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Differential expression of ARID3B in normal adult tissue and carcinomas
Gene 543 (2014) 174–180
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Differential expression of ARID3B in normal adult tissue and carcinomas Serene J. Samyesudhas a, Lynn Roy a, Karen D. Cowden Dahl a,b,c,d,⁎ a
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN, USA Department of Chemistry and Biochemistry, Notre Dame University, Notre Dame, IN, USA c Eck Institute for Global Health, Notre Dame University, Notre Dame, IN, USA d Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA b
a r t i c l e
i n f o
Article history: Received 4 January 2014 Received in revised form 28 March 2014 Accepted 2 April 2014 Available online 3 April 2014 Keywords: ARID3B Transcription factor Stratified epithelium Differentiation Immunohistochemistry
a b s t r a c t ARID3B is a DNA binding protein that is overexpressed in neuroblastoma and ovarian cancer. To understand the extent that ARID3B participates in tumor development, we assessed protein expression of ARID3B in normal adult and malignant tissues. We found that ARID3B is highly expressed in differentiated layers of squamous epithelium. We also examined expression of an alternative splice form of ARID3B and found that it has similar but not identical expression patterns to the full length ARID3B isoform. ARID3B has two closely related paralogues, ARID3A and ARID3C. Each of these 3 family members exhibits different patterns of expression. Of the ARID3 family members, ARID3B is the most widely expressed and is particularly expressed in epithelium. In addition to examining normal tissue, we investigated ARID3B expression in a variety of tumor types. Most notably we found that ARID3B expression is decreased in esophagus and stomach tumors compared to normal corresponding tissues. Our results indicate that the different patterns of ARID3B in normal tissues translate into different roles for ARID3B in carcinomas. © 2014 Elsevier B.V. All rights reserved.
1. Introduction ARID3B is a member of the AT-rich interactive domain (ARID) family of DNA binding proteins. These proteins regulate diverse cellular functions through modification of chromatin structure, histone methylation, transcriptional activation and are involved in development (Wilsker et al., 2002). ARID3B is essential for development (as mice lacking Arid3b die midgestation) (Casanova et al., 2011; Takebe et al., 2006; Webb et al., 2011). Three independent knockout mice have been generated for ARID3B. These mice have defects in vasculature, cranial mesenchyme, limbs, heart and the craniofacial region (Casanova et al., 2011; Takebe et al., 2006; Webb et al., 2011). During development ARID3B is expressed in many regions of developing embryos between embryonic days 7–10.5 (Takebe et al., 2006). Among the places ARID3B is expressed are the primitive streak, paraxial mesoderm, neural crest, brachial arches and the outflow tract of the heart (Takebe et al., 2006). ARID3B is present in mouse embryonic stem (ES) cells and increases
Abbreviations: ARID3B, AT rich interactive domain 3B; ARID3B-FL, full length isoform of ARID3B; ARID3B-Sh, short alterative splice form of ARID3B; ES, embryonic stem (cells); MEFs, mouse embryo fibroblasts; TNFα, tumor necrosis factor alpha; QRT-PCR, quantitative reverse transcribed polymerase chain reaction; cDNA, complimentary DNA; TMA(s), tissue microarray; IHC, immunohistochemistry. ⁎ Corresponding author at: 1234 Notre Dame Ave, Harper Hall A225, South Bend, IN 46530, USA. E-mail addresses: [email protected] (S.J. Samyesudhas), [email protected] (L. Roy), [email protected] (K.D. Cowden Dahl).
http://dx.doi.org/10.1016/j.gene.2014.04.007 0378-1119/© 2014 Elsevier B.V. All rights reserved.
with ES cell differentiation (Wang et al., 2006). Despite knowing the patterns of ARID3B expression in the developing mouse embryo, ARID3B expression in adult mouse and human tissues has not been examined in detail. Tissue northern blots demonstrate that ARID3B is expressed in the human spleen, thymus, prostate, testis, small intestine, colon, leukocytes, heart, brain, placenta, lungs, skeletal muscle, kidney, and pancreas (Numata et al., 1999). Of these the highest mRNA expression was found in the testis, placenta, leukocytes and spleen (Numata et al., 1999). A second paper demonstrated through northern blot that in the adult mouse, ARID3B is only expressed in the testis, prostate, thyroid, and thymus (Takebe et al., 2006). Careful examination of ARID3B expression at the protein level has not been reported. In addition to its essential role in development, ARID3B is implicated in cellular transformation and tumor progression. ARID3B is overexpressed in both neuroblastoma and ovarian cancer (Cowden Dahl et al., 2009; Kobayashi et al., 2006). ARID3B immortalizes mouse embryo fibroblasts (MEFs) and cooperates with MYCN to transform fibroblasts and promotes neuroblastoma tumor growth (Kobayashi et al., 2006). We demonstrated that ARID3B is overexpressed in serous ovarian cancer (Cowden Dahl et al., 2009). ARID3B has several functions that may cooperate in tumorigenesis. ARID3B enables its paralogue ARID3A to remain nuclear in B-cells (Kim et al., 2007); ARID3B promotes migration of MEFs and breast cancer cells (Akhavantabasi et al., 2012; Casanova et al., 2011), and induces TNFα signaling (Joseph et al., 2012). The goal of this study is to determine the normal pattern of expression for ARID3B protein in adult tissue. Additionally, we wish to identify which tumor types express ARID3B. In doing this, we will
S.J. Samyesudhas et al. / Gene 543 (2014) 174–180
gain insight into the role of ARID3B in human cancer. Collectively our data demonstrates that ARID3B expression is differentially regulated in different normal and malignant tissues.
