G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in renal cell carcinomas

G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in renal... The chronic inflammatory microenvironment within or surrounding the primary renal cell carcinoma (RCC) site promotes oncogenic transformation as well as contributes to the development of metastasis. G3BP stress granule assembly factor 1 (G3BP1) was found to be involved in the regulation of multiple cellular functions. However, its functions in RCC have not been previously explored. Here, we first showed that the expression of G3BP1 is elevated in human RCC and correlates with RCC progression. In cultured RCC cells, knockdown of G3BP1 results in inhibition of tumor cell proliferation, migration, and invasion, consistently with the alteration of epithelial–mesenchymal transition (EMT) and cell proliferative markers, including Cadherins, Vimentin, Snail, Slug, c-Myc, and cyclin D1. Remarkably, knockdown of G3BP1 dramatically impaired the signaling connection of pro-inflammatory cytokine IL-6 stimulation and downstream STAT3 activation in RCC, thus eventually contributing to the disruption of IL-6-elicited RCC migration and metastasis. In addition, in vivo orthotopic tumor xenografts results confirmed that knockdown of G3BP1 suppressed RCC tumor growth and metastasis in mice. Collectively, our findings support the notion that G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in RCC. Introduction metastatic disease is already present in about one-third of Renal cell carcinoma (RCC) is the most common solid cases at the time of diagnosis . Clear cell RCC is the most cancer of the adult kidney and accounts for ~90% of prevalent subtype of RCC. Its characteristic high meta- kidney neoplasms . More than 350,000 people are diag- static potential and resistance to traditional radiotherapy nosed with renal cell cancer worldwide, and an estimated and chemotherapy present a major challenge for mana- 2 3,4 140,000 people die from the disease each year . Many ging the disease . Although surgical intervention fol- cases of RCC are asymptomatic until the condition lowed by immunotherapy has emerged a major becomes malignant. As a result, local invasion or therapeutic option for RCC with metastasis, it has failed to demonstrate clear benefits as a therapeutic strategy for 3,5 the overall survival of RCC patients . The identification Correspondence: Hua Geng (huahuageng@gmail.com)or of molecular targets modulating RCC progression and Dan Yue (danyue0705@sina.cn) metastasis would provide useful information for tailoring Department of Urology, The Second Hospital of Tianjin Medical University, targeted treatments for patients with advanced RCC . Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300211, China Department of Microbiology, School of Medical Laboratory, Tianjin Medical The chronic inflammatory microenvironment is impli- University, Tianjin 300203, China cated to trigger cellular events that induce oncogenic Full list of author information is available at the end of the article These authors contributed equally: Yong Wang, Donghe Fu. Edited by A. Stephanou © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Wang et al. Cell Death and Disease (2018) 9:501 Page 2 of 13 STAT3 signaling in RCC metastasis will be important for uncovering the novel molecular targets for RCC immunotherapy. G3BP stress granule assembly factor 1 (G3BP1, also known as GTPase-activating protein SH3 domain-binding protein 1), is an RNA-binding protein involved in the regulation of multiple cellular functions . Previous stu- dies showed that G3BP1 regulates mRNA stability in response to extracellular stimuli, and plays an important 19–22 role in stress granule (SG) formation . In addition to its RNA-binding activity, G3BP1 promotes S-phase entry and controls cell proliferation in fibroblast . Further- more, G3BP1 regulates cell apoptosis through interaction 24,25 with p53 and affecting its cellular translocation . More recently, the overexpression of G3BP1 has been impli- cated in human cancers, including breast, gastric, colon, and liver carcinomas, suggesting the oncogenic and 26–29 functional role of G3BP1 in tumorigenesis . However, it remains unknown whether and how G3BP1 contributes to RCC progression and metastasis. In this report, we explored the expression of G3BP1 in primary RCC and its association with clinicopathological parameters. Functionally, we investigated the effects of G3BP1 on RCC cell proliferation, migration, and invasion in vitro and in vivo. We further defined the role of G3BP1 in linking inflammatory cytokine IL-6 with the activation of STAT3 signaling in RCC. Fig. 1 The expression of G3BP1 is increased in RCC. a G3BP1 expression in 16 pairs of primary RCC (T) and adjacent normal kidney Result tissues (N) were examined by Western blot. Upper panel: G3BP1 is frequently upregulated in RCC patients representative four pairs of Western blot images. Lower panel: the We first examined the expressions of G3BP1 in the 16 expression of G3BP1 was quantified by normalizing with β-actin, and all 16 pairs of RCC patient samples were analyzed using a pairs of RCC tissues and their corresponding adjacent paired t-test, p = 0.012. b G3BP1 expression was examined by normal kidney tissues by Western blotting. The results immunohistochemistry staining in a study cohort with 43 pairs of RCC showed that G3BP1 expression was significantly (p = (tumor) and adjacent normal kidney tissues (para-tumor). 0.012) increased in the RCC tissues when compared Representative G3BP1 staining of RCC and paired non-tumor sections with adjacent non-cancerous kidney tissues (Fig. 1a). To were shown, and brown signal indicated the G3BP1-positive staining. further investigate the correlation of G3BP1 expression with RCC clinicopathologic features, a cohort comprising of 43 RCC patients were subjected to immunohisto- 7,8 transformation of cells and distal metastasis . Cytokines chemical (IHC) staining with an antibody specifically are pivotal players of the tumor microenvironment that against G3BP1 (Fig. 1b). All slides were reviewed by may be contributing towards RCC pathogenesis. Inter- two pathologists at double-blinded manner for IHC leukin 6 (IL-6) is one of the most studied cancer- scoring based on the percentage and intensity of 30,31 associated cytokines, and elevated levels of IL-6 have G3BP1 staining signals . Consistent with Western been found in primary RCC cultures, RCC cell lines, as blotting results, the expression level of G3BP1 was sig- 9–12 well as in the serum from RCC patients . Primarily, IL- nificantly higher in 51.2% (22/43) of the RCC samples, in 6 activates signal transducer and activator of transcription contrast, G3BP1 expression was dramatically reduced to 3 (STAT3) signaling thus promotes tumor cell prolifera- 25.6% (11/43) in para-tumor normal tissues (p = 0.0147, tion and enhances cell invasiveness in cancers, which is in OR = 3.048, 95% CI 1.227–7.568), suggesting the line with the constitutive activation of STAT3 in RCC, expression level of G3BP1 in RCC tumors was markedly 13,14 especially in metastatic disease . Recently, blockade of elevated as compared with the adjacent normal tissues. the IL-6/STAT3 pathway was considered as a potential Further analysis revealed that the expression level of 15–17 therapeutic approach for RCC treatment . Thus, fully G3BP1 in RCC patients is significantly (p < 0.05) asso- understanding the role and mechanism of IL-6/ ciated with higher levels of TNM stages and Fuhrman Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 3 of 13 Table 1 Correlation between G3BP1 expression and functional studies (Fig. 2a and Suppl Fig. 1). The efficiency clinicopathologic characteristics in RCC patients of G3BP1 knockdown was confirmed at both mRNA and protein levels by quantification of qRT-PCR G3BP1 expression (Supplementary Fig. 1A) and Western blot (Suppl Fig. 1B), respectively. G3BP1 knockdown cells expressed N − + p Value <35% of detectable G3BP1 as compared to scramble Gender Male 29 12 17 0.159 control cells were qualified as G3BP1 knockdown and used for further experiments. The effects of G3BP1 on Female 14 9 5 RCC cell proliferation was then examined using CCK8 Age <60 16 8 8 0.907 assay. The results indicated downregulation of ≥60 27 13 14 G3BP1 significantly (p < 0.01) inhibited cell proliferation Tumor size <4 6 4 2 0.188 in both ACHN and A498 cells (Fig. 2b). Further transwell assay showed that G3BP1 knockdown strongly reduced >4, ≤76 1 5 the migrated RCC cell number when compared to the >7 31 16 15 scramble controls (Fig. 2c). Together, these data sug- TNM stage T1–2 25 20 5 0.000* gested that G3BP1 plays an important role in RCC cell T3–418 1 17 proliferation and migration in vitro. Since enhanced tumor cell migratory capacity is one of the characteristics Fuhrman grade I, II 27 17 10 0.027* of tumor metastasis, further tests based on loss-of- III, IV 16 4 12 function approach were used to examine the effects of Location Tumor 43 21 22 0.015* G3BP1 on RCC tumor cell mesenchymal phenotypes. Para-tumor 43 32 11 The results showed that G3BP1 downregulation led to the decrease of tumor cell invasion in the Matrigel inva- RCC subtype ccRCC 38 19 19 1.000 sion assay (Fig. 2d). Moreover, it was known that Non-ccRCC 5 2 3 EGFR and its ligand EGF are frequently overexpressed in Tumor side Left 19 12 7 0.095 RCC, and its signaling cascade is critical for RCC metas- 33–35 Right 24 9 15 tasis . Specifically, we found that EGF-induced che- Necrosis Negative 15 5 10 0.137 motaxis, a pivotal event for RCC metastasis, was significantly (p < 0.01) impaired in G3BP1 knockdown Positive 28 16 12 cells when compared to controls as indicated by micro- Note: G3BP1 expression (−/+) was determined by IHC staining score. The Boyden chamber assays (Fig. 2e). Collectively, these scoring system was composed of two independent parameters: percentage of results indicated that G3BP1 contributes to promoting tumor cells exhibiting positive staining (0–5: 0–1%; 1–5%; 6–10%; 11–20%; 21–50%; >50%); and intensity of IHC staining (0–2: 0, no staining; yellow-brown RCC cell proliferative, migratory, and invasive capacities, staining; strong brown staining). The final scores of two parameters were added thereby facilitating the transitioning of tumor mesenchy- to a sum and defined as the IHC staining score. 