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Downregulation of ARID1A, a component of the SWI/SNF chromatin remodeling complex, in breast cancer

Downregulation of ARID1A, a component of the SWI/SNF chromatin remodeling complex, in breast cancer Recent studies unraveled that AT-rich interactive domain-containing protein 1A (ARID1A), a subunit of the mammary SWI/SNF chromatin remodeling complex, acts as a tumor suppressor in various cancers. In this study, we first evaluated ARID1A expression by immunohistochemistry in invasive breast cancer tissue specimens and assessed the correlation with the prognosis of patients with breast cancer. Non-tumorous mammary duct epithelial cells exhibited strong nuclear ARID1A staining, whereas different degrees of loss in ARID1A immunoreactivity were observed in many invasive breast cancer cells. We scored ARID1A immunoreactivity based on the sum of the percentage score in invasive cancer cells (on a scale of 0 to 5) and the intensity score (on a scale of 0 to 3), for a possible total score of 0 to 8. Interestingly, partial loss of ARID1A expression, score 2 to 3, was significantly correlated with poor disease free survival of the patients. Subsequently, we performed siRNA-mediated ARID1A knockdown in cultured breast cancer cells followed by comprehensive gene profiling and quantitative RT-PCR. Interestingly, many genes were downregulated by partial loss of ARID1A, whereas RAB11FIP1 gene expression was significantly upregulated by partial loss of ARID1A expression in breast cancer cells. In contrast, a more than 50% reduction in ARID1A mRNA decreased RAB11FIP1gene expression. Immunoblotting also demonstrated that partial downregulation of ARID1A mRNA at approximately 20% reduction significantly increased the expression of RAB11FIP1 protein in MCF-7 cells, whereas, over 50% reduction of ARID1A mRNA resulted in reduction of RAB11FIP1 protein in cultured breast cancer cells. Recent studies reveal that RAB11FIP1 overexpression leads to breast cancer progression. Altogether, the present findings indicated that partial loss of ARID1A expression is linked to unfavorable outcome for patients with breast cancer, possibly due to increased RAB11FIP1 expression. Key words: ARID1A, breast cancer, prognosis, chromatin remodeling complex, RAB11FIP1. Introduction Condensation of chromosomal DNA by called BRG1-associated factors (BAFs), have been nucleosomes is an essential biological process to store identified as components of the human SWI/SNF-like the large amount of DNA in the nucleus, but occludes chromatin-remodeling protein complexes [2]. AT-rich many regulatory DNA elements. Chromatin interactive domain-containing protein 1A (ARID1A; remodeling complexes are critical for transcription by also known as BAF250a, p270, and B120, and facilitating access to packaged DNA [see review in 1]. SMARCF1) is a subunit of the SWI/SNF chromatin The SWI/SNF-related, matrix-associated, actin- remodeling complex, which possesses DNA binding dependent regulators of chromatin (SMARC), also activity [3-6]. ARID1A contributes to the specific http://www.jcancer.org Journal of Cancer 2017, Vol. 8 2 recruitment of its chromatin remodeling activity by University Graduate School of Medicine (specific binding to transcription factors and transcriptional approval number: 25-81). coactivator/corepressor complexes [7]. As expected Antibodies and immunohistochemical staining by the central role of ARID1A in the chromatin A mouse-specific monoclonal (clone PSG3) and remodeling machinery, the ablation of ARID1A conventional rabbit antibodies against ARID1A were results in early embryonic developmental arrest [8]. purchased from Santa Cruz Biotechnology (Santa In contrast, early pathobiological analysis Cruz, CA, USA) and GeneTex (San Antonio, TX, revealed that mutation in the ARID1A gene results in USA), respectively. We also generated a murine the development of several malignant tumors [6, 9]. monoclonal antibody against human ARID1A The high prevalence of inactivating ARID1A expressed in bacteria. The detailed procedure for mutations in endometrial and clear cell carcinomas of preparing recombinant proteins and the the ovary or uterus also suggests that loss of ARID1A establishment of the hybridoma according to the function contributes to carcinogenesis in these tumors method of Köhler and Milstein were previously [10-12]. Furthermore, growing evidence indicates that described [3]. The characterization of the generated ARID1A may have a widespread role in the antibody against human ARID1A is described in suppression of various tumors [see review in 13]. (Figure S1). A rabbit specific antibody against However, the mechanisms by which mutations in the RAB11FIP1 was purchased from GeneTex. ARID1A gene drive tumorigenesis are largely unclear. Archived pathological tissue specimens from 127 Especially, why insufficient ARID1A expression is invasive ductal carcinomas were used in this study. linked to tumorigenesis instead of cancer cell death All tissue specimens were obtained surgically, fixed in remains a mystery. In addition, the prognosis value of 10% buffered formalin, and embedded in paraffin. ARID1A expression remains debatable in many Tissues were immunostained with antibodies using cancers. the ImmPRESS™ polymerized reporter enzyme In this study, we demonstrated that partial loss staining system (Vector laboratories, Inc. Burlingame, of ARID1A is related to poor prognosis for patients CA, USA) as previously reported [15]. with invasive breast cancer. Subsequent in vitro comprehensive gene expression analysis unraveled Evaluation of immunohistochemical staining that the expression of more than 80% genes was and statistical analysis reduced by partial loss of ARID1A in breast cancer We scored the immunohistochemical staining cells. RAB11 family interacting protein 1 (RAB11FIP1, results as a percentage of ARID1A immunoreactivity also known as Rab-coupling protein (RCP)), which in breast cancer cells. The fraction of nuclear assists breast cancer progression [14], was significantly ARID1A-positive stained cancer cells was scored after increased by the partial loss of ARID1A. The present examining six high-power fields (40×) in one tissue findings suggest that partial loss of ARID1A might section for each case. The proportional intensity was drive mammary gland carcinogenesis, possibly scored on a scale of 0 to 5 (Score 0: less than 50%, score through increasing the RAB11FIP1-mediating 1: 50–75%, score 2: 75–90%, score 3: 90–95%, score 4: pathway. 