Modulation of extracellular matrix/adhesion molecule expression by BRG1 is associated with increased melanoma invasiveness
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Metastatic melanoma is an aggressive malignancy that is resistant to therapy and has a poor prognosis. The progression of primary melanoma to metastatic disease is a multi-step process that requires dynamic regulation of gene expression through currently uncharacterized epigenetic mechanisms. Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes. Understanding the mechanisms involved in the regulation of gene expression during metastasis is important for developing an effective strategy to treat metastatic melanoma. SWI/SNF enzymes are multisubunit complexes that contain either BRG1 or BRM as the catalytic subunit. We previously demonstrated that heterogeneous SWI/SNF complexes containing either BRG1 or BRM are epigenetic modulators that regulate important aspects of the melanoma phenotype and are required for melanoma tumorigenicity in vitro.
To characterize BRG1 expression during melanoma progression, we assayed expression of BRG1 in patient derived normal skin and in melanoma specimen. BRG1 mRNA levels were significantly higher in stage IV melanomas compared to stage III tumors and to normal skin. To determine the role of BRG1 in regulating the expression of genes involved in melanoma metastasis, we expressed BRG1 in a melanoma cell line that lacks BRG1 expression and examined changes in extracellular matrix and adhesion molecule expression. We found that BRG1 modulated the expression of a subset of extracellular matrix remodeling enzymes and adhesion proteins. Furthermore, BRG1 altered melanoma adhesion to different extracellular matrix components. Expression of BRG1 in melanoma cells that lack BRG1 increased invasive ability while down-regulation of BRG1 inhibited invasive ability in vitro. Activation of metalloproteinase (MMP) 2 expression greatly contributed to the BRG1 induced increase in melanoma invasiveness. We found that BRG1 is recruited to the MMP2 promoter and directly activates expression of this metastasis associated gene.
We provide evidence that BRG1 expression increases during melanoma progression. Our study has identified BRG1 target genes that play an important role in melanoma metastasis and we show that BRG1 promotes melanoma invasive ability in vitro. These results suggest that increased BRG1 levels promote the epigenetic changes in gene expression required for melanoma metastasis to proceed.
KeywordsMelanoma Melanoma Cell Metastatic Melanoma Melanoma Cell Line SW13 Cell
Melanoma is an aggressive malignancy, characterized by high potential for metastasis and notoriously resistant to chemotherapeutics [1, 2]. The prognosis for patients with melanoma is dependent on the stage of the disease as measured by tumor thickness, ulceration, and the presence of metastases . According to the American Joint Committee on Cancer staging system, Stage I melanomas are less than 1 mm thick and localized to the skin. Stage II melanomas are greater than 1 mm thick, may be ulcerated, but are still localized to the skin. In stage III, the tumor has spread to nearby lymph nodes but not yet detected at distant sites. In stage IV, the tumor has spread beyond the original area of skin and nearby lymph nodes to other organs, or to distant areas of the skin or lymph nodes. The five year survival rate for stage I, II, III, and IV is estimated to be 92%, 68%, 45%, and 11% respectively . The high mortality rate associated with metastatic melanoma and the lack of effective treatment underscores the necessity to understand the mechanisms that promote melanoma progression.
The progression from a primary tumor to metastatic melanoma is a multistep process that involves detachment from the primary tumor mass, invasion into the dermis, migration through the extracellular matrix (ECM), and vasculature and colonization of distant sites [5, 6]. Each of these steps involves cytoskeletal alterations as well as changes in the tumor cell's interactions with neighboring cells and with the ECM . The inherently high metastatic potential associated with melanoma has been attributed to the migratory nature of neural crest derived precursors that give rise to the melanocyte lineage . Metastatic potential is also dependent on pro-metastatic genetic changes such as those involving NEDD9 amplification as well as epigenetic changes that modulate the expression of genes required for each step in the process [9, 10]. Thus, the propensity for melanoma to metastasize may be intrinsically determined, permanently fixed by genetic alterations, and dynamically modulated at an epigenetic level by signals from the changing microenvironment.