0–3: Negative; 4–7: Positive. *p < 0.05 indicates statistical significance by χ test. mal phenotype. Knockdown of G3BP1 inhibited expressions of cell proliferative and EMT markers in RCC grade (Table 1). Meanwhile, no significant (p > 0.05) To further investigate the role of G3BP1 in RCC pro- associations were identified between the expression of liferation and metastasis, G3BP1 was knocked down in G3BP1 and any other clinicopathological characteristics, ACHN and A498 RCC cells, and the expression levels of including patient’s age, gender, RCC subtype, tumor size, cell proliferative and epithelial–mesenchymal transition tumor side, and necrosis (Table 1). Taken together, our (EMT) markers were examined by Western blot. As findings suggest that the expression of G3BP1 is increased shown in Figure 3, the expressions of c-Myc and cyclin D1 in RCC and its expression level is associated with the were downregulated in G3BP1 knockdown RCC cells, progression of RCC. consistent with the inhibition of cell proliferation in shG3BP1 cells (Figs. 2b, 3a, b). In addition, the expression Silencing of G3BP1 represses the mesenchymal phenotype of the epithelial cell–cell adhesion molecule E-cadherin in RCC cells in vitro was upregulated. Consistently, the expressions of In order to investigate the role of G3BP1 in RCC pro- mesenchymal markers N-cadherin, Vimentin, Snail, and gress and metastasis, two RCC cell lines (ACHN and Slug were dramatically decreased in G3BP1 knockdown A498) with different potential for metastasis were selected cells (Fig. 3a, b). Thus, it is suggested that downregulation as in vitro cell models . RCC cells with lentivirus- of G3BP1 suppresses not only cell proliferation but also mediated G3BP1 stable knockdown were used for EMT in RCC cells. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 4 of 13 Fig. 2 Knockdown of G3BP1 represses RCC mesenchymal phenotype in vitro.a ACHN and A498 cells were transduced with lentivirus-mediated G3BP1-specific shRNA (shG3BP1) or scramble control (Scr), and the efficiency of G3BP1 knockdown was examined by Western blot. b ACHN and A498 cells with G3BP1 knockdown (shG3BP1) or scramble control (Scr) were cultured in growth medium and cell proliferation was determined by CCK8 assay at indicated time points, **p < 0.01 by two-way ANOVA. c Transwell assay was performed to determine the cell migration. Left panel: representative microscopic images, Naïve: untreated; Scr: scramble control; shG3BP1: G3BP1 knockdown. Right panel: quantitative analysis of cell migration, data were presented as mean ± s.d., n = 10 (ACHN) and n = 6 (A498) respectively, **p < 0.01 by one-way ANOVA, NS: no significant difference. d Matrigel cell invasion assay was used to evaluate the cell invasive ability. Left panel: representative microscopic images, Naïve: untreated; Scr: scramble control; shG3: G3BP1 knockdown. Right panel: quantitative analysis of cell invasion, data were presented as mean ± s.d., n = 10 (ACHN) and n = 6 (A498) respectively, **p < 0.01 by one-way ANOVA, NS: no significant difference. e G3BP1 knockdown impaired EGF-induced chemotaxis. Scr: scramble control; shG3BP1: G3BP1 knockdown. Data were presented as mean ± s.d. from three independent repeats, **p < 0.01 by two-way ANOVA. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 5 of 13 expression in scramble RCC cells. In contrast, IL-6- induced STAT3 phosphorylation was dramatically abol- ished in G3BP1 knockdown cells (Fig. 4b). In addition, we found that treatment with stattic, a specific inhibitor for STAT3 activation and dimerization, exhibited a profound effect on STAT3 phosphorylation, with no effect on the expression of G3BP1 in ACHN cells, suggesting STAT3 acts as a G3BP1 downstream signaling molecule (Fig. 4c). Together, the data firmly indicated that knockdown of G3BP1 blocked IL-6-induced STAT3 activation, and G3BP1 is a bridge molecule linking IL-6 and STAT3 signaling in RCC cells. To verify the functional impact of the IL-6/G3BP1/ STAT3 signaling axis in the regulation of EMT events in RCC, we next evaluated the effects of G3BP1 depletion on RCC cell migration and invasion with presence or absence of IL-6. The results revealed that treatment of IL-6 (100 ng/ml) significantly (p < 0.01) promoted tumor cell migration (Fig. 4d) and invasion (Fig. 4e) in RCC ACHN Fig. 3 Knockdown of G3BP1 suppressed expressions of cell cells, while G3BP1 depletion strongly attenuated the IL-6- proliferative and EMT markers in RCC cells. ACHN (a) and A498 (b) induced enhancement of RCC cell migration and invasion cells were transduced with lentivirus-mediated G3BP1-specific shRNA (shG3BP1) or scramble control (Scr). Western blot analysis was (Fig. 4d, e). Thus, these results functionally indicated that conducted to determine the target molecule expressions of cell IL-6 increased RCC cell migrating and invasive abilities, proliferative markers (c-Myc and cyclinD1) and EMT markers (E- which could be declined at least partially by the knock- cadherin, N-cadherin, vimentin, snail, and slug). down of G3BP1. Knockdown of G3BP1 impaired IL-6-induced STAT3 Expression of G3BP1 is correlated with IL-6 and p-STAT3 in activation and led to suppression of RCC migration and primary RCC patients invasion Given the significance of G3BP1 in controlling IL-6/ To further explore the underlying molecular mechan- G3BP1/STAT3 signaling in RCC migration and metas- ism of G3BP1-mediated RCC EMT, we first searched for tasis in vitro, we next address the correlations of G3BP1 the oncogenic signaling pathways that can be affected by with IL-6 and STAT3 activation in our clinical study G3BP1 silencing, including STAT3, AP1, ISRE, TGFβ cohort comprising of 32 pairs of primary RCC and their (p3TP), P53, NFAT, and WNT (TOPFlash) using corresponding adjacent normal kidney tissues. Western pathway luciferase reporter system in RCC cells (Suppl blotting showed that the expressions of G3BP1, IL-6, and Fig. 2). The results strongly suggested that G3BP1 p-STAT3 were significantly (p < 0.05) elevated in RCC knockdown caused significant impairments of multiple tissues as compared to those in the corresponding adja- tumorigenic-related signaling pathways in RCC cells, cent normal kidneys (Fig. 5). In addition, the expression of including STAT3, AP1, and interferon (ISRE) signaling G3BP1 is positively correlated with IL-6 (r = 0.510, p = (Fig. 4a and Suppl Fig. 2). Evidence indicated that in 0.003) and p-STAT3 (r = 0.683, p < 0.0001) in RCC tis- response to extracellular insults, STAT3 is activated sues (Table 2), suggesting the associations of IL-6/G3BP1/ through tyrosine phosphorylation at Tyr705, and this STAT3 molecules in primary RCC. constitutive activation of STAT3 is a pivotal event in RCC 37,38 tumorigenesis and metastasis . Further Western blot- G3BP1 knockdown inhibited RCC tumor growth and ting results confirmed that G3BP1 silencing led to inhi- metastasis in vivo bition of STAT3 phosphorylation in RCC cells (Fig. 4b). To determine whether G3BP1 knockdown impacts RCC Specifically, IL-6 is one of the well-documented cytokines tumor growth and metastasis in vivo, we performed the which is abundantly expressed in RCC and enhances RCC orthotopic tumor xenografts with implantation of cell proliferation and invasiveness through activation of luciferase-labeled G3BP1 knockdown cells (ACHN- 13,14 STAT3 signaling . In line with this notion, we further shG3BP1-luciferase) and control RCC cells (ACHN-Scr- treated G3BP1 knockdown and scramble control cells luciferase) into the sub-renal capsule of adult nude mice with human recombinant IL-6 (100 ng/ml) for 48 h, and left kidney. After 8 weeks, mice were intraperitoneally Western blotting results revealed that stimulation with injected with luciferin and the bioluminescence intensity IL-6 did not only activate STAT3 but also induced G3BP1 was detected and quantified for the primary tumors as Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 6 of 13 Fig. 4 Knockdown of G3BP1 impaired IL-6-induced STAT3 activation and led to the suppression of RCC migration and invasion. a ACHN and A498 cells with stably knockdown of G3BP1 (shG3BP1) or scramble control (Scr) were co-transfected with STAT3 pathway firefly luciferase reporter (STAT3-luc) together with internal control Renilla luciferase reporter (pRL-TK) vector. Forty-eight hours after transfection, cell lysates were subjected to dual-luciferase assay. Data were obtained from three independent repeats and presented as mean ± s.d., **p < 0.01 by Student’s t-test. b ACHN cells with stably knockdown of G3BP1 (shG3) or scramble control (Scr) were treated with or without human recombinant IL-6 (100 ng/ml) for 48 h. The expressions of G3BP1, p-STAT3 (Tyr705), and total STAT3 were examined by Western blot. c ACHN cells were treated with STAT3 inhibitor stattic at the indicated concentration for 48 h, and expressions of G3BP1 and p-STAT3 (Tyr705) were determined by Western blot. d, e ACHN cells with stably knockdown of G3BP1 (shG3BP1) or scramble control (Scr) were treated with or without human recombinant IL-6 (100 ng/ml) for 48 h, and then (d) Transwell assay was performed to determine the cell migration and (e) Matrigel cell invasive assay was used to examine the cell invasion, respectively. Left panel: representative microscopic images. Right panel: quantitative analysis. Data were presented as mean ± s.d. from three independent experiments, and five random microscopic fields were acquired in each experiment for quantification, **p < 0.01 by one way ANOVA followed by Tukey’s post hoc test, NS: no significant difference. well as lung and liver metastasis using the live biolumi- Additionally, the primary RCC tumor and metastatic nescence IVIS imaging system. The results showed that a organs of liver and lung were isolated from the afore- significant (p < 0.01) reduction of primary RCC tumor size mentioned mice. The primary renal tumor volumes were was observed in mice with G3BP1 knockdown as com- measured to be dramatically decreased in the G3BP1 pared to the scramble control group (Fig. 6a, b), sug- knockdown group (Fig. 6c), and further IHC staining of gesting G3BP1 plays an important role in RCC tumor the xenograft renal tissues confirmed the knockdown of growth in vivo. In addition, bioluminescent signals indi- G3BP1 expression in the ACHN-shG3BP1 group cated that mice implanted with G3BP1 knockdown (Fig. 6d). Moreover, H&E staining of the excised livers and ACHN cells had much fewer liver and lung metastatic foci lungs clearly demonstrated that significantly (p < 0.01) detected than those in scramble controls (Fig. 6e, f, i, j), more metastatic nodules were found in the scramble thus revealing the functional role of G3BP1 in RCC control group than the G3BP1 knockdown group (Fig. 6g, metastasis. h, k, l). Collectively, the results from in vivo tumor Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 7 of 13 that knockdown of G3BP1 led to inhibition of tumor cell proliferation, migration, and invasion, as well as altera- tions of EMT markers using in vitro RCC cell line model. Consistently, our in vivo orthotopic tumor xenografts results also confirmed that knockdown of G3BP1 suppressed RCC tumor growth and metastasis in mice. Remarkably, we uncovered a novel signaling link that G3BP1 mechanistic connects upstream pro- inflammatory cytokine IL-6 stimulation to STAT3 acti- vation, and eventually contributes to downstream cellular events, including