Mechanisms Of PKM2 Accelerated Cell Migration, Cell Adhesion And The Inflammatory Cytokine Secretion In Colorectal Cancer

Colorectal cancer (CRC) is one of the most common malignancies of the digestive system worldwide, with more than 1.2 million new cases and about 600,000 deaths annually. Surgical resections of the primary tumors are the major choice for the therapy of CRC. Tumor recurrence, distant metastasis and postoperative infection induced inflammation after surgery lead to the poor prognosis of CRC patients. The process of tumour metastasis is a multistep processes involving the loosening of cell-cell contacts, the enhancement of cell-matrix adhesion and the degradation of extracellular matrix components, mainly by matrix metalloproteinases (MMPs). Cell migration and adhesion are critical parameters of tumor metastasis. Lipopolysaccharide (LPS), an outer membrane glycoprotein of Gram-negative bacteria, can induce severe sepsis, systemic inflammation and even postoperative infectious complications after CRC surgery. The levels of LPS are often elevated in the peri-operative window. However, the definite roles of LPS in tumour progression remain elusive. Clarification of the mechanisms of CRC progression, CRC metastasis and CRC progression caused by inflammatory microenvironment are important to reduce its morbidity and mortality.Tumor cells consume large amounts of glucose and produce more lactate to generate energy rather than using oxidative phosphorylation, even when oxygen is abundant. This obvious metabolic feature is termed as Warburg effect. PKM2 plays an important role in the Warburg effect. Pyruvate kinase (PK) is a rate-limiting enzyme that catalyses the conversion from phosphoenolpyruvate (PEP) to pyruvate. PKM1 and PKM2, which are the most common isoforms of PK, are encoded by exclusive alternative splicing of the PKM gene. PKM1 majorly expressed in normal cells except liver, kidney and red blood cells, but PKM2 highly expressed in tumour cells. PKM1 is not affected by allosteric regulation, exists only as a highly active tetrameric form. The conformation of PKM2 is switch from dimer with protein kinase activity and tetramer with glycolytic activity to confer a selective advantage to adapt to various environmental signals in tumour cells. PKM2 has been reported to facilitate tumour cell proliferation, neoplastic transformation, angiogenesis, and cell cycle. However, the role of PKM1 and PKM2 on tumorigenesis, metastasis and inflammatory microenvironment in CRC remain elusive.The stable PKM knockdown cell lines, stably expressing PKM1, PKM2 and stably expressing various mutations of PKM2 (K367M, R399E and K433E) were constructed. These stable cells were used to explore the roles and mechanisms of PKM1 and PKM2 on migration, cell-matrix adhesion, proliferation and inflammatory cytokines secretion. The purified GST-PKM1, GST-PKM2 mimicing the secreted PKM1 and PKM2 were used to explore the role and mechanisms on CRC migration. The main research contents and results are shown as follows:1. Lentiviral vectors bearing PKM1, PKM2, mutations of PKM2 (K367M, R399E and K433E) and the PKM short hairpin RNA (shRNA) were constructed. Lentiviral supernatants were packaged, harvested and then infected to DLD1 cells. Cells were subjected to puromycin for screening DLD-1 stable cells. Western blot and qPCR analysis showed the stable cells were successfully obtained.2. Wound healing assay and cell adhesion assay showed that PKM2 overexpression but not PKM1 drove CRC cell migration and cell adhesion. Western blotting and qPCR analysis showed that the overexpression of PKM2 significantly increased the expression of N-cadherin, MMP-2, MMP-9, Snail-2, pFAK and active β1-integrin, while E-cadherin expression was suppressed. PKM attenuation reversed these phenomena. More importantly, PKM2 overexpression significantly increased the mRNA, protein, phosphorylated levels of STAT3 and facilitated STAT3 nuclear translocation. Actinomycin D assay indicated that STAT3 upregulation by PKM2 was due to increased transcription rather than decelerated mRNA decay. The pyruvate kinase activity assay, western blot and wound healing assay were used to determine properties of PKM2 mutant stable cells. The results showed that R399E mutants, which had clearly low pyruvate kinase activity, exhibited elevated STAT3 and pSTAT3 expression levels and promoted CRC cell migration.3. The levels of TNF-α, IL-1β and PKM2 were significantly increased in a dose dependent manner after LPS treatment. By contrast, levels of IL-6 were not affected by LPS. Western blot, qPCR, ELISA and MTT assays showed that knockdown of PKM in DLD1 cells led to a significant decrease in LPS induced expression levels of PKM2, TNF-α and IL-1β and suppressed CRC cell proliferation. PKM2 overexpression obviously elevated expression levels of TNF-α and IL-1β and promoted CRC cell proliferation with LPS stimulation. However, PKM1 overexpression had negligible effects on it. The inhibition of NF-κB with NF-κB inhibitor BAY-11-7082 blocked LPS-induced PKM2 expression, suggested that NF-κB was crucial for LPS stimulated PKM2 expression. The knockdown of PKM in DLD1 cells led to the total STAT3 protein levels and nuclear localization drastically decreased, in particular with LPS treatment. However, PKM2 silencing did not change the nuclear p65 and total p65 protein levels induced by LPS. The increase of TNF-α and IL-1β mRNA caused by PKM2 overexpression was reversed along with STAT3 knockdown. ChIP assay showed that LPS promoted the binding of PKM2 to the STAT3 promoter and activated PKM2 induced STAT3 transcription upon LPS stimulation and facilitated TNF-α and IL-1β production. The R339E mutant, which possessed protein kinase activity of PKM2, obviously elevated mRNA, protein, secreted levels of TNF-α and IL-1β.4. The purified GST-PKM1, GST-PKM2 mimicking the secreted PKM1 and PKM2 were used to investigate their effect on CRC cell migration. The results showed that secreted PKM2 was able to promote colon cancer cell migration. Knockdown of PKM2 suppressed secreted PKM2 and secreted PKM2 induced cell migration. Secreted PKM2 upregulated expressions of MMP-2, MMP-9, N-cadherin and β-catenin, and downregulated E-cadherin expression. Purified PKM2 treatment also upregulated the phosphorylation level of Akt, while LY294002 (a PI3K/Akt inhibitor) significantly reversed GST-PKM2 induced expressions of E-cadherin, N-cadherin and β-catenin. In addtion, GST-PKM2 induced the expression of E-cadherin and N-cadherin was reversed by knockdown of β-catenin.In summary, we found that PKM2 facilitated colon cancer cell adhesion and migration via a mechanism involving the induction of STAT3 transcription, and this effect is dependent on its protein kinase activity rather than pyruvate kinase activity. LPS stimulated both PKM2 expression and its binding to STAT3 promoter, which further drove the expression and nuclear translocation of STAT3 to facilitate TNF-α and IL-1β production. Secreted PKM2 facilitated CRC cell migration via PI3K/Akt and Wnt/β-catenin pathway in colon cancer cells. This study suggests that PKM2 overexpression promotes CRC cell migration, cell adhesion and inflammatory cytokine production. Therefore, PKM2 may serve as a therapeutic target for CRC metastasis and surgery-induced inflammation.