Glioma-initiating cells (GICs) have stem-like cell properties thought to be sufficient for recurrence, progression and drug resistance in glioblastomas. In the present study, we defined miR-340 as a differentially expressed microRNA (miRNA) in human GICs that inhibit GIC-mediated tumorigenesis. Further, we defined tissue plasminogen activator (PLAT) as a critical direct target of miR-340 for inhibition. Among miRNAs screened we found that miR-340 expression was decreased in all human GICs and in human glioblastoma tissues, compared to human neural stem cells and normal brain tissues. miR-340 overexpression in GICs suppressed their proliferative, invasive and migratory properties in vitro, triggering cell senescence in vitro and inhibiting GIC-induced tumorigenesis in mouse brains. shRNA-mediated silencing of PLAT in GICs phenocopied the effects of miR-340 overexpression in vitro and in vivo, suggesting a potential role for tissue factor in stem-like cell function. Taken together, our results identified miR-340 as a tumor suppressor that functions in GIC to enforce PLAT blockade and ablate their stem-like functions.

The mobilization of bone marrow–derived cells (BMDC) to distant tissues before the arrival of disseminated tumor cells has been shown preclinically to facilitate metastasis through the establishment of metastatic niches. Primary tumor hypoxia has been demonstrated to play a pivotal role in the production of chemokines and cytokines responsible for the mobilization of these BMDCs, especially in breast cancer. Carbonic anhydrase IX (CAIX, CA9) expression is highly upregulated in hypoxic breast cancer cells through the action of hypoxia-inducible factor-1 (HIF1). Preclinical evidence has demonstrated that CAIX is required for breast tumor growth and metastasis; however, the mechanism by which CAIX exerts its prometastatic function is not well understood. Here, we show that CAIX is indispensable for the production of granulocyte colony-stimulating factor (G-CSF) by hypoxic breast cancer cells and tumors in an orthotopic model. Furthermore, we demonstrate that tumor-expressed CAIX is required for the G-CSF–driven mobilization of granulocytic myeloid-derived suppressor cells (MDSC) to the breast cancer lung metastatic niche. We also determined that CAIX expression is required for the activation of NF-κB in hypoxic breast cancer cells and constitutive activation of the NF-κB pathway in CAIX-depleted cells restored G-CSF secretion. Together, these findings identify a novel hypoxia-induced CAIX–NF-κB–G-CSF cellular signaling axis culminating in the mobilization of granulocytic MDSCs to the breast cancer lung metastatic niche. Cancer Res; 75(6); 1–13. ©2015 AACR.

The mobilization of bone marrow-derived cells (BMDCs) to distant tissues prior to the arrival of disseminated tumor cells has been shown preclinically to facilitate metastasis through the establishment of metastatic niches. Primary tumor hypoxia has been demonstrated to play a pivotal role in the production of chemokines and cytokines responsible for the mobilization of these BMDCs, especially in breast cancer. Carbonic Anhydrase IX (CAIX) expression is highly upregulated in hypoxic breast cancer cells through the action of Hypoxia-Inducible Factor-1 (HIF-1). Preclinical evidence has demonstrated that CAIX is required for breast tumor growth and metastasis, however, the mechanism by which CAIX exerts its prometastatic function is not well understood. Here, we show that CAIX is indispensable for the production of G-CSF by hypoxic breast cancer cells and tumors. Furthermore, we demonstrate that CAIX expression in the tumor is required for the G-CSF-driven mobilization of granulocytic myeloid-derived suppressor cells (MDSCs) to the breast cancer lung metastatic niche. We also determined that CAIX expression is required for the activation of NF-κB in hypoxic breast cancer cells and constitutive activation of the NF-κB pathway in CAIX-depleted cells restored G-CSF secretion. Together, these findings identify a novel hypoxia-induced CAIX-NF-κB-G-CSF cellular signalling axis culminating in the mobilization of granulocytic MDSCs to the breast cancer lung metastatic niche.

Cigarette smoking is a major risk factor in the development of non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the major addictive component of tobacco smoke, can induce proliferation, invasion and epithelial-mesenchymal transition (EMT) in NSCLC cell lines and promote metastasis of NSCLC in mice. Here we demonstrate that the scaffolding protein β-arrestin-1 is necessary for nicotine-mediated induction of mesenchymal genes vimentin and fibronectin as well as EMT regulators ZEB1 and ZEB2. Nicotine induced changes in cell morphology and ablate tight junctions consistent with EMT; β-arrestin-1, but not β-arrestin-2, was required for these changes. β-arrestin-1 promoted the expression of the mesenchymal genes as well as ZEB1 and ZEB2 through the mediation of the E2F1 transcription factor; this required Src kinase activity. Stimulation of multiple NSCLC cell lines with nicotine led to enhanced recruitment of β-arrestin-1 and E2F1 on vimentin, fibronectin, ZEB1 and ZEB2 promoters. Further, there was significantly more β-arrestin-1 and E2F1 associated with these promoters in human NSCLC tumors and β-arrestin-1 levels correlated with vimentin and fibronectin levels in human NSCLC samples. A549-luciferase cells lacking β-arrestin-1 showed a significantly reduced capacity for tumor growth and metastasis when orthotopically implanted into the lungs of SCID-beige mice. Taken together, these studies reveal a novel role for β-arrestin-1 in the growth and metastasis of NSCLC.

Short-term starvation or fasting can augment cancer treatment efficacy and can be effective in delaying cancer progression in the absence of chemotherapy, but the underlying molecular mechanisms of action remain elusive. Here, we describe the role of REV1, a specialized DNA polymerase involved in DNA repair, as an important signaling node linking nutrient sensing and metabolic control to cell fate. We show that REV1 is a novel binding partner of the tumor suppressor p53 and regulates its activity. Under starvation, REV1 is modified by SUMO2/3, resulting in the relief of REV1′s inhibition of p53 and enhancing p53′s effects on proapoptotic gene expression and apoptosis in breast cancer and melanoma cells. Thus, fasting in part through its effect on REV1 is a promising nontoxic strategy to increase p53-dependent cell death and to enhance the efficacy of cancer therapies. Cancer Res; 75(6); 1–12. ©2015 AACR.