promotion of RCC migration and metastasis. Together, our findings support a notion that G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in RCC. G3BP1, based on its crucial involvement in multiple biological processes and disease pathogenesis, including tumorigenesis, has emerged a target for molecular inves- Fig. 5 Expression of G3BP1 is correlated with IL-6 and p-STAT3 in 24,26,29 tigation in many oncology studies . However, there primary RCC patients. The expressions of G3BP1, IL-6, and p-STAT3 (Tyr705) in a panel of 32 pairs of primary RCC (T) and adjacent normal is still limited knowledge about its correlation with pro- kidney tissues (N) were examined by Western blot. Upper panel: gression of RCC. Overexpression of G3BP1 has been representative results from six pairs of patient samples were shown. shown to promote breast cancer cells proliferation and Lower panel: the expressions of G3BP1, p-STAT3 (Tyr705), and IL-6 induce EMT transition . Also, knockdown of G3BP1 was were quantified by normalizing with β-actin, and all 32 pairs of RCC reported to prevent tumor invasion and metastasis in patient samples were analyzed using a paired t-test. 26,29 xenograft model . Recently, Dou et al. reported that upregulation of G3BP1 was associated with enhanced cell Table 2 Correlation between G3BP1 and IL-6/pSTAT3 migration and poor survival of hepatocellular carcinoma expressions in RCC (HCC), with no significant effect on HCC cell growth . Similarly, our results demonstrated that G3BP1 was fre- G3BP1 expression Spearman’s r quently upregulated at protein levels in primary RCC Up Down Correlation p Value compared to adjacent normal tissues, and a higher level of coefficient G3BP1 expression is correlated with RCC progression. In addition, we analyzed but failed to detect the correlation IL-6 Up 21 2 0.510 0.003* of G3BP1 mRNA expression and RCC survival using The Down 4 5 Cancer Genome Altas (TCGA) database (data not shown). This suggests that G3BP1 might exert its tumorigenic pSTAT3 Up 20 0 0.683 <0.0001* function mainly at protein level, and the post- Down 5 7 transcriptional regulation of G3BP1 might be crucial for Note: The expressions of G3BP1, IL-6 and pSTAT3 were quantified and compared its role in RCC. Indeed, it remains possible that the between RCC tumor (T) and paired adjacent normal tissue (N). The relative ratio expression of G3BP1 displays a tissue-specific or cancer T/N > 1 was defined as “Up”, and T/N < 1 was defined as “Down”. *p < 0.05 indicates statistical significance by Spearman’s correlation coefficient type-specific manner. Functionally, our results demon- analysis. strated that G3BP1 plays an important role in RCC cell proliferation, migration, invasion, and metastasis both xenografts model indicated that silencing of G3BP1 could in vitro and in vivo, suggesting that G3BP1 is a critical suppress RCC tumor cell growth as well as inhibit RCC molecule in RCC progression. It is well established that cell metastasis to liver and lung. the majority of RCC arise from renal tubule epithelial cells, and EMT is a key event that occurs during the 38,39 Discussion invasion of cancers with an epithelial origin . During RCC frequently involves systemic metastasis which is EMT, cell–-cell and cell-matrix interactions are altered to the major cause of RCC mortality. However, the mole- facilitate tumor cell motility. In this content, the loose of cular basis of RCC progression remains elusive. In the adherens junction by “cadherin switching” is one of the present study, we revealed that the expression of G3BP1 is hallmark events in EMT, which is characterized by the increased in primary RCC as compared to adjacent nor- replacement of E-cadherin with N-cadherin . We found mal tissues, and G3BP1 is associated with higher levels of that G3BP1 depletion is associated with upregulation of TNM stages and Fuhrman grade. Functionally, we found E-cadherin and downregulation of N-cadherin, as well as Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 8 of 13 Fig. 6 G3BP1 knockdown inhibited RCC tumor growth and metastasis in vivo. ACHN cells were transduced with Luciferase-labeled G3BP1 knockdown (shG3BP1-luc) or scramble control (Scr-luc) were used to implant into nude mice for 8 weeks, n = 6 each group. The bioluminescence was detected by IVIS imaging system, and followed by histological examination. a Tumor growth was examined by IVIS imaging system, and representative images were shown. b Tumor growth was quantified by bioluminescence intensity. c Primary tumor volume was measured and compared. d Mouse primary renal tumors were collected and processed for IHC staining of G3BP1, and representative images were shown. e–l Liver and lung metastases were examined by IVIS bioluminescence imaging system and histological H&E staining for detection of tumor foci. e, i Tumor metastases in liver (e) and lung (i) were examined by IVIS imaging system, and representative images were shown. f, j Quantified bioluminescence in liver (f) and lung (j) metastatic sites were calculated and compared. g, k Tumor metastatic foci were evaluated by H&E staining in liver (g) and lung (k), and representative images were shown. h, l Tumor metastatic foci in liver (h) and lung (l) were quantified and compared using five random views under the microscope. All data were presented as mean ± s.e.m, n = 6, **p < 0.01 by Student’s t-test. repression of mesenchymal marker vimentin, a type III above-mentioned results from in vitro experiments, we intermediate filament related with cell-matrix interaction. also confirmed the G3BP1-mediated regulation of RCC Consistently, the expressions of E-cadherin repressors, tumor growth and metastasis using xenograft model Snail, and Slug were decreased in G3BP1 knockdown in vivo. Taken together, G3BP1 is an important molecule RCC cells. Our data clearly indicated that G3BP1 was not only involved in controlling tumor cell growth, but indeed involved in regulating EMT in RCC. Besides the also in mediating EMT in RCC. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 9 of 13 Previously, G3BP1 was reported to interact with the migration and invasion, suggesting depletion of G3BP1 SH3 domain of RasGAP and modulate Ras signaling functionally blocks the IL-6/STAT3 signaling axis and pathway . Recently, Annibaldi et al. questioned G3BP1 diminishes the RCC metastatic potential. Importantly, we being a genuine RasGAP-binding partner and suggested found that IL-6 itself can induce the G3BP1 expression in G3BP1-mediated signaling may not involve RasGAP .In RCC cells. This further supports the notion that G3BP1 is addition, G3BP1 has been shown to be involved in lung a critical mediator that links IL-6 to STAT3 activation in RCC. In line with this finding, we showed the positive cancer cell migration and metastasis via Src/FAK signal- ing pathway . In breast cancer, G3BP1 participated in the correlations between the expression of G3BP1 with IL-6 EMT and metastasis through regulating Smad signaling . and p-STAT3 in our primary RCC cohort. Collectively, In our current study, we screened several oncogenic sig- oncogenic G3BP1 serves as a signaling linkage molecule naling pathways and identified STAT3, AP1, and type 1 from upstream IL-6/EGF elicited stimulations to down- interferon (ISRE) signaling pathways were dramatically stream STAT3 signaling, eventually contributing to pro- impaired by a G3BP1 knockdown in RCC cells, indicating mote tumor cell growth and metastasis in RCC. that G3BP1 is a pivotal joint molecule connecting multi- In summary, the expression of G3BP1 is significantly ple signaling pathways. It was also suggested that higher in RCC comparing to para-tumor normal tissues, G3BP1-associated signaling pathways are diverse in can- and knockdown of G3BP1 significantly decreased tumor cers, and more importantly, to be cancer type specific. cell growth and metastasis in vitro and in vivo. In addition, Consistently, we verified that both c-Myc and cyclin D1 IL-6 can induce the expression of G3BP1, while G3BP1 as cell proliferation-associated downstream targets of depletion diminishes the IL-6-elicited STAT3 activation AP1 signaling molecules repressed in G3BP1 knockdown and leads to inhibition of tumor cell growth and invasion RCC cells. In addition, to be of particular interest is the in RCC. It was well known that RCC is resistant to con- STAT3 signaling. STAT3 signaling pathway plays critical ventional radiotherapy and chemotherapy, and immu- roles in tumor invasion and metastasis . In response to notherapies using a high dose of IL-2 and IFN-α are now 47–50 extracellular signals, STAT3 is activated by phosphoryla- applied for RCC treatment . Unfortunately, clinical tion of a conserved tyrosine residue and the phosphory- studies showed that the objective response rates are about lated STAT3 dimers undergo nuclear translocation where 5–20% for RCC patients who use IL-2 or IFN-α treatment 51,52 they regulate several “master” EMT transcriptional net- regimen . Thus, there is a pressing need to develop 38,44 works, including Snail and Slug . Remarkably, our new immunological targets for advanced treatment of 14,53–56 RCC results showed that the depletion of G3BP1 inhibited . Recently, several antibodies developed for STAT3 signaling, further suppressing the EMT pheno- targeting blockage of the IL-6/STAT3 pathway for RCC types via transcriptional repression of Snail and Slug. immunotherapy have been registered for preclinical stu- 14,57 This perhaps implies that G3BP1 functions through dies and phase I/II clinical trials . Our findings suggest STAT3 signaling and eventually contribute to RCC EMT. G3BP1 as a novel mediator of RCC tumor progression The crosstalk between tumor cells and their surround- and further extends the current knowledge about IL-6/ ing extracellular signals forms tumor microenvironment STAT3 associated mechanism in RCC carcinogenesis. and leads to activation of oncogenic signaling pathways, Indeed, targeting the molecules of IL-6/G3BP1/STAT3 including STAT3 signaling pathway. It was known that axis provides a novel and potential treatment strategy for 13,14,45 STAT3 can be robustly activated by IL-6 and EGF . RCC. It was documented that EGF and its receptor EGFR are frequently overexpressed in many forms of RCC, and Materials and methods signaling cascade through EGF/EGFR/STAT3 play an Cell culture, transfection, and treatment important role in tumor cell growth and EMT in RCC cell lines including A498 and ACHN were 33,35,46 RCC . In this study, we showed that the knockdown obtained from American Type Culture Collection of G3BP1 dramatically impaired EGF-induced RCC cell (ATCC). Cells were cultured in Eagle’s Minimum Essen- chemotaxis, an essential component of tumor dis- tial Medium (MEM) (Gibco) supplemented with 10% FBS semination during progression and metastasis, indicating (HyClone) and 1× penicillin/streptomycin at 37 °C