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the delta delta CT method was used to calculate fold differences in gene expression. 2.2. Normal and tumor tissue microarray
2. Materials and methods 2.1. Quantitative reverse transcribed polymerase chain reaction (QRT-PCR) for ARID3B isoforms on RNA from adult human organs QRT-PCR was performed on total RNA purchased from Clontech (Mountain View, CA) for a panel of human organs. 1 μg of RNA was reverse transcribed into cDNA using the High Capacity cDNA Archive kit from Applied Biosystems (Carlsbad, CA). QRT-PCR was performed using iTaq Universal Probes Supermix (Bio-Rad Laboratories, Inc., Hercules, CA) for ARID3B-Sh and ARID3B-FL. The following primers were used: ARID3B-Sh-HEX: Primer 1: 5′-TCCCACCACATGGACAACAAGCTA3′, Primer 2: 5′-TCATCCAGATTCCAGTTCGGCTGT-3′, HEX probe: 5′AAGATGCTTCCAAGGCCTCACCTTCT-3′; ARID3B FAM (ARID3B-FL), ARID3B (Applied Biosystems, HS01084919_g1); and ARID3A: Primer 1: 5′-CTT CTC CGT CAC CAG CAC-3′, Primer 2: 5′-GAC TTG TTC AGC TTC ATG CAG-3′, ARID3C: Primer 1: 5′-CAC TTT GCG GTT GAT GAC TTC-3′, Primer 2: 5′-TCT GGA TGA CCT GTT TAG CTT C-3′, and HPRTFAM (Applied Biosystems). Expression was normalized to HPRT and
The following tissue microarray (TMA) slides were obtained from US Biomax Inc. (Rockville, MD): (MTU951, ES1501, CR1501, BC01114, TH721, KD1503, BN1002a and LN801) and Imgenex (San Diego, CA) (IMH-372 and IMH-335). 2.3. Immunohistochemistry Immunohistochemistry (IHC) was performed using an ARID3B antibody from Abcam (ab50927) (Cambridge, MA) in Fig. 1. IHC in Figs. 2–4 was performed with the Bethyl Laboratories (Montgomery, TX) ARID3B IHC Antibody (IHC-00717) and ARID3B antibody from Abcam (ab50927) (Cambridge, MA). Additionally the C-terminal anti-ARID3B antibody from Bethyl Laboratories (A302 565A) that detects the last 50 amino acids that are only found in ARID3BFL was also used in Fig. 3. Deparaffinization of tissue microarray slides was performed for 1 h at 60 °C followed by three 5 minute incubations in xylene. Slides were rehydrated in an ethanol series. Antigen retrieval was done in citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) at 95 °C for
Fig. 1. ARID3B immunohistochemistry (IHC) in corresponding adult mouse and human tissues. IHC was performed on tissue microarrays (TMAs) for normal human and mouse tissues. Sections from cerebellum, salivary gland, stomach, and testis are shown. Brown staining denotes ARID3B expression. No primary control IHC on human sections is depicted in right panels. Original magnification is 20×.
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20 min. The slides were washed twice in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, and 11.9 mM phosphate buffer, pH 7.4) and then incubated for 20 min in a solution of 3% H2O2 in methanol. After rinsing three times for 5 min in PBS, immunohistochemistry was performed using the ABC kit (Vector Laboratories, Burlingame, CA) and developed with ImmPACT DAB peroxidase substrate or DAB kit (Vector Laboratories, Burlingame, CA). Slides were incubated with primary antibody overnight at 4 °C using either the anti-ARID3B IHC Antibody (1:250) (Bethyl Laboratories) or anti-ARID3B from Abcam (ab50927 at 1:100). The slides were counter-stained with Hematoxylin QS (Vector Laboratories, Burlingame, CA). Slides were dehydrated and mounted with VectaMount permanent mounting medium (Vector Laboratories, Burlingame, CA). Slides were scanned using Aperio ScanScope system (Aperio ePathology, Vista, CA) and analyzed using the Imagescope software at 20× magnification.
2.4. Tissue blot A normal human organ tissue blot (IMB-103) containing 20 μg of lysate from 11 human organs was obtained from Imgenex (San Diego, CA). The tissue blot was rehydrated, blocked, and blotted with primary antibody according to manufacturer's instructions (Imgenex (San Diego, CA)). The anti-ARID3B antibody from (A302 564A) Bethyl Laboratories (Montgomery, TX) was used at a 1:1000 dilution and the ARID3B-Sh antibody (Pacific Immunology Inc. (Ramona, CA)) was used at a dilution of 1:1000. The blot was stained with Ponceau S (0.1% Ponceau S, 1% Acetic Acid) as a loading control. The BIORAD Chemidoc XRS+ System using Imager Lab Software (Bio-Rad Laboratories, Inc., Hercules, CA) was used to image and analyze the blot.
3. Results 3.1. ARID3B protein is preferentially expressed in epithelial and secretory tissue To understand the extent to which ARID3B is involved in cancer, we began by examining the normal pattern of ARID3B expression in adult organs. We performed immunohistochemistry (IHC) for ARID3B on tissue microarrays (TMAs) containing either human or mouse organs. As a negative control we performed IHC on TMAs without primary antibodies. We observed ARID3B protein expression in several secretory cell types (stomach, salivary gland, and Leydig cells) (Fig. 1). In Supplemental Fig. 1 we provide staining controls for the various tissues examined. ARID3B is also expressed in epithelial cell types including urinary bladder and kidney (Table 1). We found that ARID3B was expressed in many epithelial and secretory tissues (Table 1). The expression pattern of ARID3B was similar between adult mouse and human tissues. Interestingly, we did not observe significant ARID3B protein expression in muscle (skeletal muscle and heart). We did detect ARID3B expression in spleen and lymph nodes, but expression in thymus was variable (Table 1). Nervous system tissues also did not have ARID3B protein expression in mice or humans (pons, cerebrum, and cerebellum) (data not shown and Fig. 1). Strikingly blood vessels in all tissues examined expressed high levels of ARID3B (data not shown). One of the most striking findings is that ARID3B is strongly expressed in stratified squamous epithelium (Fig. 2). In the three examples of stratified epithelium that we examined (skin, esophagus and cervix), we observed strong staining for ARID3B in the suprabasal layers and lower expression in the basal cells (Fig. 2). The lower staining of ARID3B in the basal cells was most noticeable in the esophagus and more variable in the skin
Fig. 2. ARID3B expression in stratified squamous epithelium. IHC was performed on TMAs for normal human skin, esophagus, and cervix. Brown staining denotes ARID3B. No primary control IHC for each tissue depicted on the right panels. Original magnification is 20×.
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Fig. 3. Expression of ARID3B isoforms in normal human organs. A. Western blot was performed on Imgenex Insta-Blot Human Tissues IMB-103 for ARID3B. This demonstrates the expression of ARID3B FL (61 kDa) (top panel) and ARID3B-Sh (28 kDa) (bottom panel). Ponceau S staining was used as a loading control. B. IHC using 2 different ARID3B antibodies. The “Total” antibody detects both ARID3-FL and ARID3B-Sh. The ARID3B-FL detects only the longer isoform. Original magnification is 20×. C. QRT-PCR was performed for the ARID3B isoforms (ARID3B-FL and ARID3B-Sh) on RNA samples from different human organs. Gene expression was normalized to HPRT and shown as fold induction over whole brain (which has the least expression). QRTPCR was performed for ARID3A (D) and ARID3C (E) gene expression was normalized to HPRT and shown as fold induction over whole brain.
