Deciphering the Role and Signaling Pathways of PKCα in Luminal A Breast Cancer Cells

Abstract

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.

Keywords: PKC; breast cancer; kinases; signaling pathways; targeted therapy.

Figure 1

Knock-down of PKCα in MCF-7 cells affects their proliferation capacity: (A) MCF-7 cells were transfected with siRNA specific for PKCα. Protein expression was assessed by WB relative to β-actin expression at the days indicated. (B) Proliferation rates of PKCα-WT and PKCα-KD MCF-7 cells were measured by fluorometric DNA quantitation. The mean difference for two comparisons against the PKCα-WT and PKCα-KD cells are shown in the Cumming estimation plots (see Materials and Methods for a more extensive explanation).

Figure 2

Transcriptomic analysis: (A) Volcano plot displaying the expression of probe sets in PKCα-KD vs. PKCα-WT MCF-7 cells. Log₂ fold changes and their corresponding p-values of all genes in the microarray were taken to construct the volcano plot. Genes up-regulated with more than 1.5-fold change with a p-value <0.05 are depicted in blue dots, and those down-regulated with identical fold change and p-value are in red dots. All other genes in the array are not significantly altered and are represented in grey dots. (B) Validation of significant up-and down-regulated genes by qPCR.

Figure 3

Functional enrichment analysis with Metascape of DEGs obtained after comparing PKCα-KD vs. PKCα-WT MCF-7 cells. Network of enriched terms colored by cluster-ID. DEGs on the left represent the up-regulated genes and on the right represent the down-regulated genes. Here, we show the five top clusters with enriched representative terms, and the numbers in brackets indicate the (Log₁₀ (p-value)). Table S2a,b show the genes included in each cluster.

Figure 4

Human phospho-kinase array comparing PKCα-KD vs. PKCα-WT MCF-7 cells. The histogram shows the fold changes of the kinases affected by the down-regulation of the expression of PKCα in MCF-7 cells. Red bars represent phosphorylated proteins showing a decrease in phosphorylation levels, whereas blue bars show an increase.

Figure 5

Critical signaling pathway analysis: (A) Protein–protein interaction network of DEGs using the STRING database [33]. DEGs obtained from the microarray analysis were colored in dark red when down-expressed, whereas up-expressed DEGs were colored in dark blue. Down- and up-activated kinases obtained from the phospho-kinase array were depicted in light red and light blue, respectively. The MCODE tool was used to identify the two main protein clusters present in the protein-protein interaction network: Cluster 1 (22 nodes and 222 edges) (B) and Cluster 2 (17 nodes and 51 edges) (C).

Figure 6

Effect of additional inhibition of the key signaling pathways identified in the arrays. The proliferation and migration capabilities of PKCα-WT and PKCα-KD MCF-7 cells were measured under different treatments: (A,B), U73122; (C,D), KT5720; (E,F), BMS 599626; (G,H), Imatinib on PKCα-WT and PKCα-KD MCF-7 cells. A heat map-type graph was used with the dark blue color corresponding to the lowest value for the DNA fold change or migration % and the dark red color to the highest value. The effect of each treatment on PKCα-KD vs. PKCα-WT MCF-7 cells at day 8 (proliferation assay) or 72 h (migration assay) is shown at the top of the heat map as the percentage of inhibition on the ability to proliferate or migrate.