Interleukin-6 Promotes Epithelial-Mesenchymal Transition and Cell Invasion through Integrin β6 Upregulation in Colorectal Cancer

The metastatic potential of colorectal cancer (CRC) is intensively promoted by the tumor microenvironment (TME) in a paracrine manner. As a pleiotropic inflammatory cytokine, Interleukin-6 (IL-6) is produced and involved in CRC, the same scenario where integrin αvβ6 also becomes upregulated. However, the relationship between IL-6 and integrin αvβ6 as well as their involvement in the crosstalk between CRC and TME remains largely unclear. In the present study, we demonstrated a positive correlation between the expression of IL-6 and integrin β6 in CRC samples. The mutually promotive interaction between CRC and TME was further determined by an indirect coculture system. CRC cells could augment the secretion of IL-6 from fibroblasts, which in return induced invasion and integrin β6 expression of CRC cells. Through the classic IL-6 receptor/STAT-3 signaling pathway, IL-6 mediated the upregulation of integrin β6, which was involved in the invasion and epithelial-mesenchymal transition of CRC cells induced by IL-6. Taken together, our results reveal a paracrine crosstalk between IL-6 signals originating from the TME and increased the integrin β6 level of CRC. IL-6 induces CRC invasion via upregulation of integrin β6 through the IL-6 receptor/STAT-3 signaling pathway. Combined inhibition of IL-6 along with integrin β6-targeted strategy may indicate new directions for antitumor strategies for CRC.


Introduction
Due to environmental causes and risk factors in modern society such as intestinal pathogens and inflammation, colorectal cancer (CRC) is among the most common malignancies [1]. Distant metastasis remains one of the leading contributors of CRC-related deaths, despite recent advancements in surgery techniques and adjuvant therapy. Therefore, a better understanding is urgently needed to investigate the mechanism of CRC metastasis so as to further improve the clinical outcomes.
Even though many vital molecular mechanisms have been revealed involved in cancer progression, it seems not realistic to prevent CRC metastasis by targeting one oncogene or signaling pathway within cancer cells [2]. The tumor consists of malignant cancer cells and stromal cells that constitute the tumor microenvironment (TME). More and more studies have discovered that the metastatic potential of cancer cells not only depends on certain oncogenes and signaling pathways but also is intensively promoted by the TME in a paracrine manner [3]. The stromal cells in the TME can be activated by cancer cells but meanwhile pro-mote the epithelial-mesenchymal transition (EMT) of cancer cells the other way round through secretion of various factors including chemokines and cytokines [4]. Interleukin-6 (IL-6), a pleiotropic inflammatory cytokine which plays a vital role in immune and inflammatory response, is as well considered as a key growth factor for malignancy. IL-6 can be produced by multiple cell types located within the TME, including tumor-infiltrating immune cells, stromal cells, and cancer cells themselves [5]. During classic signaling, IL-6 binds to IL-6 receptor (IL-6R), forming the heterohexameric complex followed by recruitment of cellular proteins and subsequent activation of signaling pathways including JAK/STAT-3, PI3K, and MAPK [6]. IL-6 has been shown to increase metastatic capability in a variety of tumor models by multiple mechanisms. IL-6 promotes cancer stemness and oncogenicity of osteosarcoma cells by upregulating a secreted phosphorylated glycoprotein osteopontin [7]. In addition to tumorigenesis, IL-6 contributes to chemoresistance and recurrence [8]. Moreover, IL-6 can serve as a prognostic indicator of poor survival [9,10] as well as a predictor of therapeutic response [11,12] in a wide range of tumors.
The best described mechanism under which IL-6 increases tumor invasion is by conferring tumor cells on an EMT phenotype, through which tumor cells transit from adherent epithelial to mobile mesenchymal states thus facilitating invasion and metastasis [13]. During the complex process of metastasis, integrins, a large family of cellular adhesion receptors, are involved in almost every step by mediating the adhesion and interaction of the invading cancer cells with surrounding cells and extracellular matrix (ECM) [14]. So far, 24 different subtypes of integrins, composed of α and β subunits, have been identified [13]. Integrin αvβ6 is the only subtype the β6 subunit could constitute, which is specifically expressed in the epithelia. Integrin αvβ6 is barely expressed in adult normal tissues but becomes substantially upregulated in the context of inflammation and malignancy, acting as an indicator of tumor progression [15]. Our previous researches have defined the important participation of integrin αvβ6 in cancer invasion, matrix metalloproteinase (MMP) secretion, and chemoresistance [16][17][18]. In addition, integrin αvβ6 plays a bidirectional regulation role between CRC cells and stromal cells [19].
Previous studies have reported that IL-6 associates with CRC progression. IL-6 is not only related to advanced tumor stages and poor survival of CRC patients [20] but also promotes the motility of CRC cells [21]. IL-6 is produced and involved in inflammation and carcinogenesis, the same scenario where integrin αvβ6 also becomes upregulated. A correlation of expression of IL-6 with integrin αvβ6 was recently reported [22]; however, many questions still remain as to whether the expression of integrin αvβ6 is mediated by the activation of the IL-6 signaling pathway rather than being a concomitant phenomenon, since IL-6 may directly induce EMT of cancer cells to promote metastasis. Furthermore, the downstream mechanism of IL-6-induced invasion as well as the relationship between IL-6 and integrin αvβ6 remains largely unknown. In this study, we analyzed the effect of IL-6 on the expression of integrin αvβ6 and further character-ized the roles of IL-6 and integrin αvβ6 in the paracrine crosstalk between the TME and CRC cells.