95–98%, and score 5: over 98% invasive cancer cells exhibited ARID1A immunoreactivity). The intensity Materials and methods score was determined by the ARID1A Ethical statements immunoreactivity of invasive cancer cells, Score 0: The paraffin-embedded tissues surgically negative; score 1: weak compared to ARID1A resected from patients with breast cancer were used immunoreactivity in adipose nuclear cells; score 2: retrospectively after being used for diagnosis. The equal intensity to adipose nuclear cells; and score 3: need for written informed consent was waived by the stronger than ARID1A immunoreactivity in adipose Institutional Review Board of the Gifu University nuclear cells. The total score was the sum of the Graduate School of Medicine. Instead, the intensity + proportional scores. Institutional Review Board requested us to inform the Curves for disease free survival were drawn patients that they could refuse the use of their tissue using the Kaplan-Meier method and the differences in specimens for this study, if they did not want to survival rates were compared using the log-rank test participate in the present study. The present study for univariate survival analysis. Multivariate analysis was conducted in accordance with the ethical was performed using the Cox proportional hazards standards of the Helsinki Declaration in 1975, after model and the R statistics system. A p value of <0.05 approval of the Institutional Review Board of the Gifu was considered statistically significant. http://www.jcancer.org Journal of Cancer 2017, Vol. 8 3 Cell culture and siRNA-mediated RNA DH-forward 5′-GAAATCCCATCACCATCTTCCAG interference G-3′; GAPDH-reverse 5′-GAGCCCCAGCCTTCTCCA TG-3′; ARID1A-forward 5′-CATGTCCTATGAGCCA A breast cancer cell line, MDA-MB-157 (ATCC AATAAGGATCC-3′; ARID1A-reverse 5′-GAATAAC HTB-24) was obtained from the ATCC (Manassas, ATCCCCGAGCTGGGTTGGAA-3′. VA, USA). MCF-7 and MDA-MB-231 breast cancer To ensure that the SYBR green was not cells were obtained from Japan Health Science incorporated into primer dimers or non-specific Research Resources Bank (JCRB) (Osaka, Japan). Cells amplicons during the real-time PCR runs, the PCR were cultured in Dulbecco’s modified Eagle’s products were analyzed by polyacrylamide gel medium (DMEM; Gibco Life Technologies, Grand electrophoresis in preliminary experiments. Single Island, NY, USA) containing 10% heat-inactivated bands at the expected sizes were obtained in all fetal bovine serum (FBS). Cells were passaged in our instances. The samples were cultured in triplicate and laboratory for no more than 6 months after the expression of each target gene was analyzed by resuscitation. -ΔΔCT the 2 method described by Livak and Schmittgen The detailed procedure for siRNA silencing of a [17] using the LightCycler system. The ΔCT values target gene has been previously described [16]. In this were normalized to GAPDH for each triplicate set in study, we employed the Origene (Origene both the Trilencer-27 Universal scrambled negative Technologies, Inc. Rockville, MD, USA) siRNAs. control siRNA-treated (control) and 5′-rArGrArUrCrArCrCrArArGrUrUrGrUrArUrGrAr si-ARID1A-treated groups. The values for the GrCrUrGGG-3′ (SR305421A), 5′-rArCrArGrArArGrA si-ARID1A -treated group were then calculated for rArUrGrArUrCrCrArUrUrUrGrUrGrGTG-3′ (SR3054 each target gene as the fold change relative to the 21B), and 5′-rArGrArCrUrArCrArArUrGrUrArUrCr mean values for the control group (control; set to 1.0). ArArCrArGrCrArACA-3′ (SR305421C). A The standard deviations were then computed for the Trilencer-27 Universal scrambled negative control triplicate sets, namely, the 3 target genes and the fold siRNA (Origene) was used as a non-silencing control. changes are presented. In addition, Student’s t-tests siRNAs were transfected into cells using were performed to determine significant differences. lipofectamine™ RNAiMAX following the P < 0.05 was considered statistically significant. manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). At 48 h after transfection, the cells were Plasmid and transfection used for subsequent studies. Expression vector containing human ARID1A cDNA under the CMV promoter [18] was kindly cDNA microarray assay, reverse transcription provided by Dr. Kato H (Rockefeller University, NY, polymerase chain reaction (RT-PCR), and NY). Cells were transfected using the quantitative real-time RT-PCR N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammo We utilized the Human Whole Genome DNA nium methylsulfate (DOTAP) transfection reagent Microarray system (SurePrint G3 Human 8x60K ver. (Boehringer Mannheim, Indianapolis, IN, USA) 2.0, Agilent Technologies, Santa Clara, CA, USA) to according to the manufacturer protocol, as previously obtain the altered gene expression profile for described [16]. After G418 selection, three MDA-MB-157 cells in which the ARID1A gene was independent ARID1A-overexpressing MCF-7 and silenced by siRNA. cDNA synthesis from the total MDA-MB-157 cell lines were obtained. RNA and subsequent PCR were performed using a RT-PCR kit (Takara, Ohtsu, Japan). The procedure Western blotting was performed according to the manufacturer’s Western blotting was performed as previously instructions, as previously described [15]. Real-time described [19], according to Towbin et al. [20]. The PCR reactions were performed using the SYBR Green separated proteins were transferred onto reaction kit according to the manufacturer’s polyvinylidene difluoride membranes (Millipore Co., instructions (Roche Diagnostics, GmbH, Mannheim, Bedford, MA, USA) and probed with an anti-ARID1A Germany) in a LightCycler (Roche Diagnostics). antibody, anti-RAB11FIP1 antibody, or anti-GAPDH cDNA (2 µL each) was diluted to a volume of 20 µL antibody (Sigma-Aldrich, St. Louis, MO, USA). with the PCR mix containing a final primer Immunoreactivity was assessed using a western concentration of 0.2 pmol. blotting detection kit (Promega, Madison, WI, USA). The following primers were used for real-time Immunoblot bands were quantified by densitometry RT-PCR: RAB11FIP1-forward 5′-AGAGCTGTCAAG using LI-COR C-DiGit Blot Scanner imaging software CCCCGACTTCATCCTG-3′; RAB11FIP1-reverse 5′-A (LI-Cor Biosciences, Lincoln, NE) and were GCTGCGCATATGCAAATGCAGGGTCCG-3′; GAP normalized to the GAPDH