Epigenetic regulation of gene expression often involves changes in chromatin structure that are catalyzed by chromatin remodeling enzymes [11, 12]. Two classes of enzymes remodel chromatin structure by catalyzing covalent histone modifications or by hydrolyzing ATP to mobilize nucleosomes . SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that have been shown to increase DNA accessibility, allowing gene specific regulators or general transcription factors to bind and to activate or repress gene expression . SWI/SNF enzymes play critical roles during organism development . Particularly relevant to melanoma is the regulatory role that SWI/SNF enzymes play in promoting neural crest migration and differentiation as well as SWI/SNF interactions with Microphthalmia -Associated Transcription Factor (MITF), a lineage survival oncogene in melanoma [15, 16, 17].
Mammalian SWI/SNF complexes are composed of the BRG1 or BRM catalytic ATPase subunit and 9-12 BRG1/BRM associated factors (BAFs) . Diverse SWI/SNF complexes are distinguished by the particular ATPase and the presence of unique BAFs . The BRG1 and BRM containing complexes have similar chromatin remodeling activity in vitro but do not necessarily have redundant functional roles in vivo . Dependent on the cellular context, BRG1 and BRM play overlapping or distinct roles in tumorigenesis. Both BRG1 and BRM expression is down-regulated in lung cancer . However, low expression of BRM has been associated with gastric cancer while high expression of BRG1 has been associated with advanced stages of gastric and prostate cancer [22, 23, 24].
Reconstitution of SWI/SNF subunits into cancer cells that lack expression typically induces a change in morphology [25, 26]. Furthermore, disruption of SWI/SNF activity by the introduction of dominant negative BRG1 and BRM into normal cells dramatically alters cell size and shape and invasiveness . These morphological changes parallel changes in the expression of cytoskeletal regulators, cell surface proteins, adhesion molecules, and enzymes that degrade the ECM [26, 27, 28, 29, 30]. Thus, SWI/SNF enzymes play an important role in regulating the expression of genes important for tumor metastasis. We previously demonstrated that BRG1 and BRM expression is variable in melanoma cell lines, such that some cell lines express elevated levels of BRG1 and BRM and a subset of cell lines are deficient in BRG1 or BRM . We found that reconstitution of BRG1 in a BRG1 deficient melanoma cell line promoted expression of MITF target genes that regulate melanogenesis and survival. Furthermore, BRG1 promoted resistance to cisplatin and down-regulation of BRG1/BRM significantly compromised tumorigenicity. An independent study determined that sequential down-regulation of BRG1 and BRM inhibits melanoma proliferation . These studies suggest that SWI/SNF enzymes are important epigenetic modulators of melanoma tumorigenicity and potentially regulate metastatic potential.
To further characterize BRG1 expression in melanoma, we assayed expression of BRG1 in patient derived metastatic melanomas. We found that BRG1 mRNA levels were significantly higher in stage IV tumors compared to stage III tumors and to normal skin. Furthermore, BRG1 protein levels were elevated in highly invasive human metastatic melanoma cell lines. We expressed BRG1 in an established melanoma cell line that lacks detectable levels of BRG1 and profiled expression of extracellular matrix and adhesion molecules. We found that BRG1 modulated the expression of a subset of cell surface receptors, adhesion proteins, and extracellular matrix remodeling enzymes. Furthermore, BRG1 altered adhesion to different ECM components and promoted invasion through matrigel. Activation of matrix metalloproteinase (MMP) 2 expression in BRG1 expressing cells was determined to contribute to the BRG1 mediated increase in invasive ability. Down-regulation of BRG1 in a highly invasive melanoma cell line resulted in decreased MMP2 expression and decreased invasive ability. We investigated the mechanisms involved in BRG1 mediated activation of MMP2 expression and found that BRG1 interacts with a transcriptional regulator of MMP2, the SP1 transcription factor, and is recruited to the matrix metalloproteinase (MMP) 2 promoter. In combination, these results suggest that BRG1 plays a role in promoting melanoma progression by regulating the expression of metastasis associated genes.