Cell Culture and Transfection. CRC cell lines HT-29 and
Colo205 and fibroblast cell line CCD-18Co from the colon were purchased from ATCC. Human normal colon mucosal epithelial cell line NCM460 was obtained from INCELL. HT-29 and Colo205 were cultured in complete Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), with essential supplementation including penicillin/streptomycin solution, while NCM460 in complete RPMI-1640 medium. CCD-18Co was cultured in ATCCformulated Eagle's Minimum Essential Medium (#30-2003) with 10% FBS. All the cells were incubated at 37°C in a 5% CO 2 humidity atmosphere. The recombinant human IL-6 was purchased from R&D Systems. For the function blocking, the specific antibody 10D5 against integrin β6 was purchased from Merck Millipore and neutralizing anti-IL-6 as well as anti-IL-6R from R&D Systems. To knock down integrin β6 (#144659), IL-6R (#106147), or STAT-3 (#116558), cells were transfected with specific siRNA (Thermo Fisher Scientific) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. For the inhibition of the STAT-3 pathway, specific STAT-3 inhibitors Stattic and Cryptotanshinone (CTS), ERK/MAPK inhibitor U0126, and PI3K inhibitor LY294002 were purchased from SelleckChem.

Primary CRC Samples.
For the immunohistochemistry (IHC) assay, a total of 155 CRC patients with intact clinical information who underwent radical operation were selected between May 2015 and June 2017 from the First Affiliated Hospital of Xi'an Jiaotong University. All the samples were histopathologically confirmed by two pathologists independently. Written informed consents were obtained from all patients. The protocol was approved by the Institutional Medical Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University.
2.3. IHC. IHC staining was performed on formalin-fixed paraffin-embedded CRC slide sections. Optimal antigen retrieval was induced in citrate buffer at pH 6.0 and 95°C for 30 min. The sections were blocked with 10% normal goat serum, followed by incubation with primary antibody against IL-6 (#6672, Abcam) or integrin β6 (Clone 442.5C4, Millipore Sigma) overnight at 4°C before incubation with secondary antibody conjugated with HRP. Finally, the staining was established by a DAB peroxidase substrate (Beyotime). The extent and intensity of IHC staining for IL-6 and integrin β6 were scored by two blinded observers under a light microscope. Briefly, the intensity of staining was graded as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong) and the extent of staining as 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), or 4 (76-100%). The product of intensity and extent was used as the final IHC score. The patients were further classified into low or high expression group based on IL-6 immunostaining with the median IHC score as the cut-off value. 2.5. Enzyme-Linked Immunosorbent Assay (ELISA). The culture medium was collected after coculture for 72 h and centrifuged to detect the concentration of supernatant IL-6 using the human IL-6 ELISA kit (Boster) according to the manufacturer's instructions.
2.6. Quantitative PCR (qPCR). Total RNA of cells was harvested using the TRIzol reagent, and cDNA was synthesized following the manufacturer's protocol (TaKaRa). qPCR was performed on a LightCycler 480 system (Roche Life Science).
2.9. Statistics. Statistical analyses were performed using GraphPad Prism 5 software. Data were presented as means ± SEM. Student's t-tests were performed for the comparison of two means while ANOVA for 3 or more data sets. The association between IHC staining of IL-6 and integrin β6 was determined by the Mann-Whitney test. Chi-squared tests were used to explore the association of IL-6 and integrin β6 expression with different clinicopathological variables. p < 0:05 was considered to be statistically significant. To further investigate whether there was a correlation between IL-6 and β6 expression, the samples were divided into high and low expression groups based on IL-6 immunostaining, and the IHC scores of integrin β6 from two groups were compared by Mann-Whitney analysis, which indicated that the IL-6-high group demonstrated higher immunostaining of integrin β6 than the IL-6-low group ( Figure 1(e)). Therefore, the expression of IL-6 was positively related to the expression of integrin β6 in CRC samples. The association of IL-6 and integrin β6 expression with clinicopathological factors is summarized in Table 1. Both IL-6 and integrin β6 expression were associated with poor tumor differentiation and advanced N stage and TNM stage. Moreover, IL-6 expression was also related to the tumor stage, while integrin β6 expression to M stage, indicating an association of IL-6 and integrin β6 expression with disease severity in the CRC patients.  (Figure 1(e)), and the critical role of integrin β6 in CRC cell migration, we hypothesized that integrin β6 might participate in IL-6-induced invasion of CRC cells. Based on qPCR assays, we found that the gene expression of integrin β6 was upregulated by IL-6 in a dosedependent manner, while integrin αv was not affected (Figures 3(a) and 3(b)). Consistently, the protein level of integrin β6 was also increased upon IL-6 stimulation, confirming that IL-6 could upregulate the expression of integrin β6 (Figures 3(c) and 3(d)). In fact, as short as 15 h treatment with exogenous IL-6 was sufficient to induce integrin β6 transcription, indicating a potential direct regulation of integrin β6 by IL-6 in CRC cells ( Supplementary Fig. 1a, b).

