Prognostic significance of SOCS3 and its biological function in colorectal cancer

Accepted Manuscript Prognostic significance of SOCS3 and its biological function in colorectal cancer

Qinjun Chu, Dan Shen, Long He, Hongwei Wang, Chunlan Liu, Wei Zhang PII: DOI: Reference:

S0378-1119(17)30463-8 doi: 10.1016/j.gene.2017.06.013 GENE 41976

To appear in:

Gene

Received date: Revised date: Accepted date:

18 September 2016 5 June 2017 7 June 2017

Please cite this article as: Qinjun Chu, Dan Shen, Long He, Hongwei Wang, Chunlan Liu, Wei Zhang , Prognostic significance of SOCS3 and its biological function in colorectal cancer, Gene (2017), doi: 10.1016/j.gene.2017.06.013

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Prognostic significance of SOCS3 and its biological function in colorectal cancer

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QINJUN CHU，M.D1,#, DAN SHEN，M.D1,#, LONG HE，M.D1, HONGWEI WANG，M.D 1, CHUNLAN LIU, M.D 1, WEI ZHANG, M.D, Ph.D 1* Department of Anesthesiology, Zhengzhou University Affiliated First Hospital,

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450052, He Nan, China

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QINJUN CHU, DAN SHEN are the co-first authors

*Correspondence to WEI ZHANG

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450052, He Nan, China

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Department of Anesthesiology, Zhengzhou University Affiliated First Hospital,

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E-mail: [email protected]

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Abstract Colorectal cancer（CRC）is a common malignant tumor, in which the

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inflammatory microenvironment plays an important role. STAT3 signaling pathway is regarded as the “bridge” between inflammation and cancer, and involved in the development of CRC. SOCS3 is a key negative feedback

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regulator of JAK/STAT signaling pathway. Studies about SOCS3 gene in CRC are rarely reported. The purpose of this study is to determine the expression of

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SOCS3 in CRC tissue and its correlation with the clinical pathological characteristics and prognosis of colorectal cancers. The effects of SOCS3 on

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biological behavior such as apoptosis, proliferation, migration, invasion and

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tumor formation in nude mice were studied. We observed that SOCS3

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expression was down-regulated in CRC tissues, while IL-6, pSTAT3 were up-regulated. Inflammatory cytokines IL-6 can promote the expression of

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STAT3 signaling pathways while inhibit the expression of SOCS3 by promoting hypermethylation of SOCS3 gene promoters. 5-Aza-cdR treatment can reverse IL-6/STAT3 signaling pathway mediated down-regulation of SOCS3 in colorectal cancer cells. Low expression of SOCS3 was correlated with lymph node metastasis and advanced clinical stage. Patients with high expression of SOCS3 in colorectal cancers often indicated a relatively good prognosis.

ACCEPTED MANUSCRIPT Overexpression of SOCS3 inhibited proliferation, migration, invasion and tumorigenic ability of CRC cells while increased cell apoptosis. This study demonstrated that IL-6/STAT3 signaling activation negatively regulated SOCS3 expression, which led to imbalance and sustained activation of STAT3

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signaling pathway. Reduced expression of SOCS3 promoted the growth and metastasis of colorectal cancer. Thus, targeting IL-6/STAT3/SOCS3 signaling pathway may become an important treatment strategy of colorectal cancer.

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Keywords: IL-6, STAT3, SOCS3, Colorectal cancer

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Introduction Colorectal cancer (CRC) is the most common gastrointestinal cancer, particularly in developed countries, and it has become the third cause of cancer death currently [1]. In recent years, with the change in the standard of

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living and eating habits, the incidence of CRC in developing countries showed an increasing trend, and the age of patients are becoming increasingly younger [2]. Etiology of colorectal cancer had confirmed many oncogenes and

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tumor suppressor genes and epigenetic regulation may directly affect the development of CRC [3]. However, the clinical treatment of CRC and the

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prognosis had still no substantial improvement in cancer mortality rates [4, 5]. Therefore, further study of the mechanism in CRC is an urgent need.

