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The Requirement of SEPT2 and SEPT7 for Migration and Invasion in Human Breast Cancer Via MEK/ERK Activation

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The Requirement of SEPT2 and SEPT7 for Migration and Invasion in Human Breast Cancer Via MEK/ERK Activation www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 38 Research Paper The requirement of SEPT2 and SEPT7 for migration and invasion in human breast cancer via MEK/ERK activation Nianzhu Zhang1,*, Lu Liu2,*, Ning Fan2, Qian Zhang1, Weijie Wang1, Mingnan Zheng3, Lingfei Ma4, Yan Li2, Lei Shi1,5 1Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, Dalian, 116044, Liaoning, P.R.China 2College of Basic Medical Sciences, Dalian Medical University, Dalian, 116044 Liaoning, P.R.China 3Department of Gynecology and Obstetrics, Dalian Municipal Central Hospital Affiliated to Dalian Medical University, Dalian, 116033, Liaoning, P.R.China 4The First Affiliated Hospital of Dalian Medical University, Dalian, 116011, Liaoning, P.R.China 5State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, P.R.China *These authors contributed equally to this work Correspondence to: Lei Shi, email: [email protected] Yan Li, email: [email protected] Lingfei Ma, email: [email protected] Keywords: septin, forchlorfenuron, breast cancer, invasion, MAPK Received: April 24, 2016 Accepted: July 28, 2016 Published: August 19, 2016 ABSTRACT Septins are a novel class of GTP-binding cytoskeletal proteins evolutionarily conserved from yeast to mammals and have now been found to play a contributing role in a broad range of tumor types. However, their functional importance in breast cancer remains largely unclear. Here, we demonstrated that pharmaceutical inhibition of global septin dynamics would greatly suppress proliferation, migration and invasiveness in breast cancer cell lines. We then examined the expression and subcellular distribution of the selected septins SEPT2 and SEPT7 in breast cancer cells, revealing a rather variable localization of the two proteins with cell cycle progression. To determine the role of both septins in mediating malignant behavior of cancer cells, we used RNA silencing to specifically deplete endogenous SEPT2 or SEPT7 in highly invasive breast cancer cell line MDA-MB-231. Our findings showed that SEPT2/7 depletion had virtually identical inhibitory effects on cellular proliferation, apoptosis, migration and invasion. Moreover, the opposite performance in migration and invasion was observed after enforced expression of SEPT2/7 in the same cell line. We further demonstrated MEK/ERK activation, but not other MAPKs and AKT, was positively correlated with the protein levels of SEPT2 and SEPT7. Additionally, in SEPT2/7-overexpressing cells, the MEK specific inhibitor U0126 was able to correct the high active status of MEK/ERK while normalizing the increased invasive behaviors of these cells. Taken together, these results strongly suggest that SEPT2 and SEPT7 are involved in breast carcinogenesis and may serve as valuable therapeutic targets for breast cancer. INTRODUCTION formation [2]. Basic elements for septin core structure have been described by forming non-polar SEPT2/6/7 hexamer Septins constitute a family of highly conserved [3], although the components might be variable depending GTP-binding proteins from yeast to human, and by far 14 on cell type and level of expression. An emerging body of members have been identified in human genome [1]. Septins studies reveals that septins maintain a conserved role in are categorized as the fourth cytoskeletal proteins and can cytokinesis and have also been implicated in a variety of assemble into higher-order cytoskeletal structures including other cellular functions such as chromosome segregation, filaments, bundles and rings by heteromeric complex DNA repair, cell polarization, migration and apoptosis www.impactjournals.com/oncotarget 61587 Oncotarget [1, 4–7]. In addition, septins are reported to be tightly behavior, including cell proliferation, migration and associated with human physiological and pathological invasion, and uncovering its underlying mechanism. events, including bacterial infection, Alzheimer disease, Parkinson disease, and male infertility [8]. RESULTS Currently, numerous studies have indicated that alterations of septin proteins, by mutation or expression FCF suppress breast cancer cell proliferation changes, strongly link with tumorigenesis [1, 9–11]. and invasion Among the septin family, perhaps the best-studied member closely associated with human cancers is In order to generally evaluate the importance of SEPT9, which was identified as an oncogene in ovarian, septin proteins in affacting breast cancer (BC) cells head and neck and prostate cancer cells [12]. Moreover, proliferation and invasion, Forchlorfenuron (FCF), promoter methylation of SEPT9 was regarded as a a