c-Myc phosphorylation by PKCζ represses prostate tumorigenesis

Abstract

Studies showing reduced PKCζ expression or enzymatic activity in different types of human cancers support the clinical relevance of PKCζ as a tumor suppressor. However, the in vivo role of PKCζ and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCζ in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCζ-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKCζ knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKCζ knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKCζ is a critical event in the control of metastasis. Collectively, these results establish PKCζ as an important tumor suppressor and regulator of c-Myc function in prostate cancer.

Fig. 1.

Loss of PKCζ cooperates with PTEN^+/− to promote invasive prostate carcinoma. (A) PKC levels were analyzed by immunoblot in prostates of PTEN^+/− and PTEN^+/−/PKCζ KO mice. (B) Representative prostate glands of PTEN^+/− and PTEN^+/−/PKCζ KO mice at 8 mo of age. (C) Genitourinary (GU) tract weight of mice at 10 mo of age and of both genotypes: PTEN^+/− (n = 23) and PTEN^+/−/PKCζ KO (n = 9) mice, ****P < 0.0001. (D) H&E staining of prostates of the indicated genotypes. (E) Quantification of the incidence of invasive carcinoma in PTEN^+/− (n = 7) and PTEN^+/−/PKCζ KO mice (n = 15). (F) α-SMA and p63 staining of prostate sections of 9-mo-old mice of the indicated genotypes (n = 5). (G) Ki67 staining of prostate sections from 9-mo-old mice of the indicated genotypes. (H) Quantification of Ki67-positive cells. Results are the mean ± SD of 10 different fields per mouse sample (n = 5; *P < 0.05). (Scale bars, 20 μm in all panels.)

Fig. 2.

Role of PKCζ in tumorigenesis of PCa cells. (A) PTEN-P2 prostate epithelial cells were infected with retrovirus expressing HA-tagged PKCζ (HA-PKCζ) or kinase-dead mutant PKCζ (HA-PKCζ-KD) and cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Cell proliferation of PKCζ-WT– or PKCζ-KD–expressing cells in the presence of 0.1% FCS. Cell number was determined by trypan blue exclusion assay. Values are the mean ± SEM of triplicate counts from three different experiments, **P < 0.01, ***P < 0.001. (C) Colony formation in soft agar by PKCζ-WT– or PKCζ-KD–expressing cells. The total number of colonies per plate was scored by counting and is represented as the mean ± SD of six plates from two independent experiments. Insets are representative pictures showing colony size differences. (D) Cell-cycle analysis of PKCζ- or PKCζ-KD–expressing cells. (E) Genes differentially expressed in PKCζ-KD vs. PKCζ-WT cells (FDR <0.01 and fold change higher than three) with human homolog in the GSE21034 dataset. Yellow color indicates high expression levels and blue indicates low expression levels. (F) Unsupervised patient clustering analysis of the human PCa dataset GSE21034 using mouse genes with significantly altered expression, with FDR <0.01 and fold change higher than three when comparing PKCζ-KD vs. PKCζ-WT. Patient signatures were classified as PKCζ-KD–like or PKCζ-like based on whether the expression levels of signature genes were similar to PKCζ-KD or PKCζ-WT samples. If the expression levels of signature genes in a patient sample correlated better with PKCζ-KD samples, the sample was classified as PKCζ-KD–like, and vice versa. The association between such classification and the disease status of the sample (normal vs. tumor) was statistically significant (Fisher’s exact test P < 0.001).

Fig. 3.