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Fig. 4. Expression of ARID3B in normal and tumor tissues. A. ARID3B expression in normal tissue and malignant tumors of the esophagus and stomach. B. ARID3B expression in benign, normal adjacent, and malignant thyroid tissues. Original magnification is 20×.
and cervix (Supplemental Fig. 2). To assess the level of ARID3B expression in squamous epithelium we repeated the IHC on esophagus and cervix without hematoxylin. We again found that ARID3B expression is higher in the suprabasal cells and is lower in basal cells, suggesting that ARID3B expression is regulated during differentiation (Supplemental Fig. 2). In conclusion, ARID3B is expressed in the epithelium (particularly in squamous epithelium), some secretory cells, spleen, and blood vessels in the adult organism. This suggests that ARID3B has important functions in the epithelium, hematopoietic cells, and endothelium. 3.2. Expression of an alternate splice form of ARID3B (ARID3B-Sh) and ARID3 subfamily members We previously reported that ARID3B contains an alternatively spliced isoform (Joseph et al., 2012). The smaller isoform (ARID3B-Sh) lacks exons 5–9 including 81% of the DNA binding domain and the REKLES
domains that are involved in ARID3 homo- and hetero-oligomerization (Kim et al., 2007). We examined tissue distribution of the product of the smaller isoform of ARID3B (ARID3B-Sh) by western blot. ARID3B-Sh is a 28 kDa protein and easily distinguished from the 61 kDa ARID3B-FL (the full length isoform) on a western blot using an antibody to amino acids 100–150 present in both isoforms. ARID3B-Sh is expressed in the brain, kidney, liver, lung, stomach, and spleen (Fig. 3). We attempted to perform IHC for ARID3B on tissue microarrays using an ARID3B-Sh specific antibody that we generated. However, this antibody produced excessive background staining. We did perform IHC using two different antibodies for ARID3B on the kidney (since this is a tissue where both ARID3B isoforms are expressed). The first antibody detects residues 100–150 of the ARID3B protein (Bethyl Laboratories anti-ARID3B IHC Antibody (IHC-00717)). These amino acids are found in both isoforms; therefore, the “Total” ARID3B expression is detected with this antibody. We used a second antibody (Bethyl laboratories anti-ARID3B Antibody
S.J. Samyesudhas et al. / Gene 543 (2014) 174–180 Table 1 ARID3B expression in adult human and mouse tissues.
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Table 2 ARID3B expression in normal and tumor tissues.
Organ
Human
Mouse
Organ
Normal
Benign
Malignant
Skin Esophagus Cervix Salivary gland Stomach Small intestine Kidney, cortex Kidney, medulla Testis (Leydig cells) Ovary Uterus (endometrium) Urinary bladder Liver Spleen Lymph node Thymus Lung Cerebellum Skeletal muscle
+ + + + + + + + + + + + + + + Variable Variable − −
+ NA NA + + + + + + + + + + + NA − + − −
Brain Small intestine Colon Rectum Salivary gland Thyroid Esophagus Stomach Cervix
− + + + + + + + +
Variable + + + + + NA NA NA
+ + + + + + + + +
NA = not available.
(A302 565A)), which detects amino acids 510–560 only found in ARID3B-FL. We found that both antibodies detect extensive expression of ARID3B in the kidney. As additional way of examining ARID3B isoform expression in tissue, we performed QRT-PCR for each isoform using isoform specific primers. RNA from a panel of normal tissues was obtained, converted to cDNA and QRT-PCR was conducted. As expected (from the IHC) we detected very little expression of both ARID3B isoforms in the whole adult brain. Therefore we normalized expression of the other tissues to the whole brain. Interestingly, ARID3B expression is relatively high in the fetal brain compared to the whole brain. In the fetal brain compared to the whole brain ARID3B-FL mRNA is increased by 6.78 fold and ARID3B-Sh is increased by 7.44 fold (Fig. 3C). Expression for both isoforms was lowest in the brain, heart, kidney and liver (Fig. 3C). Expression was highest in the placenta (not shown), lung, and spleen. Similar to the previously reported northern blots (Numata et al., 1999; Takebe et al., 2006) ARID3B was expressed highly in the placenta with a 59.3-fold induction over the whole brain for ARID3B-FL and a 45.3-fold induction over the whole brain for ARID3B-Sh. For most tissues ARID3BFL and ARID3B-Sh appeared to be expressed coordinately (Fig. 3C). Differences between the RNA and protein expression may be related to RNA or protein stability. One example of this would be that ARID3B-FL contains miR125a/b sites in its 3′UTR that are not present in the 3′UTR for ARID3B-Sh (data not shown) (Cowden Dahl et al., 2009). We think that it is unlikely that ARID3B-FL and ARID3B-Sh have overlapping or redundant functions in different tissues since ARID3B-Sh lacks 81% of the DNA binding domain (Joseph et al., 2012). Due to the high homology of ARID3B with its paralogues ARID3A and ARID3C, we examined expression of these genes in normal human tissues. We found that all three ARID3 family members have distinct patterns of expression. For consistency, all three genes were normalized to the whole brain. ARID3B appears to have the highest expression in most tissue and be the most broadly expressed. ARID3A is expressed the highest in the lung and spleen and had very low expression in other tissues (Fig. 3D). Interestingly, we observed a 22-fold expression (over the whole brain) of ARID3A in stomach tumor, which is a 9.5-fold increase than in normal stomach (Fig. 3D). This suggests that ARID3A may be overexpressed in stomach cancer. ARID3C was expressed at very low levels in most tissues (Fig. 3E). The highest level of ARID3C expression was in the liver (29-fold increase over the whole brain). In addition to the liver, ARID3C was expressed in the whole brain, cerebellum, and fetal brain (Fig. 3E). The unique patterns of expression indicate that the ARID3C subfamily members have tissue specific functions.
