Microphthalmia-associated transcription factor (MITF), the master regulator of melanocyte differentiation, promotes melanocyte lineage survival and plays a key role in melanoma progression. MITF is a basic helix loop helix leucine zipper transcripton factor that binds to a conserved M box in the promoters of tyrosinase and other melanocyte specific genes that regulate melanin synthesis and melanosome structure (Hemesath et al., 1994
). MITF also promotes expression of genes that regulate melanoma proliferation and survival and has been termed a “lineage survival oncogene,” being amplified in 10-20% of human melanomas (Garraway et al., 2005
The role that MITF plays in melanoma has been controversial. MITF can inhibit proliferation by directly activating expression of the cyclin dependent kinase inhibitors, p21CIP1 and p16INK4A (Carreira et al., 2005
; Loercher et al., 2005
). MITF can also promote survival and proliferation by activating anti-apoptotic factors, BCL2 and MLIAP (Dynek et al., 2008
; McGill et al., 2002
), TBX2 (Carreira et al., 2000
), and the cell cycle dependent kinase, CDK2 (Du et al., 2004
). It has been proposed that the levels of MITF determine whether MITF promotes differentiation and cell cycle arrest or proliferation (Carreira et al., 2006
). In addition, signaling pathways and interactions with other regulatory proteins may regulate MITF activity. We have previously determined that MITF interacts with SWI/SNF chromatin remodeling enzymes to activate differentiation specific genes (de la Serna et al., 2006a
SWI/SNF enzymes are ATP dependent multisubunit complexes that disrupt histone-DNA contacts and promote chromatin structural changes (Sif, 2004
). Mammalian SWI/SNF complexes are composed of the BRG1 or BRM catalytic ATPase subunit and 9-12 BRG1/BRM associated factors (BAFs). BRG1 and BRM have overlapping functions but are not always interchangeable and can interact with distinct transcription factors in vitro (Kadam et al., 2000
). Heterogeneous SWI/SNF complexes, generated by the alternative presence of either the BRG1 or BRM ATPase as well as by a specific BAF composition, may have distinct functions. For example, BRG1 and BRM can differentially regulate gene expression (Flowers et al., 2009
; Xu et al., 2007
) and SWI/SNF complexes containing either the BAF250A or BAF250B subunit have opposing roles in cell cycle regulation (Nagl et al., 2007
). Thus, SWI/SNF subunit composition is an important determinant of SWI/SNF specificity.
BRG1 and BRM and other components of the SWI/SNF complex have been implicated in cancer. BRG1 or BRM expression is down-regulated in a wide array of human cancers. Loss of both BRM and BRG1 expression correlates with poor prognosis of non-small cell lung cancer (Fukuoka et al., 2004
; Reisman et al., 2003
). Other SWI/SNF components are mutated, deleted, or not expressed in many human cancer cell lines (Decristofaro et al., 2001
). Inactivating mutations in the INI1 subunit occur in the majority of malignant rhabdoid tumors (Versteege et al., 1998
). Re-introduction of SWI/SNF subunits into cancer cells that lack expression induces a flat cell morphology, cell cycle arrest, apoptosis or senescence, with reversion of the transformed phenotype, indicating that loss of SWI/SNF function contributes to tumorigenicity (Dunaief et al., 1994
; Wang et al., 2005
). Conversely, components of the SWI/SNF complex interact with the androgen receptor to promote androgen dependent prostate cancer proliferation (Link et al., 2008
; Link et al., 2005
). Thus, the role that SWI/SNF plays in cancer is complex and dependent on cellular context and potentially on subunit composition.
SWI/SNF enzymes are important regulators of cellular differentiation (de la Serna et al., 2006b
). In normal cells, key regulatory factors interact with SWI/SNF enzymes to promote myogenic, myeloid, erythropoietic, adipogenic, and neuronal differentiation. SWI/SNF enzymes can also promote differentiation of cancer cells. Introduction of the BRM subunit into gastric cancer cells that are deficient in BRM promotes expression of markers of intestinal differentiation and introduction of the INI1 subunit into malignant rhabdoid tumor cells differentiates these cells along the adipogenic pathway (Caramel et al., 2008
; Yamamichi et al., 2007
The status and activity of SWI/SNF components have been characterized in many cancer types, but poorly characterized in human melanoma. A recent report indicates that BRG1 is down-regulated in primary and metastatic melanomas (Becker et al., 2009
). Moreover, it has recently been reported that down-regulation of the INI1 subunit of the SWI/SNF complex is important for bypassing oncogenic BRAF induced senescence in melanocytes and that the BRM subunit is associated with heterochromatic foci in senescing melanocytes (Bandyopadhyay et al., 2007
; Wajapeyee et al., 2008
). Because our previous work indicated that SWI/SNF enzymes are required to promote MITF mediated activation of melanocyte specific gene expression (de la Serna et al., 2006a
), we hypothesized that SWI/SNF enzymes are also critical regulators of melanoma differentiation and tumorigenicity.
We previously determined that in a tissue culture model of melanocyte differentiation, MITF promotes recruitment of BRG1 to the promoter of a melanocyte specific gene, chromatin remodeling, and activation of melanocyte specific gene expression (de la Serna et al., 2006a
). To determine the ramifications of the previously observed SWI/SNF/MITF interaction in melanoma, we characterized SWI/SNF subunit expression in normal human melanocytes and in established human melanoma cell lines. We found that normal melanocytes express high levels of all SWI/SNF subunits, including both the BRG1 and BRM catalytic subunits whereas several melanoma cell lines were markedly down-regulated in either BRG1 or BRM.
To characterize the role of SWI/SNF enzymes in the regulation of MITF target gene expression in melanoma and to distinguish between the roles played by BRG1 and BRM, we expressed BRG1 in BRG1 deficient SK-MEL5 cells. We found that expression of BRG1 enhanced melanoma differentiation and increased expression of MITF target genes that regulate pigmentation. Down-regulation of BRM in BRG1 deficient SK-MEL5 cells inhibited expression of many MITF target genes and reduced growth on soft agar. Our data indicate that both BRG1 and BRM containing SWI/SNF complexes interact with MITF to promote optimal expression of a subset of overlapping and distinct MITF target genes and that a functional SWI/SNF ATPase is required for aspects of melanoma tumorigenicity in vitro. Thus, we propose that SWI/SNF enzymes through their partnership with MITF are important epigenetic modulators in melanoma cells.