Deregulation of signaling pathways involving phosphorylation is a hallmark of malignant transformation. Degradation of phosphoproteins generates cancer-specific phosphopeptides that are associated with MHC-I and II molecules and recognized by T-cells. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8 T-cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and HLA-matched primary leukemia cells ex vivo. Healthy individuals showed surprisingly high levels of CD8 T-cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients, which correlated with clinical outcome, and was restored following allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T-cell adoptive transfer immunotherapies..

No abstract

The transcription factor NF-E2-related factor 2 (Nrf2) binds the antioxidant DNA response element (ARE) to activate important cellular cytoprotective defense systems. Recently several types of cancers have been shown to overexpress Nrf2, but its role in the cellular response to radiation therapy has yet to be fully determined. In this study, we report that single doses of ionizing radiation from 2 to 8 Gy activate ARE-dependent transcription in breast cancer cells in a dose-dependent manner, but only after a delay of five days. Clinically relevant daily dose fractions of radiation also increased ARE-dependent transcription, but again only after five days. Downstream activation of Nrf2-ARE-dependent gene and protein markers, such as heme oxygenase-1, occurred, whereas Nrf2-deficient fibroblasts were incapable of these responses. Compared with wild-type fibroblasts, Nrf2-deficient fibroblasts had relatively high basal levels of reactive oxygen species that increased greatly five days after radiation exposure. Further, in vitro clonogenic survival assays and in vivo sublethal whole body irradiation tests showed that Nrf2 deletion increased radiation sensitivity, whereas Nrf2-inducing drugs did not increase radioresistance. Our results indicate that the Nrf2-ARE pathway is important to maintain resistance to irradiation, but that it operates as a second-tier antioxidant adaptive response system activated by radiation only under specific circumstances, including those that may be highly relevant to tumor response during standard clinical dose-fractionated radiation therapy. Cancer Res; 70(21); 8886-95. ©2010 AACR.

The activation and recruitment of CD4 ؉ T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4 ؉ T cells, we have investigated phosphopeptides, a new category of tumorderived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4 ؉ T cell recognition. We first demonstrated the fine specificity of human CD4 ؉ T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome ''fingerprints.'' We then generated HLA-DR␤1 * 0101-restricted CD4 ؉ T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4 ؉ T cells provides potential new targets for cancer immunotherapy.tumor antigen ͉ tumor immunology

Nitroxyl (HNO) can inhibit the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Because of the importance of glycolysis in many malignant cells, we thus propose that HNO can adversely affect tumor growth. This hypothesis was tested using in vitro and in vivo models of breast cancer. We report here for the first time that HNO suppresses the proliferation of both estrogen receptor (ER)-positive and ER-negative human breast cancer cell lines, in a dose dependent manner. Mice treated with HNO either injected into the tumor itself or via the intraperitoneal approach had smaller xenograft tumor size. In addition to significantly decreased blood vessel density in the HNO-treated tumors, we observed lower levels of circulating serum vascular endothelial growth factor (VEGF). Accordingly, there was a decrease in total HIF-1a (hypoxia-inducible factor) protein in HNO-treated tumor cells. Further studies showed inhibition of GAPDH activity in HNO-treated human breast cancer cell lines and in HNO-treated tumor tissue derived from xenografts. One explanation for the multiplicity of actions observed after HNO treatment could be the effect from the initial inhibition of GAPDH, providing a potential therapeutic avenue based upon blocking glycolysis resulting in decreased HIF-1a, thus leading to angiogenesis inhibition. Therefore, HNO appears to act via mechanism(s) different from those of existing breast cancer drugs, making it a potential candidate to overcome known and emerging drug resistance pathways. ' 2007 Wiley-Liss, Inc.