band. http://www.jcancer.org Journal of Cancer 2017, Vol. 8 4 α5β1Integrin expression and Matrigel invasion antibodies, a conventional rabbit antibody and murine monoclonal antibody against ARID1A, on assay several tissue specimens to confirm the results. We Cell surface integrin α5β1 expression was obtained similar results with the three antibodies. evaluated by immunocytochemical staining using Representative results from the specific antibody (Biorbyt LLC. San Francisco, CA) as immunohistochemical staining are shown in Figure 1. previously reported [21]. In this study, we examined ARID1A expression in The invasiveness of the cultured cells was archived pathological tissue specimens from patients determined using 24-well BD BioCoat Matrigel with invasive ductal carcinoma. ARID1A Invasion Chamber Plates (BD Falcon) according to the immunoreactivity was scored as the sum of manufacturer protocol as previously described [16]. proportional intensity (Score 0 to 5) and intensity Briefly, 1 × 10 cells were placed in the upper score (Score 0 to 3). Subsequently, ARID1A compartment of the invasion chamber. After 48 h of immunoreactivity was classified into four groups incubation with DMEM containing 10% (lower (total score 0 or 1, 2 or 3, 4 to 6, and 7 or 8) and chamber) or 0% (upper chamber) FBS, the subsequently correlated with the prognosis of the non-invading cells were gently removed from the patients. The results are summarized in Figure 2. filter by scrubbing with a cotton-tipped swab. The Surprisingly, the disease free survival rate of patients cells on the lower surface of the filter were counted with score 2 or 3 was significantly worse than that of under a microscope. Experiments were performed in the other groups. Unexpectedly, severe loss of triplicate, and mean and standard deviation values ARID1A immunoreactivity (score 0 or 1) or no loss of were calculated. ARID1A immunoreactivity (score 7 or 8) had no significant effect on the prognosis of the patients. In Results the multivariate Cox regression model, nodal ARID1A expression in invasive breast metastasis (p=0.0017), triple negative (estrogen carcinoma tissue specimens receptor-negative, progesterone receptor-negative and HER2-negative) (p=0.043), and partial loss of In this study, we mainly used a newly generated murine monoclonal antibody to determine ARID1A ARID1A (p=0.0385), were independent prognostic expression by immunohistochemical staining. In factors. addition, we used two commercially available Figure 1. Representative immunohistochemical staining. (A) ARID1A immunoreactivity was scored according to proportional intensity (Score 0 to 5) and intensity score (Score 0 to 3) as described in Materials and methods. The scale bar represents 100 µm. The asterisk indicates ARID1A immunoreactivity of lymphocytes. http://www.jcancer.org Journal of Cancer 2017, Vol. 8 5 Figure 2. Disease-free survival curves according to total score of ARID1A immunoreactivity in invasive ductal carcinoma cells. The disease free survival rate of patients with a score of 2 or 3 (indicated as 2+3) was significantly lower than that of patients in other groups. In contrast, severe loss of ARID1A immunoreactivity (score 0 or 1) or minimal loss of ARID1A immunoreactivity (score 7 or 8) had no significant effect on the prognosis of the patients. RAB11FIP1 was overexpressed by partial loss of Partial ARID1A downregulation upregulates ARID1A, while loss of ARID1A at a detectable level RAB11FIP1 expression reduced RAB11FIP1 expression in cultured breast In order to examine the molecular mechanism cancer cells. Representative results are presented in linked to poor prognosis of patients with partial loss Figure 3. of ARID1A in breast cancer cells, we further Over 90% reduction of ARID1A gene resulted in performed in vitro experiments. By using Human significant loss of RAB11FIP1 mRNA in MCF-7 and Whole Genome DNA Microarray system (Agilent MDA-MB-231 cells (Figure 3A). Forced Technologies), we determined that an approximately overexpression of ARID1A did not significantly alter 20% reduction of ARID1A mRNA level resulted in the RAB11FIP1 mRNA expression in MCF-7 and reduction, less than 50%, of the expression of about MDA-MB-157 cells (Figure 3A). 772 genes, while it upregulated by 2 folds the Partial loss of ARID1A expression increased cell expression of 468 genes in MDA-MB-157 breast cancer surface localization of integrin α5β1 in both, MCF-7 cells (Figure S2 and Figure S3). Microarray data are and MDA-MB-157 cells. Furthermore, partial loss of deposited in the GEO public database (Array data ARID1A expression significantly increased accession number: GSE72669). Matrigel-invasion activity of MCF-7 and Interestingly, RAB11FIP1 mRNA expression was MDA-MB-157 cells. Representative images are shown significantly upregulated by the 20% reduction of in Figure 4. ARID1A mRNA level. Since RAB11FIP1 is a human breast cancer-promoting gene with Ras-activating Discussion function, we further examined the relation between ARID1A gene is somatically mutated in many ARID1A and RAB11FIP1 expression. endometrial carcinomas [11, 12]. However, a recent Subsequently, we performed dose-dependent meta-scale analysis clearly demonstrates that ARID1A siRNA downregulation of ARID1A mRNA in several gene mutations correlate with better survival in breast cancer cell lines and evaluated RAB11FIP1 uterine endometrial carcinoma [22]. Meta-scale mRNA expression. Notably, partial loss of ARID1A analysis did not indicate any prognosis values of significantly increased RAB11FIP1 mRNA expression ARID1A gene mutation in breast cancer [ 22]. In in MCF-7 and MDA-MB-231 cells. Over 50% contrast, Zhao et al. reported that ARID1A deletion reduction of ARID1A gene rather reduced RAB11FIP1 was detected as an independent prognostic factor in mRNA expression in MCF-7 and MDA-MB-231 cells. breast cancer [23]. However, mutation of the ARID1A Subsequent western-blotting analysis confirmed that gene is not frequent in breast cancer, approximately http://www.jcancer.org Journal of Cancer 2017, Vol. 8 6 5% [24]. Recently, Zhang et al. reported that promoter Unexpectedly, we determined that partial loss of hypermethylation of the ARID1A gene is responsible ARID1A immunoreactivity was significantly related for its low mRNA expression in many invasive breast to poor disease free survival rate for patients with cancers [25]. These findings encouraged us to score