Haili Wu

Cell-surface GRP78 facilitates colorectal cancer cell migration and invasion

Glucose regulated protein 78 (GRP78) is predominantly located in the endoplasmic reticulum as a molecular chaperone. It has also been found on the membranes of some cancer cells, acting as a receptor for a wide variety of ligands. However, its presence on colorectal cancer (CRC) cell surface and its role in CRC metastatic progression remain elusive. Here we reported that GRP78 was predominantly present in the form of clustering aggregates on CRC cell surfaces, and its surface abundance was strongly correlated with CRC differentiation stage. Interestingly, we observed that cell-surface GRP78 had an interaction with the ECM adhesion molecule β1-integrin and was involved in cell-matrix adhesion through regulation of focal adhesion kinase (FAK). Moreover, the present data also implicated that surface GRP78 promoted the cell invasion process, and this effect was at least partly mediated through its association with uPA-uPAR protease system. Together, our data suggests that surface GRP78 promotes CRC cell migration and invasion by regulating cell-matrix adhesion and ECM degradation, which is independent of its signaling receptor function.

Pyruvate kinase M2 facilitates colon cancer cell migration via the modulation of STAT3 signalling

Understanding the mechanisms of colorectal cancer (CRC) metastatic progression is essential to reducing its morbidity and mortality. Pyruvate kinase (PK) catalyses the final step of glycolysis and has been identified as a critical regulator of glucose consumption. However, the mechanisms and roles of PKM1 and PKM2 in the regulation of CRC cell migration and cell adhesion remain elusive. Here, we report that PKM2 rather than PKM1 drives CRC cell migration and cell adhesion, whereas PKM attenuation reverses these phenomena. Furthermore, the overexpression of PKM2 significantly increases the expression of N-cadherin, MMP-2, MMP-9, STAT3, Snail-2, pFAK and active β1-integrin, while E-cadherin expression is suppressed. More importantly, the results indicated that PKM2 overexpression facilitates STAT3 nuclear translocation, and it is required for PKM2 function in the regulation of migration and adhesion associated signalling. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities but possesses protein kinase activity, is critical for CRC cell migration and cell adhesion. Overall, this study suggests that PKM2 overexpression promotes CRC cell migration and cell adhesion by regulating STAT3-associated signalling and that PKM2 may serve as a therapeutic target for CRC metastasis.

Pyruvate kinase M2 accelerates pro-inflammatory cytokine secretion and cell proliferation induced by lipopolysaccharide in colorectal cancer

Surgery-induced inflammation has been associated with cancer recurrence and metastasis in colorectal cancer (CRC). As a constituent of gram-negative bacteria, lipopolysaccharide (LPS) is frequently abundant in the peri-operative window. However, the definite roles of LPS in tumour progression remain elusive. Here we reported that LPS treatment increased PKM expression through activation of NF-κB signalling pathway, and knockdown of PKM reversed LPS-induced TNF-α, IL-1β production and cell proliferation in CRC cells. We further showed that the PKM2 but not PKM1 mediated the pro-inflammatory and proliferative effects of LPS. Interestingly, LPS promoted PKM2 binding to the STAT3 promoter to enhance STAT3 expression and its subsequent nuclear translocation. Depletion of STAT3 decreased PKM2-induced TNF-α and IL-1β expression, indicating that STAT3 mediates the pro-inflammatory effects of PKM2. Furthermore, it is the protein kinase activity but not the pyruvate kinase activity of PKM2 that is required for inflammatory cytokine production. Collectively, our findings reveal the NF-κB-PKM2-STAT3 axis as a novel mechanism for the regulation of TNF-α and IL-1β production and suggest the importance of PKM2 as a key inflammatory mediator in inflammatory microenvironment.

PKM2 depletion induces the compensation of glutaminolysis through β-catenin/c-Myc pathway in tumor cells.

The metabolic activity in cancer cells primarily rely on aerobic glycolysis. Besides glycolysis, some tumor cells also exhibit excessive addition to glutamine, which constitutes an advantage for tumor growth. M2-type pyruvate kinase (PKM2) plays a pivotal role in sustaining aerobic glycolysis, pentose phosphate pathway and serine synthesis pathway. However, the participation of PKM2 in glutaminolysis is little to be known. Here we demonstrated that PKM2 depletion could provoke glutamine metabolism by enhancing the β-catenin signaling pathway and consequently promoting its downstream c-Myc-mediated glutamine metabolism in colon cancer cells. Treatment with 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor, got consistent results with the above. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities, plays a critical role in regulating β-catenin. Moreover, we found that overexpression of PKM2 negatively regulated β-catenin through miR-200a. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon glucose metabolism impaired.

Secreted pyruvate kinase M2 facilitates cell migration via PI3K/Akt and Wnt/β-catenin pathway in colon cancer cells

Pyruvate Kinase M2 (PKM2) is a key glycolytic enzyme, which highly expressed in tumor cells, and plays a pivotal role in the growth, survival and metabolism reprogramming of cancer cells. Besides the location of cytoplasm as a glycolytic enzyme and the location of nucleus as a protein kinase, extracellular PKM2 is present in serum and feces of tumor patients. However, little is known about the secretion of PKM2 and its significance in the progression of colon cancer. Here we demonstrated that PKM2 could be secreted from colon cancer cells, and purified PKM2 protein mimicing the secreted PKM2 was able to promote colon cancer cell migration. Moreover, PI3K/Akt and Wnt/β-catenin signaling were involved in secreted PKM2 induced colon cancer cell migration. The results reveal critical roles of secreted PKM2 in the progression of colon cancer, and indicate that PKM2 may be a therapeutic target for colon cancer.

Direct contacts with colon cancer cells regulate the differentiation of bone marrow mesenchymal stem cells into tumor associated fibroblasts.

