LMP1 promotes expression of insulin-like growth factor 1 (IGF1) to selectively activate IGF1 receptor and drive cell proliferation.

UNLABELLED: Epstein-Barr Virus (EBV) is a gammaherpesvirus that infects the majority of the human population and is linked to the development of multiple cancers, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the primary oncoprotein of EBV, and in epithelial cells it induces the expression and activation, or phosphorylation, of the epidermal growth factor receptor kinase. To identify effects on additional kinases, an unbiased screen of receptor tyrosine kinases potentially activated by LMP1 was performed. Using a protein array, it was determined that LMP1 selectively activates insulin-like growth factor 1 receptor (IGF1R). This activation takes place in fibroblast, epithelial, and nasopharyngeal cell lines that express LMP1 stably and transiently. Of note, LMP1 altered the phosphorylation, but not the expression, of IGF1R. The use of LMP1 mutants with defective signaling domains revealed that the C-terminal activating region 2 domain of LMP1 increased the mRNA expression and the secretion of the ligand IGF1, which promoted phosphorylation of IGF1R. IGF1R phosphorylation was dependent upon activation of canonical NF-κB signaling and was suppressed by IκBα and a dominant negative form of TRAF6. Inhibition of IGF1R activation with two small-molecule inhibitors, AG1024 and picropodophyllin (PPP), or with short hairpin RNA (shRNA) directed against IGF1R selectively reduced proliferation, focus formation, and Akt activation in LMP1-positive cells but did not impair LMP1-induced cell migration. Expression of constitutively active Akt rescued cell proliferation in the presence of IGF1R inhibitors. These findings suggest that LMP1-mediated activation of IGF1R contributes to the ability of LMP1 to transform epithelial cells. IMPORTANCE: EBV is linked to the development of multiple cancers in both lymphoid and epithelial cells, including nasopharyngeal carcinoma. Nasopharyngeal carcinoma is a major cancer that develops in specific populations, with nearly 80,000 new cases reported annually. LMP1 is consistently expressed in early lesions and continues to be detected within 50 to 80% of these cancers at later stages. It is therefore of paramount importance to understand the mechanisms through which LMP1 alters cell growth and contributes to tumorigenesis. This study is the first to determine that LMP1 activates the IGF1R tyrosine kinase by regulating expression of the ligand IGF1. Additionally, the data in this paper reveal that specific targeting of IGF1R selectively impacts LMP1-positive cells. These findings suggest that therapies directed against IGF1R may specifically impair the growth of EBV-infected cells.

MERTK controls melanoma cell migration and survival and differentially regulates cell behavior relative to AXL.

The receptor tyrosine kinase AXL regulates melanoma cell proliferation and migration. We now demonstrate that AXL and the related kinase MERTK are alternately expressed in melanoma and are associated with different transcriptional signatures. MERTK-positive melanoma cells are more proliferative and less migratory than AXL-positive melanoma cells and overexpression of AXL increases cell motility relative to MERTK. MERTK is expressed in up to 50% of melanoma cells and shRNA-mediated knockdown of MERTK reduces colony formation and cell migration in a CDC42-dependent fashion. Targeting MERTK also decreases cell survival and proliferation in an AKT-dependent manner. Finally, we identify a novel mutation in the kinase domain of MERTK, MERTK(P) (802S) , that increases the motility of melanoma cells relative to wild-type MERTK. Together, these data demonstrate that MERTK is a possible therapeutic target in melanoma, that AXL and MERTK are associated with differential cell behaviors, and that mutations in MERTK may contribute to melanoma pathogenesis.

Phosphoproteomic screen identifies potential therapeutic targets in melanoma.

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma.

Bone marrow monocyte PECAM-1 deficiency elicits increased osteoclastogenesis resulting in trabecular bone loss.

In our investigations of the bone marrow (BM) of PECAM-1 null (knockout, KO) mice, we observed that the trabecular bone volume and number of trabeculae were significantly reduced in femoral and tibial long bones. Further studies in vitro revealed increased numbers and size of osteoclasts, enhanced bone resorption on dentin substrates, and hypersensitivity to macrophage CSF and receptor activator of NF-kappaB ligand in BM-derived osteoclast precursor cultures from KO mice. Associations among PECAM-1, Syk, and SHP-1 were found in wild-type BM monocyte derived osteoclast-like cells. The absence of PECAM-1 and SHP-1 interactions in the KO cells leads to the dysregulation of Syk kinases and/or phosphatases, possibly SHP-1. Indeed, KO derived osteoclast-like cells exhibited increased Syk tyrosine phosphorylation levels compared with WT cells. Lastly, WT mice engrafted with marrow from KO kindred showed loss of trabecular bone analogous to KO mice, consistent with increased osteoclastogenesis.

The high-mobility group A1 gene up-regulates cyclooxygenase 2 expression in uterine tumorigenesis.

Uterine cancer is the most common cancer of the female genital tract and is the fourth most frequent cause of cancer death in women in the U.S. Despite the high prevalence of uterine cancers, the molecular events that lead to neoplastic transformation in the uterus are poorly understood. Moreover, there are limited mouse models to study these malignancies. We generated transgenic mice with high-mobility group A1 gene (HMGA1a) expression targeted to uterine tissue and all female mice developed tumors by 9 months of age. Histopathologically, the tumors resemble human uterine adenosarcoma and are transplantable. To determine whether these findings are relevant to human disease, we evaluated primary human uterine neoplasms and found that HMGA1a mRNA and protein levels are increased in most high-grade neoplasms but not in normal uterine tissue, benign tumors, or most low-grade neoplasms. We also found that HMGA1a up-regulates cyclooxygenase 2 (COX-2) expression in transgenic tumors. Moreover, both HMGA1a and COX-2 expression are up-regulated in high-grade human leiomyosarcomas. Using chromatin immunoprecipitation, HMGA1a binds directly to the COX-2 promoter in human uterine cancer cells in vivo and activates its expression in transfection experiments. We also show that blocking either HMGA1a or COX-2 in high-grade human uterine cancer cells blocks anchorage-independent cell growth in methylcellulose. These findings show that HMGA1a functions as an oncogene when overexpressed in the uterus and contributes to the pathogenesis of human uterine cancer by activating COX-2 expression. Although a larger study is needed to confirm these results, HMGA1a may be a useful marker for aggressive human uterine cancers.