3.3. ARID3B is expressed in multiple tumor types Since ARID3B expression was prominent in a number of epithelial tissues, we ascertained ARID3B expression in carcinomas. We performed IHC on TMAs containing normal, benign and malignant tissue for the indicated tumor types (Table 2). ARID3B was present in tumors of the small intestine, colon, rectum, salivary gland, thyroid, esophagus, stomach, and cervix. However, in the esophagus, stomach, and thyroid the expression of ARID3B changed during tumor progression (Fig. 4). We examined ARID3B expression in normal tissues and Grade 1 (well differentiated), Grade 2 (moderately differentiated), and Grade 3 (poorly differentiated) tumors. In the esophagus, ARID3B is very high in the suprabasal cells (Fig. 4A). Expression of ARID3B is weak in esophageal malignant tumors. We examined 68 malignant esophageal tumors and found that ARID3B was decreased in 62 of them compared to the 6 normal esophagus sections. Next we examined 42 malignant stomach tumors, of which 34 exhibited decreased ARID3B staining intensity compared to 10 normal stomach sections. Seven sections had similar staining between normal and malignant stomach and one had increased staining. In the stomach and esophagus ARID3B staining in the tumors appeared more diffuse than in the normal tissue (Fig. 4A). Finally, we were able to compare normal thyroid, normal adjacent thyroid, and malignant thyroid tumors. In the normal adjacent thyroid (follicular epithelial cells), there is strong nuclear staining for ARID3B (Fig. 4B). ARID3B was elevated in 16 out of 18 malignant thyroid tumors compared to 3 normal thyroid sections that we examined (Fig. 4B). The localization and/or expression of ARID3B are altered in cancers of the esophagus, stomach, and thyroid. 4. Discussion 4.1. ARID3B is critical for development ARID3B is a critical transcription factor that is expressed during development. Loss of ARID3B leads to embryonic lethality. It has long been appreciated that many developmental transcription factors are inappropriately regulated during cancer progression. ARID3B was originally demonstrated to bind hypophosphorylated pRB, suggesting that it may play a role in cell cycle regulation (Numata et al., 1999). However, a function of ARID3B in cell cycle has yet to be demonstrated. Even though ARID3B expression has been carefully evaluated during development, thorough analysis of ARID3B expression in the adult had not been conducted. Accumulating evidence suggests that ARID3B may be important in many disease states. Additionally, our data demonstrates that ARID3B may be required in the differentiation of adult tissues particularly squamous epithelium. 4.2. ARID3B expression is high in fetal brain yet absent in adult brain The first evidence that ARID3B has a role in human cancer was the finding that expression of ARID3B correlates with disease progression in neuroblastoma (Kobayashi et al., 2006). ARID3B has oncogenic potential as it transforms MEFs in combination with MYCN (Kobayashi
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et al., 2006). In agreement with the knockout mice and neuroblastoma data, we find that ARID3B is highly expressed in the fetal brain compared to the adult brain (Fig. 3C). This suggests that ARID3B may be important during neuronal development. Furthermore we suspect that during neural injury or cancer progression ARID3B activity may be induced in the brain. In fact ARID3B is induced by amyloid precursor protein intracellular domain in human neural cells (Muller et al., 2007). Amyloid precursor protein is cleaved into the amyloid precursor protein intracellular domain and β-amyloid in Alzheimer's patients. Therefore, elucidation of the role of ARID3B in neurons is warranted in order to better understand Alzheimer's and neuroblastoma.
2006). We conclude that knowing the normal pattern of expression of a molecule is critical to assessing its function in cancer. Furthermore, ARID3B may be an important molecular marker to monitor during progression of several tumor types. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.04.007. Conflict of interest The authors declare no conflicts of interest. Acknowledgments
4.3. Expression of ARID3B in epithelial tissues and during tumor progression This work was supported by a Walther Cancer Foundation SRC grant. We identified ARID3B as being regulated by signaling through the epidermal growth factor receptor in ovarian cancer cells (Cowden Dahl et al., 2009; Joseph et al., 2012). We further demonstrated that ARID3B is overexpressed in serous ovarian cancer (Cowden Dahl et al., 2009). To understand the importance of ARID3B in other carcinomas, we surveyed normal and tumor tissues for ARID3B expression. By QRT-PCR we found that both ARID3B isoforms are highly expressed in the kidney and their expression increases during tumorigenesis (Fig. 3C). However, further evaluation of the role of ARID3B in renal cancer needs to be conducted. ARID3B is highly expressed in the stratified squamous epithelium. ARID3B's high expression in the suprabasal layers of the stratified epithelium and limited expression in the basal cells suggest that ARID3B expression increases with differentiation (Fig. 2). Interestingly, ARID3B expression is also increased with differentiation of ES cells. Therefore, we hypothesize that in the squamous epithelium ARID3B acts as a tumor suppressor by promoting differentiation. In examining TMAs for normal and malignant tissues, we indeed found that in the esophagus and stomach ARID3B diminishes with cancer progression (Figs. 3 and 4). We postulate that whether ARID3B acts to promote cancer or as a tumor suppressor may be related to concentration of ARID3B expression and the target genes. Data from our lab suggests that high expression of ARID3B promotes activation of different gene sets than lower levels. Further studies will dissect the role of ARID3B in cancer progression. 5. Conclusions In conclusion we demonstrate for the first time that ARID3B protein is expressed in many epithelial and secretory tissues. Furthermore, ARID3B expression decreases or has altered localization with tumor progression in some tissues (esophagus and stomach). In some tissues ARID3B expression increases during tumorigenesis (thyroid, neuroblastoma and ovarian cancer) (Cowden Dahl et al., 2009; Kobayashi et al.,
