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SLC12A7 Alters Adrenocortical Carcinoma Cell Adhesion Properties to Promote an Aggressive Invasive Behavior Taylor C

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SLC12A7 Alters Adrenocortical Carcinoma Cell Adhesion Properties to Promote an Aggressive Invasive Behavior Taylor C Brown et al. Cell Communication and Signaling (2018) 16:27 https://doi.org/10.1186/s12964-018-0243-0 RESEARCH Open Access SLC12A7 alters adrenocortical carcinoma cell adhesion properties to promote an aggressive invasive behavior Taylor C. Brown1,2, Timothy D. Murtha1,2, Jill C. Rubinstein1,2, Reju Korah1,2 and Tobias Carling1,2* Abstract Background: Altered expression of Solute Carrier Family 12 Member 7 (SLC12A7) is implicated to promote malignant behavior in multiple cancer types through an incompletely understood mechanism. Recent studies have shown recurrent gene amplifications and overexpression of SLC12A7 in adrenocortical carcinoma (ACC). The potential mechanistic effect(s) of SLC12A7 amplifications in portending an aggressive behavior in ACC has not been previously studied and is investigated here using two established ACC cell lines, SW-13 and NCI-H295R. Methods: SW-13 cells, which express negligible amounts of SLC12A7, were enforced to express SLC12A7 constitutively, while RNAi gene silencing was performed in NCI-H295R cells, which have robust endogenous expression of SLC12A7. In vitro studies tested the outcomes of experimental alterations in SLC12A7 expression on malignant characteristics, including cell viability, growth, colony formation potential, motility, invasive capacity, adhesion and detachment kinetics, and cell membrane organization. Further, potential alterations in transcription regulation downstream to induced SLC12A7 overexpression was explored using targeted transcription factor expression arrays. Results: Enforced SLC12A7 overexpression in SW-13 cells robustly promoted motility and invasive characteristics (p < 0.05) without significantly altering cell viability, growth, or colony formation potential. SLC12A7 overexpression also significantly increased rates of cellular attachment and detachment turnover (p < 0.05), potentially propelled by increased filopodia formation and/or Ezrin interaction. In contrast, RNAi gene silencing of SLC12A7 stymied cell attachment strength as well as migration and invasion capacity in NCI-H295R cells. Transcription factor expression analysis identified multiple signally pathways potentially affected by SLC12A7 overexpression, including osmotic stress, bone morphogenetic protein, and Hippo signaling pathways. Conclusions: Amplification of SLC12A7 observed in ACCs is shown here, in vitro, to exacerbate the malignant behavior of ACC cells by promoting invasive capacities, possibly mediated by alterations in multiple signaling pathways, including the osmotic stress pathway. Keywords: Adrenocortical carcinoma, SLA12A7, Ezrin, Filopodia Plain english summary However, no studies have been performed to test whether Adrenal cancer is a very deadly cancer and current treat- SLC12A7 is involved in promoting adrenal cancers, and if ment options are limited, especially for patients with so, the mechanisms behind its potential effect. Using two cancers that have spread throughout the body. Previous well-studied adrenal cancer cell lines, one with very low research discovered increased expression of a gene called expression of SLC12A7 (SW-13 cells), and one with very Solute Carrier Family 12 Member 7 (SLC12A7). high expression of SLC12A7 (NCI-H295R cells), we addressed these questions in this study. * Correspondence: [email protected] Results of experiments in this study clearly show that 1Department of Surgery, Yale University School of Medicine, 333 Cedar when SLC12A7 expression was increased, the cells be- Street, TMP FMB130A, P.O. Box 208062, New Haven, CT 06520, USA 2Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, came more aggressive. Similarly, suppressing SLC12A7 333 Cedar Street, TMP FMB130A, P.O. Box 208062, New Haven, CT 06520, function resulted in the opposite response. Moreover, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Brown et al. Cell Communication and Signaling (2018) 16:27 Page 2 of 13 experiments showed that SLC12A7 promotes the malig- SLC12A7 (KCl cotransporter 4; KCC4), a member of nant behavior of adrenal cancer cells by changing their the SLC12 gene family, is a 1083 amino acid long, cell membrane attachment kinetics and consequently, trans-membrane protein that regulates cell volume via accelerates their invasive behavior. Additional experi- potassium and chloride transport [14, 15]. However, it ments also