specific conventional inhibitor for septin proteins reliable and specific biomarker for the early detection of [5, 12], was firstly applied to BC cells, MDA-MB-231 colorectal cancer [13]. As another exemplar of septin with and MCF7. Within 3 days treatment, FCF displayed oncogenic activity, downregulation of SEPT2 expression dose dependent inhibition on MDA-MB-231 and MCF7 would contribute to PPARγ activation and thus suppress proliferation determined by MTT assay (Figure 1A). Of hepatoma cell growth [14]. Additionally, some of the most note, 100 μM FCF was able to induce cytotoxicity as convincing evidence from the study of gliomas supports seen by the continually depressed cell viability and herein a tumor suppressor role for SEPT7 through negative we excluded the dose in the following tests to avoid regulation of the crucial cell-cycle regulators such as false positive results. Similarly, the ability of single BC cyclin D1 and CDK4 [15]. These findings suggested that colonies formation was also greatly impaired by FCF the septins might be critical proteins in the development treatment (50 μM) whenever the treatment initiated on of certain cancers and merit deeply exploration to further day1 or day7 during 14 days cultures (Figure 1B). The disclose the mechanisms whereby they function. link of septin inhibition with cellular apoptosis was Breast cancer is the most commonly diagnosed detected with Annexin V/PI staining based FACS analysis, cancer and the leading cause of cancer-related death demonstrating FCF would does dependently enlarge early among women worldwide [16]. Despite improvement in and total apoptotic cell population in MDA-MB-231 cell overall survival of breast cancer patients, drug resistance (Figure 1C). In the other hand, we developed a three remains a huge challenge for clinical therapy and crucial dimensional culture assay in collagen matrix to evaluate for disease recurrence and progression. Therefore, the BC cell invasive ability. With the increased doses, FCF existing situation necessitates an extensive search for was gradually suppressing MDA-MB-231 cell invading novel bio-molecules that may be acted as safe and effective into collagen matrix within 24 hours (Figure 1D). Due to drug targets. Cytoskeleton proteins are being developed high levels of Septin proteins expression was discovered as a new class of therapeutic targets for breast cancer. in Paclitaxel resistant MDA-MB-231 cells [18], we tested For instance, inhibition of microtubule by paclitaxel and the effects of septin inhibition with FCF on Paclitaxel its semi-synthetic derivatives docetaxel and cabazitaxel, reduced cell growth, showing FCF would significantly which have been widely applied to chemotherapy of but mildly enhance Paclitaxel efficiency in non-paclitaxel breast cancer, had a decent effect on preventing cancer resistant MDA-MB-231 (Supplementary Figure S1A), the cell mitosis and cytokinesis [17]. High expression levels similar observation was also determined in SEPT2 and of four septins (SEPT2, 8, 9 and 11) were verified in SEPT7 silenced cells (Supplementary Figure S1B). paclitaxel (Taxol) resistant MDA-MB-231 cells [18], which suggests the possibility that septins can modulate SEPT2 and SEPT7 expression and localization in microtubule-based breast cancer chemotherapy by their BC cells guidance towards microtubule and actin cytoskeleton dynamics, which have been quite well described in Given FCF medicated septin dynamics inhibition epithelial cells [19] and neuron cells [20]. greatly impaired BC biological behaviors; we thought Given the importance of septins as non-conventional core septin elements might be essential to explain cytoskeletons in the regulation of cytokinesis and mitosis how importance of septin proteins contribute to BC in various human cancers, we hypothesized septins are development. Herein, we decided to focus on SEPT2 and likely to be involved in the pathogenesis of breast cancer SEPT7 as they are basic elements required for structure by modulating malignant phenotypes of cancer cells. organization and conceptually may contribute to septin Moreover, the basic information concerning the role of biological functions. We detected their protein expression septin family and representative members in affecting in different BC cells by western blotting (Figure 2A), breast cancer cell biology is at present largely unknown. revealing levels of both SEPT2 and SEPT7 were obviously Herein, this study aimed at determining whether septin higher in detected breast cancer cell lines in comparing proteins could influence breast cancer cell biological with normal breast cell line MCF10A, especially in high www.impactjournals.com/oncotarget 61588 Oncotarget invasive cell line, MDA-MB-231. To further visualize Reduced SEPT2 and SEPT7 expression inhibits SEPT2 and SEPT7 subcellular distributions, we co- BC
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