Table 1. Expression of PKCε-target genes in human NSCLC cells and specimens.
Simultaneous detection of apoptosis-related genes and 2 housekeeping genes by qPCR was carried out using RT2 Profiler PCR Array plates (QIAGEN) and a RT2 SYBR Green/5-carboxy-X-rhodamine (ROX) qPCR master mix. Data were normalized to GAPDH and β-actin housekeeping genes, and the relative levels of mRNA calculated according to the ΔCt method. Genes that changed expression > 1.5-fold as a consequence of PKCε depletion were selected for further analysis. Tumor 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. Human samples, data publicly available from the Oncomine repository. Lung adenocarcinomas (LAC) were compared against normal lung tissues (NL). Data for each gene were filtered for down-regulation in lung adenocarcinoma (LAC) vs. normal lung (NL) tissue with a p<0.05. Whenever possible, multiple studies containing information for each gene were compared by meta-analysis and an associated p value was determined. p values for single-gene (Single) or meta-analysis (Meta) are shown. To establish if PKCε-regulated genes are altered in lung cancer, we carried out a Gene Set Enrichment Analysis (GSEA), as previously described (Subramanian et al., 2005). A GSEA comparing LAC (n=139) vs. NL (n=17) was run for the defined PKCε-gene set using the complete dataset for the Bhattacharjee Study (Bhattacharjee et al., 2001). Enrichment in NL is indicated by “Yes”. ns, not significant.
| Gene Symbol | Tumor cells
|
Human samples
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| CTRL | PKCε-depleted | p-value | Oncomine p-values | GSEA (Yes/No) | |||||||
| mean | S.D. | n | mean | S.D. | n | Single gene | Meta-analysis | ||||
| Up-regulated | |||||||||||
| BAG3 | 1.01 | 0.09 | 4 | 1.63 | 0.13 | 4 | 0.0035 | * | 0.05 | N/A | No |
| BAG4 | 1.00 | 0.04 | 4 | 2.05 | 0.02 | 4 | p<0.0001 | *** | ns | N/A | No |
| BAK1 | 1.00 | 0.03 | 4 | 2.33 | 0.52 | 4 | 0.0219 | * | 0.04 | 0.04 | No |
| BCL10 | 1.01 | 0.11 | 4 | 3.10 | 0.70 | 4 | 0.013 | * | ns | N/A | No |
| BCL2A1 | 1.10 | 0.32 | 4 | 3.59 | 0.01 | 4 | p<0.0001 | *** | 6.23 ×10−5 | 0.02 | No |
| BCL2L10 | 1.07 | 0.27 | 4 | 2.99 | 0.86 | 4 | 0.0386 | * | 0.018 | N/A | No |
| BCL2L11 | 1.01 | 0.09 | 4 | 1.63 | 0.19 | 4 | 0.0124 | * | ns | N/A | No |
| BIK | 1.10 | 0.32 | 4 | 8.76 | 0.24 | 4 | p<0.0001 | *** | ns | N/A | No |
| BIRC2 | 1.09 | 0.31 | 4 | 1.53 | 0.07 | 4 | 0.1038 | ns | 1.42 ×10−4 | 3.4 ×10−4 | Yes |
| BIRC4 | 1.01 | 0.08 | 4 | 1.56 | 0.04 | 4 | 0.0004 | *** | ns | N/A | No |
| BIRC8 | 1.34 | 0.63 | 4 | 5.35 | 2.55 | 4 | 0.0889 | ns | N/A | N/A | No |
| BNIP3 | 1.01 | 0.10 | 4 | 2.44 | 0.29 | 4 | 0.0017 | ** | ns | N/A | No |
| BNIP3L | 1.02 | 0.16 | 4 | 1.99 | 0.15 | 4 | 0.0022 | ** | 1.58 ×10−5 | 0.004 | Yes |
| BRAF | 1.01 | 0.08 | 4 | 3.34 | 0.35 | 4 | 0.0003 | *** | ns | N/A | No |
| NOD1 | 1.00 | 0.06 | 4 | 1.53 | 0.19 | 4 | 0.0193 | * | ns | N/A | No |
| CASP1 | 1.06 | 0.25 | 4 | 6.47 | 2.82 | 4 | 0.0524 | ns | 1.18 ×10−5 | 1.74 ×10−8 | Yes |
| CASP2 | 1.01 | 0.10 | 4 | 1.59 | 0.35 | 4 | 0.0808 | ns | 0.008 | N/A | No |
