RESUMO
Tumor progression locus 2 (Tpl2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of serine/threonine kinases. Deletion of the Tpl2 gene is associated with a significantly higher number of papillomas and cutaneous squamous cell carcinomas (cSCCs). Overexpression of hepatocyte growth factor (HGF) and its receptor MET is abundant in cSCC and can lead to increased proliferation, migration, invasion or resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. The aim of this study was to address whether the increased tumor burden in Tpl2 -/- mice is due to aberrant HGF/MET signaling. C57Bl/6 wild type (WT) and Tpl2 -/- mice were subjected to a two-stage chemical carcinogenesis protocol for one year. At the time of promotion half of the mice received 44 mg/kg capmatinib (INC 280), a pharmacological inihibitor of MET, in their diet. Tpl2-/- mice had signficantly higher tumor incidence and overall tumor burden compared to WT mice. Further, carcinogen-intiated Tpl2 -/- mice could bypass the need for promotion, as 89% of Tpl2 -/- mice given only DMBA developed papillomas. v-rasHa -transduced keratinocytes and SCCs from Tpl2 -/- mice revealed an upregulation in HGF and p-MET signaling compared to WT animals. Long-term capmatinib treatment had no adverse effects in mice and capmatinib-fed Tpl2 -/- mice had a 60% reduction in overall tumor burden. Further, no tumors from Tpl2 -/- mice fed capmatinib underwent malignant conversion. In summary targeting MET may be a potential new strategy to combat cutaneous squamous cell carcinomas that result from dysregulation in MAPK signaling.
RESUMO
Skin cancer is the most common form of cancer in the USA, with an estimated two million cases diagnosed annually. Tumor progression locus 2 (Tpl2), also known as MAP3K8, is a serine/threonine protein kinase in the mitogen-activated protein kinase signal transduction cascade. Tpl2 was identified by our laboratory as having a tumor suppressor function in skin carcinogenesis, with the absence of this gene contributing to heightened inflammation and increased skin carcinogenesis. In this study, we used gene expression profiling to compare expression levels between Tpl2 (+/+) and Tpl2 (-) (/-) keratinocytes. We identified over 2000 genes as being differentially expressed between genotypes. Functional annotation analysis identified cancer, cell growth/proliferation, cell death, cell development, cell movement and cell signaling as the top biological processes to be differentially regulated between genotypes. Further microarray analysis identified several candidate genes, including Mmp1b, Mmp2, Mmp9 and Mmp13, involved in migration and invasion to be upregulated in Tpl2 (-) (/-) keratinocytes. Moreover, Tpl2 (-/-) keratinocytes had a significant downregulation in the matrix metalloproteinase (MMP) inhibitor Timp3. Real-time PCR validated the upregulation of the MMPs in Tpl2 (-/-) keratinocytes and zymography confirmed that MMP2 and MMP9 activity was higher in conditioned media from Tpl2 (-/-) keratinocytes. Immunohistochemistry confirmed higher MMP9 staining in 12-O-tetradecanoylphorbol-13-acetate-treated skin from Tpl2 (-/-) mice and grafted tumors formed from v-ras(Ha) retrovirus-infected Tpl2 (-/-) keratinocytes. Additionally, Tpl2 (-/-) keratinocytes had significantly higher invasion, malignant conversion rates and increased endothelial cell tube formation when compared with Tpl2 (+/+) keratinocytes. In summary, our studies reveal that keratinocytes from Tpl2 (-/-) mice demonstrate a higher potential to be invasive and metastatic.