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Binding Between ROCK1 and DCTN2 Triggers Diabetes‑Associated Centrosome Amplification in Colon Cancer Cells

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Binding Between ROCK1 and DCTN2 Triggers Diabetes‑Associated Centrosome Amplification in Colon Cancer Cells ONCOLOGY REPORTS 46: 151, 2021 Binding between ROCK1 and DCTN2 triggers diabetes‑associated centrosome amplification in colon cancer cells YUAN FEI LI1, LIN JIE SHI1,2, PU WANG3, JIA WEN WANG4, GUANG YI SHI4 and SHAO CHIN LEE4 1Department of Oncology, The First Hospital, Shanxi Medical University, Taiyuan, Shanxi 030001; 2Intensive Care Unit, Shaanxi Provincial Cancer Hospital, Xian, Shaanxi 710000; 3Changzhi Medical University, Changzhi, Shanxi 030001; 4Institute of Biomedical Sciences of The School of Life Sciences, Jiangsu Normal University, Xuzhou, Jiangsu 221116, P.R. China Received January 17, 2021; Accepted May 5, 2021 DOI: 10.3892/or.2021.8102 Abstract. Type 2 diabetes increases the risk various types of Dynactin subunit 2 (DCTN2) was confirmed to be localized cancer and is associated with a poor prognosis therein. There to the centrosomes. Treating the cells with high glucose, is also evidence that the disease is associated with cancer insulin and palmitic acid increased the protein levels of metastasis. Centrosome amplification can initiate tumori‑ ROCK1 and DCTN2, promoted their binding with each genesis with metastasis in vivo and increase the invasiveness other, and triggered centrosome amplification. Disruption of of cancer cells in vitro. Our previous study reported that the protein complex by knocking down ROCK1 or DCTN2 type 2 diabetes promotes centrosome amplification via the expression partially attenuated centrosome amplification, upregulation and centrosomal translocation of Rho‑associated while simultaneous knockdown of both proteins completely protein kinase 1 (ROCK1), which suggests that centrosome inhibited centrosome amplification. These results suggested amplification is a candidate biological link between type 2 ROCK1‑DCTN2 binding as a signal for the regulation of diabetes and cancer development. In the present study, func‑ centrosome homeostasis, which is key for diabetes‑associated tional proteomics analysis was used to further investigate the centrosome amplification, and enriches our knowledge of molecular pathways underlying centrosome amplification by centrosome biology. Therefore, the ROCK1‑DCTN2 complex targeting ROCK1 binding partners. High glucose, insulin and may serve as a target for inhibiting centrosome amplification palmitic acid were used to induce centrosome amplification, both in research or future therapeutic development. and immunofluorescent staining was employed to visualize centrosomal alterations. Combined with immunoprecipita‑ Introduction tion, mass spectrometry‑based proteomics analysis was used to identify ROCK1 binding proteins, and protein complex Diabetes is a chronic disease with increasing prevalence disruption was achieved by siRNA‑knockdown. In total, worldwide (1), and exerts significant socioeconomic burden on 1,148 ROCK1 binding proteins were identified, among which society (2). Diabetes is classified into type 1 and 2, and type 2 106 proteins were exclusively associated with the treated diabetes accounts for the majority of clinical cases (1). According samples, 193 were only associated with the control samples, to the latest dataset from the World Health Organization, the and 849 were found in both the control and treated samples. number of diabetes cases increased from 108 million in 1980 Of the proteins with evidence of centrosomal localization, to 422 million in 2014 (3). In 2016, ~1.6 million mortalities were reportedly directly caused by diabetes (4). In the United States alone, the economic burden of diabetes, undiagnosed diabetes and prediabetic complications, was estimated to be 404 billion dollars in 2017 (5). Patients with type 2 diabetes Correspondence to: Dr Yuan Fei Li, Department of Oncology, The have increased levels of blood glucose and free fatty acids First Hospital, Shanxi Medical University, 85 Jiefang South Road, Taiyuan, Shanxi 030001, P.R. China such as palmitic acid. Moreover, hyperinsulinemia occurs, at E‑mail: [email protected] least at certain stages in disease development, due to insulin resistance (6). In addition to classical vascular complications Dr Shao Chin Lee, Institute of Biomedical Sciences of The School such as nephropathy and retinopathy, accumulating evidence of Life Sciences, Jiangsu Normal University, 101 Shanghai Road, Xuzhou, Jiangsu 221116, P.R. China suggests that cancer is a complication of type 2 diabetes, E‑mail: [email protected] which led to a joint meeting of oncologists and diabetologists in 2010. These experts reviewed the evidence and released a Key words: centrosome amplification, type 2 diabetes, Rho‑associated consensus report stating that type 2 