Enzastaurin has anti-tumour effects in lung cancers with overexpressed JAK pathway molecules

Abstract

Background: Enzastaurin, an oral serine-threonine kinase inhibitor, was initially developed as an ATP-competitive selective inhibitor against protein kinase Cβ. However, the mechanism by which enzastaurin contributes to tumourigenesis remains unclear.

Methods: We analysed the anti-tumour effects of enzastaurin in 22 lung cancer cell lines to ascertain the potential for enzastaurin-based treatment of lung cancer. To identify molecules or signalling pathways associated with this sensitivity, we conducted a gene, receptor tyrosine kinases phosphorylation and microRNA expression profiling study on the same set of cell lines.

Results: We identified eight genes by pathway analysis of molecules having gene-drug sensitivity correlation, and used them to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. Pathway analysis revealed that the JAK/STAT signalling pathway was one of the main ones involved in sensitivity to enzastaurin. Overexpression of JAK1 was observed in the sensitive cells by western blotting. Simultaneous administration of enzastaurin and JAK inhibitor inhibited enzastaurin-induced cell growth-inhibitory effect. Furthermore, lentiviral-mediated JAK1-overexpressing cells were more sensitive to enzastaurin than control cells.

Conclusion: Our results suggested that the JAK1 pathway may be used as a single predictive biomarker for enzastaurin treatment. The anti-tumour effect of enzastaurin should be evaluated in lung cancer with overexpressed JAK pathway molecules.

Figure 1

IC₅₀ values for 22 lung cancer cell lines responding to enzastaurin treatment by MTS assay. According to sensitivity to enzastaurin, these 22 cell lines were classified as sensitive (IC₅₀ of ⩽10 μM) or resistant (IC₅₀ of >50 μM).

Figure 2

Sixteen genes associated with enzastaurin response were established by pathway analyses and prediction of drug response using an eight-gene signature. (A) Sixteen genes (blue circle) associated with enzastaurin response and PKC (red circle) belonged to the same signal pathway. (B) Principal component analysis based on the eight-gene profile correctly distinguished the sensitive cells from the resistant ones. The colour reproduction of this figure is available at the British Journal of Cancer online.

Figure 3

JAK1, VEGFR2, VEGFR3 and miR-21 were correlated with drug response. (A and B) JAK1 and STAT3 gene expression levels were significantly higher in the sensitive cell group than in the resistant cell group. (C and D) Elevated levels of VEGFR2 and VEGFR3 expression were observed in sensitive cells. (E) Expression of MIRN21/TMEM49 was significantly higher in sensitive cells than in resistant cells, by gene-chip analysis. (F) Mature miR-21 expression was significantly higher in sensitive cells than in resistant cells by quantitative RT–PCR analysis. (G) Quantitative comparison of miR-21 and JAK1 showed a significant positive correlation between these two molecules. ^**P<0.05 when compared with the resistant cells. ^***P<0.01 when compared with the resistant cells.

Figure 4

Effect of combination therapy with enzastaurin and JAK1 expression on cell growth in lung cancer cells. (A) JAK1 expression levels were significantly higher in the sensitive cell group than in the resistant cell group, by western blotting. (B) Completed inhibition of JAK1/STAT signalling by JAK1 inhibitor in A549 cells. P-STAT3 was completely inhibited until 72 h after the treatment of 1 μM JAK inhibitor. (C) Enzastaurin treatment with JAK inhibitor for 72 h was examined in A549 cells. Each result is expressed as cell viability in treated samples compared with the untreated sample (100%) for enzastaurin alone and concurrent therapy with the 1 μM JAK inhibitor treatment. (D) The effect of JAK inhibitor treatment (1 μM) for 24 h followed by enzastaurin treatment for 72 h was examined in A549 cells. (E) Lentiviral-mediated production of JAK1 in A549 cells. Western blotting showed that JAK1 expression levels were significantly higher in two LV-JAK1 clones than in the control clones. (F) Enzastaurin treatment for 72 h was examined in LV-JAK1-A549 cells. Each result is expressed as cell viability in the treated samples compared with the untreated sample (100%) for enzastaurin therapy.

Figure 5

Association between JAK1 and miR-21 expression. (A) p-STAT3 was overexpressed after IL-6 stimulation of A549 cells for 24 h. (B) After IL-6 stimulation, miR-21 expression was significantly increased, as measured by qRT–PCR analysis. (C) MiR-21 expression of two LV-JAK1 cells was significantly higher than in the control cells, as measured by qRT–PCR analysis. Data were mean±s.d. from three independent experiments. ^*P<0.05 when compared with the respective parent cells.