human malignancies.[6] In a phase II single-agent trial of oral enzastaurin (500 mg per day) in 55 patients with advanced-stage NSCLC whose previous chemotherapy had failed, a 6-month progression-free survival rate of 13% was reported.[9]

For a better definition of the group of patients who may benefit from enzastaurin, we studied the PRKCB1 gene for sequence variations, assessed these variations' potential effects on enzastaurin efficacy, and investigated the relationship between the inhibition of PKCβ pathway phosphorylation and proliferation in lung cancer cell lines.