invasive breast cancer. In most cases in this group the ARID1A immunoreactivity of invasive breast (total score 2 or 3), a weak ARID1A immunoreactivity cancer to evaluate the prognosis value of ARID1A in intensity was detected in over 50% cancer cells. invasive breast cancers. Figure 3. Representative quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting results. siRNA treatment reduced ARID1A mRNA (representative data of quantitative RT-PCR ) and ARID1A protein (western-immunoblotting) in breast cancer MCF-7 cells. Results using SR305421A siRNA is shown. Similar results were obtained using other siRNAs (SR305421B and SR305421C). None of the three ARID1A-overexpressing MCF-7 exhibited loss or up-regulation of ARID1A mRNA (lane 1). Partial downregulation (approximately 20%) of ARID1A mRNA resulted in a significant increased (lane 2), whereas 50% ARID1A mRNA reduction caused a decrease in RAB11FIP1 gene expression in MCF-7 cells (lane 3). Moreover, >90% reduction of ARID1A gene resulted in significant loss of RAB11FIP1 mRNA in MCF-7 cells (lane 4). Values are means ± S.D. (n = 3) after 48 h. Statistical analysis was performed by using Student’s t test for unpaired observations (P<0.01). Additionally, we performed quantitative RT-PCR experiments using MDA-MB-231 cells and obtained similar results. Partial downregulation (approximately 20%) of ARID1A mRNA significantly increased the expression of RAB11FIP1 protein in MCF-7 cells (Lane 2). In contrast, >50% reduction of ARID1A mRNA resulted in reduction of RAB11FIP1 protein (Lane 3). Lane 1 shows the protein band representing control siRNA treated cells. Concentration of siRNA used in the samples run in Lanes 1 and 2 was 5nM, while 25nM in Lane 3 at final concentration, respectively. In this immunoblot, the results using a monoclonal antibody (clone PSG3) to probe ARID1A is shown. We also obtained similar results using a monoclonal antibody generated in our laboratory. The densitometry values of bands in Lane 2 are 0.726 (ARID1A) and 1.40 (RAB11FIP1), while values in Lane 3 are 0.468 (ARID1A) and 0.678 (RAB11FIP1). The densitometry data are 'fold change' as compared with control (Lane 1) after normalization with respective loading control (GAPDH). Figure 4. Representative immunocytochemical staining and invasion assay results. siRNA treatment reduced ARID1A mRNA in breast cancer MCF-7 and MDA-MB-157 cells. Results using SR305421A siRNA is shown. Similar results were obtained using other siRNAs (SR305421B and SR305421C). (A) Partial downregulation (approximately 20%) of ARID1A mRNA resulted in a significant increase of integrin α5β1 expression on surface membrane in both MCF-7 and MDA-MB-157 cells. The scale bar represents 50 µm. (B) Partial downregulation of ARID1A mRNA resulted in a significant increase of invasion activity of both MCF-7 and MDA-MB-157 cells. Values are means ± S.D. (n = 3) after 48 h. Statistical analysis was performed by using Student’s t test for unpaired observations. (P<0.01). http://www.jcancer.org Journal of Cancer 2017, Vol. 8 7 Subsequent comprehensive gene expression the present study, we also found that partial analysis unraveled that most protein-encoding genes down-regulation of ARID1A increased RAB11FIP1 were repressed by a 20% reduction of the ARID1A expression, resulted in accumulation of integrin α5β1 mRNA level in breast cancer cells. However, no tumor on breast cancer cell surface membrane, and suppressor encoding gene was identified among the significantly up-regulated invasion activity. genes repressed by the partial ARID1A-depletion in Notably, 50% or >90% reduction of ARID1A breast cancer cells. In contrast, we determined that gene significantly reduced RAB11FIP1 mRNA in RAB11FIP1 gene expression was significantly breast cancer cells in vitro. The exact molecular upregulated by the 20% reduction of the ARID1A mechanism, which was responsible for up-regulation mRNA level. In the present microarray analysis, we of RAB11FIP1 expression via partial loss of ARID1A found that 468 genes were up-regulated over 2-fold by expression, remains unclear. partial down-regulation of ARID1A. However, we We speculate that insufficient chromatin also noted that signal intensity of Immunoglobulin λ remodeling, probably caused by partial ARID1A gene expression was 0.06 (normalized value) in reduction, may alter the three-dimensional structure 20%-ARID1A-reduced MDA-MB-157 cells. Except for of the promoter region of the RAB11FIP1 gene. As a this noise, approximately 165 gene sequences were result, partial loss of ARID1A increases RAB11FIP1 up-regulated by partial down-regulation of ARID1A. expression and may result in poor prognosis in However, most of these sequences included patients with invasive breast cancer. miscRNA, lincRNAs, or unknown gene sequences, In turn, RAB11FIP1 could be a novel target for which are out of the scope of this study. Finally, we the development of therapeutics for patients with identified RAB11FIP1 as a candidate molecule in breast cancer harboring partial ARID1A loss. partial down-regulation of ARID1A-mediated breast In summary, the present study indicates the carcinogenesis. important pathobiological role of partial loss of Quantitative RT-PCR confirmed that ARID1A in invasive breast cancer. approximately 20% reduction in ARID1A mRNA Supplementary Material significantly increased RAB11FIP1 gene expression in MCF-7 breast cancer cells. We also employed two Additional File 1: other siRNA sequences to exclude off-target effects Figure S1, S3-S4. and obtained similar results. Representative data are http://www.jcancer.org/v08p0001s1.pdf shown in Fig 3A. Notably, a higher reduction in Additional File 2: ARID1A mRNA decreased RAB11FIP1 gene Figure S2. expression. Western-blotting analysis also indicated http://www.jcancer.org/v08p0001s2.xlsx that partial ARID1A reduction affected RAB11FIP1 Competing Interests expression (Fig 3B). We also performed immunohistochemical The authors have declared that no competing staining using anti-RAB11FIP1 antibody on interest exists. representative tissue specimens from patients with poor DFS (ARID1A score 2 or 3) and from patients References with no disease recurrence after 120 months (AIRD1A 1. Luger K, Dechassa ML, Tremethick DJ. New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat Rev Mol Cell score 7 or 8). 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Downregulation of ARID1A, a component of the SWI/SNF chromatin remodeling complex, in breast cancer

