PKC- ι Regulates an Oncogenic Positive Feedback Loop Between the MAPK/JNK Signaling Pathway, c-Jun/AP-1 and TNF- α in Breast Cancer

Abstract

Breast cancer is the second most common cancer in the United States and consists of 30% of all new female cancer each year. PKC iota (PKC-ι) is a bonafide human oncogene and is overexpressed in many types of cancer, including breast cancer. This study explores the role of PKC-ι in regulating the transcription factor Jun proto-oncogene (c-Jun), pro-inflammatory cytokine Tumor Necrosis Factor-alpha (TNF-α), and the Mitogen-Activated Protein Kinase/Jun N-terminal kinase (MAPK/JNK) pathway, which also exhibits an oncogenic role in breast cancer. ICA-1S, a PKC-ι specific inhibitor, was used to inhibit PKC-ι to observe the subsequent effect on the levels of c-Jun, TNF-α, and the MAPK/JNK signaling pathway. To obtain the results, cell proliferation assay, Western blotting, co-immunoprecipitation, small interfering RNA (siRNA), immunofluorescence, flow cytometry, cycloheximide (CHX) chase assay, and reverse transcription quantitative PCR (RT-qPCR) techniques were implemented. ICA-1S significantly inhibited cell proliferation and induced apoptosis in both breast cancer cell lines. Treatment with ICA-1S and siRNA also reduced the expression levels of the MAPK/JNK pathway protein, c-Jun, and TNF-α in both cell lines. PKC-ι was also found to be strongly associated with c-Jun, via which it regulated the MAPK/JNK pathway. Additionally, ICA-1S was found to promote the degradation of c-Jun and decrease the mRNA levels of c-Jun. We concluded that PKC-ι plays a crucial role in regulating breast cancer, and the inhibition of PKC-ι by ICA-1S reduces breast cancer cell proliferation and induces apoptosis. Therefore, targeting PKC-ι as a potential therapeutic target in breast cancer could be a significant approach in breast cancer research.

Keywords: MAPK/JNK pathway; TNF-α; apoptosis; breast cancer; c-Jun.

Figure 1

Dose response curve of ICA-1S in (A) BT-549 and (B) MCF-7 cell line. The results for the cell proliferation assay indicates ICA-1S has an inhibitory effect on the cell proliferation of breast cancer cell lines. The graph represents the mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001; ** p < 0.005, * p < 0.05, ns: not significant.

Figure 2

Treatment with ICA-1S induces apoptosis for both BT-549 and MCF-7 cells. (A–D) Representative Western blot images and the corresponding graph indicate that treatment with ICA-1S increases the levels of cleaved Caspase 3 and cleaved PARP, which are hallmark events in apoptosis. Expression levels were normalized to the corresponding loading controls such as $\beta$-actin and $\alpha$-tubulin. (E–H) Flow cytometry analysis using Annexin V-APC/PI staining demonstrates an increase in both early and late apoptotic cell populations with ICA-1S treatment when compared to the control group (untreated group) as observed in the individual cell dot plots for both BT-549 and MCF-7 cells. The graph represents the mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001; ** p < 0.005, * p < 0.05. ns: not significant.

Figure 3

Representative (A,C) represents the Western blot images and its corresponding graphs for BT-549 cells and (B,D) represents the western blot images and its corresponding graph for MCF-7 cells. The results demonstrate the treatment with ICA-1S decreases the levels of PKC-$\iota$ in both BT-549 and MCF-7 cells, while the level of PKC-$\zeta$ was decreased in BT-549 cells but increased in MCF-7 cells. Expression levels were normalized to the corresponding loading controls such as $\beta$-actin and $\alpha$-tubulin. The graph represents the Mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001; ** p < 0.005, ns: not significant.

Figure 4

(A,B) Western blot images and (C,D) corresponding graphical representation demonstrating the effect of ICA-1S on the phosphorylation levels and the total protein level of the MAPK/JNK pathway proteins for BT-549 and MCF-7 cells. Expression levels were normalized to the corresponding loading controls such as $\beta$-actin and $\alpha$-tubulin. The graph represents the Mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001; ** p < 0.005, * p < 0.05.

Figure 5

(A,B) Western blot images and (C,D) corresponding graphical representation demonstrating effect of PKC-$\iota$ knockdown on aPKCs in BT-549 and MCF-7 cells. Treatment with PKC-$\iota$ siRNA lowered the levels of PKC-$\iota$ in both cell lines, as well as lowered the levels of PKC-$\zeta$ in both cell lines. Expression levels were normalized to the corresponding loading controls such as $\alpha$-tubulin. The graph represents the Mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001; ** p < 0.005, * p < 0.05.

Figure 6

(A,B) Western blot images and (C,D) corresponding graphical representation demonstrating the effect PKC-$\iota$ knockdown on the expression of MAPK-JNK pathway proteins and their phosphorylated form in BT-549 and MCF-7 cells. Expression levels were normalized to the corresponding loading controls such as $\beta$-actin and $\alpha$-tubulin. Graphs represents the Mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001; ** p < 0.005, * p < 0.05.

Figure 7

(A,B) Co-immunoprecipitation of c-Jun with PKC-$\iota$ in BT-549 and MCF-7 cell line. Representative Western blot images indicate that there is a direct association between PKC-$\iota$ and c-Jun for both the cell lines. (C–F) Immunofluorescence staining and visualization at 100X magnification of c-Jun protein with 4’,6-diamidino-2-phenylindole (DAPI) staining in BT-549 and MCF-7 cell lines. Treatment with ICA-1S also reduces the mean fluorescence intensity of c-Jun when compared to the control. (G,H) Level of PKC-$\iota$ in MCF-7 and BT-549 cells. MCF-7 cells demonstrate lower levels of PKC-$\iota$, compared to BT-549 cells. The graph represents the Mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. * p < 0.05.

Figure 8

(A,B) Western blot images and (C,D) corresponding graphical representation demonstrating the expression of c-Jun protein with and without CHX treatment for BT-549 and MCF-7 cells. An enhanced decrease in the levels of c-Jun is observed after CHX treatment in the ICA-1S treatment group. Expression levels are normalized to the corresponding loading control such as $\alpha$-tubulin. Graphs represents the Mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001; ** p < 0.005, * p < 0.05.

Figure 9

Expression of c-Jun mRNA in (A) BT-549 and (B) MCF-7 cell line. The graph representing BT-549 cells demonstrate the relative quantification value (RQ) of the target gene (c-Jun) is 0.5, which indicates a twofold downregulation in comparison to the control group, following normalization with the reference gene, GAPDH. However, no significant downregulation was observed for MCF-7 cells. Graphs represent the Mean ± Standard Error of Mean (SEM) of 3 independent experiments of each cell line. *** p < 0.001, ns: not significant.

Figure 10

Schematic representation of the possible pathway via which PKC-$\iota$ regulates MAPK/JNK pathway proteins in breast cancer. The inhibition of PKC-$\iota$ leads to c-Jun degradation or reduced expression, which decreases the AP-1 activity. Decrease in AP-1 activity reduces the levels of TNF-$\alpha$, TAK-1, MKK7 and JNK. Decrease in the levels of JNK activity further decreases c-Jun expression, thereby reinforcing the oncogenic positive feedback loop. All these proteins play an oncogenic role in breast cancer proliferation and survival; therefore, reducing the levels of these proteins will lead to decreased proliferation and survival.