| CASP3 | 1.00 | 0.06 | 4 | 2.80 | 0.21 | 4 | p<0.0001 | *** | ns | N/A | No |
| CASP4 | 1.09 | 0.31 | 4 | 7.05 | 1.02 | 4 | 0.0007 | *** | 6.22 ×10−6 | 0.005 | Yes |
| CASP5 | 1.00 | 0.03 | 4 | 1.69 | 0.51 | 4 | 0.1136 | ns | 3.13 ×10−4 | 0.015 | No |
| CASP6 | 1.02 | 0.15 | 4 | 2.91 | 0.58 | 4 | 0.0095 | ** | ns | N/A | No |
| CD40 | 1.22 | 0.50 | 4 | 9.33 | 0.53 | 4 | p<0.0001 | *** | 1.17 ×10−4 | 0.019 | Yes |
| CD40LG | 1.00 | 0.00 | 4 | 1.66 | 0.51 | 4 | 0.1238 | ns | 0.011 | N/A | No |
| CIDEA | 1.34 | 0.63 | 4 | 3.00 | 0.80 | 4 | 0.0768 | ns | ns | N/A | No |
| CIDEB | 1.00 | 0.06 | 4 | 1.56 | 0.09 | 4 | 0.0011 | *** | 0.049 | N/A | No |
| DFFA | 1.00 | 0.03 | 4 | 1.58 | 0.01 | 4 | p<0.0001 | *** | 0.031 | N/A | No |
| FAS | 1.00 | 0.05 | 4 | 1.63 | 0.05 | 4 | p<0.0001 | *** | 1.05 ×10−7 | 6.32 ×10−4 | Yes |
| FASLG | 1.00 | 0.06 | 4 | 4.26 | 0.43 | 4 | 0.0001 | *** | 0.028 | 0.038 | No |
| GADD45A | 1.00 | 0.04 | 4 | 3.44 | 0.01 | 4 | p<0.0001 | *** | ns | N/A | No |
| HRK | 1.00 | 0.01 | 4 | 2.67 | 0.87 | 4 | 0.0513 | ns | 0.035 | N/A | No |
| LTA | 1.18 | 0.44 | 4 | 5.02 | 0.20 | 4 | 0.0001 | *** | 0.006 | N/A | No |
| MCL1 | 1.04 | 0.21 | 4 | 2.66 | 0.13 | 4 | 0.0003 | *** | 2.36 ×10−6 | 9.52 ×10−4 | Yes |
| NOL3 | 1.07 | 0.27 | 4 | 2.95 | 0.06 | 4 | 0.0002 | *** | ns | N/A | No |
| TNF | 1.21 | 0.49 | 4 | 2.23 | 1.17 | 4 | 0.2261 | ns | 9.79 ×10−5 | 0.006 | Yes |
| TNFRSF10A | 1.00 | 0.06 | 4 | 1.86 | 0.44 | 4 | 0.0509 | ns | N/A | N/A | No |
| TNFRSF21 | 1.00 | 0.06 | 4 | 2.33 | 0.04 | 4 | p<0.0001 | *** | ns | N/A | No |
| TNFRSF25 | 1.07 | 0.27 | 4 | 7.22 | 1.96 | 4 | 0.0105 | * | 0.016 | N/A | No |
| TNFSF10 | 1.16 | 0.42 | 4 | 4.82 | 1.04 | 4 | 0.0085 | ** | 1.05 ×10−11 | 2.17 ×10−4 | Yes |
| TNFSF8 | 1.00 | 0.04 | 4 | 2.42 | 0.36 | 4 | 0.0038 | ** | 0.01 | 0.013 | No |
| TP53BP2 | 1.00 | 0.01 | 4 | 1.55 | 0.08 | 4 | 0.0002 | *** | 0.001 | 0.001 | No |
| TP73 | 1.01 | 0.10 | 4 | 2.21 | 0.51 | 4 | 0.0296 | * | ns | N/A | No |
| TRADD | 1.00 | 0.02 | 4 | 1.65 | 0.02 | 4 | p<0.0001 | *** | 7.26 ×10−5 | N/A | No |
| TRAF4 | 1.00 | 0.03 | 4 | 2.60 | 0.57 | 4 | 0.0152 | * | ns | N/A | No |
| B2M | 1.02 | 0.15 | 4 | 2.55 | 0.01 | 4 | p<0.0001 | *** | 4.1 ×10−5 | 2.91 ×10−4 | Yes |
| Down-regulated | |||||||||||
| BCL2 | 1.16 | 0.42 | 4 | 0.09 | 0.04 | 4 | 0.0216 | * | 0.013 | 0.029 | N/A |
| BIRC3 | 1.10 | 0.33 | 4 | 0.18 | 0.04 | 4 | 0.0163 | * | 6.57 ×10−4 | 0.017 | N/A |
| CARD6 | 1.00 | 0.01 | 4 | 0.60 | 0.16 | 4 | 0.0214 | * | N/A | N/A | N/A |
| IGF1R | 1.01 | 0.09 | 4 | 0.64 | 0.04 | 4 | 0.004 | ** | 0.021 | N/A | N/A |
| PYCARD | 1.00 | 0.07 | 4 | 0.01 | 0.00 | 4 | p<0.0001 | *** | ns | N/A | N/A |
| TNFRSF11B | 1.09 | 0.31 | 4 | 0.53 | 0.20 | 4 | 0.0907 | ns | ns | N/A | N/A |
| TNFRSF1A | 1.00 | 0.06 | 4 | 0.69 | 0.22 | 4 | 0.1076 | ns | ns | N/A | N/A |
| CD27 | 1.11 | 0.34 | 4 | 0.30 | 0.17 | 4 | 0.0381 | * | 2.83 ×10−4 | 6.39 ×10−4 | N/A |
| TNFRS9 | 1.12 | 0.36 | 4 | 0.55 | 0.14 | 4 | 0.0929 | ns | 2.34 ×10−4 | 2.34 ×10−4 | N/A |
| CD70 | 1.05 | 0.22 | 4 | 0.10 | 0.02 | 4 | 0.0025 | ** | 0.002 | 0.012 | N/A |
| RPL13A | 1.00 | 0.01 | 4 | 0.61 | 0.21 | 4 | 0.0559 | ns | 0.003 | 0.002 | N/A |