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SOCS3 is an important member of the SOCS family, and is a negative

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regulatory protein in cytokine and growth factor associated signaling pathway.

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SOCS3 plays as a negative regulator in the signal transduction of janus kinase and signal transducer and activator of transcription (JAK-STAT), preventing

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malignant transformation of tumor cells and promote apoptosis of tumor cells. Therefore，activation and overexpression of SOCS3 has a potential role in tumor growth [6, 7]. Human SOCS3 gene is located in 17q25.3[8], which is composed of SOCS box, SH2 domain and N terminal kinase inhibitor (KIR) [9]. SOCS box can be raised to Elongin B/C, and then combined with the proteasome or

ACCEPTED MANUSCRIPT recruit the ubiquitin transfer enzyme system through the Elongin B/C complex to promote the degradation of target protein, and thereby block the signal transduction of cytokines [10, 11]. In addition, SOCS box can also inhibit the degradation of SOCS3 protein and maintain the stability of the protein [12, 13].

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The SH2 domain is located in the center of SOCS3, which combined with the phosphate of protein in order to mediated interaction between SOCS3 and other signal transduction molecules [14]. KIR of SOCS3 as pseudosubstrate,

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has a competitive antagonism to inhibit JAK kinase activity [13, 15]. Inflammatory mediators, such as IL-6, IL-1β and IFN-γ, induced DNA

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methylation and involved in the transition from inflammation to cancer, which are important member of tumor microenvironment [16-18]. JAK/STAT signaling

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pathway plays a role as a bridge between inflammation and cancer, and

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various cytokines including IL-6 exert biological effects in tumor cells through

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this pathway. However, the main function of SOCS protein family is to inhibit the activity of JAK/STAT signaling pathway [8, 19]. Reduced or deleted

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expression of SOCS3 promotes the expression of downstream gene, such as C-MYC and C-FOS. Thus, it leads to the abnormal expression of oncogenes and the occurrence of tumor [20]. There is close relationship between overexpression of SOCS3 and the progression of malignant tumors [21-23]. However, it is not deeply studied in colorectal cancer. In this study, we investigated the expression of SOCS3 and the

ACCEPTED MANUSCRIPT connection between SOCS3 expression and CRC characteristics and evaluated their potential relation to the clinical outcome. Besides，The effects of SOCS3 on CRC biological behavior was also explored. SOCS3 may be a molecular marker that reflects the occurrence and development of colorectal

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cancer, and can be used as an early diagnostic marker for colorectal cancer.

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Materials and methods Patients and specimens Tissue specimens were collected from 20 CRC patients who had undergone tumor resection without receiving chemotherapy or radiotherapy

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before surgery at the Zhengzhou University Affiliated First Hospital (China) between June 2014 and July 2015. All the CRC and paired adjacent normal mucosa specimens were collected in the General Surgery Department under

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protocols approved by the Institutional Review Boards of Zhengzhou University Affiliated First Hospital Medical Center. Written informed consent

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was obtained from all patients. The freshly obtained cancer tissues and adjacent normal mucosa were immediately frozen in liquid nitrogen and stored

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at -80°C prior to protein extraction.

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The tissue microarray (TMA) containing 88 paired CRC specimens was

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purchased from Xin Chao Company (Shanghai, China). Tumors were resected between July 2006 and May 2007. The final follow-up was on August 2012,

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with a median patient follow-up time for survivors of 46.62 months (range, 3–73 months). The samples were obtained from 46 men and 42 women with a mean age of 68.72 years (range, 24–90 years). Tumor staging was carried out according to the AJCC staging criteria.

ACCEPTED MANUSCRIPT Cell lines and Reagents The CRC cell lines DLD1, HT29 and SW480 were purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China) and maintained at 37°C under high humidity and 5% CO2 in DMEM (Gibco)

target

sequence

of

the

5’-GGACCAAGAGCCTACGCAT-3’.

