Abstract
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.
Highlights
BNIP3 is overexpressed in various tumors and at different stages of tumor progression.[13,14] consistent with a context-dependent role in cell death regulation, both tumor suppressor and tumor promoter roles have been described for BNIP3
We stably knocked down BNIP3 expression by small hairpin RNA in murine (B16-F10) and human (A375m) metastatic melanoma cell lines, which resulted in a significant decrease in the BNIP3 protein levels (Figure 1a, Supplementary Figure S1A) as compared with control empty vector-transduced cells
Actin cytoskeleton remodeling, which is tightly controlled by intracellular signaling complexes, does affect cell motility, hereby supporting migration, invasion and metastatic spreading, and other vital cellular processes associated with cancer cell survival and aggressiveness and constitutes a crucial aspect of tumor progression
Summary
BNIP3 is overexpressed in various tumors and at different stages of tumor progression.[13,14] consistent with a context-dependent role in cell death regulation, both tumor suppressor and tumor promoter roles have been described for BNIP3. Opposite to this, increased expression of BNIP3 in hypoxic tumors caused by the transcriptional activation of HIF-117 or downregulation of tumor suppressor pathways, like miR145,18 correlated with accelerated tumor growth and unfavorable prognosis in cancer patients.[13]. The latter studies highlight an important role for BNIP3 in carcinogenesis, there is still a considerable debate regarding the cellular processes and mechanisms regulated by this atypical BH3-only protein in cancer cells. These data disclose a previously unknown mechanism by which BNIP3 supports melanoma cell’s plasticity and aggressiveness
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