Head and neck squamous cell carcinoma (HNSCC) is driven by cancer-initiating cells (CIC), but their maintenance mechanisms are obscure. For hematopoietic stem cells, low levels of intracellular reactive oxygen species (ROSLow) is known to help sustain stemness properties. In this report, we evaluated the hypothesis that ROSLow character conferred CIC properties in HNSCC. Sphere cultures define CIC in HNSCC cell populations (HN-CIC). We found that ROSLow cells in HN-CIC defined in this manner were more numerous than in parental HNSCC cells. Further, ROSLow cells frequently coexpressed CIC surface markers such as memGrp78 and Glut3. Exploiting flow cytometry to sort cells on the basis of their ROS level, we found that isolated ROSLow cells displayed relatively more CIC properties, including quiescence, chemoresistance, in vitro malignant properties, and tumorigenicity. Pharmacological depletion of ROS modulators in cisplatin-treated HN-CIC reduced CIC properties, enhancing cell differentiation and enhancing cisplatin-induced cell death. Overall, our work defined cell subpopulations in HNSCC on the basis of differential intracellular ROS levels, which associated with stemness and chemoresistance properties. On the basis of our findings, we suggest that strategies to promote intracellular ROS levels may heighten the efficacy of conventional chemotherapy used for HNSCC treatment. Cancer Res; 74(21); 1–15. ©2014 AACR.

Head and neck squamous cell carcinoma (HNSCC) is a lethal cancer. Emerging evidence supports cancer-initiating cells (CICs) are responsible for tumor growth, chemoresistance, etc. However, the physiological mechanisms by which to maintain the characteristics of CICs in HNSCC (HN-CICs) remain elusive. For hematopoietic stem cells, low intracellular reactive oxygen species (ROSLow) level helps sustain their stemness properties. Therefore; to understand the role of ROSLow cells, which are speculated to be the HN-CICs, may reveal physiological dependencies for head and neck tumorigenesis. Previously, we have identified the existence of HN-CICs from sphere cells. Herein, we initially demonstrated that there were more ROSLow cells in sphere cells than in parental HNSCCs. Further, the ROSLow cells mostly co-expressed CICs surface markers (memGrp78, Glut3, etc.). Next, we exploited flow cytometry to sort the ROSHigh, ROSMidi and ROSLow cells. The isolated ROSLow cells exerted more CICs properties, quiescence, chemoresistance, in vitro malignancy and in vivo tumorigenicity; however, the ROSHigh cells were the most proliferative. Lastly, the pharmacological depletion of ROS modulators in cisplatin-treated HN-CICs was performed to reduce the CICs properties by enhancing cell differentiation and to sensitize the cisplatin cytotoxicity by promoting cell death. Overall, we identified the subpopulations of cells with differential intracellular ROS level harboring diverse chemoresistance, proliferative activity and stemness properties. We also demonstrated that the ROSLow cells not only exert CICs properties but mainly contribute to tumor growth and chemoresistance in HNSCCs. Modulation of low intracellular ROS level should be a future alternative HNSCC treatment in combination with conventional chemotherapy.

Peptidylarginine deiminase 2 (PAD2/PADI2) has been implicated in various inflammatory diseases and, more recently, cancer. The goal of this study was to test the hypothesis that PAD2 promotes oncogenesis using a transgenic mouse model. We found that about 37% of transgenic mice overexpressing human FLAG-PAD2 downstream of the MMTV-LTR promoter develop spontaneous neoplastic skin lesions. Molecular and histopathologic analyses of the resulting lesions find that they contain increased levels of markers for invasion, inflammation, and epithelial-to-mesenchymal transition (EMT) and that a subset of the lesions progress to invasive squamous cell carcinoma (SCC). We then stably overexpressed FLAG-PAD2 in the human SCC cell line, A431, and found that the PAD2-overexpressing cells were more tumorigenic in vitro and also contained elevated levels of markers for inflammation and EMT. Collectively, these studies provide the first genetic evidence that PAD2 functions as an oncogene and suggest that PAD2 may promote tumor progression by enhancing inflammation within the tumor microenvironment. Cancer Res; 74(21); 1–12. ©2014 AACR.

Peptidylarginine deiminase 2 (PAD2/PADI2) has been implicated in various inflammatory diseases, and more recently, cancer. The goal of this study was to test the hypothesis that PAD2 promotes oncogenesis using a transgenic mouse model. We found that ~37% of transgenic mice overexpressing human FLAG-PAD2 downstream of the MMTV-LTR promoter develop spontaneous neoplastic skin lesions. Molecular and histopathological analysis of the resulting lesions finds that they contain increased levels of markers for invasion, inflammation, and EMT, and that a subset of the lesions progress to invasive squamous cell carcinoma. We then stably overexpressed FLAG-PAD2 in the human squamous cell carcinoma cell line, A431, and found that the PAD2 overexpressing cells were more tumorigenic in vitro, and also contained elevated levels of markers for inflammation and EMT. Collectively, these studies provide the first genetic evidence that PAD2 functions as an oncogene and suggest that PAD2 may promote tumor progression by enhancing inflammation within the tumor microenvironment.

Vaccines explored for cancer therapy have been based generally on injectable vector systems used to control foreign infectious pathogens, to which the immune system evolved to respond naturally. However, these vectors may not be effective at presenting tumor-associated antigens (TAA) to the immune system in a manner that is sufficient to engender antitumor responses. We addressed this issue with a novel orally administered Salmonella-based vector that exploits a type III secretion system to deliver selected TAA in the cytosol of professional antigen-presenting cells in situ. A systematic comparison of candidate genes from the Salmonella Pathogenicity Island 2 (SPI2) locus was conducted in the vaccine design, using model antigens and a codon-optimized form of the human TAA survivin (coSVN), an oncoprotein that is overexpressed in most human cancers. In a screen of 20 SPI2 promoter:effector combinations, a PsifB::sseJ combination exhibited maximal potency for antigen translocation into the APC cytosol, presentation to CD8 T cells, and murine immunogenicity. In the CT26 mouse model of colon carcinoma, therapeutic vaccination with a lead PsifB::sseJ-coSVN construct (p8032) produced CXCR3-dependent infiltration of tumors by CD8 T cells, reversed the CD8:Treg ratio at the tumor site, and triggered potent antitumor activity. Vaccine immunogenicity and antitumor potency were enhanced by coadministration of the natural killer T-cell ligand 7DW8-5, which heightened the production of IL12 and IFNγ. Furthermore, combined treatment with p8032 and 7DW8-5 resulted in complete tumor regression in A20 lymphoma-bearing mice, where protective memory was demonstrated. Taken together, our results demonstrate how antigen delivery using an oral Salmonella vector can provide an effective platform for the development of cancer vaccines. Cancer Res; 74(21); 1–11. ©2014 AACR.