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Abstract

Recent studies unraveled that AT-rich interactive domain-containing protein 1A (ARID1A), a subunit of the mammary SWI/SNF chromatin remodeling complex, acts as a tumor suppressor in various cancers. In this study, we first evaluated ARID1A expression by immunohistochemistry in invasive breast cancer tissue specimens and assessed the correlation with the prognosis of patients with breast cancer. Non-tumorous mammary duct epithelial cells exhibited strong nuclear ARID1A staining, whereas different degrees of loss in ARID1A immunoreactivity were observed in many invasive breast cancer cells. We scored ARID1A immunoreactivity based on the sum of the percentage score in invasive cancer cells (on a scale of 0 to 5) and the intensity score (on a scale of 0 to 3), for a possible total score of 0 to 8. Interestingly, partial loss of ARID1A expression, score 2 to 3, was significantly correlated with poor disease free survival of the patients. Subsequently, we performed siRNA-mediated ARID1A knockdown in cultured breast cancer cells followed by comprehensive gene profiling and quantitative RT-PCR. Interestingly, many genes were downregulated by partial loss of ARID1A, whereas RAB11FIP1 gene expression was significantly upregulated by partial loss of ARID1A expression in breast cancer cells. In contrast, a more than 50% reduction in ARID1A mRNA decreased RAB11FIP1gene expression. Immunoblotting also demonstrated that partial downregulation of ARID1A mRNA at approximately 20% reduction significantly increased the expression of RAB11FIP1 protein in MCF-7 cells, whereas, over 50% reduction of ARID1A mRNA resulted in reduction of RAB11FIP1 protein in cultured breast cancer cells. Recent studies reveal that RAB11FIP1 overexpression leads to breast cancer progression. Altogether, the present findings indicated that partial loss of ARID1A expression is linked to unfavorable outcome for patients with breast cancer, possibly due to increased RAB11FIP1 expression. Key words: ARID1A, breast cancer, prognosis, chromatin remodeling complex, RAB11FIP1. Introduction Condensation of chromosomal DNA by called BRG1-associated factors (BAFs), have been nucleosomes is an essential biological process to store identified as components of the human SWI/SNF-like the large amount of DNA in the nucleus, but occludes chromatin-remodeling protein complexes [2]. AT-rich many regulatory DNA elements. Chromatin interactive domain-containing protein 1A (ARID1A; remodeling complexes are critical for transcription by also known as BAF250a, p270, and B120, and facilitating access to packaged DNA [see review in 1]. SMARCF1) is a subunit of the SWI/SNF chromatin The SWI/SNF-related, matrix-associated, actin- remodeling complex, which possesses DNA binding dependent regulators of chromatin (SMARC), also activity [3-6]. ARID1A contributes to the specific http://www.jcancer.org Journal of Cancer 2017, Vol. 8 2 recruitment of its chromatin remodeling activity by University Graduate School of Medicine (specific binding to transcription factors and transcriptional approval number: 25-81). coactivator/corepressor complexes [7]. As expected Antibodies and immunohistochemical staining by the central role of ARID1A in the chromatin A mouse-specific monoclonal (clone PSG3) and remodeling machinery, the ablation of ARID1A conventional rabbit antibodies against ARID1A were results in early embryonic developmental arrest [8]. purchased from Santa Cruz Biotechnology (Santa In contrast, early pathobiological analysis Cruz, CA, USA) and GeneTex (San Antonio, TX, revealed that mutation in the ARID1A gene results in USA), respectively. We also generated a murine the development of several malignant tumors [6, 9]. monoclonal antibody against human ARID1A The high prevalence of inactivating ARID1A expressed in bacteria. The detailed procedure for mutations in endometrial and clear cell carcinomas of preparing recombinant proteins and the the ovary or uterus also suggests that loss of ARID1A establishment of the hybridoma according to the function contributes to carcinogenesis in these tumors method of Köhler and Milstein were previously [10-12]. Furthermore, growing evidence indicates that described [3]. The characterization of the generated ARID1A may have a widespread role in the antibody against human ARID1A is described in suppression of various tumors [see review in 13]. (Figure S1). A rabbit specific antibody against However, the mechanisms by which mutations in the RAB11FIP1 was purchased from GeneTex. ARID1A gene drive tumorigenesis are largely unclear. Archived pathological tissue specimens from 127 Especially, why insufficient ARID1A expression is invasive ductal carcinomas were used in this study. linked to tumorigenesis instead of cancer cell death All tissue specimens were obtained surgically, fixed in remains a mystery. In addition, the prognosis value of 10% buffered formalin, and embedded in paraffin. ARID1A expression remains debatable in many Tissues were immunostained with antibodies using cancers. the ImmPRESS™ polymerized reporter enzyme In this study, we demonstrated that partial loss staining system (Vector laboratories, Inc. Burlingame, of ARID1A is related to poor prognosis for patients CA, USA) as previously reported [15]. with invasive breast cancer. Subsequent in vitro comprehensive gene expression analysis unraveled Evaluation of immunohistochemical staining that the expression of more than 80% genes was and statistical analysis reduced by partial loss of ARID1A in breast cancer We scored the immunohistochemical staining cells. RAB11 family interacting protein 1 (RAB11FIP1, results as a percentage of ARID1A immunoreactivity also known as Rab-coupling protein (RCP)), which in breast cancer cells. The fraction of nuclear assists breast cancer progression [14], was significantly ARID1A-positive stained cancer cells was scored after increased by the partial loss of ARID1A. The present examining six high-power fields (40×) in one tissue findings suggest that partial loss of ARID1A might section for each case. The proportional intensity was drive mammary gland carcinogenesis, possibly scored on a scale of 0 to 5 (Score 0: less than 50%, score through increasing the RAB11FIP1-mediating 1: 50–75%, score 2: 75–90%, score 3: 90–95%, score 4: pathway. 