in the EGF-mediated chemotaxis in RCC is probably via G3BP1. presence of 5% CO . All cultures were routinely tested for Recent studies have shown that IL-6 can be produced by absence of mycoplasma and for the retention of their RCC cells, and enhanced levels of IL-6 increases the respective morphology and growth characteristics. Spe- 9–12,14 invasiveness of RCC . Consistently, we demonstrated cific short hairpin RNA (shRNA) was designed to target in the present study, IL-6 can activate STAT3 and lead to human G3BP1 and cloned into pLKO.1 lenti-vector, enhanced RCC cell migration and invasion. Importantly, transfected into 293T cells together with lentivirus we found that G3BP1 knockdown can significantly abolish packaging plasmids, psAX2 and pMD2.G for 48 h using the effect of IL-6 on STAT3 activation, as well as RCC cell lipofectamine 2000 (Invitrogen). Lentivirus-containing Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 10 of 13 medium was cleared by centrifugation and filtration with normalized with the expression of internal control β- 0.45 µm filter and then added to culture medium of A498 actin. and ACHN cells for shRNA transduction. G3BP1 stably knockdown (shG3BP1) and scramble control cells (Scr) IHC staining were established with puromycin (10 μg/ml) selection for Tissues were formalin-fixed and embedded in paraffin 10 days. blocks and then cut into 5 µm thickness sections. RCC cells were treated with 100 ng/ml of human Tissue sections on glass slides were deparaffinized in recombinant IL-6 (Peprotech) for 48 h and subjected to xylene, and dehydrated in gradient ethanol, followed by cell migration and invasion assays while total proteins blocking of endogenous peroxidase activity in 3% hydro- were extracted for Western blotting analysis. Stattic is a gen peroxide. Slides were then submerged in boiling commercially available small molecule inhibitor of citrate buffer for antigen retrieval. UltraVision Protein STAT3 activation and dimerization (Selleck). ACHN cells Block (BD) was applied to block non-specific background were seeded in six-well plate and treated with stattic (0, 1, staining. The tissue sections were immunostained with 2.5, 4, 5.5, and 7 µM) for 48 h, and then total proteins an anti-G3BP1 antibody (Santa Cruz). After washing were extracted for Western blotting analysis. with PBS, the slides were immunoblotted using Max Vision HRP-Polymer anti-Rabbit IHC Kit (Miaxim.bio). Human patients Sections were developed by peroxidase substrate DAB Human patient study cohort collected 43 pairs of pri- Detection Kit (Miaxim.bio) and then counterstained by mary RCC tissues and corresponding para-carcinoma hematoxylin. normal tissues. All tissue samples were surgically removed For the scoring system, all slides immunostained with and paraffin-embedded in the Tianjin Medical University G3BP1 antibody were scored by two independent Second Hospital from January 2012 to December 2015 pathologists for the percentage and intensity of cells with patients’ consents and ethical committee approval. showing specific immunostaining signals. The percentage Clinical parameters including age, gender, tumor size, score grading criteria of 0–5 were as follows: 0, (0–1)% of histological type, and Fuhrman grade were collected. All tumor cells positive; 1, (1–5)% of tumor cells were posi- patients had undergone radical nephrectomy or partial tive; 2, (6–10)% of cells were positive; 3, (11–20)% of cells nephrectomy with no preoperative and postoperative were positive; 4, (21–50)% of cells were positive; and 5, adjuvant therapies. For RCC pathological classification, all >50% of cells were positive. The intensity of immunos- samples were double-blind reviewed by two independent taining level was determined by the subjective visual pathologists. scoring of the brown stain as follows: 0, no staining; 1, yellow-brown staining; 2, brown staining. The final score Protein extraction and Western blot was calculated by combining the percentage and intensity Cells were harvested and lysed in SDS lysis buffer with scores and recorded as negative (0–3) and positive 30,31 1× protease inhibitor cocktail and 1× phosphatase inhi- (4–7) . bitor cocktail (Roche). The concentration of total protein was determined by an Enhanced BCA Protein assay kit Cell proliferation assay (Thermofisher Scientific). Equal amounts of each protein Cells (1 × 10 per well) were plated in 96-well plates and sample (40 μg) were separated by electrophoresis on SDS- cultured as described above. Cell proliferation was PAGE and transferred onto polyvinylidene fluoride assessed at 24, 48, and 72 h after seeding using Cell membranes (Invitrogen). After blocking with DifcoTM Counting Kit-8 (CCK-8, Dojindo) according to the man- skim milk (Becton Dickinson), protein lysates were blot- ufacturer’s protocol. Briefly, 10 μl of the CCK-8 solution ted with anti-G3BP1 antibody (Santa Cruz, SC), anti- was added to each well of the plate and incubated at 37 °C pSTAT3 antibody (Cell Signaling Technology, CST), anti- for 1 h. The absorbance of each well was measured at 450 STAT3 antibody (CST), anti-IL6 antibody (Saierbio), anti- nm using a microplate reader. All experiments were E-cadherin antibody (SC), anti-Vimentin antibody (CST), performed in triplicates. anti-Snail antibody (CST), anti-Slug antibody (SC), anti- N-cadherin antibody (SC), anti-c-Myc antibody (SC), Cell migration assay anti-cyclinD1, or anti-β-actin antibody (SC), overnight at Cell migration was measured by transwell (8 μm pores, 4 °C, and followed by incubation of corresponding Corning) assay. Briefly, RCC cells (1 × 10 ) were seeded horseradish peroxidase-conjugated anti-mouse or anti- into the upper chamber in serum-free medium, and rabbit secondary antibodies at room temperature for 1 h. medium containing 10% FBS was added to the lower The blots were visualized using ECL system (Millipore). chamber. After 24 h of incubation at 37℃, the cells Images were then analyzed and quantified with G:box attached to the lower surface of the membrane were fixed (SYNGENE), and the expression of the target protein was with 4% PFA and stained with hematoxylin. Cell numbers Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 11 of 13 were counted in five randomly chosen fields under the detection for primary tumors and metastasis in liver and microscope. lung using the live IVIS imaging system (Perkin Elmer). The mice were then sacrificed, and primary RCC tumors Tumor cell invasion assay were isolated. Tumors were measured by largest and Transwell insert (8 μm pores, Corning) were pre-coated smallest diameters to calculate tumor volume. Primary with diluted growth factor-reduced matrigel (2.5 mg/ml, RCC tumors, livers, and lungs were also fixed in formalin and embedded in paraffin. Serial sections and H&E BD Biosciences). RCC cells (1 × 10 ) resuspended in serum-free MEM were seeded into the upper chamber, staining were then performed to examine metastasis. and MEM medium containing 10% FBS were added to the lower chamber. The cells were allowed to invade through Statistical analysis the membrane at 37 °C for 48 h. Non-migrating cells on All data were presented as the mean ± s.d. Differences in the upper surface were removed, and only those cells mean values between two groups were analyzed by t-test. attached to the lower surface of the membrane were fixed Differences in mean values among multiple groups and/or with 4% PFA and stained with hematoxylin, followed by multiple conditions were analyzed by one-way ANOVA counting in five randomly chosen fields under the or two-way ANOVA, followed by post hoc test. χ test was microscope. used for statistical analysis of the correlations between G3BP1 expression and clinicopathologic parameters. The Chemotaxis assay correlations between the expressions of G3BP1, IL-6, and Chemotaxis assay was performed using micro-Boyden p-STAT3 were analyzed using Spearman test. Differences chambers as described previously . Briefly, RCC cells with *p < 0.05 were considered as statistically significant. (2.5 × 10 ) were seeded into the upper chambers in Acknowledgements serum-free MEM, while 10% FBS and human recombi- We thanks Dr. Justin Eze Ideozu (Division of Pulmonary Medicine, nant EGF (Peprotech) with different concentrations were Northwestern University) for his scientific proofreading of the manuscript. This work was supported by the National Natural Science Foundation of China added to the lower chamber. The 8 μmof fibronectin- [grant number 81772945, 81402094, 81400321, and 81202100], the Scientific pretreated filter membrane was placed between two Research Foundation for the Returned Overseas Chinese scholars, Burea of chambers. After 12 h incubation at 37 °C, the membrane personnel of China, Tianjin, China [grant number 2016015], and Natural Science Foundation of Tianjin, China [grant number 15JCZDJC35400]. was fixed and stained with hematoxylin. The number of migrating cells was counted in five randomly chosen fields Author details under a light microscope. 1 Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300211, China. Department of Microbiology, School of Medical Laboratory, Tianjin Medical Pathway dual-luciferase reporter assay University, Tianjin 300203, China. Research Center of Molecular Biology, Inner The screening of signaling pathways by dual luciferase 4 Mongolia Medical University, Hohhot 010059, China. Research Center of Basic assay was carried out using a serial of pathway luciferase Medical Sciences, Tianjin Medical University, Tianjin 300070, China. Department of Pharmacology, Tianjin Medical University, Tianjin 300070, reporters (SABiosciences), including STAT3, AP1, ISRE, China. Department of Urology, Renji Hospital, School of Medicine in Shanghai TGFβ (p3TP), P53, NFAT, and WNT (TOPFlash). RCC Jiao Tong University, Shanghai 200127, China cells were co-transfected with pathway luciferase repor- Conflict of interest ters and the internal control Renilla luciferase reporter The authors declare that they have no conflict of interest. (pRL-TK) vector using lipofectamine 2000. Forty-eight hours after transfection, cells were harvested and analyzed by the dual-luciferase assay kit (Promega). Each experi- Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in ment was performed in triplicates, and at least three published maps and institutional affiliations. independent assays were conducted. Supplementary Information accompanies this paper at https://doi.org/ 10.1038/s41419-018-0504-2. Xenograft tumor growth and metastasis Lentivirus-mediated luciferase-labeled G3BP1 stably Received: 15 January 2018 Revised: 18 March 2018 Accepted: 20 March knockdown (ACHN-shG3BP1-luciferase) and scramble control cells (ACHN-Scr-luciferase) were used. 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G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in renal cell carcinomas

