Expanding the Use of an SS18-SSX Antibody for Molecular Assays in Synovial Sarcoma.

Synovial sarcoma is an aggressive malignancy that generally affects adolescents and young adults and is characterized by high rates of recurrence and metastasis, with a 10-year survival rate of about 50%. The fusion oncoprotein SS18-SSX, the product of a pathognomonic chromosomal translocation t(X;18), is the oncogenic driver of this sarcoma, disrupting differentiation through widespread epigenetic dysregulation. Experimental research into SS18-SSX biology has been limited by the lack of an antibody that specifically detects the endogenous fusion oncoprotein as opposed to its native SS18 or SSX components. Recently, a rabbit monoclonal antibody was developed and made commercially available, which specifically detects the fusion junction site epitope of SS18-SSX as found in at least 95% of synovial sarcomas. Here, we characterize a suite of molecular biology assays using this new antibody, both confirming existing and reporting on novel applications. We demonstrate its high sensitivity and specificity for synovial sarcoma diagnosis on patient samples through positive immunohistochemical staining on synovial sarcoma, tissue microarray, and full face sections. In addition, we demonstrate detection of the human SS18-SSX protein when expressed in a genetically engineered mouse model of synovial sarcoma. We also demonstrate nuclear staining of SS18-SSX in synovial sarcoma cells using immunofluorescence, and visualize the interaction between SS18-SSX and the BAF complex member BRG1 through a proximity ligation assay. Lastly, we confirm the interaction between SS18-SSX and promoter regions of target genes through chromatin immunoprecipitation. This antibody represents a breakthrough in sarcoma research and has value in multiple applications to expand the knowledge of synovial sarcoma biology.

Discovery of a small-molecule inhibitor of specific serine residue BAD phosphorylation.

Human BCL-2-associated death promoter (hBAD) is an apoptosis-regulatory protein mediating survival signals to carcinoma cells upon phosphorylation of Ser99, among other residues. Herein, we screened multiple small-molecule databases queried in a Laplacian-modified naive Bayesian-based cheminformatics platform and identified a Petasis reaction product as a site-specific inhibitor for hBAD phosphorylation. Based on apoptotic efficacy against mammary carcinoma cells, N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB) was identified as a potential lead compound. In vitro biochemical analyses demonstrated that NPB inhibited the phosphorylation of hBAD specifically on Ser99. NPB was observed to exert this effect independently of AKT and other kinase activities despite the demonstration of AKT-mediated BAD-Ser99 phosphorylation. Using a structure-based bioinformatics platform, we observed that NPB exhibited predicted interactions with hBAD in silico and verified the same by direct binding kinetics. NPB reduced phosphorylation of BAD-Ser99 and enhanced caspase 3/7 activity with associated loss of cell viability in various human cancer cell lines derived from mammary, endometrial, ovarian, hepatocellular, colon, prostatic, and pancreatic carcinoma. Furthermore, by use of a xenograft model, it was observed that NPB, as a single agent, markedly diminished BAD phosphorylation in tumor tissue and significantly inhibited tumor growth. Similar doses of NPB utilized in acute toxicity studies in mice did not exhibit significant effects. Hence, we report a site-specific inhibitor of BAD phosphorylation with efficacy in tumor models.

Synthesis, characterization and cytotoxicity studies of 1,2,3-triazoles and 1,2,4-triazolo [1,5-a] pyrimidines in human breast cancer cells.

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is essential for physiological functions of tissues and neovasculature. VEGFR signaling is associated with the progression of pathological angiogenesis in various types of malignancies, making it an attractive therapeutic target in cancer treatment. In the present work, we report the synthesis of 1,4-disubstituted 1,2,3-triazoles and 1,2,4-triazolo[1, 5-a]pyrimidine derivatives via copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and screened for their anticancer activity against MCF7 cells. We identified 1-(2'-ethoxy-4'-fluoro-[1,1'-biphenyl]-4-yl)-4-phenyl-1H-1,2,3-triazole (EFT) as lead cytotoxic agent against MCF7 cell lines with an IC50 value of 1.69 µM. Further evaluation revealed that EFT induces cytotoxicity on Ishikawa, MDA-MB-231 and BT474 cells with IC50 values of 1.97, 4.81 and 4.08 µM respectively. However, EFT did not induce cytotoxicity in normal lung epithelial (BEAS-2B) cells. Previous reports suggested that 1,2,3-triazoles are the inhibitors of VEGFR1 and therefore, we evaluated the effect of EFT on the expression of VEGFR1. The results demonstrated that EFT downregulates the expression of VEGFR1 in MCF7 cells. In summary, we identified a potent cytotoxic agent that imparts its antiproliferative activity by targeting VEGFR1 in breast cancer cells. The novel compound could serve as a lead structure in developing VEGFR1 inhibitors.

Hypomethylation associated enhanced transcription of trefoil factor-3 mediates tamoxifen-stimulated oncogenicity of ER+ endometrial carcinoma cells.

Tamoxifen (TAM) is widely used as an adjuvant therapy for women with breast cancer (BC). However, TAM possesses partial oestrogenic activity in the uterus and its use has been associated with an increased incidence of endometrial carcinoma (EC). The molecular mechanism for these observations is not well understood. Herein, we demonstrated that forced expression of Trefoil factor 3 (TFF3), in oestrogen receptor-positive (ER+) EC cells significantly increased cell cycle progression, cell survival, anchorage-independent growth, invasiveness and tumour growth in xenograft models. Clinically, elevated TFF3 protein expression was observed in EC compared with normal endometrial tissue, and its increased expression in EC was significantly associated with myometrial invasion. TAM exposure increased expression of TFF3 in ER+ EC cells and its elevated expression resulted in increased oncogenicity and invasiveness. TAM-stimulated expression of TFF3 in EC cells was associated with hypomethylation of the TFF3 promoter sequence and c-JUN/SP1-dependent transcriptional activation. In addition, small interfering (si) RNA-mediated depletion or polyclonal antibody inhibition of TFF3 significantly abrogated oncogenicity and invasiveness in EC cells consequent to TAM induction or forced expression of TFF3. Hence, TAM-stimulated upregulation of TFF3 in EC cells was critical in promoting EC progression associated with TAM treatment. Importantly, inhibition of TFF3 function might be an attractive molecular modality to abrogate the stimulatory effects of TAM on endometrial tissue and to limit the progression of EC.