BRG1 is highly expressed in metastatic melanoma
We and others determined that SK-MEL5 cells, derived from an axillary node melanoma, are deficient in BRG1 expression [31, 32]. To determine whether BRG1 protein levels are consistently down regulated in other metastatic melanoma cell lines, we compared BRG1 protein levels in SK-MEL5 cells with levels in two highly metastatic melanoma cell lines, A375SM and WM-266-4. The A375SM cell line was established from a lung metastasis formed by injection of parental cells into nude mice . The WM-266-4 cell line was derived from a lymph node metastasis . We found that both A375SM and WM-266-4 express high levels of BRG1 compared to SK-MEL5 cells and to normal human melanocytes (Figure. 1B). We previously reported that re-introduction of BRG1 in SK-MEL5 cells promotes pigmentation as well as increased resistance to cisplatin . As shown in Figure. 1B, BRG1 reconstituted SK-MEL5 cells express BRG1 at similar levels as A375SM and WM-266-4, which we previously estimated to be approximately 2 fold higher than that in normal melanocytes .
BRG1 modulates extracellular matrix and adhesion molecule expression in SK-MEL5 melanoma cells
A previous microarray study showed that re-expression of BRG1 in a BRG1/BRM deficient human adrenal adenocarcinoma cell line (SW13 cells), activated the expression of 80 genes and repressed the expression of 2 genes . Many of the BRG1 regulated genes were cell surface proteins and extracellular matrix remodeling enzymes or secreted proteins such as CD44, E-cadherin, matrix metalloproteinase (MMP) 2, and osteonectin (SPARC) [28, 29, 30]. Thus, re-expression of BRG1 in BRG1/BRM deficient adenocarcinoma cells alters the expression of a subset of genes, and in particular the expression of genes that potentially have important roles in regulating tumor metastasis.
BRG1 activated the expression of 10 additional genes at least two fold, including CD44, MMP9 and MMP14 (MT1-MMP) (greater than 2 fold) (Figure. 2B). Interestingly, re-expression of BRG1 also significantly inhibited the expression of 8 genes (Figure. 2C), while the remaining 53 genes on the array were not significantly affected by the expression of BRG1 (Additional file 1, Table 1). Thus our data indicate that re-expression of BRG1 in BRG1 deficient melanoma cells affects the expression of a subset of cell surface and extracellular matrix remodeling enzymes, some of which overlap (E-cadherin, CD44, and MMP2) and some which are distinct from those reported to be modulated by reconstitution of BRG1 in BRG1/BRM deficient SW13 adenocarcinoma cells. Many of the genes we found to be modulated by BRG1 (Figure. 2A, B, and 2C) encode proteins that play a role in regulating melanoma invasiveness and metastatic potential [6, 7, 37].
The most highly activated gene in BRG1 reconstituted SK-MEL5 cells was NCAM1 (Figure. 2A). NCAM1 is a cell adhesion molecule (CAM) in the immunoglobulin superfamily that is expressed at the cell surface and mediates cell to cell and cell matrix interactions . High expression of NCAM1 in malignant neoplasms, including melanoma, is associated with an aggressive tumor phenotype . Although high levels of NCAM1 have been associated with metastatic potential, the functional role of NCAM1 in melanoma has not been demonstrated, and high levels of NCAM1 have also been detected in benign nevi . Thus, the role of NCAM1 in melanoma metastasis is unclear. MCAM (MUC18), a related cell adhesion molecule is over-expressed in advanced primary and metastatic melanoma. Its expression in melanoma cell lines enhances metastatic potential in nude mice [41, 42]. We found that in addition to NCAM1, BRG1 significantly increased the expression of MCAM (Figure. 2D). Thus, re-expression of BRG1 in SK-MEL5 cells activated the expression of two related cell adhesion molecules that have been implicated in promoting tumor metastasis. We verified that the changes in NCAM1 and MCAM expression also occurred at the protein level (Figure. 2E). Interestingly, increased levels of the 140KD NCAM1 isoform was detected in BRG1 expressing cells. This isoform is associated with malignant neoplasms and induction of anti-apoptotic programs .
