Downregulation of protein kinase C gamma reduces epithelial property and enhances malignant phenotypes in colorectal cancer cells

Abstract

Loss of epithelial integrity is associated with colorectal cancer (CRC) aggressiveness. Protein kinase C (PKC) is frequently implicated in human cancers, but the role of PKCγ in CRC remains poorly understood. Here, we show that PKCγ, a conventional PKC, is expressed in normal colonic epithelium, but this is lower in dedifferentiated CRC. PKCγ expression was downregulated by SNAI1 overexpression, and low PKCγ expression was associated with poor prognosis in patients with CRC. Transient or stable knockdown of PKCγ reduced E-cadherin expression in CRC cells. PKCγ knockdown enhanced proliferation, anchorage-independent cell growth, resistance to anti-cancer drugs, and in vivo tumor growth of DLD-1 cells. We have also identified phosphorylation substrates for PKCγ. Among them, ARHGEF18, a RhoA activator that stabilizes cell-cell junctions, was phosphorylated and stabilized by PKCγ. Thus, these results suggest that the downregulation of PKCγ decreases the epithelial property of CRC cells and enhances its malignant phenotypes.

Keywords: Cancer; Molecular biology.

Graphical abstract

Figure 1

PKCγ expression is associated with the epithelial properties of CRC cells (A) The expression of PKCγ and E-cadherin in CaCo-2, WiDr, DLD-1, SW480, SW620, Lovo, and HCT116 cells was quantified using western blotting. The correlation between PKCγ and E-cadherin expression was assessed using Pearson’s product-moment correlation test. (B) Western blot analyses of PKCγ and other marker proteins in DLD-1 and SW480 cells expressing SNAI1. (C) The relative expression levels of CDH1 (E-cadherin) and PRKCG (PKCγ) normalized to that of GAPDH, were determined by qPCR analyses in DLD-1 and SW480 cells expressing SNAI1 (n = 6, each). Data represent means ± SD. Statistical analysis was performed using Student’s t test. ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S1 and S2, and Table S1.

Figure 2

PKCγ expression is lower in dedifferentiated CRCs (A) Positive control of immunohistochemical study using xenografts of DLD-1 cells suppressing PKCγ expression using the anti-PKCγ antibody. Scale bar: 100 μm. (B–D) A human CRC tissue array, which contains 36 pairs of non-tumor colon epithelium and CRC tissues, and 15 other CRC samples, was probed with the anti-PKCγ antibody. (B) Representative images of grade 2 (moderately differentiated) or grade 3 (poorly differentiated) tumors and paired non-tumor samples are shown. Scale bar: 100 μm. (C) The PKCγ expression levels were assessed using the Wilcoxon signed-rank test (36 pairs). (D) The relationship between the expression of PKCγ and the tumor grade in 51 CRC samples was assessed using Fisher exact test. See also Table S2.

Figure 3

PKCγ knockdown reduces E-cadherin expression in CRC cells (A) DLD-1 cells suppressing PKCγ (shPKCγ#1, #2) or negative control clones (shNeg#1, #2) were assessed by western blotting. (B) DLD-1 cells transfected with shPKCγ (shPKCγ#1, #2) or negative control clones (shNeg#1, #2) were assessed by western blotting for E-cadherin. Immunofluorescence staining of E-cadherin (green) with an anti-E-cadherin antibody and counter nuclear staining using Hoechst (blue) is shown. The bar indicates 25 μm. (C) SW480, WiDr, and Caco-2 cells were transfected with siPKCγ#1 and #2 and assessed using western blotting with the indicated antibodies (n = 3). (D) DLD-1 cells were transfected with siRNA for PKCγ, and after 6 days, the cells were assessed by western blotting using the indicated antibodies. (E) DLD-1 cells were transfected with siPKCγ#1 and assessed for the TOP/FOP FLASH assay. (F) Phase contrast images of DLD-1 cells transected with indicated siRNA and cultured on collagen-coated plates. The bar indicates 50 μm. (G) Western blot analysis of DLD-1 cells transfected with the indicated siRNA, cultured on collagen-coated plates with 10% serum-containing medium for 5 days, and then cultured in serum-free medium for 24 h. (B, C, and E) Data represent means ± SD. Statistical analysis was performed using Dunnett’s multiple comparison of means test (B and C) or Student’s t test (E). ∗p < 0.05; ∗∗p < 0.01. See also Figure S3.

