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CSRP2 Suppresses Colorectal Cancer Progression Via P130cas/Rac1 Axis

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CSRP2 Suppresses Colorectal Cancer Progression Via P130cas/Rac1 Axis Theranostics 2020, Vol. 10, Issue 24 11063 Ivyspring International Publisher Theranostics 2020; 10(24): 11063-11079. doi: 10.7150/thno.45674 Research Paper CSRP2 suppresses colorectal cancer progression via p130Cas/Rac1 axis-meditated ERK, PAK, and HIPPO signaling pathways Lixia Chen1,2, Xiaoli Long1,2, Shiyu Duan1,2, Xunhua Liu1,2, Jianxiong Chen1,2, Jiawen Lan1,2, Xuming Liu1, Wenqing Huang2, Jian Geng1,2 and Jun Zhou1,2 1. Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. 2. Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China. Corresponding authors: Jun Zhou, Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China; Phone: 86-2062789365; E-mail: [email protected]; [email protected]; or, Jian Geng, Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China; Phone: 86-2061648223; E-mail: geng@ smu.edu.cn. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2020.03.05; Accepted: 2020.08.21; Published: 2020.09.02 Abstract Metastasis is a major cause of death in patients with colorectal cancer (CRC). Cysteine-rich protein 2 (CSRP2) has been recently implicated in the progression and metastasis of a variety of cancers. However, the biological functions and underlying mechanisms of CSRP2 in the regulation of CRC progression are largely unknown. Methods: Immunohistochemistry, quantitative real-time polymerase chain reaction (qPCR) and Western blotting (WB) were used to detect the expression of CSRP2 in CRC tissues and paracancerous tissues. CSRP2 function in CRC was determined by a series of functional tests in vivo and in vitro. WB and immunofluorescence were used to determine the relation between CSRP2 and epithelial-mesenchymal transition (EMT). Co-immunoprecipitation and scanning electron microscopy were used to study the molecular mechanism of CSRP2 in CRC. Results: The CSRP2 expression level in CRC tissues was lower than in adjacent normal tissues and indicated poor prognosis in CRC patients. Functionally, CSRP2 could suppress the proliferation, migration, and invasion of CRC cells in vitro and inhibit CRC tumorigenesis and metastasis in vivo. Mechanistic investigations revealed a physical interaction between CSRP2 and p130Cas. CSRP2 could inhibit the activation of Rac1 by preventing the phosphorylation of p130Cas, thus activating the Hippo signaling pathway, and simultaneously inhibiting the ERK and PAK/LIMK/cortactin signaling pathways, thereby inhibiting the EMT and metastasis of CRC. Rescue experiments showed that blocking the p130Cas and Rac1 activation could inhibit EMT induced by CSRP2 silencing. Conclusion: Our results suggest that the CSRP2/p130Cas/Rac1 axis can inhibit CRC aggressiveness and metastasis through the Hippo, ERK, and PAK signaling pathways. Therefore, CSRP2 may be a potential therapeutic target for CRC. Key words: Colorectal cancer, CSRP2, Hippo, ERK, PAK Introduction Colorectal cancer (CRC) is one of the most molecular mechanisms underlying the tumorigenesis common malignant tumors worldwide and the fourth and metastasis of CRC are not entirely clear [1, 2]. leading cause of cancer-related deaths. Metastasis and Therefore, there is an urgent need to explore the recurrence are the leading causes of death in patients underlying molecular mechanisms in CRC recurrence with CRC [1]. Although many altered pathways and and metastasis. abnormal genes have been identified, the precise http://www.thno.org Theranostics 2020, Vol. 10, Issue 24 11064 Cysteine-rich protein 2 (CSRP2) is a member of All CRC cell lines were cultured in RPMI 1640 the LIM-only family of cysteine-rich proteins and medium (Gibco, Gaithersburg, MD, USA) contains two LIM domains [3]. Members of this family supplemented with 10% fetal bovine serum (Hyclone, include CSRP1, CSRP2, and CSRP3. These proteins Logan, USA) and 100 μm U/mL penicillin/ have the LIM zinc finger domain, which is generally streptomycin (Gibco) in a 37 °C humidified chamber considered the entity of protein-protein interaction containing 5% CO2. and plays a key role in a variety of biological processes, such as signal transduction, cytoskeleton Tissue preparation formation, cell proliferation, and differentiation [4]. 