E-cadherin localization to the cell junction is compromised in BRG1 reconstituted SK-MEL5 cells
Two of the most highly activated genes in BRG1 expressing SK-MEL5 cells were E-cadherin (CDH1) and catenin delta 2/neural plakophilin related armadillo protein (CTNND2) (Figure. 2A). E-cadherin is a calcium dependent transmembrane receptor that localizes to adherens junctions and mediates cell-cell adhesion. In many cancer types, loss of E-cadherin expression coincides with acquisition of an invasive phenotype and development of metastatic disease. In normal melanocytes, E-cadherin mediates melanocyte-keratinocyte interactions and loss of E-cadherin expression or a change in its cellular distribution is associated with early phases of melanoma. Furthermore, over-expression of E-cadherin in melanoma cells reduces melanoma invasiveness . Thus, expression of BRG1 in SK-MEL5 cells could potentially reduce melanoma invasiveness through up-regulation of E-cadherin. Interestingly, BRG1 also promoted expression of δ-catenin/neural plakophilin-related armadillo protein (CTNND2) (Figure. 2A), but had no effect on the expression of β-catenin or α-catenin (data not shown), two other members of armadillo/β-catenin superfamily of cell adhesion molecules. Increased expression of CTNND2 in prostate cancer has been associated with redistribution and loss of E-cadherin at the adherens junction .
BRG1 alters melanoma adhesion to different ECM components
Re-expression of BRG1 in SK-MEL5 cells resulted in an altered pattern of integrin expression (Figures. 2A and 2C). Integrins are transmembrane glycoproteins that mediate specific interactions between cells and the ECM and regulate migration . Hetero-dimers composed of α and β subunits serve as receptors with specificity for different ligands. Integrin expression is a key determinant of a cell's ability to attach to different ECM components and to migrate on these substrates. Aberrant integrin expression has been associated with melanoma progression .
MMP2 activity is up-regulated in BRG1 expressing SK-MEL5 cells and contributes to increased melanoma invasiveness
Expression of MMPs is controlled at the transcriptional and post-translational levels . Our data indicated that BRG1 promotes expression of MMP2, MMP9, and MMP14 at the protein level (Figure. 5A). MMP2 (gelatinase A, 72-kDa type IV collagenase) and MMP9 (gelatinase B, 92-kDa type IV collagenase are secreted as inactive pro-zymogens that are subsequently processed and activated. MMP14 (MT1-MMP) is a membrane bound MMP that activates MMP2 at the cell surface . Furthermore, naturally occurring tissue inhibitor of metalloproteinases (TIMPs) down-regulate MMP activity . The balance between TIMP and MMP expression is critically important in determining overall MMP activity. We found that in addition to MMPs, BRG1 also activated expression of TIMP2 and TIMP3, which would be expected to down-modulate MMP activity (Figures. 2A, 2B, and 5A).
In order to determine if re-expression of BRG1 in SK-MEL5 cells resulted in increased secretion of active MMP2 and MMP9, we performed gelatin zymography on supernatants derived from control and BRG1 expressing SK-MEL5 cells. We determined that although TIMP levels were increased, there was still a substantial increase in active MMP2 and MMP9 secreted by SK-MEL5 cells expressing BRG1 compared to BRG1 deficient SK-MEL5 cells (Figure. 5B).
The observed increase in MMP2 and MMP9 activity as well as other alterations in extracellular matrix and adhesion molecule expression suggested that BRG1 plays an important role in regulating melanoma invasiveness. To determine the overall biological consequence of BRG1 re-expression in SK-MEL5 cells, we investigated whether BRG1 promotes changes in the ability of melanoma cells to be invasive in vitro. We found that SK-MEL5 cells that express BRG1 had significantly increased ability to invade through Matrigel coated Boyden chambers (Figure. 5C).
To elucidate the mechanisms by which BRG1 promotes invasion, we treated cells with an inhibitor of MMP2/MMP9 and performed invasion assays. We found that inhibition of MMP2 and MMP9 activity partially abrogated the BRG1 mediated increase in invasive ability (Figure. 5D). Consistently, siRNA mediated down-regulation of MMP2 (Figure. 5E) also reduced the BRG1 medicated increase in invasiveness (Figure. 5F). Thus, activation of MMP2 and possibly MMP9 expression contributes to the BRG1 induced increase in SK-MEL5 invasive ability.