Figure 4

PKCγ knockdown enhances malignant phenotypes in CRC cells (A) The cell number was determined at the indicated times and the relative proliferation is shown (n = 3). (B) Cells were seeded in 6-well plates with soft agar and after 2–3 weeks, the numbers of colonies were counted (n = 3). Representative images of the colonies are also shown. (C) Cells were treated with 10 μM oxaliplatin (oxa) for 48 h and then incubated with FITC-labeled annexin V, and the percentage of annexin V-positive cells was determined (n = 3). (D) Confluent cells were starved for 24 h and the cell layers were scratched. Images at the same position were obtained before and after 19 h incubation. (E) Cells were inoculated into the flanks of nude mice. Representative images of the xenografts 28 days after inoculation are shown in the upper panels, when the xenografts were weighed (shNeg#1; n = 5. shNeg#2, shPKCg#1, #2; n = 6, each). Data represent means ± s.e.. (F) The relationship between PKCγ expression (GSE39582; Affymetrix microarray probe:206,270_at) and survival was determined and plotted using the Kaplan–Meier method. The disease-free survival rate for patients with high or low PKCγ expression is plotted as red and blue lines, respectively. (A–D) Data represent means ± SD. Statistical analysis was performed using Dunnett’s multiple comparison of means test (A, B, and D), or Student’s t test (C). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5

Identification of phosphorylation substrates of PKCγ (A) Lysates of DLD-1 cells transfected with siPKCγ or siNeg were analyzed by western blotting using an anti-phosphorylated PKC substrate antibody (phos) (left panel). Lysates of DLD-1 and SW620 cells were immunoprecipitated using an anti-phosphorylated PKC substrate antibody, separated by SDS-PAGE, and analyzed by silver staining (right panel). The indicated bands were analyzed using nano-LC–MS/MS. (B) FLAG-tagged BICD2 or (C) Halo-tagged ARHGEF18 was co-transfected with PKCγ in DLD-1 cells. Cell lysates were immunoprecipitated using anti-FLAG or Halo antibodies, and the phosphorylation levels were assessed using the anti-phosphorylated PKC substrate antibody (phos). The cell lysate before the commencement of immunoprecipitation served as the input (n = 3). (D) Halo-tagged ARHGEF18 and PKCγ was transfected in DLD-1, performed pull-down using Halo resin, treated with Halo TEV protease to elute ARHGEF18 (GEF18), and then assessed using anti-phosphorylated Ser/Thr antibody (pSer/Thr) and indicated antibodies (n = 3). (E) In vitro kinase assays were performed using purified PKCγ and substrate proteins as indicated (n = 3). (F) FLAG-tagged BICD2 or Halo-tagged ARHGEF18 was co-transfected with control siRNA (siNeg) or siPKCγ. Cell lysates were immunoprecipitated using anti-FLAG or Halo antibodies, and then the phosphorylation levels were assessed using the anti-phosphorylated PKC substrate antibody (phos) (n = 3). (G) Halo-tagged ARHGEF18 and siRNA were transfected in DLD-1 cells, followed by a pull-down using Halo resin, and then treated with Halo TEV protease to elute ARHGEF18 (GEF18). Assessment using the anti-phosphorylated PKC substrate antibody (phos) (n = 3) was then performed. (B–G) Data represent means ± SD. Statistical analysis was performed using Tukey’s multiple comparison of means test (B, C, E, and G) or Student’s t test (D and F). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Table S3.

Figure 6

PKCγ stabilizes ARHGEF18 and maintains epithelial integrity (A) DLD-1 cells were transfected with Halo-ARHGEF18 and the indicated siRNAs. After 3 days, the cells were treated with cycloheximide (200 μg/mL) and then harvested at the indicated time points. The relative levels of Halo-ARHGEF18 normalized to ACTB were analyzed using western blotting (n = 3). (B) DLD-1 cells were transfected with indicated siRNAs. After 3 days, the cells were treated with cycloheximide (200 μg/mL) and then harvested at the indicated time points. The relative levels of ARHGEF18 normalized to ACTB were analyzed using western blotting (n = 3). (C) DLD-1 or SW480 cells were transfected with Halo-ARHGEF18 (WT or S581A), following which the cells were treated with cycloheximide (200 μg/mL) and harvested at the indicated time points. The relative levels of Halo-ARHGEF18 normalized to ACTB were analyzed using western blotting (n = 3). (D) DLD-1 cells were transfected with the indicated siRNAs. After 4 days, endogenous ARHGEF18 was detected using western blotting (n = 4). (E) DLD-1 cells were transfected with the indicated siRNAs. After 5 days, the cells were fixed with methanol and subjected to immunofluorescence staining with E-cadherin and ARHGEF18 antibodies (upper panels) or F-actin (Acti-stain 488 phalloidin; green) and ARHGEF18 antibodies (red) (lower panels). Scale bar = 10 μm. (F) DLD-1 cells were transfected with the indicated siRNAs. After 6 days, the cells were fixed with methanol and subjected to immunofluorescence staining with F-actin. Scale bar = 50 μm. (G) Caco-2 cells were transfected with the indicated siRNA, mounted in Matrigel, and then examined using immunostaining with Hoechst (blue) and phalloidin (red). The percentage of normally polarized cysts per total cell cluster containing 3–10 cells is shown in the bar graph (more than 85 clusters were counted from three experimental replicates). Scale bar = 50 μm. (H) A putative mechanism of epithelial maintenance by PKCγ. PKCγ phosphorylates and stabilizes ARHGEF18, which activates Rho A at cell-cell junctions to maintain epithelial polarity. (A–D and G) Data are presented as means ± SD. Statistical analysis was performed using Dunnett’s multiple comparison of means test (A and D) or Student’s t test (B, C, and G). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S4.