Meta-analysis of oncogenic protein kinase Ciota signaling in lung adenocarcinoma

Abstract

Purpose: Atypical protein kinase Ciota (PKCiota) is an oncogene in non-small cell lung cancer (NSCLC). Here, we identify four functional gene targets of PKCiota in lung adenocarcinoma (LAC), the most prominent form of NSCLC.

Experimental design: Three independent public domain gene expression data sets were interrogated to identify genes coordinately expressed with PKCiota in primary LAC tumors. Results were validated by QPCR in an independent set of primary LAC tumors. RNAi-mediated knockdown of PKCiota and the target genes was used to determine whether expression of the identified genes was regulated by PKCiota, and whether these target genes play a role in anchorage-independent growth and invasion of LAC cells.

Results: Meta-analysis identified seven genes whose expression correlated with PKCiota in primary LAC. Subsequent QPCR analysis confirmed coordinate overexpression of four genes (COPB2, ELF3, RFC4, and PLS1) in an independent set of LAC samples. RNAi-mediated knockdown showed that PKCiota regulates expression of all four genes in LAC cells, and that the four PKCiota target genes play an important role in the anchorage-independent growth and invasion of LAC cells. Meta-analysis of gene expression data sets from lung squamous cell, breast, colon, prostate, and pancreas carcinomas, as well as glioblastoma, revealed that a subset of PKCiota target genes, particularly COPB2 and RFC4, correlate with PKCiota expression in many tumor types.

Conclusion: Meta-analysis of public gene expression data are useful in identifying novel gene targets of oncogenic PKCiota signaling. Our data indicate that both common and cell type-specific signaling mechanisms contribute to PKCiota-dependent transformation.

Fig. 1

Identification of gene coordinately expressed with PKCι in LAC. A, expression of seven genes exhibiting coordinate expression with PKCι in primary LAC tumors. Seven genes identified through meta-analysis of public domain gene expression data from LAC cases (see Table 1) were analyzed for overexpression in 15 primary LAC tumors. Four of the seven genes are significantly overexpressed in LACs when compared with matched normal controls. mRNA abundance was measured by QPCR and expressed as fold-change (tumor/normal). Boxes, upper and lower quartile values; line within each box, median value. *, significant difference between tumor and normal with a P value of <0.05. NS, not significant. B, the abundance of each of the indicated genes was determined by QPCR in 60 primary LAC and matched control samples and expressed as fold change (tumor/normal). The 60 samples were rank ordered and binned into upper tertile (H, high PKCι) and lower tertile (L, low PKCι) based on PKCι expression. The data represent the expression of each gene in the high and low PKCι groups. Columns, mean; bars, SE. *, statistically significant difference in expression of the indicated gene in the low PKCι group when compared with the high PKCι group; P < 0.05.

Fig. 2

RNAi-mediated knockdown of PKCι inhibits anchorage-independent growth of LAC cell lines in soft agar. A549, H358, and H1437 LAC cell lines were stably transfected with either a nontarget (NT) lentiviral RNAi construct of a PKCι-RNAi construct. A, expression of PKCι mRNA in NT and PKCι-RNAi cells. *, statistically significant decrease in PKCι mRNA abundance when compared with NT control. Data are expressed as % NT control; n = 4; P < 0.05. B, PKCι-RNAi leads to selective knockdown of PKCι protein expression. Immunoblot analysis of total cell lysates from A549, H358, and H1437 treated with NT or PKCι-RNAi for PKCα, PKCι, PKCζ, PKCδ, PKCε, and actin. C, RNAi-mediated knockdown of PKCι inhibits anchorage-independent growth of LAC cell lines. Anchorage-independent growth in soft agar was assessed as described previously (6). Columns, mean (n = 4); bars, SE. Data are expressed as % NT control. *, statistically significant difference from NT control; P < 0.05.

Fig. 3

PKCι regulates the expression of COPB2, ELF3, PLS1, and RFC4 in LAC cells. A549, H358, and H1437 cells transfected with either NT or PKCι-RNAi were analyzed for expression of the four genes that are coordinately overexpressed with PKCι in LAC tumors. RNAi-mediated knockdown of PKCι causes a statistically significant decrease in the expression of COPB2 (A), ELF3 (B), RFC4 (C), and PLS1 (D) in all three LAC cell lines. Data are expressed as %NT control; columns, mean (n = 4); bars, SE. *, statistically significant difference when compared with the corresponding NT control. P < 0.05.

Fig. 4

COPB2, ELF3, PLS1, and RFC4 are functionally important for LAC cell transformation. A549 cells were stably transfected with multiple independent RNAi constructs targeting COPB2 (A), ELF3 (B), PLS1 (C), RFC4 (D), or a NT control RNAi construct. Target gene mRNA abundance was determined by QPCR in A549 cells expressing NT or target gene RNAi. Columns, mean (n = 4) and are presented as % NT control; bars, SE. *, statistically significant difference when compared with NT control cells. P < 0.05. The effect of target RNAi on anchorage-independent growth in soft agar and cellular invasion through Matrigel-coated chambers was assessed as previously described (13). Data are presented as % NT control; columns, mean (n = 4); bars, SE. *, statistically significant difference from NT control; P < 0.05.