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. 2019 Apr 15;9(1):6041.
doi: 10.1038/s41598-019-42581-4.

Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes

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Differential Regulation of Gene Expression in Lung Cancer Cells by Diacyglycerol-Lactones and a Phorbol Ester Via Selective Activation of Protein Kinase C Isozymes

Mariana Cooke et al. Sci Rep. .
Free PMC article

Abstract

Despite our extensive knowledge on the biology of protein kinase C (PKC) and its involvement in disease, limited success has been attained in the generation of PKC isozyme-specific modulators acting via the C1 domain, the binding site for the lipid second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. Synthetic efforts had recently led to the identification of AJH-836, a DAG-lactone with preferential affinity for novel isozymes (nPKCs) relative to classical PKCs (cPKCs). Here, we compared the ability of AJH-836 and a prototypical phorbol ester (phorbol 12-myristate 13-acetate, PMA) to induce changes in gene expression in a lung cancer model. Gene profiling analysis using RNA-Seq revealed that PMA caused major changes in gene expression, whereas AJH-836 only induced a small subset of genes, thus providing a strong indication for a major involvement of cPKCs in their control of gene expression. MMP1, MMP9, and MMP10 were among the genes most prominently induced by PMA, an effect impaired by RNAi silencing of PKCα, but not PKCδ or PKCε. Comprehensive gene signature analysis and bioinformatics efforts, including functional enrichment and transcription factor binding site analyses of dysregulated genes, identified major differences in pathway activation and transcriptional networks between PMA and DAG-lactones. In addition to providing solid evidence for the differential involvement of individual PKC isozymes in the control of gene expression, our studies emphasize the importance of generating targeted C1 domain ligands capable of differentially regulating PKC isozyme-specific function in cellular models.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Gene expression profiling of PMA, AJH-836 and AJH-1512 in A549 lung cancer cells. (A) Structures of DAG-lactones AJH-836 and AJH-1512. (B) Multidimensional scaling plot showing the distance of each sample from each other determined by their RNA-Seq profiles. The leading logFC is the distance metric used in both dimensions and represents the average (root mean square) of the largest absolute logFC between each pair of samples. (C) Heatmap of 1772 deregulated genes in A549 cells subjected to the different treatments (Fold-change > 2; FDR < 0.05). The most significant transcript isoform for each gene was employed for heatmap visualization. The color scale at the bottom of the heatmap is used to represent expression level (green, low expression; red, high expression). (D) Number of genes differentially up-regulated and down-regulated among compounds. (E) Venn diagram of transcripts commonly modulated among treatments.
Figure 2
Figure 2
Comparative gene expression signatures among compounds and their modulated signaling pathways. (A) Top 150 PMA up-regulated genes and their overlap with the AJH-836 and AJH-1512 up-regulated genes. (B) Percentage of genes commonly modulated by AJH-836 (left pie charts) and AJH-1512 (right pie charts) in comparison to the PMA signature (all PMA-regulated genes), the top 50, and the top 12 PMA regulated genes. (C) Functional enrichment analysis of differentially expressed genes among compounds.
Figure 3
Figure 3
Functional analysis of PMA-regulated genes. (A) Network of functionally enriched pathways and genes differentially expressed by PMA treatment relative to vehicle. Red, up-regulated genes. (B) Comparative enrichment analysis of putative transcription factor binding sites (TFBS) across the promoter of PMA-regulated genes, using three different resources (InnateDB, oPOSSUM and Enrichr). (C) Heatmap comparing the PMA up- and down-regulated genes with those in human lung adenocarcinomas, using the TCGA Lung Adenocarcinoma (LUAD) dataset. (D) Up-regulation of MMP1, MMP9 and MMP10 genes in human lung adenocarcinomas relative to normal tissue, according to the TCGA Lung Adenocarcinoma (LUAD) dataset (p < 0.001).
Figure 4
Figure 4
Differential induction of genes by PMA and AJH-836. Serum-starved A549 cells were treated with either PMA (0.1 μM) or AJH-836 (1 μM) for 1 h. RNA was extracted 3 h later, reversed transcribed to cDNA, and used for Q-PCR analysis for the indicated genes. Results are expressed as fold-induction relative to cells treated with vehicle. Data represents the mean ± S.E.M. of 5 independent experiments. *p < 0.05; **p < 0.01 vs. PMA.
