In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18-36 years) and eighteen individuals without diabetes (17-35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (microg/l) and CEC (microg/l) were significantly higher in DM (55.73 +/- 29.43, 521.47 +/- 239.13, respectively) compared to controls (24.25 +/- 10.26, 262.85 +/- 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.

The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E2) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER− and ER+ breast cancer cell lines, we showed that E2 attenuated HLA-DR in two ER+ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER− lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα+ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα− VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E2 and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E2 treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E2 inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E2 in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα− breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα+ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus (RV) for the treatment of breast cancer (BrCa). In vitro, oncolysis and cytokine production were assessed in human and murine BrCa cell lines following RV exposure. Furthermore, RV-induced upregulation of tumor cell PD-L1 was evaluated. In vivo, the immunocompetent, syngeneic EMT6 murine model of BrCa was employed to determine therapeutic and tumor-specific immune responses following treatment with RV, anti-PD-1 antibodies or in combination. RV-mediated oncolysis and cytokine production were observed following BrCa cell infection and RV upregulated tumor cell expression of PD-L1. In vivo, RV monotherapy significantly reduced disease burden and enhanced survival in treated mice, and was further enhanced by PD-1 blockade. RV therapy increased the number of intratumoral regulatory T cells, which was reversed by the addition of PD-1 blockade. Finally, dual treatment led to the generation of a systemic adaptive anti-tumor immune response evidenced by an increase in tumor-specific IFN-γ producing CD8+ T cells, and immunity from tumor re-challenge. The combination of PD-1 blockade and RV appears to be an efficacious immunotherapeutic strategy for the treatment of BrCa, and warrants further investigation in early-phase clinical trials.

The ability to repurpose sunitinib for augmentation of reovirus' immunotherapeutic efficacy positions this novel combination therapy as an attractive strategy ready for clinical testing against a range of histologies, including RCC and NSCLC. Clin Cancer Res; 22(23); 5839-50. ©2016 AACR.