Down-regulation of BRG1 in WM-266-4 cells inhibits melanoma invasiveness
We found that down-regulation of BRG1 resulted in decreased MMP2 and MCAM expression (Figure. 6C) and reduced invasion through Matrigel-coated Boyden chambers (Figure. 6D). Furthermore, although BRM levels increased in BRG1 down-regulated cells, our data suggest that BRM can not compensate for these BRG1 specific functions. Thus, both a gain of function and loss of function approach show that high levels of BRG1 promote melanoma invasive ability in vitro.
SP1 interacts with BRG1 to regulate MMP2 expression in SK-MEL5 cells
Melanoma progression is a dynamic process that requires tumor cells to possess decreased adhesive interactions with surrounding cells and with the extracellular matrix at some points in the metastatic cascade and increased adhesive interactions at other times . Metastatic potential also depends on adequate vascularization and the ability to degrade components of the ECM. These processes are regulated by reversible changes in the expression of genes involved in cell attachment, motility, and proteolytic degradation of the ECM . Previous studies showed that SWI/SNF enzymes modulate expression of ECM related molecules in normal and cancer cells [27, 28, 29, 30]. Furthermore, alterations in the expression of SWI/SNF components have been implicated in oncogenesis and multiple subunits have been determined to play tumor suppressive roles . We previously characterized SWI/SNF subunit expression in melanoma cell lines and found that a subset of melanoma cell lines was depleted in either the BRG1 or BRM catalytic subunit. Restoration of BRG1 in a melanoma cell line that lacks BRG1 expression enhanced the expression of MITF target genes and promoted increased resistance to cisplatin .
To further characterize BRG1 expression in melanoma, we assayed expression in melanoma tumors. In the present study, we determined that BRG1 mRNA levels are significantly up-regulated in stage IV melanoma tumors when compared to normal skin or stage III melanoma tumors. Furthermore, primary melanoma tumors and most melanoma cell lines express high levels of BRG1 (Figure. 1B, Additional file 1) [31, 32]. A recent study indicated that BRG1 expression is increased at the protein levels in primary melanoma tumors compared to dysplastic nevi, but that there is no significant difference in BRG1 levels between primary and metastatic melanoma samples . However, this study found that there may be a tendency for negative to weak BRG1 expression to be associated with a better patient survival . In contrast, a separate study suggested that BRG1 protein expression is frequently down-regulated in primary and metastatic melanoma compared to normal skin, but that a higher proportion of metastatic melanoma tumors express BRG1 compared to primary tumors . These studies in combination with our present study suggest that BRG1 status plays a role in melanoma progression, however further investigations that utilize larger sample sizes will be required to resolve the discrepancies between the different studies.
Re-expression of BRG1 in the BRG1/BRM deficient human adrenal adenocarcinoma cell line, SW13 preferentially alters the expression of a limited number of genes that mostly encode cell surface and ECM interacting proteins . Re-introduction of BRG1 in a BRG1 deficient breast cancer cell line, ALAB also had a high impact on the expression of genes that encode cell surface and ECM interacting proteins . This observation and the correlation between high BRG1 levels and melanoma progression prompted us to study the impact of BRG1 on the expression of genes involved in adhesion and extracellular matrix remodeling in melanoma cells.
Expression of BRG1 in melanoma cells modulated the expression of a number of ECM related genes that have opposing effects on melanoma invasiveness. In particular, BRG1 activated E-cadherin expression and down-regulated the expression of MMP1 and integrins α4 and β3. Down-regulation of E-cadherin and high levels of MMP1 and integrin αVβ3 are associated with transition from the radial non-invasive to the invasive vertical growth phase and the acquisition of metastatic potential in melanoma [37, 64, 65]. However, we found that BRG1 activated expression of other MMPs and integrins as well as MCAM, all of which have been shown to be important for promoting melanoma invasive ability and tumor progression . Melanoma cells employ distinct strategies for invasion, each of which may differ in the degree of dependence on the different molecular regulators . Interestingly, a previous study showed that dominant negative BRG1 activates integrin αV expression but still inhibits the invasive ability of fibroblasts . In our studies, both a gain of function and loss of function approach indicated that BRG1 promotes melanoma invasive ability, suggesting that high levels of BRG1 promote mechanisms by which melanoma cells invade that do not rely on the induction of all known cell surface regulators.
