SMARCE1 regulates metastatic potential of breast cancer cells through the HIF1A/PTK2 pathway
© The Author(s). 2016
Received: 23 November 2015
Accepted: 22 July 2016
Published: 5 August 2016
While aberrant activation of the chromatin-remodeling SWI/SNF complexes has been associated with cancer development and progression, the role of each subunit in tumor cells is poorly defined. This study is aimed to characterize the role of SMARCE1/BAF57 in regulating metastasis of breast cancer cells.
Genetic approaches and chemical inhibitors were used to manipulate the activities of SMARCE1 and its downstream targets in multiple breast cancer cell lines. Xenograft mouse models were used to analyze the role of SMARCE1 in lung metastasis in vivo. Nonadherent culture conditions were used to elucidate the role of SMARCE1 in regulating anoikis. Chromatin immunoprecipitation (ChIP), immunoprecipitation, and immunoblotting assays were designed to dissect the mechanism of action of SMARCE1. Public databases were used to investigate the relationship between SMARCE1 deregulation and breast cancer prognosis.
SMARCE1 knockdown reduced lung metastasis of breast cancer cells and sensitized tumor cells to anoikis. In response to loss of attachment, SMARCE1 interacted with and potentiated transcriptional activity of HIF1A, resulting in rapid PTK2 activation. Both HIF1A and PTK2 were indispensable for SMARCE1-mediated protection against anoikis by promoting activation of ERK and AKT pathways while suppressing the expression of pro-apoptotic BIM protein. Expression data analysis of a large cohort of human breast tumors revealed that high expression of SMARCE1 or PTK2 is associated with poor prognosis and tumor relapse, and PTK2 expression is positively correlated with SMARCE1 expression in basal-like and luminal B subtypes of breast tumors.
SMARCE1 plays an essential role in breast cancer metastasis by protecting cells against anoikis through the HIF1A/PTK2 pathway. SMARCE1-mediated PTK2 activation likely plays a key role in promoting metastasis of basal-like and luminal B subtype of breast tumors.
Breast cancer is the most frequently diagnosed form of cancer in American women and is the second leading cause of cancer-related deaths, primarily due to the incurable nature of metastatic breast cancer . Metastasis is the process whereby cancer cells spread from the site of the original tumor to distant organs . A stepwise sequence of events is involved in the metastatic process, whereby tumor cells encounter a number of environmental challenges such as loss of attachment to the extracellular matrix, physical stress during circulation in blood/lymph vessels, and immune surveillance [3, 4]. Only a small fraction of disseminated cells are able to alter gene expression patterns to obtain new phenotypic features according to environmental cues, and ultimately survive and adapt to form metastatic lesions.
Chromatin remodeling plays a key role in tumor progression by altering gene expression patterns to facilitate rapid adaptation of tumor cells to extracellular stimuli . Aberrant chromatin remodeling activity is frequently detected in human tumors. High-throughput genome and/or exome sequencing studies revealed that the SWI/SNF (switching-defective and sucrose nonfermenting) chromatin-remodeling complexes are the most highly mutated chromatin regulatory complexes, with nearly 20 % of human cancers harboring mutations in at least one of the genes encoding SWI/SNF subunits . Human SWI/SNF complexes consist of either Brahma-related gene 1 (BRG1)/SMARCA4 or Brahma gene (BRM)/SMARCA2 subunit that possesses helicase and ATPase activity, and several BRG/BRM associated factors (BAFs) that regulate the DNA-binding specificity and affinity of the complexes [6, 7]. The cancer-associated mutations cause aberrant activation of the residual SWI/SNF complexes due to distinct subunit configurations, rather than eliminating the chromatin-remodeling activity [8–10]. Although earlier studies identified several SWI/SNF subunits (e.g., SMARCB1 and SMARCA4) as tumor repressors, emerging evidence suggests that SWI/SNF activity is essential for tumor initiation, maintenance and progression [10, 11]. Therefore, it becomes important to define the role of each SWI/SNF subunit in development and progression of various types of tumors.
