Proteins kinase Cɛ is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes - PubMed (original) (raw)

Proteins kinase Cɛ is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes

M C Caino et al. Oncogene. 2012.

Abstract

Protein kinase C (PKC)ɛ, a member of the novel PKC family, has key roles in mitogenesis and survival in normal and cancer cells. PKCɛ is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a short-hairpin RNA approach, here we established that PKCɛ is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCɛ-selective inhibitor peptide, ɛV1-2, reduced xenograft growth in mice. Both RNA interference depletion and pharmacological inhibition of PKCɛ caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCɛ-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain-containing proteins and tumor necrosis factor ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis revealed that a vast majority of the genes changed in PKCɛ-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCɛ is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCɛ may represent an attractive therapeutic target for NSCLC.

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Figures

Fig. 1

Fig. 1. PKCε is required for the growth of NSCLC cells

A) PKCε expression was analyzed by Western blot in immortalized non-tumorigenic (HBEC3) and NSCLC-derived cell lines (H358, H441, H322 and A549). Cells lines were obtained from ATCC and grown as recommended by the provider. An anti-PKCε antibody (Santa Cruz) was used at a 1:1000 dilution. B) Cells were infected with shRNA lentiviruses for PKCε (MISSION shRNA Lentiviral Transduction particles, Sigma, NM_005400 ε#1, clone ID x-741s1c1; ε#2, clone ID x-375s1c1) followed by selection with puromycin (1–2 μg/ml). MISSION non-target shRNA Lentiviral Transduction particles (Sigma, SHC002V) were used as control (CTRL). Expression of PKCε in NSCLC stable cell lines is depicted. C) Cells (5 ×104) were seeded in 12-well plates, allowed to grow in 2% FBS and counted 48 h later using a hemocytometer. D) For liquid colony formation assays cells were plated in 100 mm plates (100 cells/plate for H358 and 1000 cells/plate for A549). Medium was replaced twice a week and after 15 days colonies were stained with 0.7% methylene blue in 50% ethanol. E) To evaluate anchorage-independent growth 3 ×103 cells were plated in 0.35% agar over a 0.5% agar layer. After 10 days the plates were stained with MTS. For each well, the number of colonies was counted in 5 different fields and averaged. In all cases, data are expressed as mean ± S.E.M. of 3 individual experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

Fig. 2

Fig. 2. PKCε is required for NSCLC tumor growth in athymic nude mice

A) H358 cells expressing shRNA control (CTRL) or PKCε (ε#1 and ε#2) at 80% confluency were resuspended in serum-free medium, and then 0.1 ml containing 5 ×106 cells were injected s.c. into the flank of male athymic nude-Foxn1nu mice (Harlan Laboratories). The width and length of tumors were measured with a caliper at different times, and tumor volume calculated as Vol=π x width2 x length/6. Inset, PKCε levels at the day of inoculation. Data are expressed as mean ± S.E.M. (n=10). *, p<0.05; **, p<0.01; ***, p<0.001. A second experiment gave similar results. B) Tumors were removed and processed for immunohistochemistry, 15 days post-inoculation. Upper panels, H&E staining, lower panels, TUNEL labeling. C) H358 cells were injected s.c. in the flank of athymic mice (5 ×106 cells/mice). When tumors reached ~100 mm3 (~ 20 days post-inoculation) animals were randomized into two groups and subject to treatment with either control carrier peptide (TAT) or εV1-2 with TAT (18 mg/kg/day). Peptide delivery was achieved by weekly subcutaneous implantation of osmotic minipumps in the opposite flank. Tumor volume was expressed as the mean ± S.E.M. (n=8). *, p<0.05; **, p<0.01. A second experiment gave similar results. D) Tumors were removed 15 days after the beginning of treatment and stained for TUNEL.

Fig. 3

Fig. 3. Validation of representative PKCε target genes in NSCLC cells

H358 cells expressing shRNA control (CTRL) or shRNA for PKCε (ε#1 and ε#2) were assayed for expression of key apoptosis-related genes. A) For qPCR, RNA was isolated using the QIAGEN RNeasy kit and reverse transcribed to cDNA using random hexamers and the TaqMan Reverse Transcription kit (Applied Biosystems). Real-time PCR assays were performed in a 7300 ABI PCR System (Applied Biosystems), using TaqMan Gene expression assays and TaqMan Universal master mix. Human 18S rRNA was used as an endogenous control for normalization. The relative levels of mRNA compared to control were calculated according to the ΔCt method. B) Western blots and the corresponding densitometric analyses were carried out essentially as previously described (Oliva et al., 2008). The following primary antibodies were used: anti-Bik, anti-Bak, anti-caspase 3, anti-cIAP2, anti-Bcl2, anti-Bcl-XL (Cell Signaling, 1:1000 dilution), and anti-β-actin (Sigma, 1:50,000 dilution). Densitometric analysis of 3 independent experiments is shown. Data are expressed as the mean ± S.E.M. (n=3). *, p<0.05; **, p<0.01.

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