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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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Abstract

The chronic inflammatory microenvironment within or surrounding the primary renal cell carcinoma (RCC) site promotes oncogenic transformation as well as contributes to the development of metastasis. G3BP stress granule assembly factor 1 (G3BP1) was found to be involved in the regulation of multiple cellular functions. However, its functions in RCC have not been previously explored. Here, we first showed that the expression of G3BP1 is elevated in human RCC and correlates with RCC progression. In cultured RCC cells, knockdown of G3BP1 results in inhibition of tumor cell proliferation, migration, and invasion, consistently with the alteration of epithelial–mesenchymal transition (EMT) and cell proliferative markers, including Cadherins, Vimentin, Snail, Slug, c-Myc, and cyclin D1. Remarkably, knockdown of G3BP1 dramatically impaired the signaling connection of pro-inflammatory cytokine IL-6 stimulation and downstream STAT3 activation in RCC, thus eventually contributing to the disruption of IL-6-elicited RCC migration and metastasis. In addition, in vivo orthotopic tumor xenografts results confirmed that knockdown of G3BP1 suppressed RCC tumor growth and metastasis in mice. Collectively, our findings support the notion that G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in RCC. Introduction metastatic disease is already present in about one-third of Renal cell carcinoma (RCC) is the most common solid cases at the time of diagnosis . Clear cell RCC is the most cancer of the adult kidney and accounts for ~90% of prevalent subtype of RCC. Its characteristic high meta- kidney neoplasms . More than 350,000 people are diag- static potential and resistance to traditional radiotherapy nosed with renal cell cancer worldwide, and an estimated and chemotherapy present a major challenge for mana- 2 3,4 140,000 people die from the disease each year . Many ging the disease . Although surgical intervention fol- cases of RCC are asymptomatic until the condition lowed by immunotherapy has emerged a major becomes malignant. As a result, local invasion or therapeutic option for RCC with metastasis, it has failed to demonstrate clear benefits as a therapeutic strategy for 3,5 the overall survival of RCC patients . The identification Correspondence: Hua Geng (huahuageng@gmail.com)or of molecular targets modulating RCC progression and Dan Yue (danyue0705@sina.cn) metastasis would provide useful information for tailoring Department of Urology, The Second Hospital of Tianjin Medical University, targeted treatments for patients with advanced RCC . Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300211, China Department of Microbiology, School of Medical Laboratory, Tianjin Medical The chronic inflammatory microenvironment is impli- University, Tianjin 300203, China cated to trigger cellular events that induce oncogenic Full list of author information is available at the end of the article These authors contributed equally: Yong Wang, Donghe Fu. Edited by A. Stephanou © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Wang et al. Cell Death and Disease (2018) 9:501 Page 2 of 13 STAT3 signaling in RCC metastasis will be important for uncovering the novel molecular targets for RCC immunotherapy. G3BP stress granule assembly factor 1 (G3BP1, also known as GTPase-activating protein SH3 domain-binding protein 1), is an RNA-binding protein involved in the regulation of multiple cellular functions . Previous stu- dies showed that G3BP1 regulates mRNA stability in response to extracellular stimuli, and plays an important 19–22 role in stress granule (SG) formation . In addition to its RNA-binding activity, G3BP1 promotes S-phase entry and controls cell proliferation in fibroblast . Further- more, G3BP1 regulates cell apoptosis through interaction 24,25 with p53 and affecting its cellular translocation . More recently, the overexpression of G3BP1 has been impli- cated in human cancers, including breast, gastric, colon, and liver carcinomas, suggesting the oncogenic and 26–29 functional role of G3BP1 in tumorigenesis . However, it remains unknown whether and how G3BP1 contributes to RCC progression and metastasis. In this report, we explored the expression of G3BP1 in primary RCC and its association with clinicopathological parameters. Functionally, we investigated the effects of G3BP1 on RCC cell proliferation, migration, and invasion in vitro and in vivo. We further defined the role of G3BP1 in linking inflammatory cytokine IL-6 with the activation of STAT3 signaling in RCC. Fig. 1 The expression of G3BP1 is increased in RCC. a G3BP1 expression in 16 pairs of primary RCC (T) and adjacent normal kidney Result tissues (N) were examined by Western blot. Upper panel: G3BP1 is frequently upregulated in RCC patients representative four pairs of Western blot images. Lower panel: the We first examined the expressions of G3BP1 in the 16 expression of G3BP1 was quantified by normalizing with β-actin, and all 16 pairs of RCC patient samples were analyzed using a pairs of RCC tissues and their corresponding adjacent paired t-test, p = 0.012. b G3BP1 expression was examined by normal kidney tissues by Western blotting. The results immunohistochemistry staining in a study cohort with 43 pairs of RCC showed that G3BP1 expression was significantly (p = (tumor) and adjacent normal kidney tissues (para-tumor). 0.012) increased in the RCC tissues when compared Representative G3BP1 staining of RCC and paired non-tumor sections with adjacent non-cancerous kidney tissues (Fig. 1a). To were shown, and brown signal indicated the G3BP1-positive staining. further investigate the correlation of G3BP1 expression with RCC clinicopathologic features, a cohort comprising of 43 RCC patients were subjected to immunohisto- 7,8 transformation of cells and distal metastasis . Cytokines chemical (IHC) staining with an antibody specifically are pivotal players of the tumor microenvironment that against G3BP1 (Fig. 1b). All slides were reviewed by may be contributing towards RCC pathogenesis. Inter- two pathologists at double-blinded manner for IHC leukin 6 (IL-6) is one of the most studied cancer- scoring based on the percentage and intensity of 30,31 associated cytokines, and elevated levels of IL-6 have G3BP1 staining signals . Consistent with Western been found in primary RCC cultures, RCC cell lines, as blotting results, the expression level of G3BP1 was sig- 9–12 well as in the serum from RCC patients . Primarily, IL- nificantly higher in 51.2% (22/43) of the RCC samples, in 6 activates signal transducer and activator of transcription contrast, G3BP1 expression was dramatically reduced to 3 (STAT3) signaling thus promotes tumor cell prolifera- 25.6% (11/43) in para-tumor normal tissues (p = 0.0147, tion and enhances cell invasiveness in cancers, which is in OR = 3.048, 95% CI 1.227–7.568), suggesting the line with the constitutive activation of STAT3 in RCC, expression level of G3BP1 in RCC tumors was markedly 13,14 especially in metastatic disease . Recently, blockade of elevated as compared with the adjacent normal tissues. the IL-6/STAT3 pathway was considered as a potential Further analysis revealed that the expression level of 15–17 therapeutic approach for RCC treatment . Thus, fully G3BP1 in RCC patients is significantly (p < 0.05) asso- understanding the role and mechanism of IL-6/ ciated with higher levels of TNM stages and Fuhrman Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 3 of 13 Table 1 Correlation between G3BP1 expression and functional studies (Fig. 2a and Suppl Fig. 1). The efficiency clinicopathologic characteristics in RCC patients of G3BP1 knockdown was confirmed at both mRNA and protein levels by quantification of qRT-PCR G3BP1 expression (Supplementary Fig. 1A) and Western blot (Suppl Fig. 1B), respectively. G3BP1 knockdown cells expressed N − + p Value <35% of detectable G3BP1 as compared to scramble Gender Male 29 12 17 0.159 control cells were qualified as G3BP1 knockdown and used for further experiments. The effects of G3BP1 on Female 14 9 5 RCC cell proliferation was then examined using CCK8 Age <60 16 8 8 0.907 assay. The results indicated downregulation of ≥60 27 13 14 G3BP1 significantly (p < 0.01) inhibited cell proliferation Tumor size <4 6 4 2 0.188 in both ACHN and A498 cells (Fig. 2b). Further transwell assay showed that G3BP1 knockdown strongly reduced >4, ≤76 1 5 the migrated RCC cell number when compared to the >7 31 16 15 scramble controls (Fig. 2c). Together, these data sug- TNM stage T1–2 25 20 5 0.000* gested that G3BP1 plays an important role in RCC cell T3–418 1 17 proliferation and migration in vitro. Since enhanced tumor cell migratory capacity is one of the characteristics Fuhrman grade I, II 27 17 10 0.027* of tumor metastasis, further tests based on loss-of- III, IV 16 4 12 function approach were used to examine the effects of Location Tumor 43 21 22 0.015* G3BP1 on RCC tumor cell mesenchymal phenotypes. Para-tumor 43 32 11 The results showed that G3BP1 downregulation led to the decrease of tumor cell invasion in the Matrigel inva- RCC subtype ccRCC 38 19 19 1.000 sion assay (Fig. 2d). Moreover, it was known that Non-ccRCC 5 2 3 EGFR and its ligand EGF are frequently overexpressed in Tumor side Left 19 12 7 0.095 RCC, and its signaling cascade is critical for RCC metas- 33–35 Right 24 9 15 tasis . Specifically, we found that EGF-induced che- Necrosis Negative 15 5 10 0.137 motaxis, a pivotal event for RCC metastasis, was significantly (p < 0.01) impaired in G3BP1 knockdown Positive 28 16 12 cells when compared to controls as indicated by micro- Note: G3BP1 expression (−/+) was determined by IHC staining score. The Boyden chamber assays (Fig. 2e). Collectively, these scoring system was composed of two independent parameters: percentage of results indicated that G3BP1 contributes to promoting tumor cells exhibiting positive staining (0–5: 0–1%; 1–5%; 6–10%; 11–20%; 21–50%; >50%); and intensity of IHC staining (0–2: 0, no staining; yellow-brown RCC cell proliferative, migratory, and invasive capacities, staining; strong brown staining). The final scores of two parameters were added thereby facilitating the transitioning of tumor mesenchy- to a sum and defined as the IHC staining score. 