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Rnd3 regulation of the actin cytoskeleton promotes melanoma migration and invasive outgrowth in three dimensions.

ABSTRACT: The depth of cell invasion into the dermis is a clinical determinant for poor prognosis in cutaneous melanoma. The signaling events that promote the switch from a noninvasive to invasive tumor phenotype remain obscure. Activating mutations in the serine/threonine kinase B-RAF are prevalent in melanoma. Mutant B-RAF is required for melanoma cell invasion. The expression of Rnd3, a Rho family GTPase, is regulated by mutant B-RAF, although its role in melanoma progression is unknown. In this study, we determined the functional contribution of Rnd3 to invasive melanoma. Endogenous Rnd3 was targeted for knockdown using a doxycycline-inducible short hairpin RNA system in invasive human melanoma cells. Depletion of Rnd3 promoted prominent actin stress fibers and enlarged focal adhesions. Mechanistically, stress fiber formation induced by Rnd3 knockdown required the specific involvement of RhoA and ROCK1/2 activity but not RhoB or RhoC. Rnd3 expression in human melanoma cell lines was strongly associated with elevated extracellular signal-regulated kinase phosphorylation and invasive behavior in a three-dimensional dermal-like environment. A functional role for Rnd3 was shown in the invasive outgrowth of melanoma tumor spheroids. Knockdown of Rnd3 reduced the invasive outgrowth of spheroids embedded in collagen gels. Additionally, Rnd3 depletion inhibited collective and border cell movement out from spheroids in a ROCK1/2-dependent manner. Collectively, these findings implicate Rnd3 as a major suppressor of RhoA-mediated actin cytoskeletal organization and in the acquisition of an invasive melanoma phenotype.

SUBMITTER: Klein RM

PROVIDER: S-EPMC2657934 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Rnd3 regulation of the actin cytoskeleton promotes melanoma migration and invasive outgrowth in three dimensions.

Klein R Matthew RM Aplin Andrew E AE

Cancer research 20090224 6

The depth of cell invasion into the dermis is a clinical determinant for poor prognosis in cutaneous melanoma. The signaling events that promote the switch from a noninvasive to invasive tumor phenotype remain obscure. Activating mutations in the serine/threonine kinase B-RAF are prevalent in melanoma. Mutant B-RAF is required for melanoma cell invasion. The expression of Rnd3, a Rho family GTPase, is regulated by mutant B-RAF, although its role in melanoma progression is unknown. In this study, ...[more]

PMID: 19244113

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Project description:BNIP3 is an atypical BH3-only member of the BCL-2 family of proteins with reported pro-death as well as pro-autophagic and cytoprotective functions, depending on the type of stress and cellular context. In line with this, the role of BNIP3 in cancer is highly controversial and increased BNIP3 levels in cancer patients have been linked with both good as well as poor prognosis. In this study, using small hairpin RNA (shRNA) lentiviral transduction to stably knockdown BNIP3 (BNIP3-shRNA) expression levels in melanoma cells, we show that BNIP3 supports cancer cell survival and long-term clonogenic growth. Although BNIP3-shRNA increased mitochondrial mass and baseline levels of reactive oxygen species production, which are features associated with aggressive cancer cell behavior, it also prevented cell migration and completely abolished the ability to form a tubular-like network on matrigel, a hallmark of vasculogenic mimicry (VM). We found that this attenuated aggressive behavior of these melanoma cells was underscored by severe changes in cell morphology and remodeling of the actin cytoskeleton associated with loss of BNIP3. Indeed, BNIP3-silenced melanoma cells displayed enhanced formation of actin stress fibers and membrane ruffles, while lamellopodial protrusions and filopodia, tight junctions and adherens junctions were reduced. Moreover, loss of BNIP3 resulted in re-organization of focal adhesion sites associated with increased levels of phosphorylated focal adhesion kinase. Remarkably, BNIP3 silencing led to a drop of the protein levels of the integrin-associated protein CD47 and its downstream signaling effectors Rac1 and Cdc42. These observations underscore that BNIP3 is required to maintain steady-state levels of intracellular complexes orchestrating the plasticity of the actin cytoskeleton, which is integral to cell migration and other vital processes stimulating cancer progression. All together these results unveil an unprecedented pro-tumorigenic role of BNIP3 driving melanoma cell's aggressive features, like migration and VM.

Dataset Information

Rnd3 regulation of the actin cytoskeleton promotes melanoma migration and invasive outgrowth in three dimensions.

Publications

Rnd3 regulation of the actin cytoskeleton promotes melanoma migration and invasive outgrowth in three dimensions.

Similar Datasets

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