PKCζ controls cell movement and invasion. (A) Ingenuity knowledge-based molecular and cellular functions of the PKCζ-KD transcriptome shows significant enrichment of cell-movement genes (rank 1, P = 1.36E-14). (B and C) Invasion determined by modified Boyden chamber assay for control PTEN-P2 cells or cells expressing PKCζ or PKCζ-KD. Results are shown as mean ± SEM n = 3. **P < 0.01. (D) Cell proliferation of shNT and shPKCζ-infected DU145 cells determined by trypan blue exclusion assay. Values are mean ± SEM of triplicate counts of three different experiments. **P < 0.01. (E and F) Invasion in shNT- and shPKCζ-infected DU145 cells. Results are shown as mean ± SEM n = 3. *P < 0.05. (G) H&E-stained sections of shNT- and shPKCζ-infected DU145 cells cultured in an organotypic system. Quantification of invasion fold (Right). Results are shown as mean ± SEM n = 3. **P < 0.01. (H) Unsupervised patient clustering analysis of human PCa dataset GSE21034 using genes significantly altered in PKCζ-KD vs. PKCζ-WT mice with FDR <0.01 and fold change higher than three. Patient signatures were classified as in Fig. 2F. The association between such classification and the disease status of the sample (normal vs. metastasis) was statistically significant (Fisher’s exact test P < 0.001).

Fig. 4.

PKCζ regulates c-Myc levels. (A) Dot plot view of the distributions of c-Myc expression data in normal and PCa samples. (B) Quantification by Q-PCR of c-Myc mRNA levels in control, PKCζ, and PKCζ-KD P2 cells (Left), and in shNT and shPKCζ DU145 cells (Right). Results are shown as mean ± SEM; n = 3. *P < 0.05; **P < 0.01. (C) Immunoblot analysis of c-Myc and PKCζ levels in shNT- and shPKCζ-expressing PC3M and DU145 cells. (D) Immunostaining of c-Myc in prostate sections and quantification of staining (Right). Results are the mean ± SEM (n = 5 mice; ***P < 0.001). (Scale bar, 20 μm.) (E) Immunoblotting for c-Myc, PKCζ, and actin levels of DU145 cells infected with the indicated shRNA lentiviral vectors. (F) Cell proliferation was determined by trypan blue exclusion assay in DU145 cells infected with the indicated lentiviral knockdown vectors. Values are mean ± SEM of triplicate counts of three different experiments. *P < 0.05. (G–J) Invasion by the modified Boyden chamber assay (G and H) or by organotypic cultures (I and J) for DU145 cells infected with the indicated shRNA lentiviral vectors. Results are shown as mean ± SD n = 3. **P < 0.01. (K) shNT or shPKCζ DU145 cells were transfected with c-Myc–luciferase reporters. Luciferase activity was normalized to Renilla activity. Values are mean ± SEM of triplicate counts of two different experiments. **P < 0.01. (L) Comparisons of the distributions of Myc cumulative binding scores for the 519 genes up-regulated (FDR < 0.05) in PKCζ-KD samples (red) and the remaining 20,693 genes (blue). The proportion of up-regulated genes with high cumulative binding score (CBS) is significantly higher (Kolmogorov–Smirnov test P value = 4.3 × 10⁻¹³) than the proportion of genes with high CBS that were not up-regulated.

Fig. 5.

Functional role of c-Myc phosphorylation by PKCζ. (A) PKCζ phosphorylates c-Myc in vitro. (B) MS/MS spectra of phosphopeptides containing the pS373 site of c-Myc. Fragment ions are shown, as is the sequence coverage due to identified fragment ions. Mass-to-charge ratio, m/z. (C) Immunoblotting analysis of c-Myc phosphorylated by PKCζ in vitro. (D) The Ser-373 phosphorylation site is highly conserved among different species. (E) Immunostaining of phospho-S373–c-Myc in prostate sections of the indicated genotypes (n = 5). (Scale bar, 20 μm.) (F) DU145 cells were infected with retroviral vectors expressing c-Myc, c-Myc–S373A, or c-Myc–S373E mutants, and cell proliferation was determined by trypan blue exclusion. Values are mean ± SEM of triplicate counts of three different experiments. Cell lysates were analyzed by immunoblotting for c-Myc and actin (Upper). (G) Invasion was determined in organotypic cultures of DU145 cells expressing c-Myc, c-Myc–S373A, or c-Myc–S373E. Results (Right) are shown as mean ± SD (n = 3 ***P < 0.001). (H) H&E staining of lung sections of mice i.v. inoculated with DU145 cells expressing c-Myc or c-Myc mutants. Total numbers of lung metastatic nodules in individual mice injected with the indicated cells (Right) are shown as mean ± SEM **P < 0.01. n = 6–7 mice per group.