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Differential expression of miRNAs regulating NF-κB and STAT3 crosstalk during colitis-associated tumorigenesis
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Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Differential expression of miRNAs regulating NF-κB and STAT3 crosstalk during colitis-associated tumorigenesis Sherien M. El-Dalya,b,∗, Enayat A. Omarac, Jihan Husseina, Eman R. Younessa, Zakaria El-Khayata a
Medical Biochemistry Department, Medical Research Division, National Research Centre, Cairo, Egypt Cancer Biology and Genetics Laboratory, Centre of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt c Pathology Department, Medical Research Division, National Research Centre, Cairo, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Colitis Tumorigenesis Inflammation NF-κB STAT3 miRNAs
Inflammatory bowel disease (IBD) is mostly responsible for the development of colitis-associated colon cancer. Of the several signaling pathways involved in colonic inflammation, the activation and crosstalk between NF-κB and STAT3 serve as the pivotal regulatory hubs that regulate epithelial tumorigenesis by linking inflammation with cancer development. Understanding the molecular mechanisms regulating the crosstalk between NF-κB and STAT3 will help in targeting these signaling pathways and halt epithelial tumorigenesis. MicroRNAs (miRNAs) play important role in the regulation of NF-κB and STAT3 and function in a positive- or negative feedback loop to regulate the crosstalk of these transcription factor. In the present study we evaluated the aberrant expression of a selected panel of miRNAs (miR-181b, miR-31, miR-34a, miR-146b, miR-221, and miR-155) that regulate the crosstalk between NF-κB and STAT3 during colitisassociated tumorigenesis. We used the stepwise colorectal carcinogenesis murine model known as Azoxymethane (AOM)/Dextran sodium sulphate (DSS) to recapitulate the different stages of tumorigenesis. Our results revealed that the expression of the selected miRNAs changed dynamically in a stepwise pattern as colonic tissue transforms from normal to actively inflamed to neoplastic state, in accordance with the gradual activation of NF-κB and STAT3, suggesting that the aberrant expression of these miRNAs could function as the epigenetic switch between inflammation and colorectal tumorigenesis. We were able to elucidate the contribution of miRNAs in the NF-κB - STAT3 crosstalk during the stepwise development of colitis-associated carcinoma, and this could improve our understanding of the molecular pathology of colorectal tumorigenesis and even suggesting a therapeutic strategy by modulating the expression of these regulating miRNAs.
1. Introduction Solid tumors are mostly infiltrated with inflammatory and immune cells where inflammation is involved in the initiation, promotion, and progression stages of tumor development. Therefore cancer-related inflammation is considered as the seventh hallmark of cancer [1]. Colorectal cancer (CRC) which is one of the most diagnosed cancer types is reported to show strong connection between inflammation and carcinogenesis stages. Colitis-associated cancer (CAC) is the subtype of CRC that is associated with inflammation. CAC is a well-recognized and serious complication of inflammatory bowel disease (IBD) that arises from epithelial cells lining the colon/rectum of the gastrointestinal tract following a sustained chronic inflammatory condition [2]. In this case of inflammation, an array of pro-inflammatory mediators is constantly elevated locally and systemically. Of these inflammatory mediators, the
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two transcription factors NF-κB and STAT3 are considered as the master regulator factors that link inflammation to cancer [2,3]. Nuclear Factor-Kappa B (NF-κB) has a central role in regulating the immune (innate and adaptive) responses and inflammatory responses through modulating the expression of several inflammatory cytokines and chemokines and eventually controlling cell proliferation and malignant transformation. Therefore, deregulated NF-κB activity represents a hallmark of many types of cancer [3]. It has been documented that NF-κB is aberrantly activated in the development of colitisassociated cancer [4,5]. On the other hand, Signal Transducer and Activator of Transcription (STAT) proteins are responsible for regulating cell growth, survival and differentiation. There are 7 members of the STAT family; STAT1,2,3,4,5A,5B and 6 [6]. Among all STAT members, STAT3 is considered the prominent one that is activated by interleukin-6 (IL-6) family members and is involved in controlling cell-
Corresponding author. Department of Medical Biochemistry, Medical Research Division, National Research Centre, 33 El Buhouth St. Dokki, Cairo, Egypt. E-mail address: [email protected] (S.M. El-Daly).
https://doi.org/10.1016/j.mcp.2019.101442 Received 6 July 2019; Received in revised form 12 August 2019; Accepted 31 August 2019 0890-8508/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Sherien M. El-Daly, et al., Molecular and Cellular Probes, https://doi.org/10.1016/j.mcp.2019.101442
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loop that underlies the epigenetic switch between inflammation and colorectal tumorigenesis.