There Is a Positive Correlation between the
In contrast, we were unable to detect the expression of integrin β6 in NCM460 cells in the absence or presence of exogenous IL-6 (data not shown). Furthermore, the role of integrin β6 in IL-6-mediated cell invasion was verified by loss-of-function study. Inhibition of integrin β6 with function blocking antibody 10D5 or siRNA could remarkably suppress IL-6-induced cell invasion (Figures 3(e) and 3(f)). In addition, inhibition of IL-6R reduced IL-6-upregulated transcription and translation levels of integrin β6 (Figures 3(g) and 3(h)). These results suggested the important participation of integrin β6 in IL-6/IL-6R-induced invasion of CRC cells.  Oxidative Medicine and Cellular Longevity metastasis, and IL-6 has been shown to induce EMT in a paracrine manner in a variety of types of carcinoma including bladder [24] and pancreatic cancers [25]. Here, we show that in addition to cell invasion, IL-6 could also promote EMT of CRC cells (Figures 4(a) and 4(b)). Since IL-6 could upregulate integrin β6, the cytoplasmic domain of which is critical to EMT [26]. We therefore examined whether integrin β6 was involved in IL-6-mediated EMT in CRC cells. Inhibition of integrin β6 could inhibit the suppression of Ecadherin and the induction of Vimentin mediated by IL-6 in both HT-29 and Colo205 cells (Figures 4(a) and 4(b)), thus reversing IL-6-induced EMT.

STAT-3 Signaling Pathway Participates in IL-6-Mediated
Cell Invasion and Integrin β6 Upregulation. The STAT-3 signaling pathway plays a prominent role in diverse cellular behaviors mediated by IL-6, including tumor cell survival, invasion, and metastasis [27]. We therefore investigated whether the STAT-3 pathway was involved in IL-6- In contrast, while IL-6 could also expectedly activate ERK/MAPK and PI3K pathways in HT-29 cells, ERK/MAPK inhibitor U0126 could only inhibit IL-6-induced expression of integrin β6 in a small degree while PI3K inhibitor LY294002 had no effect (Supplementary Fig. 2a, b), indicating that MAPK and PI3K pathways were dispensable in IL-6-induced integrin β6 upregulation. Collectively, these data indicated that the STAT-3 signaling pathway was involved in IL-6-induced invasion and expression of integrin β6 in CRC cells.
3.6. Fibroblast-Derived IL-6 Promotes the Invasion and Upregulation of Integrin β6 in CRC Cells. To further investigate the crosstalk between cancer cells and TME, the indirect coculture assay was utilized, which showed that both HT-29 and Colo205 cells increased IL-6 secretion from fibroblasts (Figures 6(a) and 6(b)), and the mRNA level of IL-6 was predominantly induced in fibroblasts rather than CRC cell lines (Figures 6(c) and 6(d)), confirming that CRC cells could augment the production of IL-6 from the fibroblasts. Concomitantly, we also determined the effect of IL-6 secreted by fibroblasts on the expression of integrin β6 in CRC cells. The cancer cells in the coculture system demonstrated an upregulation of integrin β6, which could be antagonized via neutralizing IL-6 antibody (Figures 6(e) and 6(f)). Moreover, both IL-6 antibody and integrin β6 antibody inhibited the enhanced CRC cell invasion when cocultured with fibroblasts, with the combination of both function blocking antibodies presenting the most significant inhibition (Figures 6(g) and 6(h)). In contrast, integrin β6 expression of NCM460 cells was undetectable under coculture with fibroblasts (data not shown). Taken together, these data confirmed the role of IL-6 from TME in the promotion of tumor invasion through upregulation of integrin β6 in CRC cells.