The activation of MMP2 expression by BRG1 contributed to the increased invasive ability of BRG1 expressing SK-MEL5 cells (Figures. 5C and 5D). BRG1 was previously shown to regulate MMP2 expression in SW13 cells by a transcriptional mechanism that involves SP1 . Our data indicate that BRG1 activates MMP2 expression in melanoma cells by a similar mechanism involving co-activation of SP1 mediated transcription (Figure. 7). However, BRG1 inhibited the expression of integrin β3, which is also regulated by SP1 . The differential requirement for SWI/SNF function in the regulation of a transcription factor's targets has been previously observed and is not well understood [17, 67]. A recent study suggests that diverse SWI/SNF complexes and sub-complexes can be recruited to different promoters and that the functional outcome of SWI/SNF activity on specific promoters may be determined by the composition of the SWI/SNF complex and the chromatin context . Furthermore, the recent observation that SWI/SNF enzymes also regulate microRNA expression adds an additional layer of complexity to the overall impact made by SWI/SNF enzymes in the regulation of cellular gene expression profiles . Further work will be required to decipher the mechanisms by which a high level of BRG1 results in a gene expression profile that promotes melanoma invasiveness and potentially dictates metastatic potential in vivo.
A number of studies have implicated SWI/SNF subunits, including BRG1, as tumor suppressors. Mutations or down-regulation of BRG1 expression occurs in multiple human tumors and haploinsufficiency of BRG1 predisposes mice to mammary tumors . Furthermore, when re-expressed in SW13 cells, BRG1 interacts with the retinoblastoma protein (Rb) to induce a G1 cell cycle arrest . These studies have implicated BRG1 as a tumor suppressor that curbs proliferation. In contrast, our data suggest that BRG1 expression is elevated in melanoma and promotes melanoma invasiveness. Interestingly, higher levels of BRG1 have also been associated with prostate and gastric cancer invasiveness and tumor progression [23, 24]. A recent study showing that residual BRG1 expression is required for tumorigenesis to occur in INI1 deficient mice suggests that the role of BRG1 in tumorigenesis is more complex than previously thought and that the outcome of BRG1 disruption may be lineage specific . We previously reported that BRG1 interacts with MITF, the master regulator of melanocyte differentiation and lineage addiction oncogene in melanoma . In this study, we found that BRG1 promotes expression of NCAM1 and CTNND2, two markers that are highly expressed in neural crest derived cells. Thus, the contrasting role of BRG1 in melanoma may in part result from the lineage specific derivation of this cancer type.
Our study suggests that over-expression of BRG1 contributes to melanoma progression. We have determined that BRG1 mRNA levels are higher in stage IV metastatic melanomas compared to stage III melanomas and to normal skin. Furthermore, we have determined that BRG1 modulates the expression of extracellular matrix and adhesion molecules that play an important role in melanoma metastasis. Our data indicate that modulation of extracellular matrix and adhesion molecule expression by BRG1 is associated with increased melanoma invasive ability in vitro. The down-regulation of SWI/SNF components in tumorigenesis has been elegantly demonstrated in numerous studies and is further supported by mouse models . Our work adds to several other studies [23, 24, 72] that suggest the over-expression of a SWI/SNF component may also contribute to tumorigenesis.