95–98%, and score 5: over 98% invasive cancer cells exhibited ARID1A immunoreactivity). The intensity Materials and methods score was determined by the ARID1A Ethical statements immunoreactivity of invasive cancer cells, Score 0: The paraffin-embedded tissues surgically negative; score 1: weak compared to ARID1A resected from patients with breast cancer were used immunoreactivity in adipose nuclear cells; score 2: retrospectively after being used for diagnosis. The equal intensity to adipose nuclear cells; and score 3: need for written informed consent was waived by the stronger than ARID1A immunoreactivity in adipose Institutional Review Board of the Gifu University nuclear cells. The total score was the sum of the Graduate School of Medicine. Instead, the intensity + proportional scores. Institutional Review Board requested us to inform the Curves for disease free survival were drawn patients that they could refuse the use of their tissue using the Kaplan-Meier method and the differences in specimens for this study, if they did not want to survival rates were compared using the log-rank test participate in the present study. The present study for univariate survival analysis. Multivariate analysis was conducted in accordance with the ethical was performed using the Cox proportional hazards standards of the Helsinki Declaration in 1975, after model and the R statistics system. A p value of <0.05 approval of the Institutional Review Board of the Gifu was considered statistically significant. http://www.jcancer.org Journal of Cancer 2017, Vol. 8 3 Cell culture and siRNA-mediated RNA DH-forward 5′-GAAATCCCATCACCATCTTCCAG interference G-3′; GAPDH-reverse 5′-GAGCCCCAGCCTTCTCCA TG-3′; ARID1A-forward 5′-CATGTCCTATGAGCCA A breast cancer cell line, MDA-MB-157 (ATCC AATAAGGATCC-3′; ARID1A-reverse 5′-GAATAAC HTB-24) was obtained from the ATCC (Manassas, ATCCCCGAGCTGGGTTGGAA-3′. VA, USA). MCF-7 and MDA-MB-231 breast cancer To ensure that the SYBR green was not cells were obtained from Japan Health Science incorporated into primer dimers or non-specific Research Resources Bank (JCRB) (Osaka, Japan). Cells amplicons during the real-time PCR runs, the PCR were cultured in Dulbecco’s modified Eagle’s products were analyzed by polyacrylamide gel medium (DMEM; Gibco Life Technologies, Grand electrophoresis in preliminary experiments. Single Island, NY, USA) containing 10% heat-inactivated bands at the expected sizes were obtained in all fetal bovine serum (FBS). Cells were passaged in our instances. The samples were cultured in triplicate and laboratory for no more than 6 months after the expression of each target gene was analyzed by resuscitation. -ΔΔCT the 2 method described by Livak and Schmittgen The detailed procedure for siRNA silencing of a [17] using the LightCycler system. The ΔCT values target gene has been previously described [16]. In this were normalized to GAPDH for each triplicate set in study, we employed the Origene (Origene both the Trilencer-27 Universal scrambled negative Technologies, Inc. Rockville, MD, USA) siRNAs. control siRNA-treated (control) and 5′-rArGrArUrCrArCrCrArArGrUrUrGrUrArUrGrAr si-ARID1A-treated groups. The values for the GrCrUrGGG-3′ (SR305421A), 5′-rArCrArGrArArGrA si-ARID1A -treated group were then calculated for rArUrGrArUrCrCrArUrUrUrGrUrGrGTG-3′ (SR3054 each target gene as the fold change relative to the 21B), and 5′-rArGrArCrUrArCrArArUrGrUrArUrCr mean values for the control group (control; set to 1.0). ArArCrArGrCrArACA-3′ (SR305421C). A The standard deviations were then computed for the Trilencer-27 Universal scrambled negative control triplicate sets, namely, the 3 target genes and the fold siRNA (Origene) was used as a non-silencing control. changes are presented. In addition, Student’s t-tests siRNAs were transfected into cells using were performed to determine significant differences. lipofectamine™ RNAiMAX following the P < 0.05 was considered statistically significant. manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). At 48 h after transfection, the cells were Plasmid and transfection used for subsequent studies. Expression vector containing human ARID1A cDNA under the CMV promoter [18] was kindly cDNA microarray assay, reverse transcription provided by Dr. Kato H (Rockefeller University, NY, polymerase chain reaction (RT-PCR), and NY). Cells were transfected using the quantitative real-time RT-PCR N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammo We utilized the Human Whole Genome DNA nium methylsulfate (DOTAP) transfection reagent Microarray system (SurePrint G3 Human 8x60K ver. (Boehringer Mannheim, Indianapolis, IN, USA) 2.0, Agilent Technologies, Santa Clara, CA, USA) to according to the manufacturer protocol, as previously obtain the altered gene expression profile for described [16]. After G418 selection, three MDA-MB-157 cells in which the ARID1A gene was independent ARID1A-overexpressing MCF-7 and silenced by siRNA. cDNA synthesis from the total MDA-MB-157 cell lines were obtained. RNA and subsequent PCR were performed using a RT-PCR kit (Takara, Ohtsu, Japan). The procedure Western blotting was performed according to the manufacturer’s Western blotting was performed as previously instructions, as previously described [15]. Real-time described [19], according to Towbin et al. [20]. The PCR reactions were performed using the SYBR Green separated proteins were transferred onto reaction kit according to the manufacturer’s polyvinylidene difluoride membranes (Millipore Co., instructions (Roche Diagnostics, GmbH, Mannheim, Bedford, MA, USA) and probed with an anti-ARID1A Germany) in a LightCycler (Roche Diagnostics). antibody, anti-RAB11FIP1 antibody, or anti-GAPDH cDNA (2 µL each) was diluted to a volume of 20 µL antibody (Sigma-Aldrich, St. Louis, MO, USA). with the PCR mix containing a final primer Immunoreactivity was assessed using a western concentration of 0.2 pmol. blotting detection kit (Promega, Madison, WI, USA). The following primers were used for real-time Immunoblot bands were quantified by densitometry RT-PCR: RAB11FIP1-forward 5′-AGAGCTGTCAAG using LI-COR C-DiGit Blot Scanner imaging software CCCCGACTTCATCCTG-3′; RAB11FIP1-reverse 5′-A (LI-Cor Biosciences, Lincoln, NE) and were GCTGCGCATATGCAAATGCAGGGTCCG-3′; GAP normalized to the GAPDH band. http://www.jcancer.org Journal of Cancer 2017, Vol. 8 4 α5β1Integrin expression and Matrigel invasion antibodies, a