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.

Our recent studies have revealed that among the 10 different commonly used AAV serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single-mutants in liver cancer cells in vitro. In immuno-deficient mouse xenograft models, direct intra-tumor injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intra-tumor and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing pro-apoptotic genes should prove useful for the potential gene therapy of human liver cancers.

The met proto-oncogene is a member of the family of tyrosine kinase growth factor receptors. We describe the isolation and characterization of a cDNA clone (pOK) for the met receptor from a gastric carcinoma cell line. This clone differs from the published cDNA clone by the absence of 54 bp predicted to encode 18 amino acids in the extracellular domain. The pOK cDNA corresponds to the most abundant met RNA species of 8 kb expressed in human cell lines and tissue, and we show that there are in fact two 8-kb met receptor tyrosine kinase (RTK) isoforms that are generated by alternative splicing. This newly described met isoform when transiently expressed in COS cells encodes a protein of 190 kDa which corresponds in size to the p190 met alpha beta heterodimer expressed in human cell lines. Furthermore, we show that the 190-kDa product of pOK consists of the 140-kDa met beta subunit associated with the 50-kDa met alpha subunit. This finding suggests that both the alpha and beta met chains are encoded by this construct and confirms the hypothesis that a single chain precursor is cleaved to produce both subunits of met. In contrast, the previously characterized met isoform corresponds to a minor met RNA species and encodes a protein of 170 kDa that is not cleaved yet is processed in a manner that allows cell surface expression. Both met RTK isoforms are autophosphorylated in the in vitro kinase assay. These results suggest that different isoforms of the met RTK may have distinct biological activities.

The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.

Triple negative breast cancer (TNBC), defined as tumors lacking expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), are especially difficult to treat effectively. While ER+ and HER2+ breast cancer subtypes can be treated with Tamoxifen and Herceptin, respectively, there are no targeted therapies for TNBC patients. Furthermore, while only 20-30% of TNBC patients respond to chemotherapy in the neoadjuvant setting, overall outcome remains poor for non-responding patients. However, mounting evidence suggests that immune-checkpoint inhibitor immunotherapies may be especially promising for TNBC patients. We and others have shown that the presence of CD8+ T cells, a crucial component of the cytotoxic arm of the adaptive immune response, is a sign of good clinical outcome in TNBC patients. However, good outcome only correlates with CD8 +T cell invasion of the tumor parenchyma. Some patients had an accumulation of CD8+ T cells in the surrounding tumor-associated stroma, but not the tumor epithelia, and these patients responded as poorly as patients with no CD8 T cells at all. Yet how cancer associated fibroblasts (CAFs), the dominant cell type of the tumor-associated stroma, affects CD8+ T cell invasion into the tumor epithelia is still poorly understood. To identify potential stroma-dependent mechanisms which potentiate or inhibit CD8+ T cells invasion into the tumor epithelia, we performed gene expression profiling of laser-capture microdissected tumor-associated stroma (and matched epithelia) from 38 TNBC cases. Here we identify several stromal and epithelial canonical pathways as well as biomarkers that are associated with and may explain the accumulation of CD8 T cells outside of the tumor epithelia. Citation Format: Gruosso T, Gigoux M, Bertos N, Zuo D, Manem V, Monette A, Lapointe R, Haibe-Kains B, Park M. Mechanisms of CD8+ T cell immunosuppression in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-03-08.