References Akhavantabasi, S., Sapmaz, A., Tuna, S., Erson-Bensan, A.E., 2012. miR-125b targets ARID3B in breast cancer cells. Cell Structure and Function 37, 27–38. Casanova, J.C., Uribe, V., Badia-Careaga, C., Giovinazzo, G., Torres, M., Sanz-Ezquerro, J.J., 2011. Apical ectodermal ridge morphogenesis in limb development is controlled by Arid3b-mediated regulation of cell movements. Development 138, 1195–1205. Cowden Dahl, K.D., Dahl, R., Kruichak, J.N., Hudson, L.G., 2009. The epidermal growth factor receptor responsive miR-125a represses mesenchymal morphology in ovarian cancer cells. Neoplasia 11, 1208–1215. Joseph, S., Deneke, V.E., Cowden Dahl, K.D., 2012. ARID3B induces tumor necrosis factor alpha mediated apoptosis while a novel ARID3B splice form does not induce cell death. PloS One 7, e42159. Kim, D., Probst, L., Das, C., Tucker, P.W., 2007. REKLES is an ARID3-restricted multifunctional domain. The Journal of Biological Chemistry 282, 15768–15777. Kobayashi, K., Era, T., Takebe, A., Jakt, L.M., Nishikawa, S., 2006. ARID3B induces malignant transformation of mouse embryonic fibroblasts and is strongly associated with malignant neuroblastoma. Cancer Research 66, 8331–8336. Muller, T., Concannon, C.G., Ward, M.W., Walsh, C.M., Tirniceriu, A.L., Tribl, F., Kogel, D., Prehn, J.H., Egensperger, R., 2007. Modulation of gene expression and cytoskeletal dynamics by the amyloid precursor protein intracellular domain (AICD). Molecular Biology of the Cell 18, 201–210. Numata, S., Claudio, P.P., Dean, C., Giordano, A., Croce, C.M., 1999. Bdp, a new member of a family of DNA-binding proteins, associates with the retinoblastoma gene product. Cancer Research 59, 3741–3747. Takebe, A., Era, T., Okada, M., Martin Jakt, L., Kuroda, Y., Nishikawa, S., 2006. Microarray analysis of PDGFR alpha+ populations in ES cell differentiation culture identifies genes involved in differentiation of mesoderm and mesenchyme including ARID3b that is essential for development of embryonic mesenchymal cells. Developmental Biology 293, 25–37. Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D.N., Theunissen, T.W., Orkin, S.H., 2006. A protein interaction network for pluripotency of embryonic stem cells. Nature 444, 364–368. Webb, C.F., Bryant, J., Popowski, M., Allred, L., Kim, D., Harriss, J., Schmidt, C., Miner, C.A., Rose, K., Cheng, H.L., Griffin, C., Tucker, P.W., 2011. The ARID family transcription factor bright is required for both hematopoietic stem cell and B lineage development. Molecular and Cellular Biology 31, 1041–1053. Wilsker, D., Patsialou, A., Dallas, P.B., Moran, E., 2002. ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development. Cell Growth & Differentiation 13, 95–106.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Differential expression of ARID3B in normal adult tissue and carcinomas Serene J. Samyesudhas a, Lynn Roy a, Karen D. Cowden Dahl a,b,c,d,⁎ a
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN, USA Department of Chemistry and Biochemistry, Notre Dame University, Notre Dame, IN, USA c Eck Institute for Global Health, Notre Dame University, Notre Dame, IN, USA d Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA b
a r t i c l e
i n f o
Article history: Received 4 January 2014 Received in revised form 28 March 2014 Accepted 2 April 2014 Available online 3 April 2014 Keywords: ARID3B Transcription factor Stratified epithelium Differentiation Immunohistochemistry
a b s t r a c t ARID3B is a DNA binding protein that is overexpressed in neuroblastoma and ovarian cancer. To understand the extent that ARID3B participates in tumor development, we assessed protein expression of ARID3B in normal adult and malignant tissues. We found that ARID3B is highly expressed in differentiated layers of squamous epithelium. We also examined expression of an alternative splice form of ARID3B and found that it has similar but not identical expression patterns to the full length ARID3B isoform. ARID3B has two closely related paralogues, ARID3A and ARID3C. Each of these 3 family members exhibits different patterns of expression. Of the ARID3 family members, ARID3B is the most widely expressed and is particularly expressed in epithelium. In addition to examining normal tissue, we investigated ARID3B expression in a variety of tumor types. Most notably we found that ARID3B expression is decreased in esophagus and stomach tumors compared to normal corresponding tissues. Our results indicate that the different patterns of ARID3B in normal tissues translate into different roles for ARID3B in carcinomas. © 2014 Elsevier B.V. All rights reserved.
1. Introduction ARID3B is a member of the AT-rich interactive domain (ARID) family of DNA binding proteins. These proteins regulate diverse cellular functions through modification of chromatin structure, histone methylation, transcriptional activation and are involved in development (Wilsker et al., 2002). ARID3B is essential for development (as mice lacking Arid3b die midgestation) (Casanova et al., 2011; Takebe et al., 2006; Webb et al., 2011). Three independent knockout mice have been generated for ARID3B. These mice have defects in vasculature, cranial mesenchyme, limbs, heart and the craniofacial region (Casanova et al., 2011; Takebe et al., 2006; Webb et al., 2011). During development ARID3B is expressed in many regions of developing embryos between embryonic days 7–10.5 (Takebe et al., 2006). Among the places ARID3B is expressed are the primitive streak, paraxial mesoderm, neural crest, brachial arches and the outflow tract of the heart (Takebe et al., 2006). ARID3B is present in mouse embryonic stem (ES) cells and increases
Abbreviations: ARID3B, AT rich interactive domain 3B; ARID3B-FL, full length isoform of ARID3B; ARID3B-Sh, short alterative splice form of ARID3B; ES, embryonic stem (cells); MEFs, mouse embryo fibroblasts; TNFα, tumor necrosis factor alpha; QRT-PCR, quantitative reverse transcribed polymerase chain reaction; cDNA, complimentary DNA; TMA(s), tissue microarray; IHC, immunohistochemistry. ⁎ Corresponding author at: 1234 Notre Dame Ave, Harper Hall A225, South Bend, IN 46530, USA. E-mail addresses: [email protected] (S.J. Samyesudhas), [email protected] (L. Roy), [email protected] (K.D. Cowden Dahl).
http://dx.doi.org/10.1016/j.gene.2014.04.007 0378-1119/© 2014 Elsevier B.V. All rights reserved.
with ES cell differentiation (Wang et al., 2006). Despite knowing the patterns of ARID3B expression in the developing mouse embryo, ARID3B expression in adult mouse and human tissues has not been examined in detail. Tissue northern blots demonstrate that ARID3B is expressed in the human spleen, thymus, prostate, testis, small intestine, colon, leukocytes, heart, brain, placenta, lungs, skeletal muscle, kidney, and pancreas (Numata et al., 1999). Of these the highest mRNA expression was found in the testis, placenta, leukocytes and spleen (Numata et al., 1999). A second paper demonstrated through northern blot that in the adult mouse, ARID3B is only expressed in the testis, prostate, thyroid, and thymus (Takebe et al., 2006). Careful examination of ARID3B expression at the protein level has not been reported. In addition to its essential role in development, ARID3B is implicated in cellular transformation and tumor progression. ARID3B is overexpressed in both neuroblastoma and ovarian cancer (Cowden Dahl et al., 2009; Kobayashi et al., 2006). ARID3B immortalizes mouse embryo fibroblasts (MEFs) and cooperates with MYCN to transform fibroblasts and promotes neuroblastoma tumor growth (Kobayashi et al., 2006). We demonstrated that ARID3B is overexpressed in serous ovarian cancer (Cowden Dahl et al., 2009). ARID3B has several functions that may cooperate in tumorigenesis. ARID3B enables its paralogue ARID3A to remain nuclear in B-cells (Kim et al., 2007); ARID3B promotes migration of MEFs and breast cancer cells (Akhavantabasi et al., 2012; Casanova et al., 2011), and induces TNFα signaling (Joseph et al., 2012). The goal of this study is to determine the normal pattern of expression for ARID3B protein in adult tissue. Additionally, we wish to identify which tumor types express ARID3B. In doing this, we will
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gain insight into the role of ARID3B in human cancer. Collectively our data demonstrates that ARID3B expression is differentially regulated in different normal and malignant tissues.