20 pairs of fresh CRC tissues and paired normal The three CSRPs (CSRP1-3) are preferentially colorectal tissues from patients with primary CRC in expressed in muscle cells and localized in the nucleus Nanfang Hospital were analyzed by WB and qPCR. and cytoplasm. In the nucleus, CRPs can interact with (Guangzhou, China). Tissue paraffin specimens of 215 transcription factors, such as MyoD, SRF, and GATA patients with primary CRC were collected in Nanfang family proteins, to facilitate smooth (CRP1 and CRP2) Hospital from 2000 to 2005. None of these patients or striated muscle (CRP3) differentiation [5]. In the received any chemotherapy or radiotherapy before cytoplasm, they decorate filamentous actin structures the operation. The classification system (PTNM) and participate in cytoskeletal remodeling [6]. determines the stage of the disease according to the However, the exact function of these proteins remains size of the tumor, lymph node metastasis, and distant unclear. metastasis [12]. CSRP2 is widely expressed in various tissues [7]. Immunohistochemistry Previous studies have shown that CSRP2 contributes to the assembly and/or maintenance of the actin The immunohistochemical (IHC) method was backbone. It is involved in actin cytoskeleton used to detect the expression of CSRP2 protein in 215 rearrangement, and a lack of CSRP2 can promote the pairs of paraffin-embedded CRC tissues and matched migration of vascular smooth muscle cells [8]. CSRP2 adjacent normal colorectal tissues. The slices were has been studied in several tumors, including breast heated, dewaxed, rehydrated, and put into sodium [9], gastric [10], hepatocellular cancers [11] and citrate buffer (pH buffer = 6.0) for antigen repair. The lymphoblastic leukemia [6] and is involved in the slide was then soaked in 3% hydrogen peroxide to proliferation, migration and invasion of tumor cells. inhibit endogenous peroxidase activity and sealed However, the CSRP2 role is controversial in different with sheep10% FBS/PBS. After rinsing three times, tumors, and its precise cellular function needs to be the slices were incubated overnight with the first further elucidated. The expression of CSRP2 in CRC antibody (rabbit anti-CSRP2, 1:500 dilution, # and its role in the growth and progression of CRC is HPA045617; Sigma) at 4 °C. Slices were washed three unknown. times using PBS and then treated with a second In this study, we found that the expression of antibody (anti-rabbit IgG 1:2000 diluted, # 7074; Cell CSRP2 was lower in CRC and correlated with poor Signal Technology, Danvers, MA, USA) for 40 min at prognosis. Further investigations revealed that CSRP2 37 °C. After being stained with 3BI-3-diamino- could interact with p130Cas to suppress the activation benzidine (DAB), it was stained with hematoxylin, of Rac1 and then inhibit EMT and metastasis of CRC dehydrated, sealed, and observed. via activating Hippo and inhibiting ERK and PAK- IHC scores based on staining intensity and LIMK-cortactin signaling pathways. For the first time, percentage of positive tumor cells were performed by our findings provide a deeper understanding of the two independent pathologists who did not know the functions and mechanisms of CSRP2 in CRC clinical data. The staining intensity scores were 0 progression and may contribute to the development (negative), 1 (weak), 2 (medium) and 3 (strong). of new therapeutic targets for CRC. According to the percentage of the positive staining area of the specimen in the entire tumor area or the Materials and Methods entire section, the staining degree was scored as 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), 4 (76-100%). Cell culture The sum of the strength and range scores is used as FHC (CRL-1831) and 7 human CRC cell lines, the final score of the CSRP2 (0-12). The final staining including HCT116, SW620, HCT15, SW480, RKO, score of more than 7 was regarded as high expression SW1116, and LoVo, came from the Cell Bank of and the score of less than 7 was regarded as the low Chinese Academy of Sciences (Shanghai). All cells expression of CSRP2. were identified by (Str) map of short tandem repeats and passaged less than 6 months after resuscitation. http://www.thno.org Theranostics 2020, Vol. 10, Issue 24 11065 RNA extraction and qPCR a wild-type p130Cas plasmid constructed by inserting Trizol reagent (Invitrogen, Carlsbad, CA) was BCAR1 into vector PCDNA3.1 (You ming, used to extract the total RNA of cells and fresh tissues. Guangzhou, China). p130Cas-F15 is a human p130Cas PrimeScript RT kit (Promega, Madison, WI, USA) was mutant lentivirus (pSLenti-CMVBCAR1 (MUTs) used to synthesize cDNA. qPCR was performed in -PGK-Puro-WPRE) by mutating all 15 tyrosines ABI7500 Real-time PCR system (Applied Biosystems, within the common motif YXXP in the P130Cas Foster City, USA) using SYBR PreMix ex Taq substrate domain (SD) to phenylalanine(Heyuan, benchmark (Takala, Dalian, China) . The expression of Shanghai, China).POLO Deliver 3000 transfection related genes was measured by the comparative reagents were purchased from Shanghai
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