cycle progression and apoptosis. Normally, STAT3 is present in a dormant state in the cytoplasm with specific cell receptors for inflammatory cytokines, mostly IL-6. Following ligand-induced dimerization of IL-6 receptor, the associated protein tyrosine kinases JAK1 and JAK2 are activated and subsequently induces STAT3 tyrosine-705 phosphorylation, the active form of STAT3, that is translocated from the cytoplasm to the nucleus where it could induce the expression of antiapoptotic genes like Bcl-2, Bcl-xL, Mcl-1 along with important transcription factors like c-myc, c-fos, and c-jun and cell cycle controlling genes like cyclin D1, cyclin E1, and p21. Therefore, elevated levels of activated STAT3 are associated with poor cancer prognosis [7,8]. Activated STAT3is found to be significantly elevated during IBD and specifically colitis associated with high-grade dysplasia [8]. Targeting and inhibiting STAT3 activation were reported to induce apoptosis of CRC cells [9,10]. NF-κB and STAT3 are effective interactive duo that promotes CRC pathogenesis. The interaction and crosstalk between NF-κB and STAT3 plays a major role in promoting the development and progression of epithelial tumorigenesis through controlling the communication between malignant cells and inflammatory or immune cells of the microenvironment [11]. In most cases elevation in the levels of cytokines, in response to NF-κB activation, cause STAT3 activation in both malignant and immune cells. This kind of crosstalk offers great opportunity for the design of new therapeutic interventions [12,13]. MicroRNAs (miRNAs) are small endogenous non-coding RNAs with an average size of 16–22 nucleotides that have been reported to regulate the expression of large number of genes through an epigenetic post-transcriptional mode of action by binding to the 3′ UTR of the mRNA of the target gene. Therefore, miRNAs are involved in the regulation of several biological processes related to tumorigenesis by directly or indirectly regulating the expression of different oncogenes or tumor suppressor genes [14,15]. Previous literatures supported the existence of both direct and indirect regulatory mechanisms between miRNAs and the NF-κB/STAT3 signaling pathways, creating a regulatory positive or negative feedback loops between miRNAs and these signaling pathways and eventually controlling cancer progression [16,17]. For example, in case of a transient inflammatory signal an epigenetic switch between non-transformed and transformed cells occurs mediated by an inhibition of let-7 causing an elevation in the levels of its target gene IL-6 and a subsequent activation of NF-κB and STAT3, thereby forming a positive feedback loop [18]. Therefore, elucidating the biological contribution of miRNAs to the NF-κB - STAT3 interaction is essential to improve the understanding of the tumorigenesis process of colorectal cancer and even suggesting anticancer therapeutic techniques through modulating the expression of these regulating miRNAs. The intent of the present study was to evaluate the aberrant expression of miRNAs that dynamically regulate the crosstalk between NF-κB and STAT3 signaling pathways during colorectal tumorigenesis process. We assessed the differential expression of a panel of known miRNAs (miR-181b, miR-34a, miR-31, miR-146b, miR-221, miR-155) that have been reported before in several literatures to target the expression of NF-κB and/or STAT3 either directly or indirectly [16,17,19]. In our study we used the stepwise colorectal tumorigenesis murine model known as Azoxymethane (AOM)/Dextran sodium sulphate (DSS). This model is a reproducible, affordable model and most importantly it recapitulates the sequence of aberrant crypt foci to adenoma reaching carcinoma similar to human CRC [20,21]. Several studies used the AOM/DSS model to study the activation of NF-κB and/ or STAT3 signaling pathways in epithelial cells and the mechanism through which this activation contributed to tumor initiation and promotion during colorectal tumor development [22,23]. Our results revealed that the expression of the selected miRNAs seems to change in a stepwise pattern as colonic tissue transforms from normal to actively inflamed to neoplastic state in accordance with the gradual activation of NF-κB and STAT3, suggesting that the aberrant expression of these miRNAs represent important part in the feedback
2. Material and methods 2.1. Ethics statement The in vivo experiments in the present study were conducted with the approval of the Research Ethical Committee of the National Research Centre (Ethical approval No#16/300 NRC). All regulations concerning the animal model were carefully applied and strictly adhered to the rules of the animal ethics committee of the National Research Centre, Cairo, Egypt. 2.2. Establishment of AOM/DSS colitis associated carcinoma model AOM/DSS mice model was conducted following previous protocols with well established and reproducible procedures [20,24]. In our study, colitis associated carcinoma was induced in male CD-1 mice (25–30 g, 6-week old) via a single intraperitoneal injection of the carcinogen AOM (10 mg/kg) and then maintained for 7 days with a regular diet and autoclaved drinking water. One week later, mice were subjected to different cycles of DSS in drinking water. In each DSS cycle, mice received 2.5% DSS in autoclaved drinking water for 7 days followed by 14 days of recovery period. According to the number of DSS cycles, mice were separated into 4 groups (n = 12) where each group represented a phase of induction during the CAC development process (Fig. 1). Mice that have not received AOM or DSS and only received autoclaved drinking water during the whole experiment represented the vehicle group. During the animal experiment, the physical signs like change in body weight, stool consistency, bloody diarrhea and anorectal prolapse, were regularly tracked and monitored. Mice of each group were sacrificed at the determined collection point as shown in (Fig. 1). Blood was collected and colons were dissected from the ileocecal junction to anal verge, then immediately opened longitudinally and rinsed in cold PBS to remove excess feces. For each dissected colon section, part was preserved in RNALater and stored at −80°C for RNA extraction and the other part fixed in 10% phosphate-buffered formalin for histopathological and immunohistochemical analysis. 