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supplemented with 10% FBS (Gibco), 1% streptomycin, and penicillin. The SOCS3

A

siRNA

were

scrambled

siRNA

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(5’-UUCUCCGAACGUGUCACGUTT-3’) was used as a negative control. The SOCS3 coding sequences were inserted into the GV358 vector (Genechem

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Co. Ltd., Shanghai, China). SW480 cells were transfected with GV358-SOCS3 or control vector using lentivirus according to the manufacturer’s instructions.

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Stable cells are obtained by puromycin selection for 7 days 24h after

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transduction. Stattic and IL6 were purchased from Selleckchem (Houston,

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Texas, USA) and Sino Biological Inc. (Beijing, China), respectively.

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Methylation-specific PCR Methylation-specific PCR was performed to examine the methylation status of the SOCS3 promoter. The sequences of the methylated primers were: forward,

5’-TGATTAAATATTATAAGAAGGTCGGTCG-3’

5’-ACTAACTACGTACGAAACCGAAACG-3’. unmethylated

primers

The

and

sequences

were:

reverse, of

the

forward,

ACCEPTED MANUSCRIPT 5’-GTAGTGATTAAATATTATAAGAAGGTTGGTTG-3’

and

reverse,

5’-CTAACTACATACAAAACCAAAACAA-3’. Amplification was achieved in a 25μl reaction volume containing 1μl each primer, 1μl modified DNA, 12.5μl 2×Tag enzyme. The PCR reaction conditions were as follows: Initial

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denaturation at 95°C for 5min; 40 cycles of denaturation at 95°C for 40 sec, annealing at 56.5°C for 60 sec and extension at 72°C for 60 sec, then a final 10-min extension at 72°C. The PCR products were visualized on a 2%

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agarose gel using DuRed and UV illumination.

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Luciferase reporter assay

The human SOCS3 promoter region (-1084 through +1) containing the

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STAT3-SOCS3 binding sites was amplified by PCR and inserted into pGL3 sequences of

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vector (Promega). The

and

reverse,

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5′-GGAGTTCCTGGACCAGTACG-3′

PCR primers were: forward,

5′-TTCTTGTGCTTGTGCCATGT-3′. HT29 and DLD1 cells were seeded on

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24-well plates and incubated for 24h before transfection. The cells were transfected with 0.45μg reporter plasmid (pGL3-SOCS3) using Lipofectamine 2000 transfection reagent. 0.05 μg Renilla luciferase plasmid pRL-TK (Promega) was cotransfected to normalize the transfection efficiency. The transfected cells were treated with 100ng/ml IL-6, 200 ng/ml IL-6 or 5 μmol/L 5-AZA after 24h. Then, 48h after treatment, Luciferase activity was examined

ACCEPTED MANUSCRIPT using the Dual Luciferase Reporter Assay System (Promega). All of the assays were performed in triplicate. Imunohistochemistry TMA slides were constructed by using formalin-fixed, paraffin-embedded

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samples. Standard immunohistochemical procedures were performed using a GT vision III kit (Genetech, Shanghai, China). After antigen retrieval in citrate buffer (pH 6.0) for 10 minutes, the specimen slides were incubated overnight

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at 4°C with the primary antibody against SOCS3 (1:200, Abcam, UK), IL-6 (1:100, Abcam, UK) and p-STAT3 (1:100, Abcam, UK), followed by incubation

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with the secondary antibody (Genetech, Shanghai, China). Tissue sections were counterstained with Mayer’s hematoxylin. Two specialists are blinded to

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patient outcomes independently in order to evaluate the intensity and extent of

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staining. Staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate),

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and 3 (strong). Staining extent was scored ad 0 (0%), 1 (1%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (76%-100%) according to the percentage of

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positively stained cells. The two scores were multiplied to give a final score and divided into two groups: low (0-6), high (7-12).

Western blot analysis Cellular protein was extracted from tissue samples and cells using RIPA lysis buffer. The protein concentration was measured using the BCA protein

ACCEPTED MANUSCRIPT assay kit (Beyotime Biotechnology, China). The standard western blot was performed in order to transfer protein onto PVDF membranes. Membranes were then incubated with an antibody against SOCS3 (1:1000, Abcam, UK), p-STAT3 (1:1000, Abcam, UK), STAT3 (1:1000, Abcam, UK) and β-actin

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(1:1000, Cell Signaling Technology, USA) overnight at 4°C. After washing with TBST, the membranes were incubated with a secondary antibody against mouse immunoglobulin G. The proteins were detected using ECL (Pierce

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Biotechnology, USA) according to the manufacturer’s instructions.

