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Abstract
Metastasized malignant melanoma has a poor prognosis because of its intrinsic resistance to chemotherapy and radiotherapy. The central role in the melanoma transcriptional network has the transcription factor MITF (microphthalmia-associated transcription factor). It has been shown recently that the expression of MITF and some of its target genes require the SWI/SNF chromatin remodeling complex. Here we demonstrate that survival of melanoma cells requires functional SWI/SNF complex not only by supporting expression of MITF and its targets and but also by activating expression of prosurvival proteins not directly regulated by MITF. Microarray analysis revealed that besides the MITF-driven genes, expression of proteins like osteopontin, IGF1, TGFß2 and survivin, the factors known to be generally associated with progression of tumors and the antiapoptotic properties, were reduced in acute BRG1-depleted 501mel cells. Western blots and RT-PCR confirmed the microarray findings. These proteins have been verified to be expressed independently of MITF, because MITF depletion did not impair their expression. Because these genes are not regulated by MITF, the data suggests that loss of BRG1-based SWI/SNF complexes negatively affects survival pathways beyond the MITF cascade. Immunohistochemistry showed high expression of both BRM and BRG1 in primary melanomas. Exogenous CDK2, osteopontin, or IGF1 each alone partly relieved the block of proliferation imposed by BRG1 depletion, implicating that more factors, besides the MITF target genes, are involved in melanoma cell survival. Together these results demonstrate an essential role of SWI/SNF for the expression of MITF-dependent and MITF-independent prosurvival factors in melanoma cells and suggest that SWI/SNF may be a potential and effective target in melanoma therapy.
Highlights
Malignant melanoma is highly invasive and early metastasizing tumor, and its incidence has been increasing in recent years [1,2]
We demonstrate that the BRG1 knockdown suppressed proliferation of melanoma cells and both BRG1 and BRM are highly expressed in melanoma samples
Detached and remaining attached cells were separately analyzed by flow cytometry and high sub-G1 and G1 contents were observed for detached cells (Figure S2) while no G2/M and very low S phase of the cell cycle were dectected, indicating that detached cells did not proliferate and most of them succcumbed to apoptosis
Summary
Malignant melanoma is highly invasive and early metastasizing tumor, and its incidence has been increasing in recent years [1,2]. MITF is central for the transcription of genes involved in various cellular processes from embryonic development of melanocytes to metastasis of melanoma [3,4,5]. MITF expression is heterogeneous in advanced melanomas [11] but is highly expressed at the early phases of melanocyte transformation. SWI/SNF chromatin remodeling complexes are consisting of about 12 proteins, and are present in cells as several subcomplexes having only subtle differences in subunit composition [12,13,14]. They alter the local nucleosome structure at the promoter regions to regulate transcription. These complexes use the energy provided by either BRM (Brahma, SMARCA2) or BRG1
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