Protein Kinase C Alpha (PKCα) overexpression leads to a better response to retinoid acid therapy through Retinoic Acid Receptor Beta (RARβ) activation in mammary cancer cells

Abstract

Purpose: Retinoids have proved to be effective for hematologic malignancies treatment but till nowadays, their use as single agent for the solid tumor's management is still controversial. All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, exerts non-genomic interactions with different members of the protein kinase C (PKC) family, recognized modulators of different tumor progression pathways. To determine whether a group of patients could become benefited employing a retinoid therapy, in this study we have evaluated whether PKCα expression (a poor prognosis marker in breast cancer) could sensitizes mammary cells to ATRA treatment.

Methods: PKCα overexpression was achieved by stable transfection and confirmed by western blot. Transfected PKC functionality was determined by nuclear translocation-induction and confocal microscopy. In vitro proliferation was evaluated by cell counting and cell cycle distribution was analyzed by flow cytometry. In vivo studies were performed to evaluate orthotopic tumor growth and experimental lung colonization. Retinoic acid response elements (RARE) and AP1 sites-dependent activity was studied by gene reporter assays and retinoic acid receptors (RARs) were measured by RT-qPCR.

Results: Our findings suggest that high PKCα levels improve the differentiation response to ATRA in a RAR signaling-dependent manner. Moreover, RARβ expression appears to be critical to induce ATRA sensitization, throughout AP1 trans-repression.

Conclusion: Here we propose that retinoids could lead a highly personalized anticancer treatment, bringing benefits to patients with aggressive breast tumors resulting from high PKCα expression but, an adequate expression of the RARβ receptor is required to ensure the effect on this process.

Keywords: ATRA; Metastasis dissemination; PKCα; Proliferation; Tumor growth.

Fig. 1

Overexpression of PKCα in T47D and MDA-MB231 cells. a Whole cell lysates prepared from T47D and MDA-MB231 cells transfected with pGFPN1-PKCα or pGFPN1 (vector alone) were resolved on 10% SDS-PAGE and blotted with an anti-PKCα antibody (50 μg protein/lane). b Densitometric analysis of PKC overexpression. Results were expressed in fold of PKCα expression relative to vector-transfected cells expression. Each data point represents the mean ± S.D. three independent experiments. *p < 0.05 versus the respective vector-transfected cell line (ANOVA test). c T47D cells transfected with pGFPN1-PKCα or pGFPN1 vector alone were incubated with PMA (50 nM) for 10 min, fixed and PKCα-GFP localization was analyzed by confocal microscope (scale bar: 50 μm). Results are representative of three independent experiments

Fig. 2

Effect on in vitro proliferation and cell cycle progression of T47D and MDA-MB231 transfectants. a, b Cell number was assessed 96 h after the treatment with ATRA (1 µM). Each data point represents the mean ± S.D. of triplicate determinations. *p < 0.05 versus untreated T47D-Vector cells, **p < 0.01 versus untreated T47D-PKCα cells (ANOVA test). At least three independent experiments were performed with similar results. c, d T47D and MDA-MB231 cells overexpressing or not PKCα were treated with ATRA (1 µM) for 48 h and then stained with propidium iodide to analyze cycle distribution by flow cytometry. Each data point represents the mean ± S.D. of triplicate determinations. *p < 0.05 versus the respective untreated transfectant (ANOVA test). At least three independent experiments were performed with similar results