Genomic amplification of BAF57/SMARCE1, a core subunit of human SWI/SNF complexes that plays a role in interaction with transcription factors and chromatin, has been linked to high risk of recurrence of estrogen/progesterone receptor-negative breast tumors . However, the underlying molecular mechanisms remain undefined. In this study, we demonstrated that SMARCE1 plays a key role in breast cancer metastasis by protecting breast cancer cells against anoikis through the HIF1A/PTK2 pathway.
All cell lines were purchased from ATCC (Manassas, VA, USA). MDA-MB-231, HCC38, BT549 and their derivatives were maintained in Minimum Essential Media (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with 10 % fetal bovine serum (FBS), 200 units/ml penicillin-streptomycin (Cellgro, Manassas, VA, USA) and 0.5 μg/ml amphotericin B (Cellgro, Manassas, VA, USA). 184B5 cells were cultured in Mammary Epithelial Cell Growth Medium (MEGM, Lonza, Basel, Switzerland). A lung metastatic derivative of MDA-MB-231 (LM) was established as previously described . Cells stably expressing short hairpin RNA (shRNA) against SMARCE1, HIF1A, PTK2 or empty vector (EV) control were established by lentivirus transduction and selected in medium containing 2 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA). pLKO.1-HIF1AshRNA (NM_001530.x-1048s1c1), pLKO.1-SMARCE1shRNA (TRCN0000015779) and pLKO.1-PTK2shRNA (TRCN0000344599) were obtained from Open Biosystems/GE Dharmacon (Lafayette, CO, USA). Cells stably expressing focal adhesion kinase (FAK)/PTK2 or SMARCE1 were obtained by lentiviral transduction and selected in medium containing 30 μg/ml blasticidine (Sigma-Aldrich). pLX304-PTK2 (HsCD00442503) and pLX304-SMARCE1 (HsCD00440078) were purchased from DNASU Plasmid Repository (Tempe, AZ, USA). Control cells were established by lentivirus transduction to express selection marker only. Accell SMARTpool (a mixture of four short inhibitory RNA (siRNA) provided as a single reagent) for human SMARCE1 was obtained from Dharmacon (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and used to knock down SMARCE1 expression by following the manufactory’s protocol. The Accell Human Control siRNA (non-targeting) was used as control oligonucleotides.
Orthotopic xenograft model and experimental lung metastasis model (tail vein injection)
All animal studies adhered to protocols approved by the Institutional Animal Care and Use Committee of University of Tennessee Health Science Center. For the orthotopic xenograft model in NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, ME, USA), cells (7.5 × 105 cells in 10 μl phosphate-buffered saline) were surgically inoculated into the right inguinal mammary gland fat pads of 4-week-old female mice as previously described . Mice were inspected weekly for tumor appearance by visual observation and palpation. Primary tumor outgrowth was monitored twice a week using digital calipers. Tumor volume was calculated as: volume = (width2 × length)/2. Tumor and lung tissues were extracted 6 weeks after inoculation. The left lung lobes were imaged under the fluorescent microscope, fixed with 4 % paraformaldehyde, followed by tissue section (10 μm) and hematoxylin and eosin staining. Metastatic loci in lungs were quantified using ImageJ software. For experimental lung metastasis model, cells were inoculated into to 4-week-old NSG mice by tail vein injection. Whole blood was collected by cardiac puncture at various time and subjected to Ficoll-Paque separation (Ficoll-Paque PLUS, GE Healthcare Life Sciences, Piscataway, NJ, USA) to isolate circulating tumor cells. Fluorescent circulating tumor cells were counted under fluorescent microscope using a ×10 objective.
Quantitation of mRNA using qPCR
Total RNA was prepared with Trizol (Life Technologies, Grand Island, NY, USA). cDNAs for mRNA were synthesized by using iScript cDNA Synthesis Kits (Bio-Rad, Hercules, CA, USA). qPCR was performed on the CFX96™ Real-Time PCR Detection System using SYBR Green Supermix (Bio-Rad). Expression data of mRNA were normalized by the 2-ΔΔCT method to RPL13A, and presented as mean ± SE (n = 3). qPCR primers were obtained from PrimerBank .
