Anchorage dependency is a characteristic that distinguishes normalcy from the cancerous state. In normal cells, adhesion to the extracellular matrix is critical for many processes, including cell proliferation. By contrast, neoplastic cells bypass the requirement for adhesion and proliferate in an adhesion-independent manner. Integrins are the main cellular receptors for the extracellular matrix and acting via Rho family GTPases initiate actin cytoskeletal rearrangements. Our long-term objectives are to understand how integrins regulate cell proliferation and how cancerous cells overcome the requirement for adhesion. Integrins modulate growth factor-initiated signal transduction. The extracellular signal regulated kinase (ERK) cascade is an important growth factor-stimulated pathway. Growth factor-mediated ERK signaling is required for cell cycle progression and we have demonstrated that both activation and nucleocytoplasmic trafficking of ERK are regulated by integrins. Integrin-dependent ERK nucleocytoplasmic trafficking is poorly defined and the proposed experiments will provide important insights into this control. Immunofluorescence and biochemical approaches will be utilized to analyze ERK localization and activity in the nucleus in adhesion-dependent cell models.
Specific Aim 1 will determine the role of ERK import, export and cytoplasmic anchoring in adhesion control of ERK nuclear accumulation.
Specific Aim 2 will test the involvement of Rho family GTPases in ERK nucleocytoplasmic trafficking. Melanoma has the fastest rising cancer incidence rate. ERK activation and nuclear accumulation are adhesion-independent in metastatic melanoma cells. B-Raf is a component of the ERK cascade and is frequently mutated in melanoma. Expression of mutant B-Raf is sufficient to promote adhesion-independent nuclear accumulation of ERK in primary melanocytes.
Specific Aim 3 will establish the contribution of mutant B-Raf-mediated constitutive nuclear accumulation of ERK to melanocyte cell cycle progression in the absence of integrin-mediated adhesion. Fulfillment of these Aims will lead to a mechanistic understanding of integrin control of ERK localization and determine the consequences of constitutive nuclear accumulation of ERK to aberrant cell proliferation. These studies should ultimately lead to novel strategies to target aberrant cell proliferation in melanoma.
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