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the delta delta CT method was used to calculate fold differences in gene expression. 2.2. Normal and tumor tissue microarray
2. Materials and methods 2.1. Quantitative reverse transcribed polymerase chain reaction (QRT-PCR) for ARID3B isoforms on RNA from adult human organs QRT-PCR was performed on total RNA purchased from Clontech (Mountain View, CA) for a panel of human organs. 1 μg of RNA was reverse transcribed into cDNA using the High Capacity cDNA Archive kit from Applied Biosystems (Carlsbad, CA). QRT-PCR was performed using iTaq Universal Probes Supermix (Bio-Rad Laboratories, Inc., Hercules, CA) for ARID3B-Sh and ARID3B-FL. The following primers were used: ARID3B-Sh-HEX: Primer 1: 5′-TCCCACCACATGGACAACAAGCTA3′, Primer 2: 5′-TCATCCAGATTCCAGTTCGGCTGT-3′, HEX probe: 5′AAGATGCTTCCAAGGCCTCACCTTCT-3′; ARID3B FAM (ARID3B-FL), ARID3B (Applied Biosystems, HS01084919_g1); and ARID3A: Primer 1: 5′-CTT CTC CGT CAC CAG CAC-3′, Primer 2: 5′-GAC TTG TTC AGC TTC ATG CAG-3′, ARID3C: Primer 1: 5′-CAC TTT GCG GTT GAT GAC TTC-3′, Primer 2: 5′-TCT GGA TGA CCT GTT TAG CTT C-3′, and HPRTFAM (Applied Biosystems). Expression was normalized to HPRT and
The following tissue microarray (TMA) slides were obtained from US Biomax Inc. (Rockville, MD): (MTU951, ES1501, CR1501, BC01114, TH721, KD1503, BN1002a and LN801) and Imgenex (San Diego, CA) (IMH-372 and IMH-335). 2.3. Immunohistochemistry Immunohistochemistry (IHC) was performed using an ARID3B antibody from Abcam (ab50927) (Cambridge, MA) in Fig. 1. IHC in Figs. 2–4 was performed with the Bethyl Laboratories (Montgomery, TX) ARID3B IHC Antibody (IHC-00717) and ARID3B antibody from Abcam (ab50927) (Cambridge, MA). Additionally the C-terminal anti-ARID3B antibody from Bethyl Laboratories (A302 565A) that detects the last 50 amino acids that are only found in ARID3BFL was also used in Fig. 3. Deparaffinization of tissue microarray slides was performed for 1 h at 60 °C followed by three 5 minute incubations in xylene. Slides were rehydrated in an ethanol series. Antigen retrieval was done in citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) at 95 °C for
Fig. 1. ARID3B immunohistochemistry (IHC) in corresponding adult mouse and human tissues. IHC was performed on tissue microarrays (TMAs) for normal human and mouse tissues. Sections from cerebellum, salivary gland, stomach, and testis are shown. Brown staining denotes ARID3B expression. No primary control IHC on human sections is depicted in right panels. Original magnification is 20×.
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20 min. The slides were washed twice in phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, and 11.9 mM phosphate buffer, pH 7.4) and then incubated for 20 min in a solution of 3% H2O2 in methanol. After rinsing three times for 5 min in PBS, immunohistochemistry was performed using the ABC kit (Vector Laboratories, Burlingame, CA) and developed with ImmPACT DAB peroxidase substrate or DAB kit (Vector Laboratories, Burlingame, CA). Slides were incubated with primary antibody overnight at 4 °C using either the anti-ARID3B IHC Antibody (1:250) (Bethyl Laboratories) or anti-ARID3B from Abcam (ab50927 at 1:100). The slides were counter-stained with Hematoxylin QS (Vector Laboratories, Burlingame, CA). Slides were dehydrated and mounted with VectaMount permanent mounting medium (Vector Laboratories, Burlingame, CA). Slides were scanned using Aperio ScanScope system (Aperio ePathology, Vista, CA) and analyzed using the Imagescope software at 20× magnification.
2.4. Tissue blot A normal human organ tissue blot (IMB-103) containing 20 μg of lysate from 11 human organs was obtained from Imgenex (San Diego, CA). The tissue blot was rehydrated, blocked, and blotted with primary antibody according to manufacturer's instructions (Imgenex (San Diego, CA)). The anti-ARID3B antibody from (A302 564A) Bethyl Laboratories (Montgomery, TX) was used at a 1:1000 dilution and the ARID3B-Sh antibody (Pacific Immunology Inc. (Ramona, CA)) was used at a dilution of 1:1000. The blot was stained with Ponceau S (0.1% Ponceau S, 1% Acetic Acid) as a loading control. The BIORAD Chemidoc XRS+ System using Imager Lab Software (Bio-Rad Laboratories, Inc., Hercules, CA) was used to image and analyze the blot.
3. Results 3.1. ARID3B protein is preferentially expressed in epithelial and secretory tissue To understand the extent to which ARID3B is involved in cancer, we began by examining the normal pattern of ARID3B expression in adult organs. We performed immunohistochemistry (IHC) for ARID3B on tissue microarrays (TMAs) containing either human or mouse organs. As a negative control we performed IHC on TMAs without primary antibodies. We observed ARID3B protein expression in several secretory cell types (stomach, salivary gland, and Leydig cells) (Fig. 1). In Supplemental Fig. 1 we provide staining controls for the various tissues examined. ARID3B is also expressed in epithelial cell types including urinary bladder and kidney (Table 1). We found that ARID3B was expressed in many epithelial and secretory tissues (Table 1). The expression pattern of ARID3B was similar between adult mouse and human tissues. Interestingly, we did not observe significant ARID3B protein expression in muscle (skeletal muscle and heart). We did detect ARID3B expression in spleen and lymph nodes, but expression in thymus was variable (Table 1). Nervous system tissues also did not have ARID3B protein expression in mice or humans (pons, cerebrum, and cerebellum) (data not shown and Fig. 1). Strikingly blood vessels in all tissues examined expressed high levels of ARID3B (data not shown). One of the most striking findings is that ARID3B is strongly expressed in stratified squamous epithelium (Fig. 2). In the three examples of stratified epithelium that we examined (skin, esophagus and cervix), we observed strong staining for ARID3B in the suprabasal layers and lower expression in the basal cells (Fig. 2). The lower staining of ARID3B in the basal cells was most noticeable in the esophagus and more variable in the skin
Fig. 2. ARID3B expression in stratified squamous epithelium. IHC was performed on TMAs for normal human skin, esophagus, and cervix. Brown staining denotes ARID3B. No primary control IHC for each tissue depicted on the right panels. Original magnification is 20×.