Migration and invasion assays
Cells (20,000 cells/0.5 ml/well) were plated onto control membrane inserts with 8-micron pores or Matrigel-coated membrane transwell inserts (BD Biosciences, Bedford, MA, USA), which are placed in 24-well chambers filled with 0.6 ml growth medium. Twenty-four hours after plating, cells that remained on the upper surface of the membrane were removed by cotton-tipped swabs, and cells that migrated/invaded to the lower surface of the membrane were viewed under the fluorescent microscope. The percent migration was expressed as percent migration = (mean number of cells migrating through Matrigel insert membrane × 100/mean seeding control) and percent invasion was expressed as: percent invasion = (mean number of cells invading through Matrigel insert membrane × 100/mean number of cells migrating through control insert membrane.
Suspension culture and inhibitor assays
To mimic loss of attachment, cells were seeded in culture dishes coated with polyHEMA (Sigma-Aldrich) to prevent cell adherence for various period of time. The cell suspensions were placed into regular culture dishes 16 h prior to end-point viable cell counting. To identify the signaling pathways that promote survival of detached cells, a panel of chemical inhibitors were added in culture medium: PTK2/FAK inhibitor – 0.5 μM PF562271 (Selleckchem, Houston, TX, USA), ERK1/2 inhibitor – 10 μM U0126 (LC Laboratories, Woburn, MA, USA) or 10 μM PD98059 (Santa Cruz Biotechnology, Dallas, TX, USA), AKT inhibitor – 30 μM ZSTK474 (LC Laboratories), TGFBRI/II inhibitor – 10 μM LY210761 (Selleckchem), SRC inhibitor – 3 μM dasanitib (LC Laboratories) or Notch inhibitor – 5 μM DAPT (Selleckchem).
Protein extraction and immunoblotting
Protein extraction from whole cell lysates, cytosolic and nuclear fractions, and immunoblotting with luminescence detection were performed as previously described . The following antibodies were used in this study: anti-PTK2/FAK, FAK-Tyr576P/Tyr577P, ERK1/2-Thr202P/Tyr204P, ERK1/2, AKT-Tyr416P, AKT, and BCL2L11 from Cell Signaling Technologies (Boston, MA, USA); anti-HIF1A from Novus Biologicals (Littleton, CO, USA); anti-SMARCE1 and SMARCA4 from Bethyl Laboratories (Montgomery, TX, USA); anti-GAPDH from Millipore (Merck, Darmstadt, Germany) and anti-TBP from Abcam (Cambridge, MA, USA).
Chromatin immunoprecipitation (ChIP)
Cells (2 × 107) were crosslinked with 1 % formaldehyde (Pierce™ 16 % formaldehyde (w/v), methanol-free, Life Technologies) for 15 minutes. The nuclear fraction was prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Life Technologies), chromatin was sheared by sonication to obtain approximately 500 bp fragments and the soluble fraction was collected after centrifuge (12,000 rpm, 15 min). The soluble nuclear fractions were pre-cleaned with control IgG and MagnaBind Goat anti-Rabbit IgG (Life Technologies) and used for immunoprecipitation with antibodies against HIF1A, SMARCE1 or SMARCA4. Immunocomplexes were isolated with MagnaBind Goat anti-Rabbit IgG, de-crosslinked, and subjected to DNA preparation using the MiniElute PCR Purification Kit (QIAGEN, Germantown, MD, USA). qPCR was performed to detect the presence of PTK2 promoter region by using primers (Forward: 5′-CTCTTCCTCCTCCTGCCTCT-3′; reverse 5′-GTTCGGGGAAGACAGAAA GG-3′). To detect protein-protein interaction, the immunocomplexes were dissolved in Laemmli Sample Buffer (Bio-Rad) and subjected to immunoblotting assay.