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CCK-8 assays

Cells were plated in 96-well plates in triplicate before measurement.

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CCK-8 Cell Proliferation Assay Kit (Invitrogen, USA) was used to evaluate cell

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proliferation. 10μL of CCK-8 solution was added into each well and incubated

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for 2h at 37°C. Absorbance was calculated on a Gen5 microplate reader

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(BioTek, USA) at 450 nm.

Flow cytometry

HT29 and SW480 cells were trypsinized and washed in phosphate buffered saline (PBS). The number of apoptotic cells was analyzed using the Annexin V-APC/PI Apoptosis Kit (eBioscience, CA, USA) according to the manufacturer’s instructions.

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Cell scratch-wound assay For cell migration, CRC cells were seeded in six-well plates. A cell scratch-wound was generated using a 10-μL tip. The wounded cells were

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photographed for measuring gap sizes at 0h and 24h.

Invasion assays

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The invasion ability of treated CRC cells were evaluated by using Transwell assay. Cells (5×104) were seeded into the upper chambers with

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Matrigel (BD, CA, USA), and the lower chamber was filled with 10% FBS. After 24h incubation, migrated cells on the lower membrane were fixed, stained and

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counted under a microscope.

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Tumorigenesis in nude mice

All protocols for the animal studies were approved by Zhengzhou Affiliated

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University

First

Hospital

Animal

Care

guidelines.

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overexpressed SW480 cells, HT29-SiSOC3 cells and controls (1×106; n=5/group) were subcutaneously injected into nude mice (6-week-old) respectively. Four weeks later, tumor volume and weight were measured.

Statistical analysis

ACCEPTED MANUSCRIPT The statistical significance of differences between experimental groups were determined by the Pearson chi-square test and Fisher’s exact test. The Kaplan-Meier method was used to calculate the survival rates. A log-rank test was used to compare the survival curves. A Cox proportional hazards model

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was applied to evaluate the hazard ratios for the variables. A P value less than 0.05 was considered statistically significant. All statistical analyses were carried out with the SPSS 21.0 statistical software package (SPSS Inc.,

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Chicago, IL).

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Result

Expression of pSTAT3 and SOCS3 in human colorectal cancer

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Among the 20 paired cases for IHC analysis, IL-6 and p-STAT3

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expression was significantly higher in cancerous tissues than in adjacent

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normal mucosa (P<0.001，Figure 1). However, the expression of SOCS3 was significantly decreased in tumor tissue (Figure 1). Subsequent western blotting

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also confirmed that STAT3 was significantly activated in tumor tissues (Figure 2), while the expression of SOCS3 was decreased as compared to colorectal tissue (Figure 2). From those data we inferred that p-STAT3 and SOCS3 may act as oncogenes or tumor suppressor genes in the CRC respectively.

IL-6/STAT3 signaling activation negatively regulated SOCS3 expression

ACCEPTED MANUSCRIPT To confirm the correlation between STAT3 and SOCS3 in colorectal cancer, we chose HT29，DLD1 cells and SW480 cells, because HT29 and DLD1 had relatively higher expression of SOCS3 while SW480 had relatively lower expression of SOCS3 according to our studies. HT29 and DLD1 cells

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were treated by IL-6 (100ng/ml), which is agonist of IL-6/Jak-2/STAT3 signaling pathway. SW480 cells were treated by Stattic (10uM), which is inhibitor of the IL-6/Jak-2/STAT3 signaling pathway. Western blots showed that

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STAT3 is obviously activated and SOCS3 expression is significantly decreased in DLD1 and HT29 cells treated by IL-6 (Figure 3). Conversely, the expression

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of SOCS3 is increased in SW480 cells treated by Stattic (Figure 3). Our research showed that IL-6 regulated SOCS3 by acting on the STAT3 signaling

