Inhibition of CCL7 derived from Mo-MDSCs prevents metastatic progression from latency in colorectal cancer.

In colorectal cancer (CRC), overt metastases often appear after years of latency. But the signals that cause micro-metastatic cells to remain indolent, thereby enabling them to survive for extended periods of time, are unclear. Immunofluorescence and co-immunoprecipitation assays were used to explore the co-localization of CCL7 and CCR2. Immunohistochemical (IHC) assays were employed to detect the characters of metastatic HT29 cells in mice liver. Flow cytometry assays were performed to detect the immune cells. Bruberin vivo MS FX Pro Imager was used to observe the liver metastasis of CRC in mice. Quantitative real-time PCR (qRT-PCR) and western blot were employed to detect the expressions of related proteins. Trace RNA sequencing was employed to identify differentially expressed genes in MDSCs from liver micro-M and macro-M of CRC in mice. Here, we firstly constructed the vitro dormant cell models and metastatic dormant animal models of colorectal cancer. Then we found that myeloid-derived suppressor cells (MDSCs) were increased significantly from liver micro-metastases to macro-metastases of CRC in mice. Moreover, monocytic MDSCs (Mo-MDSC) significantly promoted the dormant activation of micro-metastatic cells compared to polymorphonuclear MDSCs (PMN-MDSC). Mechanistically, CCL7 secreted by Mo-MDSCs bound with membrane protein CCR2 of micro-metastatic cells and then stimulated the JAK/STAT3 pathway to activate the dormant cells. Low-dose administration of CCL7 and MDSCs inhibitors in vivo could significantly maintain the CRC metastatic cells dormant status for a long time to reduce metastasis or recurrence after radical operation. Clinically, the level of CCL7 in blood was positively related to the number of Mo-MDSCs in CCR patients, and highly linked with the short-time recurrence and distant metastasis. CCL7 secreted by Mo-MDSCs plays an important role in initiating the outgrowth of metastatic latent CRC cells. Inhibition of CCL7 might provide a potential therapeutic strategy for the prevention of metastasis recurrence.

PRMT5 regulates colorectal cancer cell growth and EMT via EGFR/Akt/GSK3ß signaling cascades.

Emerging evidence shows that type II protein arginine methyltransferase 5 (PRMT5) serves as an oncoprotein and plays a critical role in many types of human cancer. However, the precise role and function of PRMT5 in human colorectal cancer (CRC) growth and epithelial-mesenchymal transition (EMT) are still unclear, and the related molecular mechanism and signaling axis remains largely obscure. Here, we show that PRMT5 is highly expressed in CRC cell lines and tissues. Using PRMT5 stable depletion cell lines and specific inhibitor, we discover that down-regulation of PRMT5 by shRNA or inhibition of PRMT5 activity by specific inhibitor GSK591 markedly suppresses CRC cell proliferation and cell cycle progression, which is closely associated with PRMT5 enzyme activity. Moreover, PRMT5 regulates CRC cell growth and cycle progression via activation of Akt, but not through ERK1/2, PTEN, and mTOR signaling pathway. Further study shows that PRMT5 controls EMT of CRC cells by activation of EGFR/Akt/GSK3ß signaling cascades. Collectively, our results reveal that PRMT5 promotes CRC cell proliferation, cell cycle progression, and EMT via regulation of EGFR/Akt/GSK3ß signaling cascades. Most importantly, our findings also suggest that PRMT5 may be a potential therapeutic target for the treatment of human colorectal cancer.

GFPT2 promotes metastasis and forms a positive feedback loop with p65 in colorectal cancer.

As a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP), which is responsible for glycosylation, Glutamine fructose-6-phosphate amidotransferase 2 (GFPT2) is involved in human breast and lung tumorigenesis. However, whether GFTP2 is associated with tumor metastasis remains unclear. Here, we found that GFPT2 promoted the proliferation, migration, invasion and metastasis of colorectal cancer (CRC) cells. Mechanically, p65 acted as an upstream transcription factor of GFPT2 and regulated its expression and function. In turn, GFPT2 enhanced the glycosylation of p65, which led to the nuclear translocation of p65 and then activated NF-κB pathway. Thus, GFTP2 and p65 formed a positive feedback loop to promote the progression of CRC. In addition, GFPT2 was up-regulated in CRC tissues and closely related with liver metastasis (P<0.0001) and tumor stage (P=0.0184). High expression of GFPT2 predicted poor prognosis for CRC patients. Moreover, GFTP2 expression was positively linked with O-linked N-acetylglucosamine transferase in CRC tissues. Our study reveals a new mechanism of GFPT2 in CRC metastasis and provides a new target therapeutic target to deter metastasis.

KNK437 restricts the growth and metastasis of colorectal cancer via targeting DNAJA1/CDC45 axis.