diabetes increases the risk protein kinase 1, dynactin subunit 2, colon cancer cells, functional of cancer at various sites, including the colon (7). Moreover, proteomics type 2 diabetes has been associated with a poor cancer prog‑ nosis (8), as well as metastasis (9). At present, cancer accounts for a large proportion of the mortality of patients with type 2 2 LI et al: BINDING BETWEEN ROCK1 AND DCTN2 TRIGGERS CENTROSOME AMPLIFICATION. diabetes (10). However, why and how type 2 diabetes promotes proliferation (22), regulates apoptosis (23) and enhances cancer development remains unknown. migration and invasiveness (24). In humans, the ROCK1 geno‑ The centrosome is the smallest organelle in the eukaryotic type was found to be associated with colon cancer in men (25), cell, which serves as a microtubule‑organization center, and and is a candidate prognostic marker for vulvar cancer (26). is a key regulator of cell division. Centrosome amplification Our previous studies revealed that ROCK1 signaling promotes is defined as >2 centrosomes in a single cell. It has been diabetes‑associated centrosome amplification (6,20). However, reported to occur in various cancer types, and is recognized as inhibiting ROCK1 only partially suppresses centrosome ampli‑ a typical feature of cancer cells (11). Experimental data have fication, indicating that other factors are involved. Therefore, indicated that centrosome amplification is associated with the aim of the present study was to investigate ROCK1 binding tumorigenesis and tumor metastasis. For example, genetically proteins in order to identify key signaling molecules involved modified cells with centrosome amplification can form tumors in centrosome amplification. in recipient animals, with the potential for metastasis (12,13), while genetically modified mice with centrosome amplifica‑ Materials and methods tion can spontaneously develop tumors (14,15). Furthermore, in Barrett's esophagus tumorigenesis, a multistep progression Reagents and cell lines. All chemical reagents were purchased model of human cancer, centrosome amplification occurs in a from Sigma‑Aldrich (Merck KGaA). Cell culture media and stepwise manner before neoplasia (16). It has also been shown supplements were obtained from Gibco (Thermo Fisher that genetically modified cells with centrosome amplification Scientific, Inc.). HCT116 human colon cancer cells were kindly exhibit increased invasive potential (17). However, the obvious gifted by Dr B. Vogelstein of the Johns Hopkins University limitation is that genetically modified models do not accu‑ School of Medicine (Baltimore, USA). IEC‑6, which is a rately reflect reality, as human cancers are not considered to non‑cancerous immortalized rat colon epithelial cell line, was commonly be caused by transgenesis. purchased from the Chinese Academy of Sciences Beijing Cell Since type 2 diabetes is associated with DNA damage (18), Bank. The cells were maintained in the lab in low‑glucose which can cause centrosome amplification (19), it was hypoth‑ medium. esized that type 2 diabetes may favor centrosome amplification. Indeed, when compared between non‑diabetic subjects and Cell culture and treatments. Cells were cultured in DMEM diabetic patients, a 2.6‑fold increase in centrosome ampli‑ (low glucose, 5 mM) supplemented with 50 U/ml penicillin, fication was observed in the peripheral blood mononuclear 50 µg/ml streptomycin and 10% (v/v) fetal calf serum, and cells of diabetic patients (6). In vitro experiments have iden‑ maintained at 37˚C (5% CO2) in a humidified incubator. Cells tified that the pathophysiological factors in type 2 diabetes, in the experimental group were treated with high glucose including high glucose, insulin and free fatty acids (such as (15 mM), insulin (5 nM) and palmitic acid (150 µM). Cells palmitic acid), are potent inducers, and that the centrosomal treated for 48 h were used for the assessment of the centro‑ translocation of Rho‑associated protein kinase 1 (ROCK1) some amplification level; cells treated for 30 h were used is a signal for diabetes‑associated centrosome amplifica‑ for immunoprecipitation and western blot analyses. Palmitic tion (6). Moreover, inhibition of ROCK1 partially attenuated acid, the most common saturated free fatty acid, is frequently centrosome amplification (6,20). These results suggest that used to investigate the biological activities of free fatty acids, centrosome amplification may be a candidate mechanism for particularly their adverse effects (27). Palmitic acid was conju‑ the association between type 2 diabetes and cancer develop‑ gated to BSA; both reagents were mixed (molar ratio, 3:1) and ment, and that type 2 diabetes may present a natural model incubated for 1 h before use. to investigate the roles of centrosome amplification in cancer. However, to confirm this conclusion with substantial Immunofluorescent staining and confocal
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