Discussion
The prognosis of CRC patients with recurrent or metastatic disease remains poor, as the treatment at later stages is always an obstacle. Since tumor progression is driven not only by dysregulation of genes in cancer cells but also by various kinds of stromal cells, it has become a research hotspot to consider the paracrine crosstalk between cancer cells and TME so as to develop novel treatment strategies combining anticancer and antistroma therapies [28].
Regarding the role of TME in a tumor, the major IL-6producing cancer-associated fibroblasts (CAFs) promote cancer stemness and induce an immune adaptive inflammatory response, thus favoring CRC progression [29]. It is well known that CAFs facilitate malignancy by secreted factors and components of ECM. In accordance, our current study shows that IL-6 secretion from fibroblasts is much more abundant than that from CRC cells, but on the other hand, CRC cells augment IL-6 secretion from fibroblasts that in return facilitates the invasion of cancer cells, forming a positive mutual enhancement between CRC and TME. The bidirectional intercellular communications participate in response to environmental cues and are necessary in regard   [30]. Multiple growth factors secreted by cancer cells, such as transforming growth factor-beta (TGF-β) [19] and platelet-derived growth factors [31], induce the transformation of fibroblasts. Fur-thermore, tumor-derived extracellular vesicles (EVs), including exosomes, are involved in tumor-fibroblast communication by transferring principal factors. EVs contain functional cellular components, such as proteins and microRNAs, which not only promote the differentiation of fibroblast into CAF but also induce chemokine secretion 7 Oxidative Medicine and Cellular Longevity from fibroblasts in both proximal surrounding and distal sites [32]. In addition, tumor-derived exosomal effects in fibroblasts are also influenced by the aggressiveness of cancer cells. The molecules encapsulated in EVs derived from cancer cells with high-metastatic property contribute to an appropriate TME creation for tumor metastasis [33]. Further investigations are still needed to uncover the specific mechanisms of CRC cells to promote IL-6 secretion from fibroblasts. In addition to fibroblast-derived IL-6, we verify that exogenous IL-6 induces CRC cell invasion via IL-6R, consistent with previous study that anti-IL-6R antibody suppresses angiogenesis and inhibits the interaction between tumor and stroma [34].
As an indicator of aggressiveness, integrin β6 promotes EMT [35] and metastasis of CRC cells to the liver [36]. For the underlying mechanism, our study shows that IL-6/IL-6R induces the mRNA and protein levels of integrin β6 in a concentration-dependent manner. Besides, IL-6 is sufficient to induce integrin β6 transcription in a short time frame of stimulation, indicating the existence of a direct regulation. What is more important, integrin β6 is critical to IL-6induced EMT and aggressiveness as inhibition of integrin β6 by function blocking antibody or siRNA could reverse the facilitation of invasion mediated by IL-6 in CRC cells. In contrast, we were unable to detect integrin β6 in NCM460 cells under exogenous IL-6 stimulation or coculture with fibroblasts, indicating that IL-6-dependent upregulation of integrin β6 is a feature of CRC cells rather than normal colon epithelial cells. This is consistent with the fact that integrin β6 is barely expressed in normal epithelia but becomes significantly upregulated, acting as a tumor promoter, in the context of malignancy. Liang et al. [22] showed that IL-6 and integrin β6 expression could be used as a predictor of poor overall survival of CRC; here, we not only validate the positive relationship between the expression of IL-6 and integrin β6 besides their association with disease severity of CRC patients in another single-center study but also show for the first time that IL-6 upregulates integrin β6 expression. In addition to classic signaling, transsignaling also plays an important role in the TME to recruit tumor-associated stromal cells [37]; therefore, further study is needed to investigate whether the IL-6 transsignaling pathway is involved in the process here. It should be noted that antagonizing IL-6 by neutralizing antibody in the coculture