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pathway, which inhibited the activation of SOCS3 and promoted the

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expression of STAT3. Because previous study found that IL-6 could induce

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methylation modification in colorectal cancer, we took whether IL-6 could induce hypermethylation of the SOCS3 gene into consideration. We used MSP

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to analyze the methylation level of SOCS3 gene in DLD1, HT29 and SW480 cell lines. We found the methylation level of SOCS3 gene in DLD1 and HT29 cells increased after IL-6 incubation for 24h. In contrast, Stattic treatment for 24h could decrease the methylation level of the SOCS3 gene in SW480 cells (Figure S1A). To further confirm that SOCS3 expression loss was caused by promoter methylation, we incubated HT29 cells with 5-AZA, an inhibitor of

ACCEPTED MANUSCRIPT DNA methylation, and IL-6 and 5-AZA treatments simultaneously could reverse IL-6 mediated repression of SOCS3 expression (Figure S1B). To observe the effect of pSTAT3 on SOCS3 promoter activity, we next transfected HT29 and DLD1 cells with a reporter vector encoding Luciferase under control of the

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SOCS3 promoter (PGL3-SOCS3). SOCS3 promoter activity was decreased after IL-6 treatment and had dose dependent. Nevertheless, 5-AZA could abolish pSTAT3-mediated down-regulation of SOCS3 in HT29 and DLD1 cells

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(Figure S1C). The above results demonstrated IL-6/STAT3 signaling activation negatively regulated SOCS3 expression by affecting the methylation

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modification the SOCS3 promoter.

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Association between SOCS3 expression and clinicopathological parameters in

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colorectal cancer

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To determine the clinicopathologic significance of SOCS3 expression, immunohistochemistry of a TMA containing 88 cases of primary CRC paired

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with noncancerous tissue was performed. 36 (40.9%) showed positive staining in CRC tissue, while 75 (85.2%) showed positive staining in normal mucosa (Table 1). The expression of SOCS3 was significantly higher in normal tissue than the tissues of colorectal cancer, and the difference was statistically significant (P=0.03). Next, we examined the correlation between SOCS3 and clinical parameters among CRC patients. In colorectal cancer, SOCS3

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was

significantly

associated

with

tumor

size

(P=0.001),

differentiation (P=0.005), N stage (P<0.001) and TNM stage (P<0.001) (table 1).

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Low SOCS3 expression was associated with poor clinical outcomes of colorectal cancer

Survival analysis was performed on 88 patients. The postoperative

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median survival time was 24 months in the negative expression of SOCS3, while the postoperative median survival time was 42 months in the positive

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expression of SOCS3. The Kaplan-Meier plots showed that patients with positive tumor SOCS3 expression had a better OS rate than those with

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SOCS3 tumor low-expression (Figure 4). We used the Cox proportional

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hazards model in order to conduct a multivariate analysis for all the significant

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variables. The results demonstrated that negative SOCS3 expression was a

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significant independent prognostic factor for shorter survival (Table 2).

SOCS3 inhibited proliferation and induced CRC cell apoptosis To further elucidate the role of SOCS3 during CRC development, we selected SW480 and HT29 cells which had relatively low and high expression of SOCS3 as our cell models. Protein level of SOCS3 was effectively enhanced after infection with overexpression lentivirus in SW480, and SOCS3

ACCEPTED MANUSCRIPT knockdown in HT29 was efficient after transfection with siSOCS3 compared to scramble group. (Fig. S2). The CCK-8 assay showed that the knockdown of SOCS3 significantly increased survival and proliferation (Figure 5A). Analysis of apoptosis revealed that the knockdown of SOCS3 decreased apoptosis

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(Figure 5C). However, overexpression of SOCS3 by gene transfer did the

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opposite (Figure 5B, D).

ACCEPTED MANUSCRIPT Altered expression of SOCS3 affected migratory and invasive ability of CRC cells in vitro and tumorigenecity in vivo In the cell scratch-wound assay and transwell assay, the knockdown of SOCS3 promoted the flattening and spreading of HT29 cells and increased the

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numbers of invaded HT29 cells (Figure 6A, C), whereas forced SOCS3 expression attenuated the migratory and invasive ability of SW480 cells (Figure 6B, D). And we performed western blotting on invasion related proteins.