A total of 5 × 105 cells were plated in polyHEMA-coated dishes and caspase-3 levels were measured at indicated time points by using the Caspase-Glo® 3/7 assay kit (Promega, Madison, WI, USA). Caspase activity was calculated as per manufacturer protocol, normalized to cell numbers.
Nucleosome scanning assay (NUSA)
106 cells were placed under suspension culture in polyHEMA-coated dishes for 0, 0.5, 1 and 2 hours. Nuclei were isolated, treated with Atlantis double-strand specific DNase (dsDNase) and subjected to DNA purification according to the EZ Nucleosomal DNA Prep Kit (Zymo Research, Irvine, CA, USA). qPCR was performed to determine nucleosome positioning on the PTK2 promoter. Overlapping primers were designed from −150 to +1589 relative to start site of PTK2 promoter to generate amplicons of approximately 150 bp, the size of DNA covered by one nucleosome. DNA amount was calculated according to a standard curve (qPCR CTs vs. various concentrations of template) generated for each primer and normalized to qPCR CTs of DNA purified from equal number of nuclei untreated with dsDNase.
Analysis of variance (ANOVA) and post hoc least significant difference analysis or t tests were performed using GraphPad Prism 5 software (Graphpad, San Diego, CA, USA). p values < 0.05 (*) were considered statistically significant. Data from two or three independent experiments with replicates are presented as means ± standard deviation (SD).
SMARCE1 knockdown reduces lung metastasis of breast cancer in vivo
SMARCE1 knockdown reduces lung colonization of tumor cells inoculated through tail vein
SMARCE1 knockdown sensitizes tumor cells to anoikis
To determine whether SMARCE1-mediated anoikis resistance can be extended to other breast cancer cell lines, we examine the effect of SMARCE1 knockdown in HCC38 (Fig. 3c), a triple-negative breast cancer cell line. We found that SMARCE1 knockdown resulted in significant reduction of the viability of cells in suspension culture. In addition, we examined the effect of SMARCE1 overexpression in BT549, a triple-negative breast cancer cell line that lacks SMARCE1 expression due to a biallelic inactivating mutation that causes a frameshift . As expected, no SMARCE1 mRNA or protein was detected in BT549 cells (Fig. 3d). Enforced SMARCE1 expression in BT549 substantially increased the viability of cells in suspension culture. To validate the observations with SMARCE1-shRNA, we examined the effect of SMARCE1 siRNA (Accell SMARTpool, GE Dharmacon) or control oligonucleotides on anoikis in LM and HCC38 cell lines. As showed in Fig. 3e, SMARCE1 siRNA (SMARCE1-i) treatment resulted in decreased expression of SMARCE1 protein and enhanced activation of caspase 3/7 in cells under suspension culture. These results demonstrate that SMARCE1 plays role in anoikis resistance of breast cancer cells.
SMARCE1-mediated anoikis resistance requires activation of PTK2, ERK and AKT pathways
SMARCE1 collaborates with HIF1A to activate PTK2 transcription in detached cells
By inspecting the transcription factor binding sequences in the proximal promoter region of PTK2 gene (Fig. 6b), we found a potential HIF1A/ARTN binding site located in the binding region of SWI/SNF subunits (i.e., SMARCA4 and SMARCB1) identified by ENCODE TFBS ChIP-seq analysis . Normoxic HIF1A activation has been linked to anoikis resistance in breast cancer cells [21, 23]. In addition, SWI/SNF complexes were reported to play a role in modulating HIF1A-mediated transcription activation in cells under hypoxia [24, 25]. Therefore, we speculated that SMARCE1 regulates PTK2 gene transcription through HIF1A in detached cells. By using ChIP-qPCR assay, we showed that cell detachment induced recruitment of SMARCE1, HIF1A, and BRG1/SMARCA4 to the PTK2 promoter region (−874 to −575 bp) in LM-EV cells, which was abolished by SMARCE1 knockdown (Fig. 6b). In addition, reciprocal immunoprecipitation assays, we detected HIF1A and SMARCE1 proteins in the immunoprecipitated SMARCE1 and HIF1A complexes, respectively (Fig. 6c). These results suggest that SMARCE1 activity is required for HIF1A-mediated PTK2 transcription activation in detached cells.