Fig. 3

Effect of PKCα overexpression on tumor growth and lung colonization in a murine experimental model. a Whole cell lysates prepared from LM3-Vector or LM3-PKCα cells were resolved on 10% SDS-PAGE and blotted with anti-PKCα antibodies (50 μg/lane). Actin expression level was used as protein loading control. A representative experiment is shown. b Cell number was assessed 96 h after ATRA (1 µM) treatment. Each data point represents the mean ± S.D. of triplicate determinations, *p < 0.05 versus the respective untreated LM3 transfectant, ^#p < 0.05 versus untreated LM3-Vector cells (ANOVA test). At least three independent experiments were performed with similar results. c LM3-PKCα and LM3-Vector transfected cells were harvested from subconfluent monolayers and orthotopically inoculated into the fat pad of BALB/c mice. Fifteen days later, animals receive a silastic pellet containing ATRA (10 mg) or an empty pellet as control. Tumor diameters were measured twice a week and used to calculate tumor volume. Each data point represents the mean ± S.D. (n = 6), *p < 0.05 versus ATRA-treated LM3-PKCα cells (ANOVA test). Two independent experiments were performed with similar results. d Effect of ATRA on histopathological features of LM3-PKCα and LM3-Vector tumors. Tumors were fixed in 10% formalin and slides were stained with hematoxylin and eosin (D: Normal dermis, U: Ulcerated dermis). e LM3 cells, overexpressing or not PKCα, were injected in the lateral tail vein of mice carrying a subcutaneous silastic pellet containing ATRA (10 mg) or an empty pellet as control. Each data point represents the number of lung nodules per animal. The figure shows the results of one experiment representative of three independent assays. *p < 0.05 versus untreated LM3-PKCα cells (Kruskall-Wallis test)

Fig. 4

Evaluation of RARE an AP-1-dependent activity through reporter gene assays. a, b T47D and MDA-MB231 cells overexpressing or not PKCα were transfected with the TK luciferase plasmid containing the retinoic acid response element. Cells were co-transfected with an expression vector encoding renilla luciferase in a 1:10 ratio. After transfection cells were treated with ATRA (1 µM) and luciferase/renilla ratio was determined. Each data point represents the mean ± S.D. of triplicate determinations. **p < 0.01 versus the respective untreated transfectant, ^#p < 0.05 versus ATRA-treated T47D-Vector cells (ANOVA test). Three independent experiments were performed with similar results. c, d T47D and MDA-MB231 overexpressing or not PKCα were transfected with TK luciferase plasmid containing AP-1 sites. Cells were co-transfected with expression plasmids encoding renilla in a 1:10 ratio. After transfection cells were treated or not with ATRA (1 µM) and luciferase/renilla ratio was determined. Each data point represents the mean ± S.D. of triplicate determinations. *p < 0.05 and **p < 0.01 versus the respective untreated T47D transfectant (ANOVA test). Three independent experiments were performed with similar results

Fig. 5

Modulation of RAR isotypes expression in T47D and MDA-MB231 trasnfectants. T47D and MDA-MB231 cells transfected with PKCα or the empty vector as control were treated or not with ATRA (1 µM) and the different RAR isotypes expression was analyzed by qPCR (RARα panels a and d, RARβ panels b–e and RARγ panels c–f). *p < 0.05 and **p < 0.01 versus the respective untreated transfectant (ANOVA test). Three independent experiments were performed with similar results

Fig. 6

Role of RARβ on MDA-MB231 response to ATRA treatment. a MDA-MB231 cells were transfected with RARβ or with the empty vector as control. RARβ expression levels were evaluated by RT-qPCR. **p < 0.01 versus vector transfected cells (Student’s t test). b Cell number was assessed 96 h after the treatment with ATRA (1 µM). Each data point represents the mean ± S.D. of triplicate determinations, *p < 0.05 versus MDA-Vector cells that received the same treatment (ANOVA test). At least three independent experiments were performed with similar results. c MDA-MB231 expressing or not RARβ were transfected with the TK luciferase plasmid containing the retinoic acid response element. Cells were co-transfected with an expression vector encoding renilla luciferase in a 1:10 ratio. After transfection cells were treated with ATRA (1 µM) and luciferase/renilla ratio was determined. Each data point represents the mean ± S.D. of triplicate determinations. *p < 0.05 versus the respective untreated transfectant (ANOVA test). Three independent experiments were performed with similar results. d MDA-MB231 expressing or not RARβ were transfected with the TK luciferase plasmid containing AP1 sites. Cells were co-transfected with expression plasmids encoding renilla in a 1:10 ratio. After transfection, cells were treated with ATRA (1 µM) and luciferase/renilla ratio was determined. Each data point represents the mean ± S.D. of triplicate determinations. *p < 0.05 versus MDA-Vector cells that received the same treatment (ANOVA test). Three independent experiments were performed with similar results