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The results showed a notably elevated protein level of MMP-2 and MMP-9 for HT29/ siSOCS3 cells as compared with scramble cells (Fig. S2). Conversely,

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the expression levels of MMP-2 and MMP-9 were reduced in SW480/SOCS3 overexpression cells.

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Xenograft tumors in nude mice formed by knocking down SOCS3 gene

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stably transfected HT29 cells and SOCS3 gene stably transfected SW480

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cells and the controls. As Figure 7 shown, the knockdown of SOCS3 decreased the sizes and weight of tumors. Conversely, the overexpression of

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SOCS3 increased the sizes and weight of tumors. Those results indicated that the overexpression of SOCS3 effectively inhibited the CRC formation.

Discussion SOCS3 is one of the important members of the SOCS family including SOCS1-7. Colorectal inflammatory microenvironment played an important role

ACCEPTED MANUSCRIPT in the occurrence and development of colorectal cancer. IL-6 secreted by tumor and stromal cells was an important member of tumor inflammatory microenvironment. It binded with receptor and played a biological function through the following 3 signal pathways: JAK2/STAT3, MEK/ERK and

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PI3K/AKT [24]. Among them, the STAT3 signal pathway is closely related to the occurrence of colorectal cancer, angiogenesis and tumor metastasis [25, 26]. SOCS3 is an important signal inhibition factor in JAK2/STAT3 signal

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pathway. The activation of STAT3 in normal cells is transient and rapid [27], while reduction or deletion of SOCS3 expression resulted in sustained

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activation of STAT3 in many malignant tumors [28, 29]. Our study indicated that the expression of IL-6 and pSTAT3 in CRC tissues was significantly higher

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than that in adjacent tissues, whereas the expression of SOCS3 is significantly

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decreased or absent in CRC tissues. Our results were as same as the

not clear.

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previous reports [15, 30]. However, the mechanism of abnormal change is still

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IL-6/STAT3 is currently recognized as an important signaling pathway in the malignant transformation from inflammation to cancer. The activation of this signaling pathway promotes the transformation of colitis to colorectal cancer [31]. The previous study showed that SOCS3 protein could be induced under the stimulation of cytokines, which can be a negative feedback regulation to inhibit SOCS3 signaling pathway [32, 33]. SOCS3 protein

ACCEPTED MANUSCRIPT inhibited the activation of JAK/STAT3 signaling pathway by negative feedback regulation of tyrosine phosphorylation of STAT3, which inhibits the growth of tumor cells. To explore the relationship between IL-6, STAT3 and SOCS3, we examed the expression of IL-6, pSTAT3 and SOCS3 in DLD1, HT29 and

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SW480 treated by IL-6 and Stattic respectively. Our results demonstrated that inflammatory cytokines IL-6 can promote the expression of STAT3 signaling pathways and STAT3 activation promoted hypermethylation of SOCS3 gene

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promoters. 5-Aza-cdR treatment can reverse IL-6/STAT3 signaling pathway mediated down-regulation of SOCS3 in colorectal cancer cells. We also

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observed that phosphorylated STAT3 suppressed SOCS3’s transcription activity by dual luciferase report gene tests. Our findings showed firstly

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IL-6/STAT3 signaling activation negatively regulated SOCS3 expression.

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There have been no reports on the study of SOCS3 in large sale clinical

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samples of colorectal cancer. In our study, SOCS3 expression in CRC tissue was significantly lower than that in the paired normal tissues and was

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negatively correlated with lymph node metastasis and TNM staging of colorectal cancer. Kaplan-Meier analysis showed that SOCS3 expression, regional lymph node metastasis and TNM stage significantly affected the postoperative survival time of patients. On multivariate analysis, low expression of SOCS3 is an independent prognostic factor for OS in CRC after surgical resection. Our results provided evidence to some extent that SOCS3