As an inhibitor of heat shock proteins (HSPs), KNK437 has been reported to play an anti-tumor role in several cancers. But its therapeutic effect and mechanisms in colorectal cancer (CRC) remain unclear. Here, KNK437 sharply inhibited the level of DnaJ heat shock protein family (Hsp40) member A1 (DNAJA1), followed by DNAJB1, but had little effect on the levels of HSP27, HSP105, HSP90, and HSP70 in CRC cells. DNAJA1 promoted CRC cell proliferation in vitro and tumor growth and metastasis in vivo. Mechanistically, DNAJA1 was activated by E2F transcription factor 1 (E2F1) and then promoted cell cycle by stabilizing cell division cycle protein 45 (CDC45), which could be reversed by KNK437. DNAJA1 was significantly upregulated in CRC tissues and positively correlated with serosa invasion, lymph node metastasis. High level of DNAJA1 predicted poor prognosis for CRC patients. Its expression was highly linked with E2F1 and CDC45 in CRC tissues. More importantly, KNK437 significantly suppressed the growth of DNAJA1 expressing tumor in vivo. The combined treatment of KNK437 with 5-FU/L-OHP chemotherapy reduced liver metastasis of CRC. These data reveal a novel mechanism of KNK437 in anti-tumor therapy of CRC and provides a newly therapeutic strategy with potential translation to the CRC patients.

MicroRNA-147b Promotes Proliferation and Invasion of Human Colorectal Cancer by Targeting RAS Oncogene Family (RAP2B).

BACKGROUND: MicroRNAs (miRNAs), a class of small-regulatory RNA molecules, were closely involved in the pathogenesis of a broad-spectrum of colorectal cancer (CRC). But role of miR-147b in CRC still remains unclear. METHODS: Real-time RT-PCR or Western blotting was utilized to detect the expressions of miR-147b and RAP2B in CRC cell lines and tissues. Luciferase reporter assays were conducted to detect the associations between miR-147b and 3'UTRs of RAP2B. A series of assays were performed to evaluate the effect of miR-147b on proliferation, migration, and invasion of CRC in vitro and in vivo. RESULTS: We found that the level of miR-147b was significantly lower in CRC tissues than in normal tissues (p = 0.0006). Enforced expression of miR-147b led to suppression of CRC cell proliferation in vitro and tumor growth in vivo. Specifically, miR-147b promoted proliferation by arresting CRC cells in the G1/G0 phase. Mechanically, RAP2B was identified as a direct target gene of miR-147b and RAP2B rescued the suppression of proliferation and invasion reduced by miR-147b in CRC cells. CONCLUSIONS: miR-147b not only plays important roles in the regulation of cell proliferation and tumor growth in CRC, which might be a potential prognostic marker or therapeutic target for CRC.

miR-21 is involved in transforming growth factor ß1-induced chemoresistance and invasion by targeting PTEN in breast cancer.

Transforming growth factor ß1 (TGF-ß1) has been associated with poor outcomes in patients with breast cancer. However, the functions and underlying molecular mechanisms of TGF-ß1 in breast cancer remain unknown. Therefore, the present study aimed to identify the effects of components of the TGF-ß/microRNA (miR-)21/phosphatase and tensin homolog (PTEN) signaling axis in breast cancer. TGF-ß1 was identified to upregulate the expression of miR-21, and miR-21 was demonstrated to be significantly upregulated in breast cancer tissues compared with benign proliferative breast disease. In addition, the expression of miR-21 was significantly associated with increased TGF-ß1 and clinical characteristics in patients, including tumor grade and lymph node metastasis (all P<0.05). Furthermore, in the breast cancer MCF-7 cell line, TGF-ß1 was revealed to induce the expression of miR-21 in a dose- and time-dependent manner. The results of the present study additionally demonstrated that increased miR-21, in response to TGF-ß1 signaling, was associated with tumor invasion and chemoresistance in vitro. In addition, suppression of PTEN was mediated by TGF-ß1-induced expression of miR-21 in breast cancer cells and using a miR-21 inhibitor revitalized the expression of PTEN. The results of the present study explored the functions of TGF-ß1-stimulated expression of miR-21 to suppress the PTEN axis, which promotes breast cancer progression and chemoresistance.

MicroRNA-221 targeting PI3-K/Akt signaling axis induces cell proliferation and BCNU resistance in human glioblastoma.