0–3: Negative; 4–7: Positive. *p < 0.05 indicates statistical significance by χ test. mal phenotype. Knockdown of G3BP1 inhibited expressions of cell proliferative and EMT markers in RCC grade (Table 1). Meanwhile, no significant (p > 0.05) To further investigate the role of G3BP1 in RCC pro- associations were identified between the expression of liferation and metastasis, G3BP1 was knocked down in G3BP1 and any other clinicopathological characteristics, ACHN and A498 RCC cells, and the expression levels of including patient’s age, gender, RCC subtype, tumor size, cell proliferative and epithelial–mesenchymal transition tumor side, and necrosis (Table 1). Taken together, our (EMT) markers were examined by Western blot. As findings suggest that the expression of G3BP1 is increased shown in Figure 3, the expressions of c-Myc and cyclin D1 in RCC and its expression level is associated with the were downregulated in G3BP1 knockdown RCC cells, progression of RCC. consistent with the inhibition of cell proliferation in shG3BP1 cells (Figs. 2b, 3a, b). In addition, the expression Silencing of G3BP1 represses the mesenchymal phenotype of the epithelial cell–cell adhesion molecule E-cadherin in RCC cells in vitro was upregulated. Consistently, the expressions of In order to investigate the role of G3BP1 in RCC pro- mesenchymal markers N-cadherin, Vimentin, Snail, and gress and metastasis, two RCC cell lines (ACHN and Slug were dramatically decreased in G3BP1 knockdown A498) with different potential for metastasis were selected cells (Fig. 3a, b). Thus, it is suggested that downregulation as in vitro cell models . RCC cells with lentivirus- of G3BP1 suppresses not only cell proliferation but also mediated G3BP1 stable knockdown were used for EMT in RCC cells. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 4 of 13 Fig. 2 Knockdown of G3BP1 represses RCC mesenchymal phenotype in vitro.a ACHN and A498 cells were transduced with lentivirus-mediated G3BP1-specific shRNA (shG3BP1) or scramble control (Scr), and the efficiency of G3BP1 knockdown was examined by Western blot. b ACHN and A498 cells with G3BP1 knockdown (shG3BP1) or scramble control (Scr) were cultured in growth medium and cell proliferation was determined by CCK8 assay at indicated time points, **p < 0.01 by two-way ANOVA. c Transwell assay was performed to determine the cell migration. Left panel: representative microscopic images, Naïve: untreated; Scr: scramble control; shG3BP1: G3BP1 knockdown. Right panel: quantitative analysis of cell migration, data were presented as mean ± s.d., n = 10 (ACHN) and n = 6 (A498) respectively, **p < 0.01 by one-way ANOVA, NS: no significant difference. d Matrigel cell invasion assay was used to evaluate the cell invasive ability. Left panel: representative microscopic images, Naïve: untreated; Scr: scramble control; shG3: G3BP1 knockdown. Right panel: quantitative analysis of cell invasion, data were presented as mean ± s.d., n = 10 (ACHN) and n = 6 (A498) respectively, **p < 0.01 by one-way ANOVA, NS: no significant difference. e G3BP1 knockdown impaired EGF-induced chemotaxis. Scr: scramble control; shG3BP1: G3BP1 knockdown. Data were presented as mean ± s.d. from three independent repeats, **p < 0.01 by two-way ANOVA. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 5 of 13 expression in scramble RCC cells. In contrast, IL-6- induced STAT3 phosphorylation was dramatically abol- ished in G3BP1 knockdown cells (Fig. 4b). In addition, we found that treatment with stattic, a specific inhibitor for STAT3 activation and dimerization, exhibited a profound effect on STAT3 phosphorylation, with no effect on the expression of G3BP1 in ACHN cells, suggesting STAT3 acts as a G3BP1 downstream signaling molecule (Fig. 4c). Together, the data firmly indicated that knockdown of G3BP1 blocked IL-6-induced STAT3 activation, and G3BP1 is a bridge molecule linking IL-6 and STAT3 signaling in RCC cells. To verify the functional impact of the IL-6/G3BP1/ STAT3 signaling axis in the regulation of EMT events in RCC, we next evaluated the effects of G3BP1 depletion on RCC cell migration and invasion with presence or absence of IL-6. The results revealed that treatment of IL-6 (100 ng/ml) significantly (p < 0.01) promoted tumor cell migration (Fig. 4d) and invasion (Fig. 4e) in RCC ACHN Fig. 3 Knockdown of G3BP1 suppressed expressions of cell cells, while G3BP1 depletion strongly attenuated the IL-6- proliferative and EMT markers in RCC cells. ACHN (a) and A498 (b) induced enhancement of RCC cell migration and invasion cells were transduced with lentivirus-mediated G3BP1-specific shRNA (shG3BP1) or scramble control (Scr). Western blot analysis was (Fig. 4d, e). Thus, these results functionally indicated that conducted to determine the target molecule expressions of cell IL-6 increased RCC cell migrating and invasive abilities, proliferative markers (c-Myc and cyclinD1) and EMT markers (E- which could be declined at least partially by the knock- cadherin, N-cadherin, vimentin, snail, and slug). down of G3BP1. Knockdown of G3BP1 impaired IL-6-induced STAT3 Expression of G3BP1 is correlated with IL-6 and p-STAT3 in activation and led to suppression of RCC migration and primary RCC patients invasion Given the significance of G3BP1 in controlling IL-6/ To further explore the underlying molecular mechan- G3BP1/STAT3 signaling in RCC migration and metas- ism of G3BP1-mediated RCC EMT, we first searched for tasis in vitro, we next address the correlations of G3BP1 the oncogenic signaling pathways that can be affected by with IL-6 and STAT3 activation in our clinical study G3BP1 silencing, including STAT3, AP1, ISRE, TGFβ cohort comprising of 32 pairs of primary RCC and their (p3TP), P53, NFAT, and WNT (TOPFlash) using corresponding adjacent normal kidney tissues. Western pathway luciferase reporter system in RCC cells (Suppl blotting showed that the expressions of G3BP1, IL-6, and Fig. 2). The results strongly suggested that G3BP1 p-STAT3 were significantly (p < 0.05) elevated in RCC knockdown caused significant impairments of multiple tissues as compared to those in the corresponding adja- tumorigenic-related signaling pathways in RCC cells, cent normal kidneys (Fig. 5). In addition, the expression of including STAT3, AP1, and interferon (ISRE) signaling G3BP1 is positively correlated with IL-6 (r = 0.510, p = (Fig. 4a and Suppl Fig. 2). Evidence indicated that in 0.003) and p-STAT3 (r = 0.683, p < 0.0001) in RCC tis- response to extracellular insults, STAT3 is activated sues (Table 2), suggesting the associations of IL-6/G3BP1/ through tyrosine phosphorylation at Tyr705, and this STAT3 molecules in primary RCC. constitutive activation of STAT3 is a pivotal event in RCC 37,38 tumorigenesis and metastasis . Further Western blot- G3BP1 knockdown inhibited RCC tumor growth and ting results confirmed that G3BP1 silencing led to inhi- metastasis in vivo bition of STAT3 phosphorylation in RCC cells (Fig. 4b). To determine whether G3BP1 knockdown impacts RCC Specifically, IL-6 is one of the well-documented cytokines tumor growth and metastasis in vivo, we performed the which is abundantly expressed in RCC and enhances RCC orthotopic tumor xenografts with implantation of cell proliferation and invasiveness through activation of luciferase-labeled G3BP1 knockdown cells (ACHN- 13,14 STAT3 signaling . In line with this notion, we further shG3BP1-luciferase) and control RCC cells (ACHN-Scr- treated G3BP1 knockdown and scramble control cells luciferase) into the sub-renal capsule of adult nude mice with human recombinant IL-6 (100 ng/ml) for 48 h, and left kidney. After 8 weeks, mice were intraperitoneally Western blotting results revealed that stimulation with injected with luciferin and the bioluminescence intensity IL-6 did not only activate STAT3 but also induced G3BP1 was detected and quantified for the primary tumors as Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 6 of 13 Fig. 4 Knockdown of G3BP1 impaired IL-6-induced STAT3 activation and led to the suppression of RCC migration and invasion. a ACHN and A498 cells with stably knockdown of G3BP1 (shG3BP1) or scramble control (Scr) were co-transfected with STAT3 pathway firefly luciferase reporter (STAT3-luc) together with internal control Renilla luciferase reporter (pRL-TK) vector. Forty-eight hours after transfection, cell lysates were subjected to dual-luciferase assay. Data were obtained from three independent repeats and presented as mean ± s.d., **p < 0.01 by Student’s t-test. b ACHN cells with stably knockdown of G3BP1 (shG3) or scramble control (Scr) were treated with or without human recombinant IL-6 (100 ng/ml) for 48 h. The expressions of G3BP1, p-STAT3 (Tyr705), and total STAT3 were examined by Western blot. c ACHN cells were treated with STAT3 inhibitor stattic at the indicated concentration for 48 h, and expressions of G3BP1 and p-STAT3 (Tyr705) were determined by Western blot. d, e ACHN cells with stably knockdown of G3BP1 (shG3BP1) or scramble control (Scr) were treated with or without human recombinant IL-6 (100 ng/ml) for 48 h, and then (d) Transwell assay was performed to determine the cell migration and (e) Matrigel cell invasive assay was used to examine the cell invasion, respectively. Left panel: representative microscopic images. Right panel: quantitative analysis. Data were presented as mean ± s.d. from three independent experiments, and five random microscopic fields were acquired in each experiment for quantification, **p < 0.01 by one way ANOVA followed by Tukey’s post hoc test, NS: no significant difference. well as lung and liver metastasis using the live biolumi- Additionally, the primary RCC tumor and metastatic nescence IVIS imaging system. The results showed that a organs of liver and lung were isolated from the afore- significant (p < 0.01) reduction of primary RCC tumor size mentioned mice. The primary renal tumor volumes were was observed in mice with G3BP1 knockdown as com- measured to be dramatically decreased in the G3BP1 pared to the scramble control group (Fig. 6a, b), sug- knockdown group (Fig. 6c), and further IHC staining of gesting G3BP1 plays an important role in RCC tumor the xenograft renal tissues confirmed the knockdown of growth in vivo. In addition, bioluminescent signals indi- G3BP1 expression in the ACHN-shG3BP1 group cated that mice implanted with G3BP1 knockdown (Fig. 6d). Moreover, H&E staining of the excised livers and ACHN cells had much fewer liver and lung metastatic foci lungs clearly demonstrated that significantly (p < 0.01) detected than those in scramble controls (Fig. 6e, f, i, j), more metastatic nodules were found in the scramble thus revealing the functional role of G3BP1 in RCC control group than the G3BP1 knockdown group (Fig. 6g, metastasis. h, k, l). Collectively, the results from in vivo tumor Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 7 of 13 that knockdown of G3BP1 led to inhibition of tumor cell proliferation, migration, and invasion, as well as altera- tions of EMT markers using in vitro RCC cell line model. Consistently, our in vivo orthotopic tumor xenografts results also confirmed that knockdown of G3BP1 suppressed RCC tumor growth and metastasis in mice. Remarkably, we uncovered a novel signaling link that G3BP1 mechanistic connects upstream pro- inflammatory cytokine IL-6 stimulation to STAT3 acti- vation, and eventually contributes to downstream cellular events, including promotion