2.3. Histopathological detection of aberrant crypt foci (AFC) Changes in the morphology of colon crypts are detected by methylene blue staining that positively stain AFC [25]. Formalin-fixed tissue section (5–7 μm thick) stained with 0.2% methylene blue solution for 10 min were visualized using a light Zeiss Axiostar plus microscope (Zeiss Inc., Goettingen, Germany) equipped with digital camera (PowerShot A20, canon, USA). ACF characterization was conducted by evaluating the enlarged and morphologically abnormal crypts relative to normal mucosa and the increased pericryptal space and irregular lumen. The histologic characterization of ACFs varied from nearly normal morphology to those with severe epithelial dysplasia and invasion. 2.4. Immunohistochemical (IHC) analysis for NF-κB and STAT3 Formalin-fixed tissue slides from all groups were deparaffinized, rehydrated, and then pre-treated with 1% H2O2/PBS for 15–20 min to quench the activity of endogenous peroxidases. After blocking with bovine serum albumin, slides were incubated with primary antibodies (1:100) in PBS containing 1% BSA and incubated overnight at 4 °C. For detection of NF-κB expression we used NF-κB/p65 polyclonal antibody (Thermo Fischer Scientific, Catalog # 51–0500), and for evaluating the active form of STAT3 we used STAT3 (phosphor Tyr705) antibody (Gene Tex #GTX118000) to detect only the phosphorylated form of STAT3. Following overnight incubation, slides were washed with PBS 2
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Fig. 1. Schematic overview of the AOM/DSS colitis-associated carcinoma model. Each rectangle represents one week. AOM (10 mg/kg) is injected on week 1. At the beginning of the second week different cycles of DSS are received, where each cycle consists of 2.5% DSS administered to mice in their drinking water followed by two weeks of autoclaved water. The number of groups represented the number of DSS cycles.
were purchased from Qiagen as following: miR-181b-1-3p (Mm_miR181b-1*_1 Cat# MS00032368), miR-34a (Mm_miR-34a_1 Cat# MS00001428), miR-221 (Mm_miR-221_2 Cat# MS00032585), miR146b (Mm_miR-146b_1 Cat#MS00001645), miR-155 (Mm_miR-155_1 Cat# MS00001701), miR-31 (Mm_miR-31_1 Cat# MS00001407), and RNU6-2_11 (Cat# MS00033740). For each miRNA, RT-PCR was performed in triplicate for each sample and the Ct values were calculated using automatic threshold setting. The average expression levels (ΔCt) were calculated following normalization to the internal control RNU6. Relative expression levels of each miRNA in tissue samples were evaluated by normalization to mean ΔCt value of control group and then represented as 2- ΔΔCt or relative expression level.
and then biotinylated secondary antibodies suitable for each primary antibody were added and incubated at room temperature for 2 h. Signals were developed and detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich). Slides were examined and images were captured using a light Zeiss Axiostar plus microscope (Zeiss Inc., Goettingen, Germany) equipped with digital camera (PowerShot A20, canon, USA). A manual semi-quantitative scoring based on staining intensity was applied; where (0) is no staining, (1) is weak staining representing 10% of cells are stained, (2) is moderate staining representing 20–50% of cells are stained, and (3) is strong staining where more than 50% of cells are stained. 2.5. Evaluating level of serum interlukin-6 (IL-6)
2.8. Statistical analysis The level of the pro-Inflammatory cytokine IL-6 in the serum of mice from all groups were meausred using mouse ELISA kit (BioVision, Catalog #K4144) following the manufacturer's protocol.
Data of the present study were analyzed using GraphPad Prism version 6.03. The expression levels of miRNAs were represented as Boxand-whisker plots. One-way ANOVA and a Dunnett multiple comparison tests were used to evaluate the significant differences in miRNAs expression between the different groups of the stepwise tumorigenesis process. p value of < 0.05 was considered statistically significant.
2.6. RNA isolation and reverse transcription Total RNA enriched with small sequence RNAs (miRNAs) was extracted from colonic tissue samples preserved in RNAlater. Disruption and homogenization of colon tissues were conducted by Qiagen TissueLyser (cat#85300) using stainless steel beads 5 mm (cat# 69989). Following homogenization, RNA was extracted from the homogenate using miRNeasy™ RNA isolation kit (Qiagen, Germany) following the manufacturer's protocol. The concentration and purity of eluted RNA was evaluated using a Nanodrop1000 spectrophotometer (Thermo Scientific, Wilmington, USA). All extracted RNA samples from all tissue samples of all groups were reverse-transcribed to first-strand complementary DNA (cDNA) using Qiagen miScript II RT Kit (Qiagen, Cat# 218161).
