The Roles Of PURA And RIP3 Underlying Tumorigenesis In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is the most prevalent pathological type of esophageal cancer in China. Lymphatic metastasis commonly occurs in ESCC patients, and thus, these patients have a poor prognosis; the five-year survival rate of ESCC patients is no more than 10%. Programmed cell death (apoptosis) is one of the most important processes of cell death in multicellular organisms. The reduction of apoptosis can promote the formation and progression of cancer. Caspase-8 is a member of the cysteine protease family; it can be recruited to form death-inducing signaling complex (DISC) when cells are treated with an exogenous apoptosis signal, and activated Caspase-8 further induces apoptosis. Caspase-8L, which lacks the catalytic domain, is an isoform of Caspase-8. It competitively binds to DISC in Jurkat cells, thereby inhibiting apoptosis. Purine-rich element binding protein alpha (PURa) is a key player during the regulation of cell cycle and oncogenic transformation. PURa binds to both DNA and RNA directly or indirectly and functions as the initiation of DNA replication and the control of transcription, mRNA transport and translation. In this study, a PURa protein-overexpressing ESCC cell line (KYSE 510-PURa) and the control cell line KYSE 510-pCMV6 were constructed through the transfection of the plasmid pCMV6-PURa or empty pCMV6 vector. Overexpression of PURa promoted the expression of Caspase-8L and its cytoplasmic localization as well as inhibited the activity of Caspase-8. In contrast, inhibition of PURa expression enhanced Caspase-8 activity. In general, the PURa protein-overexpressing ESCC cell line was constructed as an indirect Caspase-8L overexpression cell model, and using this cell model, we found that the high expression and cytoplasmic localization of Caspase-8L inhibited the activity of Caspase-8.Invasion and metastasis are the critical biological characteristics that are related to the poor prognosis of cancer. Epithelial-mesenchymal transition (EMT) is one of the most important steps in the migration of tumor cells. However, little is known about the invasion and metastasis of ESCC, and the relationship between EMT and PURa has not been reported. The transcriptome analysis showed many of the genes that are related to cell adherin were obviously changed in KYSE 510-PURa cells. The western blotting and immunofluorescent staining results showed that the expression of E-cadherin was reduced; however, the Vimentin, N-cadherin and Snail levels were increased in the KYSE 510-PURa cells. Most strikingly, the cell morphologies were changed into fibroblast-like cells, and the cells had acquired the ability to migrate and invade. Our data suggest that the regulation of PURa expression in ESCC cells may serve as a key factor in inducing esophageal EMT.Cisplatin is a commonly used chemotherapeutic drug in the treatment of tumors, as it can cause the death of cancer cells. Receptor-interacting protein kinase 3 (RIP3) plays an important role during apoptosis and necrosis. Transient or stable knockdown of RIP3 expression in KYSE 140 cells reduced their sensitivity to cisplatin. In contrast, the overexpression of RIP3 in KYSE 510 and MEF cells enhanced the sensitivity of these cells to cisplatin. A stable knockout of RIP3, the cell line HEK293T-RIP3KO, was obtained by the CRISPR/Cas9 technique. Cell activity was detected in cells transfected with wild type, kinase domain mutation RIP3 or empty vector followed by a 24-h treatment with different concentrations of cisplatin, and the results showed that overexpression of RIP3 can enhance sensitivity to cisplatin; this effect was not dependent on RIP3 kinase activity. The western blotting results showed that the expression of CDC37, c-JUN, FOSL1 and POLD1 and the phosphorylation of JNK, ERK and AKT, which are the key molecules involved in the DNA damage repair pathway, decreased in HEK293T-RIP3KO cells after the overexpression of RIP3. In conclusion, RIP3 was likely to enhance cell sensitivity to cisplatin by influencing the DNA damage repair pathway, and this phenomenon might be universal in different cells. Thus, RIP3 may be a potential drug target to influence sensitivity to cisplatin of different cell types.