PPP5C promotes cell proliferation and survival in human prostate cancer by regulating of the JNK and ERK1/2 phosphorylation

Abstract

Background: Prostate cancer (PCa) is one of the most common malignancies and a major leading cause of cancer-related deaths in males. And it is necessary to explore new molecular targets to enhance diagnosis and treatment level of the PCa. Serine/threonine protein phosphatase 5 (PPP5C) is a vital molecule that Involve in complex cell physiological activity.

Purpose: The objective of this study was to detecte the expression level of PPP5C in the tissue of prostate cancer patients and further discussed the PPP5C biological function and mechanisms on the PCa.

Methods: The expression level of PPP5C was analyzed by immunohistochemistry and ONCOM-INE datasets. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to silence the expression of PPP5C in prostate cancer cell. Cell viability and proliferation were measured using MTT and colony formation, and the cell cycle and apoptosis was analyszed by flow cytometry. The changes of downstream protein level and protein phosphorylation level were detected by western blot.

Results: PPP5C was highly expressed in PCa tissue as analyzed by immunohistochemistry and ONCOMINE datasets. PPP5C Knockdown inhibited cell proliferation and colony formation in PCa cells. Flow cytometry analysis showed that DU145, PC3 and 22RV1 PCa cells deprived of PPP5C were arrested in G0/G1 phase and became apoptotic. Western blot analysis indicated that PPP5C knockdown could promote JNK and ERK phosphorylation.

Conclusion: Our study indicated that the PPP5C may become a new potential diagnostic biomarker and therapeutic target for the PCa.

Keywords: ERK; JNK; PPP5C; prostate cancer; tumorigenesis.

Figure 1

PPP5C is overexpressed in PCa tissue and ONCOMINE microarray database. Notes: (A) Representative immunohistochemical images showing PPP5C expression in PCa and paired paracancer tissues. (B) The mRNA level of PPP5C in PCa tissue is significantly higher than that in normal prostate tissue in Wallace prostate dataset (P<0.0001), Vanaja prostate dataset (P=0.001), Welsh prostate dataset (P=0.016), and Arredouani prostate dataset (P=0.017) by the ONCOMINE microarray database. (C) The PPP5C expression in metastatic PCa tissue is higher than that in primary PCa tissue in the Yu prostate dataset (P=0.0006), Holzbeierlein prostate dataset (P=0.0003), Ramaswamy multi-cancer prostate dataset (P<0.0001), and Ramaswamy multi-cancer 2 prostate dataset (P=0.0002) by the ONCOMINE microarray database. Abbreviations: PCa, prostate cancer; PPP5C, serine/threonine protein phosphatase 5.

Figure 2

Lentivirus-mediated interference downregulates PPP5C expression in PCa cells. Notes: (A) PPP5C expression in DU145, PC3, LNCaP, and 22RV1 cells was detected by qPCR. (B) PPP5C expression in DU145, PC3, LNCaP, and 22RV1 cells was detected by Western blot. (C) DU145, PC3, and 22RV1 cells were examined by fluorescence microscopy after 72-hour lentivirus infection. (D) Knockdown efficiency of PPP5C mRNA level in DU145, PC3, and 22RV1 infected by Lv-shPPP5C was determined by real-time qPCR (^***P<0.001). (E) The protein level of PPP5C was downregulated in DU145, PC3, and 22RV1 cells. Con: no lentivirus-mediated shRNA; Lv-shCon: lentivirus-mediated non-silencing shRNA; Lv-shPPP5C: lentivirus-mediated non-silencing shPPP5C. Data are presented as mean ± SD from at least 3 experiments. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PCa, prostate cancer; PPP5C, serine/threonine protein phosphatase 5.