suggest a role for osmotic stress as a driving has also been demonstrated that amplified expression of force for these changes. SLC12A7 promotes the malignant behavior of several In conclusion, this study demonstrates that SLC12A7 different cancer types. SLC12A7 is overexpressed in plays an important role in adrenal cancer by promoting gynecological and breast cancers and overexpression of tumor invasion, and as such, could represent a marker SLC12A7 and other SLC12 gene family members has of tumor aggressiveness to serve as a potential thera- been shown to be associated with local tumor invasion, peutic target. lymph node metastases, and poor clinical outcomes. Furthermore, SCL12A7 has been shown to promote in Background vitro tumor cell invasion [16–19], potentially mediated Adrenocortical carcinoma (ACC) is a rare and an aggres- through interactions with Ezrin (EZR), a membrane sive cancer. Its estimated incidence ranges from 0.7 to 2.0 cytoskeleton/extra-cellular matrix linker [19]. Based on cases per million people per year [1]. Tumor occurrence the previous findings by our group and others, we has a bimodal age distribution, with the first peak ob- sought to determine the phenotypic effects of SLC12A7 served in childhood and a second, higher peak observed overexpression upon ACC malignant behavior. during the 5th and 6th decades of life [2, 3]. Patients usu- ally present with symptoms of abdominal pain and/or ex- Methods cessive hormone production, while some are incidentally Cell culture, vector transfection, RNAi gene silencing, diagnosed by cross-sectional imaging [3]. For patients with gene expression analysis, and Western blot detection European Network for the Study of Adrenal Tumors ACC cell culture and vector transfection were performed (ENSAT) stage I-III tumors surgery is the primary treat- as previously described [20]. Briefly, the human ACC cell ment modality, with en bloc, R0 resection providing the lines SW-13 and NCI-H295R (authenticated and supplied only chance for cure. In addition to surgery, external beam by American Type Cell Collection) were maintained radiation, Mitotane, chemotherapy, and targeted therapies under sterile conditions in a humidified incubator at are used as either primary, or as adjuvant therapy [4]. 37.0 C with 5% CO2. SW-13 cells were grown in Dulbec- Three molecular pathways are frequently found co’s Modified Eagle Medium (DMEM) supplemented with disrupted in ACC and play a significant role in tumor 10% certified fetal bovine serum (FBS) and 10,000 U/mL origin and/or progression. Activation of canonical Wnt/ penicillin/streptomycin; designated as complete medium beta-catenin signaling occurs frequently in ACC via mu- (CM). NCI-H295R cells were grown in DMEM/F12 sup- tations of the proto-oncogene CTNNB1 (β-Catenin), plemented with 5% NuSerum, 10,000 U/mL penicillin/ which in turn stimulates target gene transcription and streptomycin, 5 μg/ml of insulin, 5 μγ/ml of transferrin, promotes tumor formation [5]. Somatic mutations in the and 5 ng/ml of selenium (all reagents from Applied tumor suppressor gene TP53 is found in approximately Biosystems); designated complete medium as well (CM). 20–35% of cases and are associated with more aggressive In general, cell strains underwent no more than 10 pas- tumors. Furthermore, Li Fraumeni Syndrome, which is sages before experiments were performed. caused by germline TP53 mutations, is often associated Myc-DDK tagged pCMV6-Entry and pCMV6-Entry/ with childhood ACCs [1, 3]. Overexpression of insulin SLC12A7-ORF plasmid expression vectors (Origene) growth factor II (IGF-II) via alteration of gene copy num- were transfected into SW-13 cells using Lipofectamine ber and/or gene imprinting is one of the most frequently 3000 (ThermoFisher) according to the manufacturer’s observed molecular events associated with ACC [3, 5]. recommendations in 6-well plates with cells grown to Gene copy number variations (CNVs) occur frequently in 70–80% confluence. Stable clones of pCMV6-Entry and ACC and promote the malignant development of these pCMV6-Entry/SLC12A7 vectors were selected in CM tumors [6–10]. Two studies utilizing whole-exome sequen- containing 800 μg/ml G-418 (Life Technologies). Mul- cing (WES) methods identified the 5p13.33 chromosome tiple clones were then pooled into populations to avoid location to be the most recurrently amplified region in the clonal variability. Selected SW-13 cell populations were ACC genome [11, 12]. Solute Carrier Family 12 Member 7 designated SW-13/V (pCMV6 vector-transfected) and (SLC12A7), a recently characterized proto-oncogene, is SW-13/S (pCMV6/SLC12A7-transfected)
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