HIF1A activates PTK2 transcription and downstream activation of survival pathways in detached cells
SMARCE1 knockdown sensitizes non-tumorigenic mammary epithelial cells to anoikis
Higher SMARCE1 and PTK2 expression is associated with poor prognosis of patients with basal-like or luminal B subtype of breast tumors
Genomic alterations contribute to deregulation of SWI/SNF complexes in breast cancer
Despite the prevalent notion that SWI/SNF complexes are tumor suppressors , emerging evidence suggests that genetic alterations of subunit genes can produce aberrant SWI/SNF complexes with oncogenic function . These findings promoted us to examine the nature and frequency of genetic alterations in SMARCE1 and other SWI/SNF subunits specifically in breast cancer by using the TCGA database. The result revealed that approximately 30 % of breast tumors harbor genetic alterations in one or more of the genes encoding SWI/SNF subunits (Fig. 11c). Intriguingly, gene amplification is the most frequent genetic event for multiple SWI/SNF subunits in breast cancer, rather than inactive mutations that were reported in other tumors . SMARCE1 is the second most frequently amplified gene among all the SWI/SNF subunits. This finding implicates an oncogenic role of SWI/SNF complexes in human breast cancer. If a SWI/SNF subunit plays a critical role in promoting progression of breast cancer, we speculate a positive correlation between its expression and poor prognosis of breast cancer patients. Therefore, we examined the relationship between the expression of each SWI/SNF subunit and interval of RFS of breast cancer patients by using the online Kaplan-Meier survival analysis tool . This analysis revealed that higher expression of nine (out of 31) SWI/SNF subunits (i.e., BCL7C, BRD7, ACTL6A, SMARCE1, SMARCA5, SMARCAL1, SMARCA4, SS18, and SMARCC1) is associated with shorter interval of RFS of breast patients (log-rank p < 0.05, n = 3550). Overall these results from genetic and expression data analysis suggest that deregulation of SWI/SNF activity likely play a key role in promoting breast cancer progression.
Loss of adhesion to proper extracellular matrix (ECM) triggers a specific form of apoptosis termed anoikis in normal epithelial cells , which plays a fundamental role in morphogenesis of the mammary gland ductal system . Circumvention of anoikis contributes to breast tumor development and is a prerequisite for metastases by allowing cells to survive loss or alterations of ECM [3, 18]. In mammary epithelial cells, the dynamic and magnitude of activation of pro-apoptotic genes (e.g., BIM and BMF) or pro-survival proteins (e.g., PTK2) were shown to play a critical role in determining the fates of detached cells, anoikis or survival [3, 18, 33, 34]. PTK2 is one of the most important signaling proteins recruited into focal adhesions upon cell-ECM contact, where PTK2 is rapidly autophosphorylated to enable the recruitment of other scaffold and signaling molecules to activate the downstream cell survival signaling pathways (e.g., PI3K/Akt and MAPK/ERK pathways) [20, 34, 35]. Emerging evidence suggests that PTK2 plays a key role in protecting tumor cells against anoikis by sustaining survival signaling [18, 20]. However, how PTK2 is activated in detached cells have not been definitively resolved. Our studies revealed a key role of SMARCE1 in promoting survival of detached cells by facilitating rapid PTK2 transcription activation through HIF1A, the master regulator of hypoxia-responsive genes. Multiple kinases were reported to activate PTK2 through phosphorylation at Tyr576/577 in detached cells, including SRC family kinases and PTK6 [18, 36]. We found that SRC inhibition by dasatinib had no effect on viability of LM and BT549 cells under suspension. This observation suggests that PTK2 phosphorylation in detached breast cancer cells is likely sustained by a SRC-independent mechanism.
