Tumor-Suppressive Effect of Metformin via the Regulation of M2 Macrophages and Myeloid-Derived Suppressor Cells in the Tumor Microenvironment of Colorectal Cancer.

Myeloid-derived suppressor cells (MDSCs) and M2 macrophages in the tumor microenvironment contribute to tumor progression by inducing immune tolerance to tumor antigens and cancer cells. Metformin, one of the most common diabetes drugs, has shown anti-inflammatory and anti-tumor effects. However, the effects of metformin on inflammatory cells of the tumor microenvironment and its underlying mechanisms remain unclarified. In this study, we investigated the effect of metformin on M2 macrophages and MDSCs using monocyte THP-1 cells and a dextran sodium sulfate (DSS)-treated ApcMin/+ mouse model of colon cancer. Metformin decreased the fractions of MDSCs expressing CD33 and arginase, as well as M2 macrophages expressing CD206 and CD163. The inhibitory effect of metformin and rapamycin on MDSCs and M2 macrophages was reversed by the co-treatment of Compound C (an AMP-activated protein kinase (AMPK) inhibitor) or mevalonate. To examine the effect of protein prenylation and cholesterol synthesis (the final steps of the mevalonate pathway) on the MDSC and M2 macrophage populations, we used respective inhibitors (YM53601; SQLE inhibitor, FTI-277; farnesyl transferase inhibitor, GGTI-298; geranylgeranyl transferase inhibitor) and found that the MDSC and M2 populations were suppressed by the protein prenylation inhibitors. In the DSS-treated ApcMin/+ mouse colon cancer model, metformin reduced the number and volume of colorectal tumors with decreased populations of MDSCs and M2 macrophages in the tumor microenvironment. In conclusion, the inhibitory effect of metformin on MDSCs and M2 macrophages in the tumor microenvironment of colon cancers is mediated by AMPK activation and subsequent mTOR inhibition, leading to the downregulation of the mevalonate pathway.

MRI assessment of glutamine uptake correlates with the distribution of glutamine transporters and cancer stem cell markers.

Glutamine provides carbon and nitrogen for macromolecular synthesis and participates in adenosine triphosphate (ATP) generation, anabolic metabolism, redox homeostasis, cell signaling, and cancer stem cell (CSC) metabolism. New treatment strategies targeting glutamine metabolism in cancer have emerged recently. We previously reported the magnetic resonance imaging (MRI) assessment of glutamine uptake by tumors and activated glutamine metabolism in CSC. In the present study, using MRI, we determined the correlation between glutamine uptake and the distribution of glutamine transporters, namely ASCT2 and SLC38A2 (SNAT2), glutaminase (GLS), and CSC markers, such as CD44 and CD166, in a mouse xenograft model of HT29 human colorectal cancer cells. MRI data revealed an obvious change in intensity following glutamine administration. Immunohistochemistry (IHC) results indicated that ASCT2 staining was stronger in regions that exhibited high glutamine uptake (p = 0.0079). Significant differences were found in the IHC staining intensities of SNAT2, GLS, and CSC markers in the areas of high and low glutamine uptake (p = 0.0079, p = 0.0159 and p = 0.0079, respectively). We also investigated the effect of an ASCT2 inhibitor on the uptake of glutamine using MRI. A statistically significant difference in the initial glutamine uptake was found after ASCT2 inhibitor administration. To conclude, glutamine uptake is positively correlated with the distribution of ASCT2 and certain CSC markers.

Metformin and Niclosamide Synergistically Suppress Wnt and YAP in APC-Mutated Colorectal Cancer.

The Wnt and Hippo pathways are tightly coordinated and understanding their reciprocal regulation may provide a novel therapeutic strategy for cancer. Anti-helminthic niclosamide is an effective inhibitor of Wnt and is now in a phase II trial for advanced colorectal cancer (CRC) patients. We found that Axin2, an authentic target gene of canonical Wnt, acts as aYAP phosphorylation activator in APC-mutated CRC. While niclosamide effectively suppresses Wnt, it also inhibits Hippo, limiting its therapeutic potential for CRC. To overcome this limitation, we utilized metformin, a clinically available AMPK activator. This combinatory approach not only suppresses canonical Wnt activity, but also inhibits YAP activity in CRC cancer cells and in patient-derived cancer organoid through the suppression of cancer stemness. Further, combinatory oral administration suppressed in vivo tumorigenesis and the cancer progression of APC-MIN mice models. Our observations provide not only a reciprocal link between Wnt and Hippo, but also clinically available novel therapeutics that are able to target Wnt and YAP in APC-mutated CRC.

mTOR Signaling Combined with Cancer Stem Cell Markers as a Survival Predictor in Stage II Colorectal Cancer.

PURPOSE: Wnt and mammalian target of rapamycin (mTOR) are major molecular signaling pathways associated with the development and progression of tumor, as well as the maintenance and proliferation of cancer stem cells (CSCs), in colorectal cancer (CRC). Identifying patients at risk of poor prognosis is important to determining whether to add adjuvant treatment in stage II CRC and thus improve survival. In the present study, we evaluated the prognostic value of Wnt, mTOR, and CSC markers as survival predictors in stage II CRC. MATERIALS AND METHODS: We identified 148 cases of stage II CRC and acquired their tumor tissue. Tissue microarrays for immunohistochemical staining were constructed, and the expressions of CD166, CD44, EphB2, ß-catenin, pS6 were evaluated using immunohistochemical staining. RESULTS: The expressions of CD166 (p=0.045) and pS6 (p=0.045) and co-expression of pS6/CD166 (p=0.005), pS6/CD44 (p=0.042), and pS6/CD44/CD166 (p=0.013) were negatively correlated with cancer-specific survival. Cox proportional hazard analysis showed the combination of CD166/pS6 [hazard ratio, 9.42; 95% confidence interval, 2.36-37.59; p=0.002] to be the most significant predictor related with decreased cancer-specific survival. In addition, co-expression of CD44/CD166 (p=0.017), CD166/ß-catenin (p=0.036), CD44/ß-catenin (p=0.001), and CD44/CD166/ß-catenin (p=0.001) were significant factors associated with liver metastasis. CONCLUSION: Specific combinations of CSC markers and ß-catenin/mTOR signaling could be a significant predictor of poor survival in stage II CRC.