ACCEPTED MANUSCRIPT might be a novel biomarker for molecular targeted therapy of colorectal cancer. SOCS3 plays a negative regulation in JAK/STAT signaling pathway, which prevented cell malignant transformation and promote the apoptosis of tumor cells. The activation and overexpression of SOCS3 have a potential role in the

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growth of tumor [6, 7]. Consistent with the studies, our findings showed that the overexpression of SOCS3 inhibited proliferation and promoted apoptosis in CRC cells. SOCS3 can inhibit the migration and invasion of hepatocellular

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carcinoma cells by down-regulation of FAK kinase activity besides the effects on tumor cell growth [34]. In the present study, the overexpression of SOCS3

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inhabited migratory and invasive abilities of CRC cells in vitro. Besides， Tumorigenicity assay also showed that the overexpression of SOCS3

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attenuates the tumor formation in vivo. All this results proved that SOCS3

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played an important role of inhibiting tumor progression.

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To our best knowledge, this is the first study that IL-6/STAT3 signaling activation negatively regulated SOCS3 expression, which affects the

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tumorigenesis and tumor progression in colorectal cancer. We also identified SOCS3 as a tumor suppressor gene that inhibits cell proliferation, migration, invasion, and Xenograft tumor formation. SOCS3 is an independent prognostic indicator of outcome for CRC patients and can be used as a biological molecular marker for the diagnosis and prognosis of CRC.

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CONFLICTS OF INTEREST

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The authors declare that they have no conflicts of interest.

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Stoye J, Hussey DJ, et al. Identification of the CIMP-like subtype and aberrant methylation of members of the chromosomal segregation and spindle assembly pathways in esophageal adenocarcinoma. Carcinogenesis. 2016; 37(4):356-365. 18. Zhang BG, Hu L, Zang MD, Wang HX, Zhao W, Li JF, Su LP, Shao Z, Zhao X, Zhu ZG, Yan M and Liu B. Helicobacter pylori CagA induces tumor

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AKT-NFkappaB pathway in gastric cancer development. Oncotarget. 2016; 7(9):9788-9800. 19. Elliott J. SOCS3 in liver regeneration and hepatocarcinoma. Molecular

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international journal of experimental cellular physiology, biochemistry, and

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pharmacology. 2016; 38(5):1796-1802. 22. Gordon P, Okai B, Hoare JI, Erwig LP and Wilson HM. SOCS3 is a

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ACCEPTED MANUSCRIPT Table 1 Association between SOCS3 expression and clinicopathological parameters in CRC.

Total

SOCS3 expression

P value

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Low（N=52） High(N=36) Sex 46

31

Female

42

21

Age 31

≥65

57

18 34

<5

CE 10

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Tumor size

≥5

0.0885 13 23 0.005*

42

36

10

0

PT

78

Moderate Poor

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Differentiation Well +

21

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<65

15

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Male

0.097

0.001*

47

20

27

41

32

9

Location

0.826

Right

37

15

12

Others

51

27

24

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Vascular

0.156

invasion No

84

51

33

Yes

4

1

3

T1+T2

9

7

T3+T4

79

45

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T stage

2

34

41

N1+N2

34

11

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54

M1

2

I +II

23 0.086

52

34

0

2 <0.001*

52

40

12

36

12

24

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III+IV

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TNM stage

<0.001*

13

PT

86

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M stage M0

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N stage N0

0.229

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Table 2 multivariate analysis of different prognostic parameters in 88 CRC patients. Multivariate analysis 95% CI

Vascular invasion

0.025*

1

Yes

2.653

2.352-2.843

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No

1

High

0.576

N stage

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1

M0 M1

4.324

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M stage

<0.001*

2.013-6.940

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N1+N2

0.483-0.670

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Low

<0.001*

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SOCS3

N0

P value

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HR

<0.001*

1

20.465

7.039-189.74

TNM stage

<0.001*

I +II

1

III+IV

47.863

16.703-113.79

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Figure 1. Representative photograph of IL-6, p-STAT3 and SOCS3 expression in normal colorectal and colorectal cancer specimens. Strong immunostaining

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of IL-6 and p-STAT3 in primary colorectal cancer is noted. However, strong immunostaining of SOCS3 in normal colorectal tissues is noted. Original

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magnification 100×.