HIF1A activation under normal oxygen tension has been linked to anoikis resistance of breast cancer cells driven by ERBB2 oncogene , and circulating breast tumor cells from patients with metastatic breast cancer . SWI/SNF complexes were shown to regulate transcription activation of a subset of HIF1A target genes under hypoxia . However, it is unclear whether SWI/SNF complex is required for HIF1A transcription activity in detached cells under normal oxygen tension. Our study provides evidence suggesting an essential role of SMARCE1-based chromatin remodeling activity in breast cancer metastasis by facilitating HIF1A-mediated PTK2 transcription activation in detached cells under normoxia. Future study is warranted to evaluate the anti-metastasis potential of approaches targeting SMARCE1-HIF1A interaction.
According to the database of Project Achilles that performed genome-wide pooled shRNA screens across hundreds of cancer cell lines to identify genes essential for tumor cell proliferation, SMARCE1 knockdown showed limited effect on proliferation of breast cancer cells . Consistently, we found that SMARCE1 knockdown had no significant effect on proliferation of two breast cancer cell lines (i.e., MDA-MB-231 and HCC38) in vitro and growth of orthotopic xenografts derived from MDA-MB-231 cells, while significantly enhanced anoikis sensitivity of breast cancer cells. These findings suggest that SMARCE1 is dispensable for proliferation of breast cancer cells under normal growth condition, but required for cells to survive anoikis. Whether SMARCE1 plays a role to protect cells against stress-induced cell death in general warrants further investigation.
In contrast to colorectal, melanoma and lung cancer where inactive mutations of SWI/SNF subunits frequently occur, breast tumors have low frequency of inactive mutations [6, 10, 11]. By inspecting the TCGA database we found that approximately 30 % breast tumors harbor genetic alterations (i.e., gene amplification, deletion, and mutation) in one or more of the SWI/SNF subunits, with gene amplification being the most frequent event. This finding implicates an oncogenic role of SWI/SNF complexes in breast cancer. Consistently, we found that higher expression of several SWI/SNF subunits is associated with shorter interval of RFS of breast cancer patients. Our findings are likely relevant to basal-like and luminal B breast tumors, because a positively correlated expression between SMARCE1 and PTK2 is evident in these subtypes of tumors.
This study establishes a novel role of SMARCE1 in regulating metastatic potential of breast cancer cells by promoting survival of detached cells through the HIF1A/PTK2 pathway.
BAF57, BRG/BRM-associated factor 57; BIM, BCL2-interacting mediator of cell death; BRG1, Brahma-related gene 1; BRM, Brahma gene; ChIP, chromatin immunoprecipitation; dsDNase, double-strand-specific DNase; ECM, extracellular matrix; EV, empty vector; FAK, focal adhesion kinase; KD, knockdown; LM, lung metastatic cell line derived from MDA-MB-231; MEGM, mammary epithelial cell growth medium; NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; NUSA, nucleosome-scanning assay; qPCR, quantitative polymerase chain reaction; RFS, recurrence-free survival; shRNA, short hairpin RNA; siRNA, short inhibitory RNA; SWI/SNF, switching defective/sucrose nonfermenting complex; TCGA, The Cancer Genome Atlas
We thank Dr. Mauricio J. Reginato (Drexel University College of Medicine, Philadelphia, PA, USA) for his critical review of this manuscript.
This work was supported by National Institutes of Health grants, CA140346 (to MF), CA138488 (to TS) and CA133322 (to LMP).
Availability of data and materials
AS carried out the mouse model studies, anoikis studies, the chromatin immunoprecipitation studies, and drafted and revised the manuscript. MB participated in the anoikis studies and drafted the manuscript. TS, ZW and LP participated in the design of the study and drafted the manuscript. MF conceived of the study, carried out mouse model studies, and drafted and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All the animal studies adhered to the Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) guidelines of University of Tennessee Health Science Center.
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