conventional rabbit antibody and murine monoclonal antibody against ARID1A, on assay several tissue specimens to confirm the results. We Cell surface integrin α5β1 expression was obtained similar results with the three antibodies. evaluated by immunocytochemical staining using Representative results from the specific antibody (Biorbyt LLC. San Francisco, CA) as immunohistochemical staining are shown in Figure 1. previously reported [21]. In this study, we examined ARID1A expression in The invasiveness of the cultured cells was archived pathological tissue specimens from patients determined using 24-well BD BioCoat Matrigel with invasive ductal carcinoma. ARID1A Invasion Chamber Plates (BD Falcon) according to the immunoreactivity was scored as the sum of manufacturer protocol as previously described [16]. proportional intensity (Score 0 to 5) and intensity Briefly, 1 × 10 cells were placed in the upper score (Score 0 to 3). Subsequently, ARID1A compartment of the invasion chamber. After 48 h of immunoreactivity was classified into four groups incubation with DMEM containing 10% (lower (total score 0 or 1, 2 or 3, 4 to 6, and 7 or 8) and chamber) or 0% (upper chamber) FBS, the subsequently correlated with the prognosis of the non-invading cells were gently removed from the patients. The results are summarized in Figure 2. filter by scrubbing with a cotton-tipped swab. The Surprisingly, the disease free survival rate of patients cells on the lower surface of the filter were counted with score 2 or 3 was significantly worse than that of under a microscope. Experiments were performed in the other groups. Unexpectedly, severe loss of triplicate, and mean and standard deviation values ARID1A immunoreactivity (score 0 or 1) or no loss of were calculated. ARID1A immunoreactivity (score 7 or 8) had no significant effect on the prognosis of the patients. In Results the multivariate Cox regression model, nodal ARID1A expression in invasive breast metastasis (p=0.0017), triple negative (estrogen carcinoma tissue specimens receptor-negative, progesterone receptor-negative and HER2-negative) (p=0.043), and partial loss of In this study, we mainly used a newly generated murine monoclonal antibody to determine ARID1A ARID1A (p=0.0385), were independent prognostic expression by immunohistochemical staining. In factors. addition, we used two commercially available Figure 1. Representative immunohistochemical staining. (A) ARID1A immunoreactivity was scored according to proportional intensity (Score 0 to 5) and intensity score (Score 0 to 3) as described in Materials and methods. The scale bar represents 100 µm. The asterisk indicates ARID1A immunoreactivity of lymphocytes. http://www.jcancer.org Journal of Cancer 2017, Vol. 8 5 Figure 2. Disease-free survival curves according to total score of ARID1A immunoreactivity in invasive ductal carcinoma cells. The disease free survival rate of patients with a score of 2 or 3 (indicated as 2+3) was significantly lower than that of patients in other groups. In contrast, severe loss of ARID1A immunoreactivity (score 0 or 1) or minimal loss of ARID1A immunoreactivity (score 7 or 8) had no significant effect on the prognosis of the patients. RAB11FIP1 was overexpressed by partial loss of Partial ARID1A downregulation upregulates ARID1A, while loss of ARID1A at a detectable level RAB11FIP1 expression reduced RAB11FIP1 expression in cultured breast In order to examine the molecular mechanism cancer cells. Representative results are presented in linked to poor prognosis of patients with partial loss Figure 3. of ARID1A in breast cancer cells, we further Over 90% reduction of ARID1A gene resulted in performed in vitro experiments. By using Human significant loss of RAB11FIP1 mRNA in MCF-7 and Whole Genome DNA Microarray system (Agilent MDA-MB-231 cells (Figure 3A). Forced Technologies), we determined that an approximately overexpression of ARID1A did not significantly alter 20% reduction of ARID1A mRNA level resulted in the RAB11FIP1 mRNA expression in MCF-7 and reduction, less than 50%, of the expression of about MDA-MB-157 cells (Figure 3A). 772 genes, while it upregulated by 2 folds the Partial loss of ARID1A expression increased cell expression of 468 genes in MDA-MB-157 breast cancer surface localization of integrin α5β1 in both, MCF-7 cells (Figure S2 and Figure S3). Microarray data are and MDA-MB-157 cells. Furthermore, partial loss of deposited in the GEO public database (Array data ARID1A expression significantly increased accession number: GSE72669). Matrigel-invasion activity of MCF-7 and Interestingly, RAB11FIP1 mRNA expression was MDA-MB-157 cells. Representative images are shown significantly upregulated by the 20% reduction of in Figure 4. ARID1A mRNA level. Since RAB11FIP1 is a human breast cancer-promoting gene with Ras-activating Discussion function, we further examined the relation between ARID1A gene is somatically mutated in many ARID1A and RAB11FIP1 expression. endometrial carcinomas [11, 12]. However, a recent Subsequently, we performed dose-dependent meta-scale analysis clearly demonstrates that ARID1A siRNA downregulation of ARID1A mRNA in several gene mutations correlate with better survival in breast cancer cell lines and evaluated RAB11FIP1 uterine endometrial carcinoma [22]. Meta-scale mRNA expression. Notably, partial loss of ARID1A analysis did not indicate any prognosis values of significantly increased RAB11FIP1 mRNA expression ARID1A gene mutation in breast cancer [ 22]. In in MCF-7 and MDA-MB-231 cells. Over 50% contrast, Zhao et al. reported that ARID1A deletion reduction of ARID1A gene rather reduced RAB11FIP1 was detected as an independent prognostic factor in mRNA expression in MCF-7 and MDA-MB-231 cells. breast cancer [23]. However, mutation of the ARID1A Subsequent western-blotting analysis confirmed that gene is not frequent in breast cancer, approximately http://www.jcancer.org Journal of Cancer 2017, Vol. 8 6 5% [24]. Recently, Zhang et al. reported that promoter Unexpectedly, we determined that partial loss of hypermethylation of the ARID1A gene is responsible ARID1A immunoreactivity was significantly related for its low mRNA expression in many invasive breast to poor disease free survival rate for patients with cancers [25]. These findings encouraged us to score invasive breast cancer. In most cases in this group the ARID1A immunoreactivity of invasive breast (total score 2 or 