of RCC migration and metastasis. Together, our findings support a notion that G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in RCC. G3BP1, based on its crucial involvement in multiple biological processes and disease pathogenesis, including tumorigenesis, has emerged a target for molecular inves- Fig. 5 Expression of G3BP1 is correlated with IL-6 and p-STAT3 in 24,26,29 tigation in many oncology studies . However, there primary RCC patients. The expressions of G3BP1, IL-6, and p-STAT3 (Tyr705) in a panel of 32 pairs of primary RCC (T) and adjacent normal is still limited knowledge about its correlation with pro- kidney tissues (N) were examined by Western blot. Upper panel: gression of RCC. Overexpression of G3BP1 has been representative results from six pairs of patient samples were shown. shown to promote breast cancer cells proliferation and Lower panel: the expressions of G3BP1, p-STAT3 (Tyr705), and IL-6 induce EMT transition . Also, knockdown of G3BP1 was were quantified by normalizing with β-actin, and all 32 pairs of RCC reported to prevent tumor invasion and metastasis in patient samples were analyzed using a paired t-test. 26,29 xenograft model . Recently, Dou et al. reported that upregulation of G3BP1 was associated with enhanced cell Table 2 Correlation between G3BP1 and IL-6/pSTAT3 migration and poor survival of hepatocellular carcinoma expressions in RCC (HCC), with no significant effect on HCC cell growth . Similarly, our results demonstrated that G3BP1 was fre- G3BP1 expression Spearman’s r quently upregulated at protein levels in primary RCC Up Down Correlation p Value compared to adjacent normal tissues, and a higher level of coefficient G3BP1 expression is correlated with RCC progression. In addition, we analyzed but failed to detect the correlation IL-6 Up 21 2 0.510 0.003* of G3BP1 mRNA expression and RCC survival using The Down 4 5 Cancer Genome Altas (TCGA) database (data not shown). This suggests that G3BP1 might exert its tumorigenic pSTAT3 Up 20 0 0.683 <0.0001* function mainly at protein level, and the post- Down 5 7 transcriptional regulation of G3BP1 might be crucial for Note: The expressions of G3BP1, IL-6 and pSTAT3 were quantified and compared its role in RCC. Indeed, it remains possible that the between RCC tumor (T) and paired adjacent normal tissue (N). The relative ratio expression of G3BP1 displays a tissue-specific or cancer T/N > 1 was defined as “Up”, and T/N < 1 was defined as “Down”. *p < 0.05 indicates statistical significance by Spearman’s correlation coefficient type-specific manner. Functionally, our results demon- analysis. strated that G3BP1 plays an important role in RCC cell proliferation, migration, invasion, and metastasis both xenografts model indicated that silencing of G3BP1 could in vitro and in vivo, suggesting that G3BP1 is a critical suppress RCC tumor cell growth as well as inhibit RCC molecule in RCC progression. It is well established that cell metastasis to liver and lung. the majority of RCC arise from renal tubule epithelial cells, and EMT is a key event that occurs during the 38,39 Discussion invasion of cancers with an epithelial origin . During RCC frequently involves systemic metastasis which is EMT, cell–-cell and cell-matrix interactions are altered to the major cause of RCC mortality. However, the mole- facilitate tumor cell motility. In this content, the loose of cular basis of RCC progression remains elusive. In the adherens junction by “cadherin switching” is one of the present study, we revealed that the expression of G3BP1 is hallmark events in EMT, which is characterized by the increased in primary RCC as compared to adjacent nor- replacement of E-cadherin with N-cadherin . We found mal tissues, and G3BP1 is associated with higher levels of that G3BP1 depletion is associated with upregulation of TNM stages and Fuhrman grade. Functionally, we found E-cadherin and downregulation of N-cadherin, as well as Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 8 of 13 Fig. 6 G3BP1 knockdown inhibited RCC tumor growth and metastasis in vivo. ACHN cells were transduced with Luciferase-labeled G3BP1 knockdown (shG3BP1-luc) or scramble control (Scr-luc) were used to implant into nude mice for 8 weeks, n = 6 each group. The bioluminescence was detected by IVIS imaging system, and followed by histological examination. a Tumor growth was examined by IVIS imaging system, and representative images were shown. b Tumor growth was quantified by bioluminescence intensity. c Primary tumor volume was measured and compared. d Mouse primary renal tumors were collected and processed for IHC staining of G3BP1, and representative images were shown. e–l Liver and lung metastases were examined by IVIS bioluminescence imaging system and histological H&E staining for detection of tumor foci. e, i Tumor metastases in liver (e) and lung (i) were examined by IVIS imaging system, and representative images were shown. f, j Quantified bioluminescence in liver (f) and lung (j) metastatic sites were calculated and compared. g, k Tumor metastatic foci were evaluated by H&E staining in liver (g) and lung (k), and representative images were shown. h, l Tumor metastatic foci in liver (h) and lung (l) were quantified and compared using five random views under the microscope. All data were presented as mean ± s.e.m, n = 6, **p < 0.01 by Student’s t-test. repression of mesenchymal marker vimentin, a type III above-mentioned results from in vitro experiments, we intermediate filament related with cell-matrix interaction. also confirmed the G3BP1-mediated regulation of RCC Consistently, the expressions of E-cadherin repressors, tumor growth and metastasis using xenograft model Snail, and Slug were decreased in G3BP1 knockdown in vivo. Taken together, G3BP1 is an important molecule RCC cells. Our data clearly indicated that G3BP1 was not only involved in controlling tumor cell growth, but indeed involved in regulating EMT in RCC. Besides the also in mediating EMT in RCC. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 9 of 13 Previously, G3BP1 was reported to interact with the migration and invasion, suggesting depletion of G3BP1 SH3 domain of RasGAP and modulate Ras signaling functionally blocks the IL-6/STAT3 signaling axis and pathway . Recently, Annibaldi et al. questioned G3BP1 diminishes the RCC metastatic potential. Importantly, we being a genuine RasGAP-binding partner and suggested found that IL-6 itself can induce the G3BP1 expression in G3BP1-mediated signaling may not involve RasGAP .In RCC cells. This further supports the notion that G3BP1 is addition, G3BP1 has been shown to be involved in lung a critical mediator that links IL-6 to STAT3 activation in RCC. In line with this finding, we showed the positive cancer cell migration and metastasis via Src/FAK signal- ing pathway . In breast cancer, G3BP1 participated in the correlations between the expression of G3BP1 with IL-6 EMT and metastasis through regulating Smad signaling . and p-STAT3 in our primary RCC cohort. Collectively, In our current study, we screened several oncogenic sig- oncogenic G3BP1 serves as a signaling linkage molecule naling pathways and identified STAT3, AP1, and type 1 from upstream IL-6/EGF elicited stimulations to down- interferon (ISRE) signaling pathways were dramatically stream STAT3 signaling, eventually contributing to pro- impaired by a G3BP1 knockdown in RCC cells, indicating mote tumor cell growth and metastasis in RCC. that G3BP1 is a pivotal joint molecule connecting multi- In summary, the expression of G3BP1 is significantly ple signaling pathways. It was also suggested that higher in RCC comparing to para-tumor normal tissues, G3BP1-associated signaling pathways are diverse in can- and knockdown of G3BP1 significantly decreased tumor cers, and more importantly, to be cancer type specific. cell growth and metastasis in vitro and in vivo. In addition, Consistently, we verified that both c-Myc and cyclin D1 IL-6 can induce the expression of G3BP1, while G3BP1 as cell proliferation-associated downstream targets of depletion diminishes the IL-6-elicited STAT3 activation AP1 signaling molecules repressed in G3BP1 knockdown and leads to inhibition of tumor cell growth and invasion RCC cells. In addition, to be of particular interest is the in RCC. It was well known that RCC is resistant to con- STAT3 signaling. STAT3 signaling pathway plays critical ventional radiotherapy and chemotherapy, and immu- roles in tumor invasion and metastasis . In response to notherapies using a high dose of IL-2 and IFN-α are now 47–50 extracellular signals, STAT3 is activated by phosphoryla- applied for RCC treatment . Unfortunately, clinical tion of a conserved tyrosine residue and the phosphory- studies showed that the objective response rates are about lated STAT3 dimers undergo nuclear translocation where 5–20% for RCC patients who use IL-2 or IFN-α treatment 51,52 they regulate several “master” EMT transcriptional net- regimen . Thus, there is a pressing need to develop 38,44 works, including Snail and Slug . Remarkably, our new immunological targets for advanced treatment of 14,53–56 RCC results showed that the depletion of G3BP1 inhibited . Recently, several antibodies developed for STAT3 signaling, further suppressing the EMT pheno- targeting blockage of the IL-6/STAT3 pathway for RCC types via transcriptional repression of Snail and Slug. immunotherapy have been registered for preclinical stu- 14,57 This perhaps implies that G3BP1 functions through dies and phase I/II clinical trials . Our findings suggest STAT3 signaling and eventually contribute to RCC EMT. G3BP1 as a novel mediator of RCC tumor progression The crosstalk between tumor cells and their surround- and further extends the current knowledge about IL-6/ ing extracellular signals forms tumor microenvironment STAT3 associated mechanism in RCC carcinogenesis. and leads to activation of oncogenic signaling pathways, Indeed, targeting the molecules of IL-6/G3BP1/STAT3 including STAT3 signaling pathway. It was known that axis provides a novel and potential treatment strategy for 13,14,45 STAT3 can be robustly activated by IL-6 and EGF . RCC. It was documented that EGF and its receptor EGFR are frequently overexpressed in many forms of RCC, and Materials and methods signaling cascade through EGF/EGFR/STAT3 play an Cell culture, transfection, and treatment important role in tumor cell growth and EMT in RCC cell lines including A498 and ACHN were 33,35,46 RCC . In this study, we showed that the knockdown obtained from American Type Culture Collection of G3BP1 dramatically impaired EGF-induced RCC cell (ATCC). Cells were cultured in Eagle’s Minimum Essen- chemotaxis, an essential component of tumor dis- tial Medium (MEM) (Gibco) supplemented with 10% FBS semination during progression and metastasis, indicating (HyClone) and 1× penicillin/streptomycin at 37 °C in the