3. Results 3.1. Identification and characterization of aberrant crypt foci (ACF) The histopathological manifestation of aberrant crypt foci (ACF) is used to evaluate the earliest alterations in crypt morphology in the adenoma-carcinoma sequence [25–27]. In the present study, the colon tissue sections of control vehicle group showed crypts with uniform size and shape and round lumen with no sign of ACF (Fig. 2). However, the alteration in the crypts morphology started to be detected as early as Group I that received AOM and one cycle of DSS. In this group crypts showed slight altered morphology and ACF appeared sporadically distributed in few regions along normal crypts. As the number of DSS cycles increase the progressive alterations in the colonic crypts were observed. The morphology of crypts in mice received AOM and 2–3 cycles of DSS (Group II and III) appeared hyperplastic and heavily stained, crypts were round shaped with thick walls and narrow lumen. Colon sections of mice received AOM and 4 cycles of DSS (Group IV)
2.7. Analysis of miRNAs expression by real time-PCR Tissue levels of the selected panel of miRNAs were evaluated by Real-time PCR using miScript SYBR green PCR kit (Qiagen, Cat#218075). RT-PCR was performed in duplicate with cycling conditions; 95 °C for 15 min, followed by 40 cycles of 94 °C for 15s, 55 °C for 30s, and 70 °C for 30s. RT-PCR primers (miScript Primer Assay) 3
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Fig. 2. Morphological characterization of ACF in distal colon of mice treated with AOM followed by different cycles of DSS for monitoring tumor development process. A) Colonic tissue sections of vehicle group stained with methylene blue showing uniform size and shape of colonic crypts with regular colonic architecture. B) Colonic tissue sections of Group I received one cycle of DSS showing slight altered morphology of the colonic crypts. C) Colonic tissue sections of Group II received two cycles of DSS showing moderate altered morphology and staining of aberrant crypts. D) Colonic tissue sections of Group III where mice received three cycles of DSS showing aberrant crypts heavily stained with methylene blue and the presence of large focal areas of abnormal crypts were detected. E) Colonic tissue sections of Group IV that received four cycles of DSS showing many focal areas of abnormal crypts that intensely stained blue, individual crypts have thickened epithelium accompanied with nuclear elongation and stratification indicating signs of dysplasia. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
characterized by many dark staining aberrant crypts assembled together in cluster as foci, and several individual aberrant crypts show signs of dysplasia with nuclear stratification and elongation (Fig. 2). 3.2. Elevation of Interleukin-6 level in serum Interleukin-6 (IL-6) is a pro-inflammatory cytokine and is considered as a key regulator in the development of colitis-associated cancer. In our study the level of Il-6 significantly increased in all groups that received AOM and DSS and represent different stages of colon carcinogenesis (Group I - IV) in comparison to the vehicle group. However, no significant differences were detected in the level of IL-6 between these four groups (Fig. 3). 3.3. Activation of NF-κB and STAT3 during the stepwise progression of colitis-associated colorectal cancer Fig. 3. Serum levels of IL‐6 measured by ELISA in the different groups of the AOM/DSS‐induced colon tumorigenesis. Statistically significant differences between the different cycle groups and the vehicle group were evaluated using one-way ANOVA followed by Dunnett multiple comparison test. *P value ˂0.05, **P value ˂0.001.
Activation of NF-κB and STAT3 during the stepwise colon tumorigenesis were evaluated in our study at the protein level by immunohistochemistry using polyclonal antibodies against the NF-κB -subunit p65 (Fig. 4) and p-STAT3 (Fig. 5). In our study, colon tissue sections revealed faint staining of NF-κB in normal epithelial cells (scoring between 0 and 1). Meanwhile, the expression of NF-κB was slightly activated in Group I that received AOM followed by one cycle of 4
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Fig. 4. Immunohistochemical staining for the NF-κB subunit p65 in colon tissue sections. (A) Colonic tissue sections of vehicle group showing faint immunostaining of NF-κB (scoring 0–1). (B) Colonic tissue sections of Group I where mice received one cycle of DSS showing mild immunopositive staining of NF-κB (scoring 1). (C) and (D) Colonic tissue sections of Group II and Group III that received two and three cycles of DSS, respectively, showing increased immunopositive staining (scoring 2). (E) Colonic tissue sections of Group IV that received four cycle of DSS showed strong immunopositive staining in the nuclei of the aberrant crypts (scoring 3).
κB/p-STAT3 staining and the malignant transformation of epithelial cells, indicating that these two markers are implicated in the stepwise tumorigenesis process of CRC.
