Inhibition of HGF/MET signaling decreases overall tumor burden and blocks malignant conversion in Tpl2-related skin cancer

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-ras(Ha) -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.</p

Purification, Cloning, and Characterization of the CEL I Nuclease^†

CEL I, isolated from celery, is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg2+ and Zn2+ for activity, with a pH optimum at neutral pH. We have purified CEL I 33 000-fold to apparent homogeneity. A key improvement is the use of α-methyl-mannoside in the purification buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatured, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifies a new family of neutral pH optimum, magnesium-stimulated, mismatch duplex-recognizing nucleases, within the S1 superfamily