My laboratory is interested in understanding molecular mechanisms of skin cancer melanoma tumorigenesis and progression, and basic cell and molecular biology of epidermal melanocytes.
BRAF oncogene-induced autophagy as melanoma tumor suppressor mechanism
Activating mutations in NRAS and BRAF are found frequently in cutaneous melanomas. Because concurrent mutations of both BRAF and RAS are extremely rare, it is thought that transformation by RAS and BRAF occurs through a common mechanism. Also, there is evidence for a relationship of synthetic lethality between NRAS and BRAF oncogenes that leads to selection against cells with a hyperactive mitogen-activated protein kinase (MAPK) pathway. However, it is not known whether the hyperactivation of the MAPK pathway by overexpression of either oncogene alone could also inhibit melanoma tumorigenesis.
Research in my laboratory showed that in melanoma cells with oncogenic BRAF (mBRAF), high levels of mBRAF induce hyperactivation of ERK and senescence-like phenotype and trigger autophagy by inhibiting the mammalian target of rapamycin complex (mTORC) signaling. Growth inhibition and cell death caused by high mBRAF levels can be partially rescued by downregulation of BRAF protein or inhibition of autophagy, but not by inhibition of the MAPK or apoptotic pathways. Quantitative immunohistochemical analysis (AQUA) of human melanomas and cell lines showed a significant positive correlation between the levels of BRAF protein and autophagy marker light chain 3 (LC3).
We are currently investigating mechanisms of autophagy induction by BRAF oncogene.
Melanoma transdifferentiation and tumor progression: crosstalk between BRAF and Notch signaling
My laboratory has previously identified expression of neuronal marker MAP2 in melanoma. This marker is both:
- Activated in cutaneous primary melanoma; and
- Inversely associated with melanoma tumor progression.
We also showed that ectopic expression of MAP2 in metastatic melanoma cells inhibits cell growth by inducing mitotic spindle defects and apoptosis. However, molecular mechanisms of regulation of MAP2 gene expression in melanoma are not understood.
Recently, we showed that in melanoma cells neuronal marker MAP2 expression is induced by the demethylating agent 5-aza-2′-cytidine, and MAP2 promoter is progressively methylated during melanoma progression, indicating that epigenetic mechanisms are involved in silencing of MAP2 in melanoma. Because MAP2 promoter activity levels in melanoma cell lines also correlated with activating mutation in BRAF, a gene that is highly expressed in neurons, we hypothesized that BRAF signaling is involved in MAP2 expression. We showed that hyperactivation of BRAF-MEK signaling activates MAP2 expression in melanoma cells by two independent mechanisms, promoter demethylation or down-regulation of neuronal transcription repressor HES1.
We are investigating the role of HES1 target genes in melanoma transdifferentiation and melanoma tumor progression.
Protein-protein interactions in melanosome biogenesis
By virtue of the presence of multiple protein-protein interaction and signaling domains, PDZ proteins play important roles in assembling protein complexes that participate in diverse cell biological processes. We showed earlier that PDZ protein GIPC is involved in melanosomal protein trafficking. GIPC is a versatile PDZ protein that binds a variety of target proteins in different cell types.
In previous studies we showed that, in epidermal melanocytes, GIPC interacts with newly synthesized melanosomal protein TRP1 in the Golgi region and proposed that this interaction may facilitate intracellular trafficking of TRP1. However, since GIPC contains a single PDZ domain and no other known protein interaction motifs, it is not known how GIPC-TRP1 interaction affects melanosome biogenesis and/or melanin pigmentation.
Recently, we showed that in human primary melanocytes GIPC interacts with AKT-binding protein APPL (adaptor protein containing pleckstrin homology, leucine zipper and phosphotyrosine binding domains), which readily co-precipitates with newly synthesized TRP1. Knockdown of either GIPC or APPL inhibits melanogenesis by decreasing tyrosinase protein levels and enzyme activity. In melanocytes, APPL exists in a complex with GIPC and phospho-AKT. Inhibition of AKT phosphorylation using a PI3-kinase inhibitor abolishes this interaction and results in retardation TRP1 in the Golgi.
Ongoing research is aimed at understanding the dynamics of these interactions and their role in melanosome biogenesis.
Calcium homeostasis in melanocytes and role of transient receptor potential Melastatin 1 (TRPM1) glutamate receptor signaling skin pigmentation
Transient receptor potential melastatin (TRPM) is a subfamily of ion channels that are involved in sensing taste, ambient temperature, low pH, osmolarity, and chemical ligands. Melastatin 1/TRPM1, the founding member, was originally identified as melanoma metastasis suppressor based on its expression in normal pigment cells in the skin and the eye but not in aggressive, metastasis-competent melanomas.
The role of TRPM1 and its regulation in normal melanocytes and in melanoma progression is not understood. Here, we studied the relationship of TRPM1 expression to growth and differentiation of human epidermal melanocytes. TRPM1 expression and intracellular Ca(2+) levels are significantly lower in rapidly proliferating melanocytes compared to the slow growing, differentiated melanocytes.
Recently, we showed that lentiviral shRNA-mediated knockdown of TRPM1 results in reduced intracellular Ca(2+) and decreased Ca(2+) uptake suggesting a role for TRPM1 in Ca(2+) homeostasis in melanocytes. TRPM1 knockdown also resulted in a decrease in tyrosinase activity and intracellular melanin pigment. Expression of the tumor suppressor p53 by transfection or induction of endogenous p53 by ultraviolet B radiation caused repression of TRPM1 expression accompanied by decrease in mobilization of intracellular Ca(2+) and uptake of extracellular Ca(2+).
Ongoing research is aimed at understanding the role of glutamate receptors in modulating TRPM1 activity and regulation of melanin pigmentation.
|Vijay Setaluri, PhD
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