Cancer testis antigens (CTA) represent attractive targets for targeted radiotherapy and imaging as their expression is restricted to cancer and germ cells. NY-ESO-1, a member of the CTA family, is highly immunogenic and expressed in multiple tumor types including carcinoma of bladder, liver lung. The aim of this study was to develop radioimmunoconjugates (RIC) to target NY-ESO-1 protein in cancer cells. Anti-NY-ESO-1 antibodies were modified by addition of DTPA for 111In-labelling or, in the presence of Iodogen, were 123I-labelled. Delivery of radiolabeled immunoconjugates across the cell membrane was achieved using a protein transfection (PT) reagent (SAINT-PhD) and by chemical linkage with the cell-penetrating and nuclear-localizing peptide, TAT (YGRKKRRQRRR). Cellular internalization, distribution and efflux of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT were investigated in cell fractionation and retention assays. It was shown that protein transfection reagent has promoted the cellular uptake of RICs into SK-MEL-37 and both of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT was retained longer in SK-MEL-37 cells in comparison to their isotope control RIC. In clonogenic assays, 111In-DTPA-anti-NY-ESO-1-TAT-PT significantly reduced surviving fraction of SK-MEL-37 cells. Cytotoxicity was inversely proportional to specific activity and the concentration of cells exposed to 111In-DTPA-anti-NY-ESO-1-TAT-PT. siRNA knock down of NY-ESO-1 resulted in partial reversal of 111In-DTPA-anti-NY-ESO-1-TAT-PT associated cytotoxicity. These promising results obtained from the in vitro study has brought the probe further into in vivo study. In preliminary biodistribution studies in SK-MEL-37 xenograft-bearing mice, tumour:muscle ratio for 111In-DTPA-anti-NY-ESO-1-TAT-PT was statistically significant compared to the control RIC 48 h post injection. This clearly indicated that the probe can be delivered into tumour in in vivo model and the successful uptake of radioactivity increased the chance of causing cytotoxicity to tumour cells through DNA damage. All of these findings have suggested that intracellular cancer associated antigen NY-ESO-1 can be reached by protein transfection reagent and cell penetrating peptide and initiates DNA damage through radio-isotope mediated cytotoxicity. Therefore, it represents a novel approach to the treatment of CTA-expressing cancers.

Endometrial cancer is the fourth most common cancer in women and the most common gynecological malignancy. While patient outcome has improved for the majority of cancers, the outlook for endometrial cancer has steadily decreased. In order to address this problem, we must better understand the different mechanisms involved in endometrial cancer development and progression. To this end, we quantified expression of 667 miRNAs in four endometrioid adenocarcinoma and four serous adenocarcinoma using Taqman Low Density Arrays (TLDAs). miR-888 was one of the most highly overexpressed miRNAs in both endometrial cancer subtypes. Analysis of miR-888 expression across multiple cancer types using the The Cancer Genome Atlas database revealed that miR-888 was selectively expressed in endometrial cancer, with a significant association to invasive and high grade tumors. In addition, miR-888 was most predominantly expressed in endometrial carcinosarcoma, a rare but deadly form of endometrial cancer. Therefore, we conclude that miR-888 expression marks an aggressive endometrial tumor phenotype. One of the top predicted targets of miR-888 by TargetScan is the progesterone receptor (PR). PR is a potent tumor suppressor of the endometrium whose expression is often lost in advanced endometrial cancers. We quantified PR mRNA expression in a panel of endometrial tumors and found a statistically significant, negative correlation between miR-888 and PR mRNA expression. Furthermore, overexpression of miR-888 in endometrial cancer cell lines was capable of decreasing PR at the protein level. To determine if miR-888 directly targets PR, we cloned each of the four miR-888 binding sites downstream of Renilla luciferase into the psiCHECK2 reporter vector. miR-888 overexpression was capable of decreasing luciferase activity for all four binding sites, with the second and third binding sites producing the most prominent results. Here we describe a novel mechanism by which miR-888 inhibits PR mRNA translation to negatively regulate PR expression in endometrial tumors. To determine the endogenous function of miR-888 in human cells, we quantified miR-888 in a panel of 21 normal human tissues. Interestingly, miR-888 was highly expressed in testes, with minimal or absence of expression in all other tissues investigated. The restricted expression pattern of miR-888 in testes and cancer suggested that miR-888 may qualify as a novel cancer-testis (CT) antigen. CT-antigens are a large class of genes that demonstrate selective expression normally in testes germ cells and abnormally in various types of cancer. Furthermore, CT-antigen genes are predominantly located on the X chromosome and are part of evolutionarily novel multicopy gene families. Indeed, miR-888 is part of a multicopy, primate-specific miRNA gene family located on the X-chromosome. Furthermore, miRNA in situ hybridization localized miR-888 expression to the early stages of spermatogenesis, as is often observed for CT antigens. Together, these data identify miR-888 as the first miRNA CT antigen and expand the CT antigen field to noncoding RNAs.