DSS, where a moderate immunoreactivity for NF-κB subunit p65 was detected (scoring 1). With the increase in the number of DSS cycles given to mice and the subsequent transformation of epithelial cells from well-to poorly-differentiated adenoma, the staining of NF-κB gradually increased reaching its maximum expression in Group IV (scoring 3) where a significant immunoreactivity and nuclear translocation of NFκB was detected (Fig. 4). Our data suggest that NF-κB is constitutively activated during the gradual transformation of epithelial cells. Analysis of STAT3 activation in intestinal epithelial cells during the tumorigenesis process was evaluated by immunohistochemical staining of p-STAT3. The same pattern of gradual increase in the staining intensity that detected for NF-κB was also observed for p-STAT3 immunostaining where the scoring ranged from 0 to 3 as the staining intensity gradually increased (Fig. 5). Colonic tissue sections of vehicle group showed no staining of p-STAT3 (scoring 0) however, transformed epithelial cells in colonic tissue sections of the groups received AOM and DSS cycles showed positive immunostaining for p-STAT3 in both cytoplasm and nucleus where the staining intensity increased as more DSS cycles were received (scoring ranged from 1 to 3). In our study, there was a detected positive correlation between the intensity of NF-
3.4. Differential expression of miRNAs during the stepwise tumorigenesis process Using RT-PCR, we verified the differential expression levels of the six selected miRNAs in the different groups that represent stages of tumor development, in comparison to vehicle group. Our results revealed a significant dynamic change in the expression of miRNAs during the stepwise tumor development process (Fig. 6). The miRNAs expression profiles dynamically changed either by overexpression (miR181b, miR-31, miR-146b, miR-221, miR-155) or by suppression (miR34a) during the four stages of colitis associated tumorigenesis and there was a dependent increase/decrease in the expression of this panel of miRNAs associated with the tumor development stage. Worth to note, the significance in the aberrant expression was mostly observed in the late stages of tumorigenesis (Group III and IV) in comparison with vehicle control. 5
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Fig. 5. Immunohistochemical images of pSTAT3 in colon tissue sections of mice treated with AOM followed by different cycles of DSS. A) Vehicle control group showing immunonegative staining of pSTAT3 (scoring 0). (B) Colonic tissue sections of Group I where mice received one cycle of DSS showing moderate immunopositive brown staining of p-STAT3 (scoring 2). (C), (D), and (E) Colonic tissue sections of Group II, III and IV that received two, three and four cycles of DSS, respectively, showing strong immunopositive staining of p-STAT3 as indicated by the brown granular deposits in the cytoplasms and nuclei (scoring 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4. Discussion
In our endeavor to clarify and understand the molecular markers controlling the activity of IL-6/NF-κB/STAT3 cooperative signaling pathway during the inflammatory tumorigenesis process, we evaluated the differential expression of a panel of miRNAs that directly or indirectly target NF-κB and/or STAT3 (Fig. 7). Our results revealed that the expression of the selected miRNAs seems to change in a stepwise pattern as colonic tissue transforms from normal to actively inflamed to neoplastic state in accordance with the gradual activation of NF-κB and STAT3. The gradual and significant up-regulation of miR-181b that is detected in our study during the different stages of colorectal tumorigenesis process was in accordance with several studies that considered miR-181b as a critical link between inflammation and cancer [19,30]. The clinical significance of miR-181b was investigated in several cancer types and it was shown to be inversely correlated with the survival rate of colon and gastric cancer patients [31,32]. In colorectal cancer, STAT3 was reported to up-regulate miR-181b through directly binding to multiple sites in the miR-181b promoter region, which in turn enhanced the expression of this miRNA [19]. The elevation in the level of miR-181b in turn enhances NF-κB activity through directly targeting and inhibiting the tumor suppressor cylindromatosis (CYLD) which is responsible for negatively modulating NF-κB activity [19,33] (Fig. 7). Therefore, the STAT3-miR-181b- CYLD- NF-κB link is an important part of the positive feedback loop that underlies the epigenetic switch
Over the past few years, several studies reported the vital role of miRNAs in the molecular pathogenesis of colorectal cancer, it was even suggested that the expression profiles of miRNAs can classify tumor types more accurately than gene expression profiles [28,29]. While transcription factors like NF-κB and STAT3 are associated with cancer progression, the network crosstalk connecting and regulating these transcription factors with miRNAs are still under investigation. In this study we provide strong evidence that the differential expression of miRNAs that control the crosstalk of NF-κB and STAT3 is important for the inflammatory process responsible for colorectal tumorigenesis. Using the AOM/DSS murine model of colitis associated tumorigenesis we were able to recapitulate the different stages of tumor development. According to our results, the growth and morphological features of ACF evaluated in colon tissue sections of the different groups support the sequential tumorigenesis process that originates from normal epithelial cells to aberrant crypts and preneoplastic lesions and eventually the traditional sequence of colitis development. This sequential transformation of epithelial cells was accompanied by a significant elevation in the level of serum IL-6 as detected from ELISA measurement (Fig. 3) and gradual consecutive activation of NF-κB and STAT-3 as can be detected from colonic tissue immunostaining (Figs. 4 and 5). 6
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Fig. 6. Box-and-whisker plots of the relative expression levels of the six selected miRNAs in the 4 groups representing the stepwise development of colitis associated tumorigenesis. The relative expression was calculated using the 2−ΔΔCt method. Statistically significant differences between the different cycle groups and the vehicle group were evaluated using one-way ANOVA followed by Dunnett multiple comparison test. *P value ˂0.05, **P value ˂0.001.
