T cells directed to endogenous tumor antigens are powerful mediators of tumor regression. Recent immunotherapy advances have identified effective interventions to unleash tumor-specific T-cell activity in patients who naturally develop them. Eliciting T-cell responses to a patient’s individual tumor remains a major challenge. Radiation therapy can induce immune responses to model antigens expressed by tumors, but it remains unclear whether it can effectively prime T cells specific for endogenous antigens expressed by poorly immunogenic tumors. We hypothesized that TGFβ activity is a major obstacle hindering the ability of radiation to generate an in situ tumor vaccine. Here, we show that antibody-mediated TGFβ neutralization during radiation therapy effectively generates CD8+ T-cell responses to multiple endogenous tumor antigens in poorly immunogenic mouse carcinomas. Generated T cells were effective at causing regression of irradiated tumors and nonirradiated lung metastases or synchronous tumors (abscopal effect). Gene signatures associated with IFNγ and immune-mediated rejection were detected in tumors treated with radiation therapy and TGFβ blockade in combination but not as single agents. Upregulation of programmed death (PD) ligand-1 and -2 in neoplastic and myeloid cells and PD-1 on intratumoral T cells limited tumor rejection, resulting in rapid recurrence. Addition of anti–PD-1 antibodies extended survival achieved with radiation and TGFβ blockade. Thus, TGFβ is a fundamental regulator of radiation therapy’s ability to generate an in situ tumor vaccine. The combination of local radiation therapy with TGFβ neutralization offers a novel individualized strategy for vaccinating patients against their tumors. Cancer Res; 75(11); 1–11. ©2015 AACR.

Lin28, a key factor for cellular reprogramming and generation of induced pluripotent stem cell (iPSC), makes a critical contribution to tumorigenicity by suppressing Let-7. However, it is unclear whether Lin28 is involved in regulating cancer stem–like cells (CSC), including in oral squamous carcinoma cells (OSCC). In this study, we demonstrate a correlation between high levels of Lin28B, Oct4, and Sox2, and a high percentage of CD44+ALDH1+ CSC in OSCC. Ectopic Lin28B expression in CD44−ALDH1−/OSCC cells was sufficient to enhance Oct4/Sox2 expression and CSC properties, whereas Let7 co-overexpression effectively reversed these phenomena. We identified ARID3B and HMGA2 as downstream effectors of Lin28B/Let7 signaling in regulating endogenous Oct4 and Sox2 expression. Let7 targeted the 3′ untranslated region of ARID3B and HMGA2 and suppressed their expression, whereas ARID3B and HMGA2 increased the transcription of Oct4 and Sox2, respectively, through promoter binding. Chromatin immunoprecipitation assays revealed a direct association between ARID3B and a specific ARID3B-binding sequence in the Oct4 promoter. Notably, by modulating Oct4/Sox2 expression, the Lin28B–Let7 pathway not only regulated stemness properties in OSCC but also determined the efficiency by which normal human oral keratinocytes could be reprogrammed to iPSC. Clinically, a Lin28Bhigh-Let7low expression pattern was highly correlated with high levels of ARID3B, HMGA2, OCT4, and SOX2 expression in OSCC specimens. Taken together, our results show how Lin28B/Let7 regulates key cancer stem–like properties in oral squamous cancers. Cancer Res; 75(12); 1–13. ©2015 AACR.

Lin28, a key factor for cellular reprogramming and generation of induced pluripotent stem cell (iPSC), makes a critical contribution to tumorigenicity by suppressing Let-7. However, it is unclear whether Lin28 is involved in regulating cancer stem-like cells (CSC) including in oral squamous carcinoma cells (OSCC). In this study, we demonstrate a correlation between high levels of Lin28B, Oct4, and Sox2, and a high percentage of CD44+ALDH1+ CSC in OSCC. Ectopic Lin28B expression in CD44−ALDH1−/OSCC cells was sufficient to enhance Oct4/Sox2 expression and CSC properties, whereas Let7 co-overexpression effectively reversed these phenomena. We identified ARID3B and HMGA2 as downstream effectors of Lin28B/Let7 signaling in regulating endogenous Oct4 and Sox2 expression. Let7 targeted the 3’UTR of ARID3B and HMGA2 and suppressed their expression, while ARID3B and HMGA2 increased the transcription of Oct4 and Sox2, respectively, through promoter binding. ChIP assays revealed a direct association between ARID3B and a specific ARID3B-binding sequence in the Oct4 promoter. Notably, by modulating Oct4/Sox2 expression, the Lin28B/Let7 pathway not only regulated stemness properties in OSCC but also determined the efficiency by which normal human oral keratinocytes could be reprogrammed to iPSC. Clinically, a Lin28Bhigh-Let7low expression pattern was highly correlated with high levels of ARID3B, HMGA2, OCT4 and SOX2 expression in OSCC specimens. Taken together, our results show how Lin28B/Let7 regulates key cancer stem-like properties in oral squamous cancers.

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression. Cancer Res; 75(11); 1–10. ©2015 AACR.

Metastatic breast cancer is the second leading cause of cancer-related deaths among women. Triple-negative breast cancer (TNBC) is a highly aggressive subcategory of breast cancer and currently lacks well-defined molecular targets for effective targeted therapies. Disease relapse, metastasis, and drug resistance render standard chemotherapy ineffective in the treatment of TNBC. Because previous studies coupled β3 integrin (ITGB3) to epithelial–mesenchymal transition (EMT) and metastasis, we exploited β3 integrin as a therapeutic target to treat TNBC by delivering β3 integrin siRNA via lipid ECO-based nanoparticles (ECO/siβ3). Treatment of TNBC cells with ECO/siβ3 was sufficient to effectively silence β3 integrin expression, attenuate TGFβ-mediated EMT and invasion, restore TGFβ-mediated cytostasis, and inhibit three-dimensional organoid growth. Modification of ECO/siβ3 nanoparticles with an RGD peptide via a PEG spacer enhanced siRNA uptake by post-EMT cells. Intravenous injections of RGD-targeted ECO/siβ3 nanoparticles in vivo alleviated primary tumor burden and, more importantly, significantly inhibited metastasis. In the span of 16 weeks of the experiments and observations, including primary tumor resection at week 9 and release from the treatment for 4 weeks, the mice bearing orthotopic, TGFβ-prestimulated MDA-MB-231 tumors that were treated with RGD-targeted ECO/siβ3 nanoparticles were free of metastases and relapse, in comparison with untreated mice. Collectively, these results highlight ECO/siβ3 nanoparticles as a promising therapeutic regimen to combat TNBC. Cancer Res; 75(11); 1–10. ©2015 AACR.