3), a weak ARID1A immunoreactivity cancer to evaluate the prognosis value of ARID1A in intensity was detected in over 50% cancer cells. invasive breast cancers. Figure 3. Representative quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting results. siRNA treatment reduced ARID1A mRNA (representative data of quantitative RT-PCR ) and ARID1A protein (western-immunoblotting) in breast cancer MCF-7 cells. Results using SR305421A siRNA is shown. Similar results were obtained using other siRNAs (SR305421B and SR305421C). None of the three ARID1A-overexpressing MCF-7 exhibited loss or up-regulation of ARID1A mRNA (lane 1). Partial downregulation (approximately 20%) of ARID1A mRNA resulted in a significant increased (lane 2), whereas 50% ARID1A mRNA reduction caused a decrease in RAB11FIP1 gene expression in MCF-7 cells (lane 3). Moreover, >90% reduction of ARID1A gene resulted in significant loss of RAB11FIP1 mRNA in MCF-7 cells (lane 4). Values are means ± S.D. (n = 3) after 48 h. Statistical analysis was performed by using Student’s t test for unpaired observations (P<0.01). Additionally, we performed quantitative RT-PCR experiments using MDA-MB-231 cells and obtained similar results. Partial downregulation (approximately 20%) of ARID1A mRNA significantly increased the expression of RAB11FIP1 protein in MCF-7 cells (Lane 2). In contrast, >50% reduction of ARID1A mRNA resulted in reduction of RAB11FIP1 protein (Lane 3). Lane 1 shows the protein band representing control siRNA treated cells. Concentration of siRNA used in the samples run in Lanes 1 and 2 was 5nM, while 25nM in Lane 3 at final concentration, respectively. In this immunoblot, the results using a monoclonal antibody (clone PSG3) to probe ARID1A is shown. We also obtained similar results using a monoclonal antibody generated in our laboratory. The densitometry values of bands in Lane 2 are 0.726 (ARID1A) and 1.40 (RAB11FIP1), while values in Lane 3 are 0.468 (ARID1A) and 0.678 (RAB11FIP1). The densitometry data are 'fold change' as compared with control (Lane 1) after normalization with respective loading control (GAPDH). Figure 4. Representative immunocytochemical staining and invasion assay results. siRNA treatment reduced ARID1A mRNA in breast cancer MCF-7 and MDA-MB-157 cells. Results using SR305421A siRNA is shown. Similar results were obtained using other siRNAs (SR305421B and SR305421C). (A) Partial downregulation (approximately 20%) of ARID1A mRNA resulted in a significant increase of integrin α5β1 expression on surface membrane in both MCF-7 and MDA-MB-157 cells. The scale bar represents 50 µm. (B) Partial downregulation of ARID1A mRNA resulted in a significant increase of invasion activity of both MCF-7 and MDA-MB-157 cells. Values are means ± S.D. (n = 3) after 48 h. Statistical analysis was performed by using Student’s t test for unpaired observations. (P<0.01). http://www.jcancer.org Journal of Cancer 2017, Vol. 8 7 Subsequent comprehensive gene expression the present study, we also found that partial analysis unraveled that most protein-encoding genes down-regulation of ARID1A increased RAB11FIP1 were repressed by a 20% reduction of the ARID1A expression, resulted in accumulation of integrin α5β1 mRNA level in breast cancer cells. However, no tumor on breast cancer cell surface membrane, and suppressor encoding gene was identified among the significantly up-regulated invasion activity. genes repressed by the partial ARID1A-depletion in Notably, 50% or >90% reduction of ARID1A breast cancer cells. In contrast, we determined that gene significantly reduced RAB11FIP1 mRNA in RAB11FIP1 gene expression was significantly breast cancer cells in vitro. The exact molecular upregulated by the 20% reduction of the ARID1A mechanism, which was responsible for up-regulation mRNA level. In the present microarray analysis, we of RAB11FIP1 expression via partial loss of ARID1A found that 468 genes were up-regulated over 2-fold by expression, remains unclear. partial down-regulation of ARID1A. However, we We speculate that insufficient chromatin also noted that signal intensity of Immunoglobulin λ remodeling, probably caused by partial ARID1A gene expression was 0.06 (normalized value) in reduction, may alter the three-dimensional structure 20%-ARID1A-reduced MDA-MB-157 cells. Except for of the promoter region of the RAB11FIP1 gene. As a this noise, approximately 165 gene sequences were result, partial loss of ARID1A increases RAB11FIP1 up-regulated by partial down-regulation of ARID1A. expression and may result in poor prognosis in However, most of these sequences included patients with invasive breast cancer. miscRNA, lincRNAs, or unknown gene sequences, In turn, RAB11FIP1 could be a novel target for which are out of the scope of this study. Finally, we the development of therapeutics for patients with identified RAB11FIP1 as a candidate molecule in breast cancer harboring partial ARID1A loss. partial down-regulation of ARID1A-mediated breast In summary, the present study indicates the carcinogenesis. important pathobiological role of partial loss of Quantitative RT-PCR confirmed that ARID1A in invasive breast cancer. approximately 20% reduction in ARID1A mRNA Supplementary Material significantly increased RAB11FIP1 gene expression in MCF-7 breast cancer cells. We also employed two Additional File 1: other siRNA sequences to exclude off-target effects Figure S1, S3-S4. and obtained similar results. Representative data are http://www.jcancer.org/v08p0001s1.pdf shown in Fig 3A. Notably, a higher reduction in Additional File 2: ARID1A mRNA decreased RAB11FIP1 gene Figure S2. expression. Western-blotting analysis also indicated http://www.jcancer.org/v08p0001s2.xlsx that partial ARID1A reduction affected RAB11FIP1 Competing Interests expression (Fig 3B). We also performed immunohistochemical The authors have declared that no competing staining using anti-RAB11FIP1 antibody on interest exists. representative tissue specimens from patients with poor DFS (ARID1A score 2 or 3) and from patients References with no disease recurrence after 120 months (AIRD1A 1. Luger K, Dechassa ML, Tremethick DJ. New insights into nucleosome and chromatin structure: an ordered state or a disordered affair? Nat Rev Mol Cell score 7 or 8). 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Journal of Cancer – Pubmed Central

Published: Jan 1, 2017

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