PPA1 regulates tumor malignant potential and clinical outcome of colon adenocarcinoma through JNK pathways

Abstract

Colorectal cancer (CRC) represents one of the most prevalent malignancies and the third leading cause of cancer death worldwide. Inorganic pyrophosphatase (PPA1) is an enzyme that catalyzes the hydrolysis of pyrophosphate to inorganic phosphate, therefore participates in the energy metabolism. Proteomic studies have demonstrated the up-regulated expression of PPA1 in various tumors, however, its expression pattern in CRC hasn't been reported. In the current study, we used RT-qCR, Western Blot and immunohistochemical (IHC) staining to explore the expression of PPA1 in 113 paired colon cancer tissues and adjacent normal tissues, which revealed that PPA1 was correlated with lymph node metastasis. The prognostic value of PPA1 was confirmed by Kaplan-Meier survival analysis and Cox regression analysis. We further purified PPA1 and obtained the phosphor-JNK1 protein and performed enzymatic studies, which identified that PPA1 can directly dephosphorylate pJNK1, while showed no catalytic activity towards pERK or p-p38 proteins. Moreover, overexpression of PPA1 enhanced cell viability through JNK-p53 signaling pathways, and it may also prevent cell apoptosis by inhibiting Bcl-2 and Caspase-3 cleavage. To our knowledge, this is the first study demonstrated the expression and clinical significance of PPA1 in colon cancer, which also provided evidence that figuring out PPA1 specific inhibitors can be invaluable in the future chemotherapy development towards colon cancer.

Keywords: JNK; PPA1; colon cancer; prognosis; proliferation.

Figure 1. Expression of PPA1 in colon adenocarcinoma tissues

(A) RT-qPCR results showed higher mRNA levels of PPA1 in cancer tissues than that in adjacent normal tissues (P=0.005). (B) Western Blot revealed the different expression levels of PPA1 protein in tumor tissues (T) and adjacent normal tissues (AN). The fold changes were labelled under the bands using AN as control. P1-P7 refer to the patient's number from whom we obtained the fresh-frozen tissues. (C) IHC of tumor tissues showed different immunoreactivities. Scale bar: 100μm. (D) Patients with advanced lymph node statues exhibited higher PPA1 levels as determined by IHC evaluation. (E) High expression of PPA1 indicated poorer overall survival of colon adenocarcinoma patients (P=0.005).

Figure 2. Kaplan-Meier survival curve with respect to clinicopathological characteristics of colon cancer patients

Older age (A), ascending tumor location (B), poor pathological differentiation (C), higher T stage (D), positive lymph node (E) and advanced TNM stage (F) were associated with poor clinical outcome.

Figure 3. PPA1 promotes proliferation in colon cancer cell lines

(A) Western Bolt results showed that PPA1 was higher expressed in colon cancer cell lines than that in normal epithelial CCD-18Co cells. SW480 cells showed highest PPA1 expression while HT29 showed lowest PPA1 expression. (B) Upon PPA1-silencing, the pJNK level was significantly increased without changes in total JNK protein level. Meanwhile, PPA1 knock-down increased Bax and p53 accumulation, Bcl-2 phosphorylation as well as Caspase-3 cleavage. In contrast, overexpression of PPA1 showed reciprocal regulation towards these signaling proteins. No significant change of pERK or p-p38 levels was observed. Knock-down of PPA1 in SW480 cells inhibited cell proliferation (C), while overexpression of PPA1 in HT29 cells showed opposite effect (D). Data presented were results from three repeated experiments.

Figure 4. Phosphor-JNK level is negatively correlated with PPA1 expression in colon adenocarcinoma tissues

Representative IHC images of pJNK in primary tumor tissues. (A) IRS was scored as 0, with negative expression. (B) IRS was scored as 2, identified as (+) immunoreactivity. (C) IRS was scored as 6, defined as (++) expression. (D) IRS was scored as 9, and the staining was classified as (+++). (E) Logistic regression curve showed the negative association between PPA1 and pJNK levels (r=0.610, P<0.001).

Figure 5. Purification and enzymatic characterization of PPA1

(A) SDS-PAGE gels with Coomassie staining showed the high purity of PPA1-WT, PPA1-D117A and JNK1 proteins. (B) The synthesized phosphor-peptides and corresponding phosphor-sites were listed. (C) PPA1 can dephosphorylate pJNK phosphor-peptides (reaction time was 10min), while its inactive mutant PPA1-D117A (reaction time was 90min) almost abolished its activity. (D) PPA1 can directly dephosphorylate pJNK1 protein which was generated by in vitro methods. Data presented were results from three repeated experiments.

Figure 6. PPA1 enhances cancer cell viability through inhibiting JNK signaling pathways

(A) CCK-8 assay showed that PPA1 transfection enhanced the cell proliferation of cancer cells, while no significant effect was identified upon PPA1 inactive mutant transfection. In contrast, PPA1-silencing significantly down-regulated the cell proliferation, while JNK inhibitor can impair this antiproliferation effect. (B) Western Blot results demonstrated that PPA1 can modulate p53, Bax, p-Bcl-2 and cleaved Caspase-3 levels by recruiting JNK as an intermediate signaling molecular. Histogram showed the quantification results calculated by Image J Software from three experiments. (C) Schematically illustrations about the possible molecular and pathways involved in the PPA1 functions towards cancer transformation and development.