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Fig. 3. Expression of ARID3B isoforms in normal human organs. A. Western blot was performed on Imgenex Insta-Blot Human Tissues IMB-103 for ARID3B. This demonstrates the expression of ARID3B FL (61 kDa) (top panel) and ARID3B-Sh (28 kDa) (bottom panel). Ponceau S staining was used as a loading control. B. IHC using 2 different ARID3B antibodies. The “Total” antibody detects both ARID3-FL and ARID3B-Sh. The ARID3B-FL detects only the longer isoform. Original magnification is 20×. C. QRT-PCR was performed for the ARID3B isoforms (ARID3B-FL and ARID3B-Sh) on RNA samples from different human organs. Gene expression was normalized to HPRT and shown as fold induction over whole brain (which has the least expression). QRTPCR was performed for ARID3A (D) and ARID3C (E) gene expression was normalized to HPRT and shown as fold induction over whole brain.
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Fig. 4. Expression of ARID3B in normal and tumor tissues. A. ARID3B expression in normal tissue and malignant tumors of the esophagus and stomach. B. ARID3B expression in benign, normal adjacent, and malignant thyroid tissues. Original magnification is 20×.
and cervix (Supplemental Fig. 2). To assess the level of ARID3B expression in squamous epithelium we repeated the IHC on esophagus and cervix without hematoxylin. We again found that ARID3B expression is higher in the suprabasal cells and is lower in basal cells, suggesting that ARID3B expression is regulated during differentiation (Supplemental Fig. 2). In conclusion, ARID3B is expressed in the epithelium (particularly in squamous epithelium), some secretory cells, spleen, and blood vessels in the adult organism. This suggests that ARID3B has important functions in the epithelium, hematopoietic cells, and endothelium. 3.2. Expression of an alternate splice form of ARID3B (ARID3B-Sh) and ARID3 subfamily members We previously reported that ARID3B contains an alternatively spliced isoform (Joseph et al., 2012). The smaller isoform (ARID3B-Sh) lacks exons 5–9 including 81% of the DNA binding domain and the REKLES
domains that are involved in ARID3 homo- and hetero-oligomerization (Kim et al., 2007). We examined tissue distribution of the product of the smaller isoform of ARID3B (ARID3B-Sh) by western blot. ARID3B-Sh is a 28 kDa protein and easily distinguished from the 61 kDa ARID3B-FL (the full length isoform) on a western blot using an antibody to amino acids 100–150 present in both isoforms. ARID3B-Sh is expressed in the brain, kidney, liver, lung, stomach, and spleen (Fig. 3). We attempted to perform IHC for ARID3B on tissue microarrays using an ARID3B-Sh specific antibody that we generated. However, this antibody produced excessive background staining. We did perform IHC using two different antibodies for ARID3B on the kidney (since this is a tissue where both ARID3B isoforms are expressed). The first antibody detects residues 100–150 of the ARID3B protein (Bethyl Laboratories anti-ARID3B IHC Antibody (IHC-00717)). These amino acids are found in both isoforms; therefore, the “Total” ARID3B expression is detected with this antibody. We used a second antibody (Bethyl laboratories anti-ARID3B Antibody
S.J. Samyesudhas et al. / Gene 543 (2014) 174–180 Table 1 ARID3B expression in adult human and mouse tissues.
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Table 2 ARID3B expression in normal and tumor tissues.
Organ
Human
Mouse
Organ
Normal
Benign
Malignant
Skin Esophagus Cervix Salivary gland Stomach Small intestine Kidney, cortex Kidney, medulla Testis (Leydig cells) Ovary Uterus (endometrium) Urinary bladder Liver Spleen Lymph node Thymus Lung Cerebellum Skeletal muscle
+ + + + + + + + + + + + + + + Variable Variable − −
+ NA NA + + + + + + + + + + + NA − + − −
Brain Small intestine Colon Rectum Salivary gland Thyroid Esophagus Stomach Cervix
− + + + + + + + +
Variable + + + + + NA NA NA
+ + + + + + + + +
NA = not available.
(A302 565A)), which detects amino acids 510–560 only found in ARID3B-FL. We found that both antibodies detect extensive expression of ARID3B in the kidney. As additional way of examining ARID3B isoform expression in tissue, we performed QRT-PCR for each isoform using isoform specific primers. RNA from a panel of normal tissues was obtained, converted to cDNA and QRT-PCR was conducted. As expected (from the IHC) we detected very little expression of both ARID3B isoforms in the whole adult brain. Therefore we normalized expression of the other tissues to the whole brain. Interestingly, ARID3B expression is relatively high in the fetal brain compared to the whole brain. In the fetal brain compared to the whole brain ARID3B-FL mRNA is increased by 6.78 fold and ARID3B-Sh is increased by 7.44 fold (Fig. 3C). Expression for both isoforms was lowest in the brain, heart, kidney and liver (Fig. 3C). Expression was highest in the placenta (not shown), lung, and spleen. Similar to the previously reported northern blots (Numata et al., 1999; Takebe et al., 2006) ARID3B was expressed highly in the placenta with a 59.3-fold induction over the whole brain for ARID3B-FL and a 45.3-fold induction over the whole brain for ARID3B-Sh. For most tissues ARID3BFL and ARID3B-Sh appeared to be expressed coordinately (Fig. 3C). Differences between the RNA and protein expression may be related to RNA or protein stability. One example of this would be that ARID3B-FL contains miR125a/b sites in its 3′UTR that are not present in the 3′UTR for ARID3B-Sh (data not shown) (Cowden Dahl et al., 2009). We think that it is unlikely that ARID3B-FL and ARID3B-Sh have overlapping or redundant functions in different tissues since ARID3B-Sh lacks 81% of the DNA binding domain (Joseph et al., 2012). Due to the high homology of ARID3B with its paralogues ARID3A and ARID3C, we examined expression of these genes in normal human tissues. We found that all three ARID3 family members have distinct patterns of expression. For consistency, all three genes were normalized to the whole brain. ARID3B appears to have the highest expression in most tissue and be the most broadly expressed. ARID3A is expressed the highest in the lung and spleen and had very low expression in other tissues (Fig. 3D). Interestingly, we observed a 22-fold expression (over the whole brain) of ARID3A in stomach tumor, which is a 9.5-fold increase than in normal stomach (Fig. 3D). This suggests that ARID3A may be overexpressed in stomach cancer. ARID3C was expressed at very low levels in most tissues (Fig. 3E). The highest level of ARID3C expression was in the liver (29-fold increase over the whole brain). In addition to the liver, ARID3C was expressed in the whole brain, cerebellum, and fetal brain (Fig. 3E). The unique patterns of expression indicate that the ARID3C subfamily members have tissue specific functions.
