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Trop-2 is a transmembrane signal transducer that can induce cancer growth. Using antibody targeting and N-terminal Edman degradation, we show here that Trop-2 undergoes cleavage in the first thyroglobulin domain loop of its extracellular region, between residues R87 and T88. Molecular modeling indicated that this cleavage induces a profound rearrangement of the Trop-2 structure, which suggested a deep impact on its biological function. No Trop-2 cleavage was detected in normal human tissues, whereas most tumors showed Trop-2 cleavage, including skin, ovary, colon, and breast cancers. Coimmunoprecipitation and mass spectrometry analysis revealed that ADAM10 physically interacts with Trop-2. Immunofluorescence/confocal time-lapse microscopy revealed that the two molecules broadly colocalize at the cell membrane. We show that ADAM10 inhibitors, siRNAs and shRNAs abolish the processing of Trop-2, which indicates that ADAM10 is an effector protease. Proteolysis of Trop-2 at R87-T88 triggered cancer cell growth both in vitro and in vivo. A corresponding role was shown for metastatic spreading of colon cancer, as the R87A-T88A Trop-2 mutant abolished xenotransplant metastatic dissemination. Activatory proteolysis of Trop-2 was recapitulated in primary human breast cancers. Together with the prognostic impact of Trop-2 and ADAM10 on cancers of the skin, ovary, colon, lung, and pancreas, these data indicate a driving role of this activatory cleavage of Trop-2 on malignant progression of tumors.

The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.

a b s t r a c tMelanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogenactivated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.

This study explores the BRAF-MITF-PGC-1α axis and compares metabolic and functional changes occurring in primary and metastaticBRAF melanoma cell lines. BRAF mutations in homo/heterozygosis were found to be correlated to high levels of pERK, to downregulate PGC-1α/β, MITF and tyrosinase activity, resulting in a reduced melanin synthesis as compared to BRAFwt melanoma cells. In this scenario,BRAF switches on a metabolic reprogramming in melanoma, leading to a decreased OXPHOS activity and increased glycolytic ATP, lactate, HIF-1α and MCT4 levels. Furthermore, the induction of autophagy and the presence of ER stress markers in BRAF metastatic melanoma cells suggest that metabolic adaptations of these cells occur as compensatory survival mechanisms. For the first time, we underline the role of peIF2α as an important marker of metastatic behaviour in melanoma. Our results suggest the hypothesis that inhibition of the glycolytic pathway, inactivation of peIF2α and a reduction of basal autophagy could be suitable targets for novel combination therapies in a specific subgroup of metastatic melanoma.

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC‐1 staining. Signaling pathways involved in H2O2 induced apoptosis were analyzed with Western blot. H2O2 induced apoptosis of MSCs in a dose‐ and time‐dependent manner. H2O2 induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H2O2 caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H2O2 activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs. J. Cell. Biochem. 111: 967–978, 2010. © 2010 Wiley‐Liss, Inc.

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The beta genus of human papillomaviruses (ß-HPV) includes approximately 50 different viral types that are subdivided into five species (ß-1 through ß-5). Nonmelanoma cancers may involve some ß-1 and ß-2 HPV types, but the biology of most ß-HPV types and their possible connections to human disease are still little characterized. In this study, we studied the effects of ß-3 type HPV49 in a novel transgenic (Tg) mouse model, using a cytokeratin K14 promoter to drive expression of the E6 and E7 genes from this virus in the basal skin epidermis and the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7-Tg mice). Viral oncogene expression only marginally increased cellular proliferation in the epidermis of Tg animals, compared with wild-type littermates, and we observed no spontaneous tumor formation during their entire lifespan. However, we found that K14 HPV49 E6/E7-Tg mice were highly susceptible to upper digestive tract carcinogenesis upon initiation with 4-nitroquinoline 1-oxide (4NQO). This was a selective effect, as the same mice did not exhibit any skin lesions after chronic UV irradiation. Opposite results were observed in an analogous Tg model expressing the ß-2 HPV38 E6 and E7 oncogenes at the same anatomic sites. While these mice were highly susceptible to UV-induced skin carcinogenesis, as previously shown, they were little affected by 4NQO treatment. Overall, our findings highlight important differences in the biologic properties of certain ß-type HPV that affect their impact on carcinogenesis in an anatomic site-specific manner.