Background:Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance.Methods:We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining.Results:The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients.Conclusions:miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist, than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth factor (CTGF)/CCN2. Tumor cell-derived CCL4 could induce fibroblasts to express CTGF/CCN2, which could support 4T1.3 clone proliferation under hypoxic culture conditions. Thus, the CCL4-CCR5 axis can contribute to breast cancer metastasis to bone by mediating the interaction between cancer cells and fibroblasts in bone cavity. Sasaki et al. 3

Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)—the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the β-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinib-sensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.

The CD74-Neuregulin1 (NRG1) fusion gene was recently identified in invasive mucinous adenocarcinoma, a malignant type of lung adenocarcinoma, and is considered to be a novel driver gene aberration. However, pathogenic functions of the CD74-NRG1 fusion gene are unknown, and the mechanism underlying the initiation of cancer stem cells (CSCs) and their maintenance in tumors with oncogenic fusion genes is still unclear. In this study, we observed that expression of the CD74-NRG1 fusion gene has an activity to increase the population of cells with CSC properties. CD74-NRG1 expression facilitated sphere formation of not only cancer cells but also non-cancerous lung epithelial cells. Using a limiting dilution assay in a xenograft model, we showed that expression of the CD74-NRG1 fusion gene enhanced tumor initiation. We observed that CD74-NRG1 expression stimulates phosphorylation of ErbB2/3 and activates the phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB signaling pathway. Furthermore, we found that levels of the secreted insulin-like growth factor 2 (IGF2) were increased, and phosphorylation levels of the receptor for IGF2, IGF1 receptor (IGF1R), were enhanced in cells expressing CD74-NRG1 in an NF-κB activity-dependent manner. These findings suggest that the NF-κB activity stimulated by CD74-NRG1 induces the IGF2 autocrine/paracrine circuit. In addition, tumor sphere formation induced by the CD74-NRG1 fusion gene was suppressed by inhibitors of ErbB2, PI3K, or NF-κB, or an anti-IGF2 antibody. Our study thus provides a rationale for developing important treatment options to block the signals that contribute to the CSC properties, ErbB/PI3K/Akt/NF-κB pathway, and IGF2 circuit, and to eradicate tumors and prevent their recurrence.