system partially inhibits invasion and integrin β6 expression of CRC cells, indicating that TME may secrete other growth factors to nourish and enhance CRC aggressiveness [38]. In fact, in regard to the crosstalk of integrin β6 with paracrine signals, the SDF-1/CXCR4 axis is able to induce directional migration and liver metastasis of CRC cells by upregulating integrin αvβ6 [39]. On the other hand, integrin αvβ6 of CRC cells activates fibroblasts via TGF-β signaling followed by increased production of SDF-1 from CAFs [19]. One important characteristic of IL-6 is the persistent activation in the whole process of inflammatory bowel disease and CRC, while the upregulations of other cytokines like TGF-β1, IL-10, and IL-23 only show up primarily during CRC development [38]. Therefore, even though the interaction between cancer cells and TME is dynamic and complicated, we speculate that IL-6 already starts to play an important role during the early stages of inflammatory bowel disease and CRC, thus sustaining CRC progression all the time till the rise of other paracrine signals as well as integrin pathways. Most anticancer therapies are currently developed to specifically target cancer cells, but the tumor stroma can promote the resistance of cancer cells to such therapies, eventually leading to fatal recurrence and metastasis. Our work now moves forward a further step in this complex crosstalk between CRC cells and TME, showing the mutually promotive roles of IL-6 and integrin β6 in CRC progression.
The discovery of TME-derived IL-6-mediated signaling pathways is helpful for further understanding the mechanism underlying CRC metastasis, which may contribute to the development of therapeutic strategies. Among the signaling pathways, STAT-3 is the main transactivator downstream of IL-6 signaling, and IL-6 is involved in the activation of oncogenic STAT-3 in many kinds of cancer [40]. Therefore, we investigated whether IL-6-induced upregulation of integrin β6 is mediated by STAT-3 signaling. IL-6 stimulation leads to phosphorylation of STAT-3 (Tyr 705), and inhibition of STAT-3 by specific inhibitors or siRNA abrogates IL-6-induced invasion and integrin β6 upregulation, indicating the participation of this classical signaling cascade in response to IL-6. Aberrant activities of STAT-3 contribute - transient STAT-3 activation here is still efficient to induce integrin β6 upregulation in CRC cells. The combination of STAT-3 and integrin β6 expression has been reported to have a better prognostic performance than either alone in gallbladder carcinoma [43]. STAT-3 has been shown to be involved in promoting integrin β6 transcription in oral squamous cell carcinoma [44] and prostate cancer cells [45]. Moreover, Niu et al. [46] recently characterized the region located at the core promoter of integrin β6 and demonstrated the binding sites for transcription factor STAT-3. The present study now shows that STAT-3 is a critical transcription factor involved in IL-6-induced upregulation of integrin β6 in CRC cells. In our prior study, we found that the MAP-K/Ets-1 signaling pathway was also involved in the dysregulation of integrin β6 [47]; however, through pathway inhibitors, we found that ERK/MAPK and PI3K pathways were dispensable in IL-6-induced integrin β6 expression, suggesting the preferred role of the STAT-3 pathway in IL-6-mediated CRC progression. Our future work will further characterize the mechanisms of the crosstalk between CRC and TME so as to provide valuable candidates for preventing or treating CRC progression.

Conclusions
In summary, our results reveal a novel link between paracrine signals originating from TME and increased integrin β6 level of CRC. We for the first time discover that integrin β6 becomes upregulated upon IL-6 stimulation through the IL-6R/STAT-3 pathway, and integrin β6 plays a vital role in IL-6-induced EMT and invasion of CRC cells. These findings have potential therapeutic implications for targeted therapy.
Since integrin β6 participates in the paracrine crosstalk between the TME and CRC cells, combined inhibition of IL-6 signaling along with integrin β6-targeted strategies may indicate new directions for antitumor strategies for CRC.

Data Availability
The raw data related to this paper may be requested from the corresponding author.