Figure 5
Figure 5
Effect of PKC isozyme RNAi on PMA-induced expression of metalloprotease genes. A549 cells were transfected with RNAi duplexes for PKCα, PKCδ or PKCε. Twenty-four h later cells were serum starved for an extra 24 h period, and then stimulated with PMA (0.1 μM), AJH-836 (1 μM) or vehicle for 1 h. RNA was extracted 3 h later and reversed transcribed to cDNA. (A) Representative Western blot for PKC isozymes subjected to RNAi depletion. (B) Effect of PKC isozyme RNAi on basal levels of MMP1, MMP9 and MMP10 as determined by Q-PCR. PMA was included as a positive control for induction. Results are expressed as fold-induction relative to cells transfected with non-target control (NTC) RNAi duplex. Data represents the mean ± S.E.M. of 3 independent experiments. (C) Effect of PKC isozyme RNAi depletion on the induction of MMP1, MMP9 and MMP10 by PMA. Results are expressed as percentage of response relative to NTC (dotted line). Data represents the mean ± S.E.M. of 3–5 independent experiments. (D) Metalloprotease gene induction by PMA was determined in the presence of either 3 μM GF109203X (GF) or Gö6976 (), added 30 min before and during PMA treatment. Data represents the mean ± S.E.M. of 3 independent experiments. Full-length blots are presented in Supplementary Fig. 4. (E) Effect of PKC isozyme RNAi depletion on the induction of MMP1, MMP9 and MMP10 by AJH-836. Results are expressed as percentage of response relative to NTC (dotted line). Data represents the mean ± S.E.M. of 3–5 independent experiments.
Figure 6
Figure 6
Effect of PKC isozyme RNAi depletion on PMA- and AJH-836-induced expression of FST and CCL20. A549 cells were transfected with RNAi duplexes for PKCα, PKCδ or PKCε. Twenty-four h later cells were serum starved for an extra 24 h period, and then stimulated with either PMA (0.1 μM) or vehicle for 1 h. RNA was extracted 3 h later and reversed transcribed to cDNA. (A) Effect of PKC isozyme RNAi on basal levels of FST and CCL20 as determined by Q-PCR. PMA was included as a positive control for induction. Results are expressed as fold-induction relative to cells transfected with non-target control (NTC) RNAi duplex. Data represents the mean ± S.E.M. of 3 independent experiments. (B) Effect of PKC isozyme RNAi depletion on induction of FST by PMA and AJH-836. Results are expressed as percentage of response relative to NTC (dotted line). Data represents the mean ± S.E.M. of 3–5 independent experiments (C) Effect of PKC isozyme RNAi depletion on induction of CCL20 by PMA and AJH-836. Results are expressed as percentage of response relative to NTC (dotted line). Data represents the mean ± S.E.M. of 3–5 independent experiments.
Figure 7
Figure 7
PKCα induces MMP-9 production in A549 cells. (A) Cells were treated with PMA (0.1 μM), AJH-836 (1 μM) or vehicle for 1 h and at different times the conditioned medium was collected. MMP-9 activity was determined using zymograms, as described in “Materials and Methods”. As a loading control for the conditioned media, Commassie Blue stained genes are included. (B) MMP-9 activity was determined at 16 h after treatment with PMA in A549 cells subjected to PKC isozyme RNAi. Representative experiments are shown. Similar results were observed in 2 additional experiments. (C) MMP-9 activity was determined at 16 h after treatment with PMA or AJH-836 in A549 cells, in the presence of either 3 μM GF109203X (GF) or Gö6976 (), added 30 min before and during PMA treatment. A representative experiment is shown. Similar results were observed in at least two separate experiments. Full-length blots are presented in Supplementary Fig. 5.

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References

    1. Griner EM, Kazanietz MG. Protein kinase C and other diacylglycerol effectors in cancer. Nat Rev Cancer. 2007;7:281–294. doi: 10.1038/nrc2110. - DOI - PubMed
    1. Newton AC. Protein kinase C: perfectly balanced. Crit Rev Biochem Mol Biol. 2018;53:208–230. doi: 10.1080/10409238.2018.1442408. - DOI - PMC - PubMed
    1. Cooke M, Magimaidas A, Casado-Medrano V, Kazanietz MG. Protein kinase C in cancer: The top five unanswered questions. Mol Carcinog. 2017;56:1531–1542. doi: 10.1002/mc.22617. - DOI - PMC - PubMed
    1. Garg R, et al. Protein kinase C and cancer: what we know and what we do not. Oncogene. 2014;33:5225–5237. doi: 10.1038/onc.2013.524. - DOI - PMC - PubMed
    1. Isakov N. Protein kinase C (PKC) isoforms in cancer, tumor promotion and tumor suppression. Semin Cancer Biol. 2018;48:36–52. doi: 10.1016/j.semcancer.2017.04.012. - DOI - PubMed

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