Figure 2. Expression of p-STAT3, STAT3 and SOCS3 in colorectal cancer and

SOCS3

protein

expression

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adjacent normal tissues. Western blotting analysis of p-STAT3, STAT3 and in

four

representative

paired

colorectal

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tumor/normal tissue pairings, actin is used as the loading control.

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Figure 3. The effect of IL-6 and Stattic on SOCS3 in colorectal cancer cell lines by Western blotting. The expression of p-STAT3 increased, while the

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expression of SOCS3 decreased in DLD1 and HT29 cells treated by IL-6 (100ng/ml). It is the opposite in the SW480 treated by Stattic.

Figure 4. A Kaplan-Meier analysis with a log rank test of overall survival in patients according to levels of SOCS3 as determined by immunohistochemial staining. OS rates of patients with SOCS3-positive primary tumors is

ACCEPTED MANUSCRIPT significantly higher than that of patients with SOCS3-negative primary tumor (log-rank test, P<0.001).

Figure 5. SOCS3 induces colorectal cancer cell proliferation inhibition and cell

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apoptosis. A, B: The CCK8 is used to examine the effect of SOCS3 on cell growth. C, D: The flow cytometry is used to examine the effect of SOCS3 on

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cell apoptosis.

Figure 6. Influence of SOCS3 expression on colorectal cancer cell migration

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and invasion. The wound healing migration assay were carried out in HT29 transfected with SiSOCS3 and SW480 transfected with SOCS3 expression

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vector. Transwell invasion assay with matragel were performed in HT29 and

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SW480 cells. Magnification, 200×.

Figure 7. SOCS3 suppresses proliferation of colorectal cancer cells in vivo.

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Tumor volume and weight of xenograft tumors in nude mice were harvested in different treatment groups.

Figure S1. STAT3 represses SOCS3 expression through methylation modification. A. The SOCS3 gene promoter methylation status was analyzed in DLD1 and HT 29 cells after IL-6 (100ng/ml) and SW480 after Stattic (10μM)

ACCEPTED MANUSCRIPT treatment for 24h using MSP. B. 5-AZA-CdR reversed the expression of IL-6 mediated SOCS3 in HT29 cell lines. C. HT29 and DLD1 cells were transfected with reporter plasmid. Then, 24h after the transfection, the cells were treated with IL-6 (100ng/ml and 200ng/ml) or 5-AZA (5μM) for 48h, and the luciferase

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activity was measured.

Figure S2. SOCS3 knockdown in HT29 was efficient after transfection with

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siSOCS3, and protein level of SOCS3 was effectively enhanced after

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transfection with expression vector in SW480 compared to Scramble group. Protein expression levels of MMP-2 and MMP-9 were analyzed in HT29 and

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SW480 cells 48 h after transfection using western blots.

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Figure 1.

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Figure 3.

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Figure 2.

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Figure 4.

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Figure 5.

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Figure 6.

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Figure 7.

ACCEPTED MANUSCRIPT Abbreviations

IL-6：Interleukin 6 JAK: Janus Kinase

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STAT3：Signal Transducer and Activator of Transcription 3 SOCS3: Suppressor Of Cytokine Signaling 3

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CRC: Colorectal cancer

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Highlights

SOCS3 was down-regulated in CRC tissues. IL-6 can promote the

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expression of STAT3 while inhibit SOCS3 by promoting methylation of SOCS3 gene promoters. Patients with high expression of SOCS3 often indicated a relatively good prognosis. Overexpression of SOCS3 inhibited proliferation,

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migration, invasion and tumorigenic ability of CRC cells while increased cell apoptosis. This study demonstrated that IL-6/STAT3 signaling activation

sustained

activation

of

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negatively regulated SOCS3 expression, which led to imbalance and STAT3

signaling

pathway.

Thus,

targeting

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IL-6/STAT3/SOCS3 signaling pathway may become an important treatment

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strategy of colorectal cancer.