SK-MEL5 and WM-2664 melanoma cells were from the ATCC. A375SM melanoma cells were a kind gift from Dr. Menashe Bar-Eli (M.D. Anderson Cancer Center). SK-MEL5 cells expressing an empty vector or BRG1 were described in . Human melanocytes were from Cascade Biologics (Portland, Oregon, USA) or Yale Cell Culture Core Facility (New Haven, Connecticut, USA). With the exception of melanocytes, all cells were grown in DMEM supplemented with 10% FBS. Human melanocytes were grown in Media 254 with added growth supplements (Cascade Biologics). The MMP2/MMP9 inhibitor, 4-Biphenylylsulfonyl)amino-N-hydroxy-3-phenylpropionamide (BiPS) was from Calbiochem (San Diego, CA, USA) and was used at 10 μM.
RNA Isolation and Quantitative Real-time PCR
Total RNA was isolated with the Qiagen RNeasy mini kit and reverse transcribed as described . Quantitative real-time PCR was performed in SYBR Green Master Mix (Qiagen, Germantown, Maryland) with an Applied Biosystems Prism 7500 PCR system and analyzed with the SDS software as described . MCAM and GAPDH primers were purchased from SABiosciences (Frederick, MD, USA).
Tumor qPCR Arrays
The Tissue Scan Melanoma qPCR Arrays (MERT501) containing cDNAs from normal skin, stage III, and stage IV melanomas were obtained from Origene Technologies (Rockville, MD, USA). The primers used to detect BRG1 (SMARCA4) were from SABiosciences (Frederick, MD, USA). BRG1 levels were normalized by amplifying with primers to Human β-actin (Forward: CAGCCATGTACGTTGCTATCCAGG) and (Reverse: AGGTCCAGACGCAGGATGGCATG). The results were averaged from values obtained by running three PCR arrays. Statistical significance was determined by utilizing a Mann-Whitney Wilcoxon test.
Extracellular Matrix and Adhesion focused qPCR Arrays
Extracellular Matrix and Adhesion molecule RT2 Profiler PCR Arrays were purchased from SABiosciences (Fedrick, MD). The primer sets in this array are described in http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-013A.html. CT values obtained for 84 extracellular matrix and adhesion molecule gene expression were normalized to a value obtained by averaging the CT values of four different housekeeping genes. For each primer set, the fold change in SK-MEL5+BRG1 cells was determined relative to values obtained in control SK-MEL5 cells +empty vector. Average values were obtained from four PCR arrays with cDNA from control cells (from three different samples) and an additional four PCR arrays with cDNA from SK-MEL5 cells +BRG1 (from three different samples). Statistical significance was calculated using the student's t test.
The Tubulin antibody was from Sigma (St. Louis, Missouri, USA). FLAG M2 antibody and FLAG M2-Agarose were from Sigma. The E-cadherin, CTNND2, and MCAM antibodies were from BD Biosciences (San Jose, CA, USA), The BRG1, NCAM1 and SP1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The MMP2 and MMP9 antibodies were from Cell Signaling (Beverly, MA, USA). The MMP14 antibody was from Millipore (Temecula, CA, USA). The TIMP3 antibody was from Abcam (Cambridge, MA, USA). Control IgG antibody used for ChIPs was from Millipore (Billerica, MA, USA).
Cell extracts and immunoblot analysis
Cells were lysed in 20mMTris (pH 7.4),150 mM NaCl, 2 mM EDTA, 1% Triton X, 10% glycerol, supplemented with a protease inhibitor cocktail (Sigma). SDS-PAGE and Western blotting were carried out as described .
Cells were incubated in fetal bovine calf serum (Invitrogen, Carlsbad, CA, USA) for 10 minutes at room temperature to block nonspecific antibody binding and then with the primary antibody or an isotype matched IgG control for 20 minutes at 4°C. After one wash with FACS buffer (PBS+0.5%BSA, 5% fetal calf serum, 0.1% sodium azide, cells were incubated with secondary antibody for 20 minutes at 4°C, then washed twice with FACs buffer. Cells were re-suspended in 0.1% paraformaldehyde then loaded onto a FACS-Calibur (BD Biosciences, San Jose, CA, USA). Data was analyzed using Cell Quest Pro (BD Biosciences). Statistical significance was calculated using the student's t test.
Immunocytochemistry was performed as described  using an E-cadherin antibody (BD Biosciences) and goat anti-mouse-Alexa Fluor568 (Molecular Probes). Images were taken with a Nikon Eclipse TE2000-U fluorescence microscope at 60× magnification.
