Combination of a fusogenic glycoprotein, pro-drug activation and oncolytic HSV as an intravesical therapy for superficial bladder cancer.

BACKGROUND: There are still no effective treatments for superficial bladder cancer (SBC)/non-muscle invasive bladder cancer. Following treatment, 20% of patients still develop metastatic disease. Superficial bladder cancer is often multifocal, has high recurrences after surgical resection and recurs after intravesical live Bacillus Calmette-Guérin. Oncovex(GALV/CD), an oncolytic herpes simplex virus-1, has shown enhanced local tumour control by combining oncolysis with the expression of a highly potent pro-drug activating gene and the fusogenic glycoprotein. METHODS: In vitro fusion/prodrug/apoptotic cell-based assays. In vivo orthotopic bladder tumour model, visualised by computed microtomography. RESULTS: Treatment of seven human bladder carcinoma cell lines with the virus resulted in tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. Oncovex(GALV/CD) and mitomycin C showed a synergistic effect, whereas the co-administration with cisplatin or gemcitabine showed an antagonistic effect in vitro. Transitional cell cancer (TCC) cells follow an apoptotic cell death pathway after infection with Oncovex(GALV/CD) with or without 5-FC. In vivo results showed that intravesical treatment with Oncovex(GALV/CD) + prodrug (5-FC) reduced the average tumour volume by over 95% compared with controls. DISCUSSION: Our in vitro and in vivo results indicate that Oncovex(GALV/CD) can improve local tumour control within the bladder, and potentially alter its natural history.

From Tpr-Met to Met, tumorigenesis and tubes.

The receptor for hepatocyte growth factor (HGF)/scatter factor (SF), Met, controls a program of invasive epithelial growth through the coordination of cell proliferation and survival, cell migration and epithelial morphogenesis. This process is important during embryogenesis and for organ regeneration in the adult. However, when deregulated the HGF/SF-Met signaling axis contributes to tumorigenesis and metastasis. Studies on the oncogenic activation of the Met receptor have shed light on the molecular mechanisms underlying the oncogenic activation of receptor tyrosine kinase (RTKs). More than a decade ago, work on the Met related oncogene, Tpr-Met, revealed the mechanism for activation of RTK-derived oncogenes generated following chromosomal translocation. More recently, studies on the mechanisms of downregulation of the Met RTK highlight a role for loss of downregulation in RTK oncogenic activation.

Oncogenic activation of the Met receptor tyrosine kinase fusion protein, Tpr-Met, involves exclusion from the endocytic degradative pathway.

Multiple mechanisms of dysregulation of receptor tyrosine kinases (RTKs) are observed in human cancers. In addition to gain-of-function, loss of negative regulation also contributes to oncogenic activation of RTKs. Negative regulation of many RTKs involves their internalization and degradation in the lysosome, a process regulated through ubiquitination. RTK oncoproteins activated following chromosomal translocation, are no longer transmembrane proteins, and are predicted to escape lysosomal degradation. To test this, we used the Tpr-Met oncogene, generated following chromosomal translocation of the hepatocyte growth factor receptor (Met). Unlike Met, Tpr-Met is localized in the cytoplasm and also lacks the binding site for Cbl ubiquitin ligases. We determined whether subcellular localization of Tpr-Met, and/or loss of its Cbl-binding site, is important for oncogenic activity. Presence of a Cbl-binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter their transforming activity. In contrast, plasma membrane targeting allows Tpr-Met to enter the endocytic pathway, and Tpr-Met transforming activity as well as protein stability are decreased in a Cbl-dependent manner. We show that transformation by Tpr-Met is in part dependent on its ability to escape normal downregulatory mechanisms. This provides a paradigm for many RTK oncoproteins activated following chromosomal translocation.

Regulation of gli activity by all-trans retinoic acid in mouse keratinocytes.

Sonic hedgehog (Shh) signaling is essential for many normal developmental processes. The Shh signal is interpreted by the Gli transcription factors. Elevated Gli-1 expression has been associated with several neoplasms, including basal cell carcinoma. All-trans retinoic acid (RA) has strong effects on epidermal growth and differentiation and has been used for the treatment of various epithelial disorders. In this report, we show that RA can inhibit Gli activity in immortalized murine keratinocytes in a RA receptor-specific manner. This inhibition may occur, at least in part, through sequestration of the transcriptional coactivator cyclic AMP-responsive element-binding protein-binding protein and suggests a novel effect of retinoid excess on Shh signaling.

Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein.

The c-Cbl protooncogene is a negative regulator for several receptor tyrosine kinases (RTKs) through its ability to promote their polyubiquitination. Hence, uncoupling c-Cbl from RTKs may lead to their deregulation. In testing this, we show that c-Cbl promotes ubiquitination of the Met RTK. This requires the c-Cbl tyrosine kinase binding (TKB) domain and a juxtamembrane tyrosine residue on Met. This tyrosine provides a direct binding site for the c-Cbl TKB domain, and is absent in the rearranged oncogenic Tpr-Met variant. A Met receptor, where the juxtamembrane tyrosine is replaced by phenylalanine, is not ubiquitinated and has transforming activity in fibroblast and epithelial cells. We propose the uncoupling of c-Cbl from RTKs as a mechanism contributing to their oncogenic activation.