metastatic colorectal cancer. The authors of this study proved that repression of miR-34a by STAT3 is required for tumor progression. The miR-34a deficient mice with colitis-associated intestinal tumors displayed significant up-regulation of p-STAT3 and IL-6 receptor and progressed efficiently to invasive carcinomas more than wild type mice. This data indicates that reduction of miR-34a level, which is common in different cancer types, is essential for the IL-6/STAT3 activation
between inflammation and colorectal cancer. Another miRNA that has is reported in our study to be differentially expressed during the tumorigenesis process is the tumor suppressor miR-34a. The link between suppression of miR-34a and colorectal cancer development was represented by IL-6 receptor/STAT3/miR-34a feedback loop which was first reported by Rokavec et al., 2014 [34] and considered as a vital feedback that occurs in both primary tumor and 7
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221. The mechanism that is suggested through which up-regulation of miR-221 contributes to the constitutive activation of NF-κB and STAT3, is through the direct targeting and inhibition of miR-221 to the PDZLIM domain protein (PDLIM2) [47]. PDLIM2 is a gene that is known to be essential for restraint NF-κB activity through intranuclear sequestration and degradation of the p65 subunit of the NF-κB [48]. At the same time PDLIM2 is responsible for the polyubiquitination and/or proteasomal degradation of STAT3 [49]. Therefore inhibiting the expression of this gene by miR-221 enhances the NF-κB/STAT3 activity. To prove the efficiency of this positive feedback loop in tumor progression, delivering miR-221 antagonism to CRC cell line or to colitisassociated cancer mice significantly suppressed the activity of NFκB and STAT3 and subsequently reduced inflammation, cell proliferation and increased apoptosis as proved previously [47], this suggested mechanism can delineate the link between our detected stepwise elevation in miR-221 and the increase in the activity of NF-κB-STAT3 crosstalk. miR-155 is one of the well-studied oncomiRs with elevated expression in several solid and hematologic malignancies where it is involved in proliferation and cell survival. It is even documented that the overexpression of miR-155 could be sufficient to trigger and promote malignant transformation [50,51]. miR-155 plays an essential role in the activation of immune cells (B- and T-cells) and therefore it acts as a link between inflammation and cancer [52,53]. In the study by Gerloff et al., 2015 [54] NF-κB is found to be a potential regulator and inducer of miR-155 via direct promoter binding and this regulation is even enhanced by activated STAT5 in acute myeloid leukemia [54]. A relation between miR-155 and STAT3 has also been found where the overexpression of miR-155 caused a constitutive activation of STAT3 as a result of the direct targeting and inhibition of the Suppressor of Cytokine Signaling 1 gene (SOCS1) [55–57]. SOCS1 is a tumor suppressor that functions as a negative feedback regulator of STAT3 signaling by preventing the JAK-STAT3 binding activity [58,59]. These direct and indirect links between the expression of miR-155 and NF-κB-STAT3 activation is linked in our study with the different stages of CAC. In conclusion, in our study we were able to provide strong evidence that the differential expression of miRNAs that control the crosstalk of NF-κB and STAT3 is important for the inflammatory process responsible for colorectal tumorigenesis. Identifying the molecular regulators of the NF-κB and STAT3 crosstalk could improve the understanding of the molecular pathology of colorectal tumorigenesis and provide novel strategies to target these signaling pathways.
Fig. 7. Schematic view of the six selected miRNAs regulating the crosstalk of NF-κB/IL-6/STAT3 signaling pathway. (NF-κB) Nuclear Factor-Kappa B; (STAT3) Signal Transducer and Activator of Transcription; (IL-6) Interlukin-6; (SOCS1) Suppressor of Cytokine Signaling 1; (CYLD) Cylindromatosis; (PDLIM2) PDZ-LIM domain protein; (JAK) The Janus kinase; (TRAF6) TNF Receptor Associated Factor 6; (IRAK1) interleukin 1 receptor associated kinase 1.