Zymography was performed as previously described . Control SK-MEL5 and SK-MEL5+BRG1 cells were cultured in serum free medium for 36 hours. Conditioned medium was collected, normalized to cell number, and subjected to electrophoresis in a polyacrylamide gel containing 1 mg/ml gelatin. After electrophoresis, the gel was washed in 2.5% Triton X-100 for 1 hour at room temperature to remove the SDS and then incubated for 24 hours at 37°C in a buffer consisting of 5 mM CaCl2 and 1 μM ZnCl2. The gel was stained in 0.25% Coomasie Blue for 30 minutes, de-stained in methanol/acetic acid solution and photographed on a light box. Proteolytic activity was detected as white bands against a blue background.
Acell SMART Pool siRNAs targeting BRG1 (E-010431), MMP2 (E-005959), SP1 (E-026959), and red non-targeting siRNAs (D-001960) were purchased from Dharmacon Inc. (Chicago, Il., USA) and used to transfect melanoma cells according to the manufacturer's instructions. WM-266-4 cells were transfected with control or siRNA targeting BRG1. BRG1 expressing SK-MEL5 cells were transfected with control or siRNA targeting MMP2 or SP1.
Adhesion assays were performed as previously described . 96 well plates were coated with laminin (10 ug/ml), collagen (type 1) (20 ugm/ml), or fibronectin (20 ugm/ml), and incubated at 4°C overnight. The plates are then washed with Wash buffer (DMEM with 0.1% BSA) and blocked in DMEM with 0.5% BSA for 45-60 minutes at 37°C. 2 × 104 cells were added to each well and incubated at 37°C for 30 minutes. Non-adherent cells were removed by washing three times with Wash buffer. The cells are then fixed with paraformaldehyde and incubated for 10-15 minutes and washed once with Wash buffer. The cells were stained with crystal violet for 10 minutes, washed with water, and dried. 2% SDS was added and the plates were incubated at room temperature for 30 minutes. Absorbance was read at 550 nm. Statistical significance was calculated using the student's t test.
Matrigel Invasion Assay
Invasion assays were performed using matrigel coated chambers (BD Biosciences, Bedford, MA, USA) as recommended by the manufacturer. SK-MEL5 cells expressing an empty vector or BRG1 were seeded in serum free media at a density of 1.25 × 105 cells per well on top of control or matrigel inserts. Media containing 5% FBS was used as a chemoattractant. After incubation for 16 hours, non-invading cells were removed from the upper surface and invading cells were stained with 1% Toluidine Blue and counted. Multiple fields were counted in triplicate membranes with a microscope at 20× magnification. The data shown is from two independent experiments done in triplicate. For studies involving inhibition of MMP2/MMP9, cells were pre-treated with10 μM 4-Biphenylylsulfonyl)amino-N-hydroxy-3-phenylpropionamide (BiPS) (Calbiochem, San Diego, CA, USA) for 3 hours and then plated onto the Boyden chambers in media containing 10 μM BiPS. For knockdown studies, invasion assays were performed 120 hours after transfection of control or siRNAs targeting BRG1. Statistical significance was calculated using a student's t test.
Co-immunoprecipitations were performed as previously described .
Chromatin Immunoprecipitations were performed as previously described  using FLAG to detect FLAG-BRG1 or IgG as a control. The primers used to detect the MMP2 promoter were (Forward: GGGGAAAAGAGGTGGAGAAA) and (Reverse: CGCCTGAGGAAGTCTGGAT). CD25 primers were previously described . Statistical significance was calculated using the student's t test.
ILD was supported by the National Institute of Environmental Health Sciences; Grant number: 5K22ES12981, Ohio Cancer Research Associates, American Cancer Society, Ohio Division We would like to thank Dr. Kathryn Eisenmann (Biochemistry and Cancer Biology, University of Toledo College of Medicine) for helpful suggestions and Sean Linkes (University of Toledo Flow Cytometry Core Facility) for help with FACS analysis.
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