Figure 2

Lentivirus-mediated interference downregulates PPP5C expression in PCa cells. Notes: (A) PPP5C expression in DU145, PC3, LNCaP, and 22RV1 cells was detected by qPCR. (B) PPP5C expression in DU145, PC3, LNCaP, and 22RV1 cells was detected by Western blot. (C) DU145, PC3, and 22RV1 cells were examined by fluorescence microscopy after 72-hour lentivirus infection. (D) Knockdown efficiency of PPP5C mRNA level in DU145, PC3, and 22RV1 infected by Lv-shPPP5C was determined by real-time qPCR (^***P<0.001). (E) The protein level of PPP5C was downregulated in DU145, PC3, and 22RV1 cells. Con: no lentivirus-mediated shRNA; Lv-shCon: lentivirus-mediated non-silencing shRNA; Lv-shPPP5C: lentivirus-mediated non-silencing shPPP5C. Data are presented as mean ± SD from at least 3 experiments. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PCa, prostate cancer; PPP5C, serine/threonine protein phosphatase 5.

Figure 3

Lentivirus-mediated knockdown of PPP5C suppresses the viability of PCa cells. Notes: (A) After infection with Lv-shCon or Lv-shPPP5C, the growth curves of DU145, PC3, and 22RV1 cells were determined by MTT. Data are presented as mean ± SD from at least 3 experiments (^***P<0.001). (B) Representative images of DU145, PC3, and 22RV1 cell colony formation under micro and macro views in the Con, Lv-shCon, and Lv-shPPP5C groups. (C). The number of colonies formed in DU145, PC3, and 22RV1 cells treated as in (B). Data are presented as mean ± SD from at least 3 experiments (^**P<0.01, ^***P<0.001). Con: no lentivirus-mediated shRNA; Lv-shCon: lentivirus-mediated non-silencing shRNA; Lv-shPPP5C: lentivirus-mediated non-silencing shPPP5C. Abbreviations: PCa, prostate cancer; PPP5C, serine/threonine protein phosphatase 5; OD, optical density.

Figure 4

Disruption of PPP5C expression results in cell cycle arrest in PCa cells. Notes: (A) Flow cytometry analysis showing cell cycle distribution of DU145, PC3, and 22RV1 cells infected by Lv-shCon or Lv-shPPP5C. (B) Cell percentages in G0/G1, S, and G2/M of DU145, PC3, and 22RV1 cells treated as in (A) are analyzed statistically (^*P<0.05, ^***P<0.001). Con: no lentivirus-mediated shRNA; Lv-shCon: lentivirus-mediated non-silencing shRNA; Lv-shPPP5C: lentivirus-mediated non-silencing shPPP5C. Abbreviations: PCa, prostate cancer; PPP5C, serine/threonine protein phosphatase 5.

Figure 5

Apoptotic cells are increased after PPP5C silencing. Notes: (A) Apoptosis of DU145, PC3, and 22RV1 is analyzed in Con, LV-shCon, and LV-shPPP5C groups using flow cytometry and annexin V/PI staining. (B) Early apoptosis (V+/PI−) and late apoptosis (V+/PI+) of the DU145, PC3, and 22RV1 cells are statistically analyzed (^*P<0.05, ^***P<0.001). Con: no lentivirus-mediated shRNA; Lv-shCon: lentivirus-mediated non-silencing shRNA; Lv-shPPP5C: lentivirus-mediated non-silencing shPPP5C. Abbreviations: PI, propidium iodide; PPP5C, serine/threonine protein phosphatase 5.

Figure 6

Protein level transformation is analyzed by Western blot. Notes: The phosphorylation level of JNK and ERK1/2 had increased and the non-phosphorylation was invariable in PPP5C downregulation group. The P38 protein level is always consistent with phosphorylation or non-phosphorylation after PPP5C knockdown in the 3 groups, using GAPDH as the loading control. Con: no lentivirus-mediated shRNA; Lv-shCon: lentivirus-mediated non-silencing shRNA; Lv-shPPP5C: lentivirus-mediated non-silencing shPPP5C. Abbreviations: ERK, extracellular signal-regulated protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun NH2-terminal kinase; PPP5C, serine/threonine protein phosphatase 5.

Figure 7

The working model of PPP5C in prostate cancer. Note: The PPP5C can inhibit the phosphorylation level of the JNK and the ERK, and then to impact the proliferation, cell cycle, and apoptosis by a direct or indirect way. Abbreviations: ERK, extracellular signal-regulated protein kinase; JNK, c-Jun NH2-terminal kinase; PPP5C, serine/threonine protein phosphatase 5.