involved in tumor progression, this link was confirmed in our study where the gradual significant reduction in miR-34a level was in accordance with the gradual NF-κB - STAT3 activation. Previous reports labeled miR-31 as an oncomiR in colorectal cancer with major clinical significance. The elevated expression of miR-31 was associated with advanced tumor stage, as its level at TNM stage III and IV was much higher than that detected at stage I and II in CRC patients [35–37]. Moreover, the overexpression of miR-31 was progressively increased during the transformation from normal to IBD-related neoplasia [38] which is similar to our detected results, revealing that miR31 expression is important for the development and progression of CAC. The up-regulation of miR-31 contributes to several pathways controlling CRC like the activation of Wnt, BMP and TGFb pathways. Additionally, a tight association between miR-31 induction and STAT3 pathway activation in intestinal tissues was also reported, where an active STAT3 signaling significantly elevated the expression of miR-31 in intestinal epithelial cells [39]. The elevated expression of miR-31 is reported to accompany an elevation in NF- κB activity in intestinal tissue of rats [40], at the same time an active state of NF-κB can enhance the expression of miR-31 through the binding of the p65 subunit of NF-κB to the putative binding sites in the promoter element of miR31, offering a positive feedback loope [41]. Aberrant expression of miR-146b-5p is documented in several cancer types however, the molecular mechanism of miR-146b-5p differs in each type. In human glioma miR-146b functions as a tumor suppressor [42] meanwhile, the expression of miR-146b-5p is significantly elevated in breast, lung, thyroid and colorectal cancer. The elevated levels of miR-146b is positively associated with tumor stage progression [43–45] and this can be observed in our study by the gradual elevation of miR-146b expression level during each stage of tumor development. miR-146b was identified as a direct STAT3 target and its expression is induced by activated STAT3. Previously it was reported that during STAT3 induction of miR-146b a negative feedback circuit inhibiting the activity of NF-κB occurs [46]. However, in our study this negative feedback loop was not detected, suggesting that miR-146b regulatory circuit could be subverted in AOM/DSS induced model of inflammatory bowel disease allowing the growth and progression of tumor cells contingent on the NF-κB/IL-6/STAT3 pathway. Another miRNA that is detected in our study to be elevated during AOM/DSS tumorigenesis process and has oncogenic properties is miR-
Conflicts of interest The authors declare that there is no conflict of interests regarding the publication of this manuscript. Acknowledgments This research was financially supported by the National Research Centre, Cairo, Egypt (NRC project grant No# 11010129). References [1] F. Colotta, P. Allavena, A. Sica, C. Garlanda, A. Mantovani, Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability, Carcinogenesis (2009), https://doi.org/10.1093/carcin/bgp127. [2] R.J. Xavier, D.K. Podolsky, Unravelling the pathogenesis of inflammatory bowel disease, Nature (2007), https://doi.org/10.1038/nature06005. [3] J.A. Didonato, F. Mercurio, M. Karin, NF-κB and the link between inflammation and cancer, Immunol. Rev. (2012), https://doi.org/10.1111/j.1600-065X.2012. 01099.x. [4] F.R. Greten, L. Eckmann, T.F. Greten, J.M. Park, Z.W. Li, L.J. Egan, M.F. Kagnoff, M. Karin, IKKβ links inflammation and tumorigenesis in a mouse model of colitisassociated cancer, Cell (2004), https://doi.org/10.1016/j.cell.2004.07.013. [5] E. Viennois, F. Chen, D. Merlin, NF-κB pathway in colitis-associated cancers, Transl. Gastrointest. Cancer 2 (1) (2013) 21–29, https://doi.org/10.3978/j.issn.22244778.2012.11.01. [6] C. Shrinkage, M. Blebbing, F. Condensation, Jak/Stat Signaling: IL-6 Receptor
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Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Differential expression of miRNAs regulating NF-κB and STAT3 crosstalk during colitis-associated tumorigenesis Sherien M. El-Dalya,b,∗, Enayat A. Omarac, Jihan Husseina, Eman R. Younessa, Zakaria El-Khayata a
Medical Biochemistry Department, Medical Research Division, National Research Centre, Cairo, Egypt Cancer Biology and Genetics Laboratory, Centre of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt c Pathology Department, Medical Research Division, National Research Centre, Cairo, Egypt b
A R T I C LE I N FO
A B S T R A C T
Keywords: Colitis Tumorigenesis Inflammation NF-κB STAT3 miRNAs
Inflammatory bowel disease (IBD) is mostly responsible for the development of colitis-associated colon cancer. Of the several signaling pathways involved in colonic inflammation, the activation and crosstalk between NF-κB and STAT3 serve as the pivotal regulatory hubs that regulate epithelial tumorigenesis by linking inflammation with cancer development. Understanding the molecular mechanisms regulating the crosstalk between NF-κB and STAT3 will help in targeting these signaling pathways and halt epithelial tumorigenesis. MicroRNAs (miRNAs) play important role in the regulation of NF-κB and STAT3 and function in a positive- or negative feedback loop to regulate the crosstalk of these transcription factor. In the present study we evaluated the aberrant expression of a selected panel of miRNAs (miR-181b, miR-31, miR-34a, miR-146b, miR-221, and miR-155) that regulate the crosstalk between NF-κB and STAT3 during colitisassociated tumorigenesis. We used the stepwise colorectal carcinogenesis murine model known as Azoxymethane (AOM)/Dextran sodium sulphate (DSS) to recapitulate the different stages of tumorigenesis. Our results revealed that the expression of the selected miRNAs changed dynamically in a stepwise pattern as colonic tissue transforms from normal to actively inflamed to neoplastic state, in accordance with the gradual activation of NF-κB and STAT3, suggesting that the aberrant expression of these miRNAs could function as the epigenetic switch between inflammation and colorectal tumorigenesis. We were able to elucidate the contribution of miRNAs in the NF-κB - STAT3 crosstalk during the stepwise development of colitis-associated carcinoma, and this could improve our understanding of the molecular pathology of colorectal tumorigenesis and even suggesting a therapeutic strategy by modulating the expression of these regulating miRNAs.