EGF-mediated chemotaxis in RCC is probably via G3BP1. presence of 5% CO . All cultures were routinely tested for Recent studies have shown that IL-6 can be produced by absence of mycoplasma and for the retention of their RCC cells, and enhanced levels of IL-6 increases the respective morphology and growth characteristics. Spe- 9–12,14 invasiveness of RCC . Consistently, we demonstrated cific short hairpin RNA (shRNA) was designed to target in the present study, IL-6 can activate STAT3 and lead to human G3BP1 and cloned into pLKO.1 lenti-vector, enhanced RCC cell migration and invasion. Importantly, transfected into 293T cells together with lentivirus we found that G3BP1 knockdown can significantly abolish packaging plasmids, psAX2 and pMD2.G for 48 h using the effect of IL-6 on STAT3 activation, as well as RCC cell lipofectamine 2000 (Invitrogen). Lentivirus-containing Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 10 of 13 medium was cleared by centrifugation and filtration with normalized with the expression of internal control β- 0.45 µm filter and then added to culture medium of A498 actin. and ACHN cells for shRNA transduction. G3BP1 stably knockdown (shG3BP1) and scramble control cells (Scr) IHC staining were established with puromycin (10 μg/ml) selection for Tissues were formalin-fixed and embedded in paraffin 10 days. blocks and then cut into 5 µm thickness sections. RCC cells were treated with 100 ng/ml of human Tissue sections on glass slides were deparaffinized in recombinant IL-6 (Peprotech) for 48 h and subjected to xylene, and dehydrated in gradient ethanol, followed by cell migration and invasion assays while total proteins blocking of endogenous peroxidase activity in 3% hydro- were extracted for Western blotting analysis. Stattic is a gen peroxide. Slides were then submerged in boiling commercially available small molecule inhibitor of citrate buffer for antigen retrieval. UltraVision Protein STAT3 activation and dimerization (Selleck). ACHN cells Block (BD) was applied to block non-specific background were seeded in six-well plate and treated with stattic (0, 1, staining. The tissue sections were immunostained with 2.5, 4, 5.5, and 7 µM) for 48 h, and then total proteins an anti-G3BP1 antibody (Santa Cruz). After washing were extracted for Western blotting analysis. with PBS, the slides were immunoblotted using Max Vision HRP-Polymer anti-Rabbit IHC Kit (Miaxim.bio). Human patients Sections were developed by peroxidase substrate DAB Human patient study cohort collected 43 pairs of pri- Detection Kit (Miaxim.bio) and then counterstained by mary RCC tissues and corresponding para-carcinoma hematoxylin. normal tissues. All tissue samples were surgically removed For the scoring system, all slides immunostained with and paraffin-embedded in the Tianjin Medical University G3BP1 antibody were scored by two independent Second Hospital from January 2012 to December 2015 pathologists for the percentage and intensity of cells with patients’ consents and ethical committee approval. showing specific immunostaining signals. The percentage Clinical parameters including age, gender, tumor size, score grading criteria of 0–5 were as follows: 0, (0–1)% of histological type, and Fuhrman grade were collected. All tumor cells positive; 1, (1–5)% of tumor cells were posi- patients had undergone radical nephrectomy or partial tive; 2, (6–10)% of cells were positive; 3, (11–20)% of cells nephrectomy with no preoperative and postoperative were positive; 4, (21–50)% of cells were positive; and 5, adjuvant therapies. For RCC pathological classification, all >50% of cells were positive. The intensity of immunos- samples were double-blind reviewed by two independent taining level was determined by the subjective visual pathologists. scoring of the brown stain as follows: 0, no staining; 1, yellow-brown staining; 2, brown staining. The final score Protein extraction and Western blot was calculated by combining the percentage and intensity Cells were harvested and lysed in SDS lysis buffer with scores and recorded as negative (0–3) and positive 30,31 1× protease inhibitor cocktail and 1× phosphatase inhi- (4–7) . bitor cocktail (Roche). The concentration of total protein was determined by an Enhanced BCA Protein assay kit Cell proliferation assay (Thermofisher Scientific). Equal amounts of each protein Cells (1 × 10 per well) were plated in 96-well plates and sample (40 μg) were separated by electrophoresis on SDS- cultured as described above. Cell proliferation was PAGE and transferred onto polyvinylidene fluoride assessed at 24, 48, and 72 h after seeding using Cell membranes (Invitrogen). After blocking with DifcoTM Counting Kit-8 (CCK-8, Dojindo) according to the man- skim milk (Becton Dickinson), protein lysates were blot- ufacturer’s protocol. Briefly, 10 μl of the CCK-8 solution ted with anti-G3BP1 antibody (Santa Cruz, SC), anti- was added to each well of the plate and incubated at 37 °C pSTAT3 antibody (Cell Signaling Technology, CST), anti- for 1 h. The absorbance of each well was measured at 450 STAT3 antibody (CST), anti-IL6 antibody (Saierbio), anti- nm using a microplate reader. All experiments were E-cadherin antibody (SC), anti-Vimentin antibody (CST), performed in triplicates. anti-Snail antibody (CST), anti-Slug antibody (SC), anti- N-cadherin antibody (SC), anti-c-Myc antibody (SC), Cell migration assay anti-cyclinD1, or anti-β-actin antibody (SC), overnight at Cell migration was measured by transwell (8 μm pores, 4 °C, and followed by incubation of corresponding Corning) assay. Briefly, RCC cells (1 × 10 ) were seeded horseradish peroxidase-conjugated anti-mouse or anti- into the upper chamber in serum-free medium, and rabbit secondary antibodies at room temperature for 1 h. medium containing 10% FBS was added to the lower The blots were visualized using ECL system (Millipore). chamber. After 24 h of incubation at 37℃, the cells Images were then analyzed and quantified with G:box attached to the lower surface of the membrane were fixed (SYNGENE), and the expression of the target protein was with 4% PFA and stained with hematoxylin. Cell numbers Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2018) 9:501 Page 11 of 13 were counted in five randomly chosen fields under the detection for primary tumors and metastasis in liver and microscope. lung using the live IVIS imaging system (Perkin Elmer). The mice were then sacrificed, and primary RCC tumors Tumor cell invasion assay were isolated. Tumors were measured by largest and Transwell insert (8 μm pores, Corning) were pre-coated smallest diameters to calculate tumor volume. Primary with diluted growth factor-reduced matrigel (2.5 mg/ml, RCC tumors, livers, and lungs were also fixed in formalin and embedded in paraffin. Serial sections and H&E BD Biosciences). RCC cells (1 × 10 ) resuspended in serum-free MEM were seeded into the upper chamber, staining were then performed to examine metastasis. and MEM medium containing 10% FBS were added to the lower chamber. The cells were allowed to invade through Statistical analysis the membrane at 37 °C for 48 h. Non-migrating cells on All data were presented as the mean ± s.d. Differences in the upper surface were removed, and only those cells mean values between two groups were analyzed by t-test. attached to the lower surface of the membrane were fixed Differences in mean values among multiple groups and/or with 4% PFA and stained with hematoxylin, followed by multiple conditions were analyzed by one-way ANOVA counting in five randomly chosen fields under the or two-way ANOVA, followed by post hoc test. χ test was microscope. used for statistical analysis of the correlations between G3BP1 expression and clinicopathologic parameters. The Chemotaxis assay correlations between the expressions of G3BP1, IL-6, and Chemotaxis assay was performed using micro-Boyden p-STAT3 were analyzed using Spearman test. Differences chambers as described previously . Briefly, RCC cells with *p < 0.05 were considered as statistically significant. (2.5 × 10 ) were seeded into the upper chambers in Acknowledgements serum-free MEM, while 10% FBS and human recombi- We thanks Dr. Justin Eze Ideozu (Division of Pulmonary Medicine, nant EGF (Peprotech) with different concentrations were Northwestern University) for his scientific proofreading of the manuscript. This work was supported by the National Natural Science Foundation of China added to the lower chamber. The 8 μmof fibronectin- [grant number 81772945, 81402094, 81400321, and 81202100], the Scientific pretreated filter membrane was placed between two Research Foundation for the Returned Overseas Chinese scholars, Burea of chambers. After 12 h incubation at 37 °C, the membrane personnel of China, Tianjin, China [grant number 2016015], and Natural Science Foundation of Tianjin, China [grant number 15JCZDJC35400]. was fixed and stained with hematoxylin. The number of migrating cells was counted in five randomly chosen fields Author details under a light microscope. 1 Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300211, China. Department of Microbiology, School of Medical Laboratory, Tianjin Medical Pathway dual-luciferase reporter assay University, Tianjin 300203, China. Research Center of Molecular Biology, Inner The screening of signaling pathways by dual luciferase 4 Mongolia Medical University, Hohhot 010059, China. Research Center of Basic assay was carried out using a serial of pathway luciferase Medical Sciences, Tianjin Medical University, Tianjin 300070, China. Department of Pharmacology, Tianjin Medical University, Tianjin 300070, reporters (SABiosciences), including STAT3, AP1, ISRE, China. Department of Urology, Renji Hospital, School of Medicine in Shanghai TGFβ (p3TP), P53, NFAT, and WNT (TOPFlash). RCC Jiao Tong University, Shanghai 200127, China cells were co-transfected with pathway luciferase repor- Conflict of interest ters and the internal control Renilla luciferase reporter The authors declare that they have no conflict of interest. (pRL-TK) vector using lipofectamine 2000. Forty-eight hours after transfection, cells were harvested and analyzed by the dual-luciferase assay kit (Promega). Each experi- Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in ment was performed in triplicates, and at least three published maps and institutional affiliations. independent assays were conducted. Supplementary Information accompanies this paper at https://doi.org/ 10.1038/s41419-018-0504-2. Xenograft tumor growth and metastasis Lentivirus-mediated luciferase-labeled G3BP1 stably Received: 15 January 2018 Revised: 18 March 2018 Accepted: 20 March knockdown (ACHN-shG3BP1-luciferase) and scramble control cells (ACHN-Scr-luciferase) were used. 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Cell Death & DiseaseSpringer Journals

Published: May 2, 2018

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