MicroRNAs (miRNAs) are short regulatory RNAs that negatively regulate protein biosynthesis at the post-transcriptional level and participate in the pathogenesis of different types of human cancers, including glioblastoma. In particular, the levels of miRNA-221 are overexpressed in many cancers and miRNA-221 exerts its functions as an oncogene. Nevertheless, the roles of miRNA-221 in carmustine (BCNU)-resistant glioma cells have not been totally elucidated. In the present study, we explored the effects of miRNA-221 on BCNU-resistant glioma cells and the possible molecular mechanisms by which miRNA-221 mediated the cell proliferation, survival, apoptosis and BCNU resistance were investigated. We found that miR-221 was overexpressed in glioma cells, including BCNU-resistant cells. Moreover, we found that miR-221 regulated cell proliferation and BCNU resistance in glioma cells. Overexpression of miR-221 led to cell survival and BCNU resistance and reduced cell apoptosis induced by BCNU, whereas knockdown of miR-221 inhibited cell proliferation and prompted BCNU sensitivity and cell apoptosis. Further investigation revealed that miR-221 down-regulated PTEN and activated Akt, which resulted in cell survival and BCNU resistance. Overexpression of PTEN lacking 3'UTR or PI3-K/Akt specific inhibitor wortmannin attenuated miR-221-mediated BCNU resistance and prompted cell apoptosis. We propose that miR-221 regulated cell proliferation and BCNU resistance in glioma cells by targeting PI3-K/PTEN/Akt signaling axis. Our findings may provide a new potential therapeutic target for treatment of glioblastoma.

[miR-221 mediates epithelial-mesenchymal transition-related gene expressions via regulation of PTEN/Akt signaling in drug-resistant glioma cells].

OBJECTIVE: To investigate the correlation between miR-221 and epithelial-mesenchymal transition (EMT) in drug-resistant glioma cells. METHODS: The expression levels of miR-221, PTEN, p-Akt, E-cadherin, vimentin, and MRP1 were quantitatively analyzed in Z1 cells (primary drug-resistant cells), Z2 cells (drug-sensitive cells) and Z2-BCNU cells (drug-resistant cells) using fluorescent real-time PCR and Western blotting. RESULTS: The expression levels of PTEN were significantly increased in Z2 cells compared with Z1 and Z2-BCNU cells which overexpressed miR-221 and vimentin. The expression levels of vimentin, p-Akt and MRP1 were significantly decreased in Z2 cells overexpressing E-cadherin. CONCLUSION: MiR-221 regulates the expression of EMT-related genes through down-regulation of PTEN and activation of PI3-K/Akt signaling.

Epithelial-to-mesenchymal transition is involved in BCNU resistance in human glioma cells.

Chemotherapy has been considered as an effective treatment for malignant glioma; however, it becomes increasingly ineffective with tumor progression. Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire morphologic and molecular alterations that facilitate tumor metastasis and progression. Emerging evidence associates chemoresistance with the acquisition of EMT in cancer. However, it is not clear whether this phenomenon is involved in glioma. We used the previously established human glioma cell lines SWOZ1, SWOZ2 and SWOZ2-BCNU to assess cellular morphology, molecular changes, migration and invasion. We found that BCNU-resistant cells showed multiple drug resistance and phenotypic changes consistent with EMT, including spindle-shaped morphology and enhanced pseudopodia formation. Decreased expression of the epithelial adhesion molecule E-cadherin and increased expression of the mesenchymal marker vimentin were observed in BCNU-resistant SWOZ1 and SWOZ2-BCNU cells compared to SWOZ2 cells. Migratory and metastatic potentials were markedly enhanced in SWOZ1 and SWOZ2-BCNU cells compared to SWOZ2 cells. These data suggest that there is a possible link between drug resistance and EMT induction in glioma cells. Gaining further insight into the mechanisms underlying chemoresistance and EMT may enable the restoration of chemosensitivity or suppression of metastasis.

Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer.

AIM: To study the correlation between high metastasis-associated protein 1 (MTA1) expression and lymphangiogenesis in colorectal cancer (CRC) and its role in production of vascular endothelial growth factor-C(VEGF-C). METHODS: Impact of high MTA1 and VEGF-C expression levels on disease progression and lymphovascular density (LVD, D2-40-immunolabeled) in 81 cases of human CRC was evaluated by immunohistochemistry. VEGF-C mRNA and protein expressions in human LoVo and HCT116 cell lines were detected by real-time polymerase chain reaction and Western blotting, respectively, with a stable expression vector or siRNA. RESULTS: The elevated MTA1 and VEGF-C expression levels were correlated with lymph node metastasis and Dukes stages (P < 0.05). Additionally, high MTA1 expression level was correlated with a large tumor size (P < 0.05). A significant correlation was found between MTA1 and VEGF-C protein expressions in tumor cells (r = 0.371, P < 0.05). Similar to the VEGF-C expression level, high MTA1 expression level was correlated with high LVD in CRC (P < 0.05). Furthermore, over-expression of MTA1 significantly enhanced the VEGF-C mRNA and protein expression levels, whereas siRNAs - knocked down MTA1 decreased the VEGF-C expression level. CONCLUSION: MTA1, as a regulator of tumor-associated lymphangiogenesis, promotes lymphangiogenesis in CRC by mediating the VEGF-C expression.