Loss Of PCBP1 Increases LC3B Expression And Enhances Autophagy

Objective: Poly C binding protein 1(PCBP1), a nucleotides-binding protein with molecule weight of 38 Kilodalton(KD), is named by its high affinity for sequence-specific interaction with polycytosine in nucleic acids. PCBP1 is believed to have multiple roles in gene expression, including gene transcription, pre-m RNA splicing, m RNA stabilization, translational enhancement and silence. As a tumor suppressor, PCBP1 is widely downregulated in tumor tissues, including colorectal cancer,lung cancer,cervical carcinoma, breast and liver cancer, and its deletion is correlated with the development of those tumors. Ovarian carcinoma is the most malignant of gynecology oncology. However, the roles of PCBP1 involved in ovarian tumorigenesis have rarely been reported and still need to be further investigated. Autophagy is a conserved catabolic pathway characterized by the double-membrane vesicles formation(autophagosomes) that can swallow cytoplasmic proteins and organelles, and then conjugate to lysosomes for their luminal content degradation. Recently, autophagy has been disclosed as a key effecter in tumor progression and cancer treatment, although it?s precise function largely remains elusive. Generally, autophagy putatively functions as a cytoprotective manner to favor stresses adaptation and to prevent cell death. For example cells in the center of tumors are deprived of nutrition and have higher autophagic flux than cells in the margins of tumors to prevent their death. So far, it still remains elusive that which factor modulates starvation-induced autophagy and determines the eventual destiny of tumor cells. To disclose the role of PCBP1 in ovarian tumorigenesis, we first applied the IHC to confirm the correlation between PCBP1 expression level and ovarian tumor progression. We then use proliferation, autophagy and apoptosis assayes, and m RNA stability experiment to clarify the underlying mechanism of PCBP1 suppressing tumor formation under starvation conditions. Finally, we further validate the relationship between PCBP1 expression level and autophagy level in ovarian tumor samples by IHC detected p62 protein levels. Collectively, we expect that the expression status of PCBP1 could be a predictive biomarker, and antiautophagic administration would be an alternative therapeutical strategy for patients with low level of PCBP1 expression and high autophagy level.Results: 1. We firstly validate that PCBP1 is significantly lower expression in ovarian tumor samples than adjacent cancer tissues(p < 0.0001), and its downregulation or deletion is positively correlated with the ovarian tumor stages(p < 0.0062), clinic stages(p < 0.0317) and metastasis(p < 0.0317). 2. Our results indicate that PCBP1 overexpression attenuates tumor cell growth upon serum-free culture conditions in A2780 and DLD-1 cell lines(p < 0.001).Notably, the autophagic degradation inhibitor, chloroquine(CQ), could mimic this repressive effect in tumor cell growth(p < 0.001). 3. Autophagy assays demonstrated that overexpression of PCBP1 blocked autophagic flux of tumor cells upon starvation in A2780 and DLD-1 cell lines, while PCBP1 downregulation in turn refueled this autophagic flux, for cell survival. Mechanistically, PCBP1 attenuated microtubule-associated protein Light chain 3B(LC3B) m RNA stability to suppress LC3 B gene expression, resulting in the autophagy inhibition. 4. We also demonstrate that overexpression of PCBP1 strongly triggered the caspase 3 and 8-mediated apoptosis of tumor cells and down-regulated anti-apoptotic Bcl-2 expression under starvation conditions, which could be further synergized by autophagic inhibitor CQ, indicating that PCBP1 not only represses tumor cell autophagy, but also promotes them to apoptosis. 5. Clinically, our results confirm that SQSTM1/p62 have significantly lower expression in ovarian tumor sample than adjacent tumor sample(p < 0.002). But its expression level is not correlated with the development of tumor. Importantly, SQSTM1/p62 as an autophagic degradation substrate is also positively correlated with PCBP1 expression level in clinic ovarian tumor samples(R2 = 0.506, p < 0.01). This suggests that PCBP1 deletion promotes tumor cell autophagy level result in the ovarian tumorigenesis.Conclusion: Taken together, our results disclose a novel mechanism of PCBP1 in suppressing autophagy-mediated cell survival and suggested that inhibition of tumor cell autophagy by PCBP1 overexpression or with autophagic inhibitors CQ treatment could be an effective therapeutical strategy to colon and ovary tumors with PCBP1 downregulation or deletion.