3.3. ARID3B is expressed in multiple tumor types Since ARID3B expression was prominent in a number of epithelial tissues, we ascertained ARID3B expression in carcinomas. We performed IHC on TMAs containing normal, benign and malignant tissue for the indicated tumor types (Table 2). ARID3B was present in tumors of the small intestine, colon, rectum, salivary gland, thyroid, esophagus, stomach, and cervix. However, in the esophagus, stomach, and thyroid the expression of ARID3B changed during tumor progression (Fig. 4). We examined ARID3B expression in normal tissues and Grade 1 (well differentiated), Grade 2 (moderately differentiated), and Grade 3 (poorly differentiated) tumors. In the esophagus, ARID3B is very high in the suprabasal cells (Fig. 4A). Expression of ARID3B is weak in esophageal malignant tumors. We examined 68 malignant esophageal tumors and found that ARID3B was decreased in 62 of them compared to the 6 normal esophagus sections. Next we examined 42 malignant stomach tumors, of which 34 exhibited decreased ARID3B staining intensity compared to 10 normal stomach sections. Seven sections had similar staining between normal and malignant stomach and one had increased staining. In the stomach and esophagus ARID3B staining in the tumors appeared more diffuse than in the normal tissue (Fig. 4A). Finally, we were able to compare normal thyroid, normal adjacent thyroid, and malignant thyroid tumors. In the normal adjacent thyroid (follicular epithelial cells), there is strong nuclear staining for ARID3B (Fig. 4B). ARID3B was elevated in 16 out of 18 malignant thyroid tumors compared to 3 normal thyroid sections that we examined (Fig. 4B). The localization and/or expression of ARID3B are altered in cancers of the esophagus, stomach, and thyroid. 4. Discussion 4.1. ARID3B is critical for development ARID3B is a critical transcription factor that is expressed during development. Loss of ARID3B leads to embryonic lethality. It has long been appreciated that many developmental transcription factors are inappropriately regulated during cancer progression. ARID3B was originally demonstrated to bind hypophosphorylated pRB, suggesting that it may play a role in cell cycle regulation (Numata et al., 1999). However, a function of ARID3B in cell cycle has yet to be demonstrated. Even though ARID3B expression has been carefully evaluated during development, thorough analysis of ARID3B expression in the adult had not been conducted. Accumulating evidence suggests that ARID3B may be important in many disease states. Additionally, our data demonstrates that ARID3B may be required in the differentiation of adult tissues particularly squamous epithelium. 4.2. ARID3B expression is high in fetal brain yet absent in adult brain The first evidence that ARID3B has a role in human cancer was the finding that expression of ARID3B correlates with disease progression in neuroblastoma (Kobayashi et al., 2006). ARID3B has oncogenic potential as it transforms MEFs in combination with MYCN (Kobayashi
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et al., 2006). In agreement with the knockout mice and neuroblastoma data, we find that ARID3B is highly expressed in the fetal brain compared to the adult brain (Fig. 3C). This suggests that ARID3B may be important during neuronal development. Furthermore we suspect that during neural injury or cancer progression ARID3B activity may be induced in the brain. In fact ARID3B is induced by amyloid precursor protein intracellular domain in human neural cells (Muller et al., 2007). Amyloid precursor protein is cleaved into the amyloid precursor protein intracellular domain and β-amyloid in Alzheimer's patients. Therefore, elucidation of the role of ARID3B in neurons is warranted in order to better understand Alzheimer's and neuroblastoma.
2006). We conclude that knowing the normal pattern of expression of a molecule is critical to assessing its function in cancer. Furthermore, ARID3B may be an important molecular marker to monitor during progression of several tumor types. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.04.007. Conflict of interest The authors declare no conflicts of interest. Acknowledgments
4.3. Expression of ARID3B in epithelial tissues and during tumor progression This work was supported by a Walther Cancer Foundation SRC grant. We identified ARID3B as being regulated by signaling through the epidermal growth factor receptor in ovarian cancer cells (Cowden Dahl et al., 2009; Joseph et al., 2012). We further demonstrated that ARID3B is overexpressed in serous ovarian cancer (Cowden Dahl et al., 2009). To understand the importance of ARID3B in other carcinomas, we surveyed normal and tumor tissues for ARID3B expression. By QRT-PCR we found that both ARID3B isoforms are highly expressed in the kidney and their expression increases during tumorigenesis (Fig. 3C). However, further evaluation of the role of ARID3B in renal cancer needs to be conducted. ARID3B is highly expressed in the stratified squamous epithelium. ARID3B's high expression in the suprabasal layers of the stratified epithelium and limited expression in the basal cells suggest that ARID3B expression increases with differentiation (Fig. 2). Interestingly, ARID3B expression is also increased with differentiation of ES cells. Therefore, we hypothesize that in the squamous epithelium ARID3B acts as a tumor suppressor by promoting differentiation. In examining TMAs for normal and malignant tissues, we indeed found that in the esophagus and stomach ARID3B diminishes with cancer progression (Figs. 3 and 4). We postulate that whether ARID3B acts to promote cancer or as a tumor suppressor may be related to concentration of ARID3B expression and the target genes. Data from our lab suggests that high expression of ARID3B promotes activation of different gene sets than lower levels. Further studies will dissect the role of ARID3B in cancer progression. 5. Conclusions In conclusion we demonstrate for the first time that ARID3B protein is expressed in many epithelial and secretory tissues. Furthermore, ARID3B expression decreases or has altered localization with tumor progression in some tissues (esophagus and stomach). In some tissues ARID3B expression increases during tumorigenesis (thyroid, neuroblastoma and ovarian cancer) (Cowden Dahl et al., 2009; Kobayashi et al.,
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