Tumor-stroma interactions are referred to as essential events in tumor progression. There has been growing attention that bone marrow-derived mesenchymal stem cells (BMSCs) can travel to tumor stroma, where they differentiate into tumor-associated fibroblast (TAF)-like cells, a predominant tumor-promoting stromal cell. However, little is definitively known about the contributors for this transition. Here, using an in vitro direct co-culture model of colon cancer cells and BMSCs, we identify that colon cancer cells can induce adjoining BMSCs to exhibit the typical characteristic of TAFs, with increased expression of α-smooth muscle actin (α-SMA). Importantly, the present data also reveals that activated Notch signaling mediates transformation of BMSCs to TAFs through the downstream TGF-β/Smad signaling pathway.

Migration Suppression of Small Cell Lung Cancer by Polysaccharides from Nostoc commune Vaucher

Nostoc commune Vauch., classified into cyanobacteria, has been always well appreciated as a healthy food and medicine worldwide owing to its rich nutrition and potent bioactivities. Nevertheless, the inhibitory effect of polysaccharides from N. commune Vauch. (NVPS) against cancer cell progression and metastasis is still being unraveled. The results in this study showed that NVPS remarkably suppressed cell migration through blocking the epithelial-mesenchymal transition program in NCI-H446 and NCI-H1688 human small cell lung cancer cells. The inhibitory effects were attributed to the suppression of integrin β1/FAK signaling through regulating cell-matrix adhesion. Furthermore, NVPS treatment could increase E-cadherin expression, but down-regulate N-cadherin, Vimentin, and MMP-9 expression, which resulted in the blockage of STAT3 nuclear translocation and JAK1 signaling. These findings suggest that NVPS may be a good candidate for development as a possible antitumor agent against small cell lung cancer.

Overexpression of PKM2 promotes mitochondrial fusion through attenuated p53 stability

M2-type pyruvate kinase (PKM2) contributes to the Warburg effect. However, it remains unknown as to whether PKM2 has an inhibitory effect on mitochondrial function. We report in this work that PKM2 overexpression inhibits the expression of Drp1 and results in the mitochondrial fusion. The ATP production was found to be decreased, the mtDNA copy number elevated and the expression level of electron transport chain (ETC) complex I, III, V depressed in PKM2 overexpressed cells. PKM2 overexpression showed a decreased p53 protein level and a shorter p53 half-life. In contrast, PKM2 knockdown resulted in increased p53 expression and prolonged half-life of p53. PKM2 could directly bind with both p53 and MDM2 and promote MDM2-mediated p53 ubiquitination. The dimeric PKM2 significantly suppressed p53 expression compared with the other PKM2 mutants. The reverse relationship between PKM2 and Drp1 was further confirmed in a large number of clinical samples. Taken together, the present results highlight a new mechanism that link PKM2 to mitochondrial function, based on p53-Drp1 axis down regulation, revealing a novel therapeutic target in patients with abnormal mitochondria.

Apigenin Restrains Colon Cancer Cell Proliferation via Targeted Blocking of Pyruvate Kinase M2-Dependent Glycolysis

Apigenin (AP), as an anticancer agent, has been widely explored. However, the molecular targets of apigenin on tumor metabolism are unclear. Herein, we found that AP could block cellular glycolysis through restraining the tumor-specific pyruvate kinase M2 (PKM2) activity and expression and further significantly induce anti-colon cancer effects. The IC50 values of AP against HCT116, HT29, and DLD1 cells were 27.9 ± 2.45, 48.2 ± 3.01 and 89.5 ± 4.89 μM, respectively. Fluorescence spectra and solid-phase AP extraction assays proved that AP could directly bind to PKM2 and markedly inhibit PKM2 activity in vitro and in HCT116 cells. Interestingly, in the presence of d-fructose-1,6-diphosphate (FBP), the inhibitory effect of AP on PKM2 was not reversed, which suggests that AP is a new allosteric inhibitor of PKM2. RT-PCR and Western blot assays showed that AP could ensure a low PKM2/PKM1 ratio in HCT116 cells via blocking the β-catenin/c-Myc/PTBP1 signal pathway. Hence, PKM2 represents a novel potential target of AP against colon cancer.

A positive feedback loop between GRP78 and VPS34 is critical for GRP78-mediated autophagy in cancer cells

ABSTRACT Autophagy and GRP78 overexpression are two important means by which tumor cells resist microenvironmental stress and chemotherapeutic drugs; however, the relationship between autophagy and GRP78 remains unclear. Here, we found that forced expression of GRP78 in tumor cells promoted autophagy, which was indicated by alterations in the levels of autophagy related proteins, such as increased VPS34 and LC3‐II, and decreased p62 and LC3‐I. Consistently, GRP78 knockdown suppressed tumor cell autophagy. Our results further demonstrated that GRP78‐induced autophagy was mediated by VPS34, and that UPR‐associated autophagy was also involved. GRP78‐overexpressing cells treated with VPS34 siRNA reversed the autophagy induced by GRP78. Importantly, the expression of microRNA‐143 (miR‐143) was decreased in GRP78‐overexpressing cells, and the increased expression of VPS34 was reversed by treatment with miR‐143 mimic. This demonstrated that miR‐143 plays a key role in GRP78s mediation of VPS34 expression. In addition, GRP78 acetylation was also involved in the occurrence of autophagy through upregulating VPS34. In turn, high expression of VPS34 promoted GRP78 transcription by modulating the GRP78 transcription factor ATF6. Moreover, VPS34 could enhance GRP78 protein stability by inhibiting GRP78 degradation via the ubiquitin‐proteasome pathway. Collectively, the results revealed a positive feedback loop between GRP78 and VPS34 in tumor cells that might be important for autophagy during tumor development.