Purpose Gastric cancers (GC) may harbor a small subset of cells with cancer stem cell (CSC) properties including chemotherapy (CT) resistance. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. Here we examine the role of HH signaling in CD44(+) GC cells. Experimental Design GC cell lines, tumor xenografts, and patient tumors were examined. Results GC cell lines AGS, MKN-45, and NCI-N87 grown as spheroids or sorted for CD44(+) were found to have upregulation of HH pathway proteins. HH inhibition using Smo shRNA or vismodegib (VIS) decreased spheroid formation and colony formation. CD44(+) cells, compared to unselected cells, were also resistant to 5-fluorouracil and cisplatin CT, and this resistance was reversed in vitro and in xenografts with Smo shRNA or VIS. CD44(+) cells also had significantly more migration, invasion, and anchorage-independent growth, and these properties could all be blocked with HH inhibition. Clinical tumor samples from a phase II trial for advanced GC of CT with or without VIS were analyzed for CD44 expression. In the CT alone group, high CD44 expression was associated with decreased survival, while in the CT plus VIS group, high CD44 expression was associated with improved survival. Conclusions HH signaling maintains CSC phenotypes and malignant transformation phenotypes in CD44(+) GC cells, and HH inhibition can block CT resistance in CD44(+) cells. GC is a heterogeneous disease, and the strategy of combining CT with HH inhibition may only be effective in the subset with high CD44 levels.

Non-thermal atmospheric pressure plasma (NTAPP) is an ionized gas at room temperature and has potential as a new apoptosis-promoting cancer therapy that acts by generating reactive oxygen species (ROS). However, it is imperative to determine its selectivity and standardize the components and composition of NTAPP. Here, we designed an NTAPP-generating apparatus combined with a He gas feeding system and demonstrated its high selectivity toward p53-mutated cancer cells. We first determined the proper conditions for NTAPP exposure to selectively induce apoptosis in cancer cells. The apoptotic effect of NTAPP was greater for p53-mutated cancer cells; artificial p53 expression in p53-negative HT29 cells decreased the pro-apoptotic effect of NTAPP. We also examined extra- and intracellular ROS levels in NTAPP-treated cells to deduce the mechanism of NTAPP action. While NTAPP-mediated increases in extracellular nitric oxide (NO) did not affect cell viability, intracellular ROS increased under NTAPP exposure and induced apoptotic cell death. This effect was dose-dependently reduced following treatment with ROS scavengers. NTAPP induced apoptosis even in doxorubicin-resistant cancer cell lines, demonstrating the feasibility of NTAPP as a potent cancer therapy. Collectively, these results strongly support the potential of NTAPP as a selective anticancer treatment, especially for p53-mutated cancer cells.

Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a late side effect of thoracic radiotherapy. The purpose of our study was to gain further insight into the development of RIPF.Experimental Design/Results: Here, we observed that irradiation of mouse lungs induced collagen deposition, particularly around blood vessels, in the early phase of RIPF. Such deposition subsequently became evident throughout the irradiated tissues. Accompanied by the collagen deposition, vascular EndMT (endothelial-to-mesenchymal transition) began to develop in the early phase of RIPF, before the appearance of EMT (epithelial-tomesenchymal transition) of alveolar epithelial (AE) II cells in the substantive fibrotic phase. Concomitant with the EndMT, we detected vascular endothelial cell (EC)-specific hypoxic damage in the irradiated lung tissues. In human pulmonary artery endothelial cells (HPAEC), the radiation-induced EndMT via activation of TGFb-R1/Smad signaling was dependent on HIF1a expression. A novel HIF1a inhibitor, 2-methoxyestradiol (2-ME), inhibited the irradiation-induced EndMT via downregulation of HIF1a-dependent Smad signaling. In vivo, 2-ME inhibited the vascular EndMT, and decreased the collagen deposition associated with RIPF. Furthermore, HIF1a-related EndMT was observed also in human RIPF tissues.Conclusions: We provide the first evidence that an EndMT occurs in RIPF development and that the EndMT may be effectively inhibited by modulating vascular EC-specific hypoxic damage.

Overexpression of NQO1 is associated with poor prognosis in human cancers including breast, colon, cervix, lung and pancreas. Yet, the molecular mechanisms underlying the pro-tumorigenic capacities of NQO1 have not been fully elucidated. Here we show a previously undescribed function for NQO1 in stabilizing HIF-1α, a master transcription factor of oxygen homeostasis that has been implicated in the survival, proliferation and malignant progression of cancers. We demonstrate that NQO1 directly binds to the oxygen-dependent domain of HIF-1α and inhibits the proteasome-mediated degradation of HIF-1α by preventing PHDs from interacting with HIF-1α. NQO1 knockdown in human colorectal and breast cancer cell lines suppresses HIF-1 signalling and tumour growth. Consistent with this pro-tumorigenic function for NQO1, high NQO1 expression levels correlate with increased HIF-1α expression and poor colorectal cancer patient survival. These results collectively reveal a function of NQO1 in the oxygen-sensing mechanism that regulates HIF-1α stability in cancers.

It remains controversial whether targeting tumour vasculature can improve radiotherapeutic efficacy. We report that radiation-induced endothelial-to-mesenchymal transition (EndMT) leads to tumour vasculature with abnormal SMA+NG2+ pericyte recruitment during tumour regrowth after radiotherapy. Trp53 (but not Tgfbr2) deletion in endothelial cells (ECs) inhibited radiation-induced EndMT, reducing tumour regrowth and metastases with a high CD44v6+ cancer-stem-cell (CSC) content after radiotherapy. Osteopontin, an EndMT-related angiocrine factor suppressed by EC-Trp53 deletion, stimulated proliferation in dormant CD44v6+ cells in severely hypoxic regions after radiation. Radiation-induced EndMT significantly regulated tumour-associated macrophage (TAM) polarization. CXCR4 upregulation in radioresistant tumour ECs was highly associated with SDF-1+ TAM recruitment and M2 polarization of TAMs, which was suppressed by Trp53 deletion. These EndMT-related phenomena were also observed in irradiated human lung cancer tissues. Our findings suggest that targeting tumour EndMT might enhance radiotherapy efficacy by inhibiting the re-activation of dormant hypoxic CSCs and promoting anti-tumour immune responses.