1. Introduction Solid tumors are mostly infiltrated with inflammatory and immune cells where inflammation is involved in the initiation, promotion, and progression stages of tumor development. Therefore cancer-related inflammation is considered as the seventh hallmark of cancer [1]. Colorectal cancer (CRC) which is one of the most diagnosed cancer types is reported to show strong connection between inflammation and carcinogenesis stages. Colitis-associated cancer (CAC) is the subtype of CRC that is associated with inflammation. CAC is a well-recognized and serious complication of inflammatory bowel disease (IBD) that arises from epithelial cells lining the colon/rectum of the gastrointestinal tract following a sustained chronic inflammatory condition [2]. In this case of inflammation, an array of pro-inflammatory mediators is constantly elevated locally and systemically. Of these inflammatory mediators, the
∗
two transcription factors NF-κB and STAT3 are considered as the master regulator factors that link inflammation to cancer [2,3]. Nuclear Factor-Kappa B (NF-κB) has a central role in regulating the immune (innate and adaptive) responses and inflammatory responses through modulating the expression of several inflammatory cytokines and chemokines and eventually controlling cell proliferation and malignant transformation. Therefore, deregulated NF-κB activity represents a hallmark of many types of cancer [3]. It has been documented that NF-κB is aberrantly activated in the development of colitisassociated cancer [4,5]. On the other hand, Signal Transducer and Activator of Transcription (STAT) proteins are responsible for regulating cell growth, survival and differentiation. There are 7 members of the STAT family; STAT1,2,3,4,5A,5B and 6 [6]. Among all STAT members, STAT3 is considered the prominent one that is activated by interleukin-6 (IL-6) family members and is involved in controlling cell-
Corresponding author. Department of Medical Biochemistry, Medical Research Division, National Research Centre, 33 El Buhouth St. Dokki, Cairo, Egypt. E-mail address: [email protected] (S.M. El-Daly).
https://doi.org/10.1016/j.mcp.2019.101442 Received 6 July 2019; Received in revised form 12 August 2019; Accepted 31 August 2019 0890-8508/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Sherien M. El-Daly, et al., Molecular and Cellular Probes, https://doi.org/10.1016/j.mcp.2019.101442
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loop that underlies the epigenetic switch between inflammation and colorectal tumorigenesis.
cycle progression and apoptosis. Normally, STAT3 is present in a dormant state in the cytoplasm with specific cell receptors for inflammatory cytokines, mostly IL-6. Following ligand-induced dimerization of IL-6 receptor, the associated protein tyrosine kinases JAK1 and JAK2 are activated and subsequently induces STAT3 tyrosine-705 phosphorylation, the active form of STAT3, that is translocated from the cytoplasm to the nucleus where it could induce the expression of antiapoptotic genes like Bcl-2, Bcl-xL, Mcl-1 along with important transcription factors like c-myc, c-fos, and c-jun and cell cycle controlling genes like cyclin D1, cyclin E1, and p21. Therefore, elevated levels of activated STAT3 are associated with poor cancer prognosis [7,8]. Activated STAT3is found to be significantly elevated during IBD and specifically colitis associated with high-grade dysplasia [8]. Targeting and inhibiting STAT3 activation were reported to induce apoptosis of CRC cells [9,10]. NF-κB and STAT3 are effective interactive duo that promotes CRC pathogenesis. The interaction and crosstalk between NF-κB and STAT3 plays a major role in promoting the development and progression of epithelial tumorigenesis through controlling the communication between malignant cells and inflammatory or immune cells of the microenvironment [11]. In most cases elevation in the levels of cytokines, in response to NF-κB activation, cause STAT3 activation in both malignant and immune cells. This kind of crosstalk offers great opportunity for the design of new therapeutic interventions [12,13]. MicroRNAs (miRNAs) are small endogenous non-coding RNAs with an average size of 16–22 nucleotides that have been reported to regulate the expression of large number of genes through an epigenetic post-transcriptional mode of action by binding to the 3′ UTR of the mRNA of the target gene. Therefore, miRNAs are involved in the regulation of several biological processes related to tumorigenesis by directly or indirectly regulating the expression of different oncogenes or tumor suppressor genes [14,15]. Previous literatures supported the existence of both direct and indirect regulatory mechanisms between miRNAs and the NF-κB/STAT3 signaling pathways, creating a regulatory positive or negative feedback loops between miRNAs and these signaling pathways and eventually controlling cancer progression [16,17]. For example, in case of a transient inflammatory signal an epigenetic switch between non-transformed and transformed cells occurs mediated by an inhibition of let-7 causing an elevation in the levels of its target gene IL-6 and a subsequent activation of NF-κB and STAT3, thereby forming a positive feedback loop [18]. Therefore, elucidating the biological contribution of miRNAs to the NF-κB - STAT3 interaction is essential to improve the understanding of the tumorigenesis process of colorectal cancer and even suggesting anticancer therapeutic techniques through modulating the expression of these regulating miRNAs. The intent of the present study was to evaluate the aberrant expression of miRNAs that dynamically regulate the crosstalk between NF-κB and STAT3 signaling pathways during colorectal tumorigenesis process. We assessed the differential expression of a panel of known miRNAs (miR-181b, miR-34a, miR-31, miR-146b, miR-221, miR-155) that have been reported before in several literatures to target the expression of NF-κB and/or STAT3 either directly or indirectly [16,17,19]. In our study we used the stepwise colorectal tumorigenesis murine model known as Azoxymethane (AOM)/Dextran sodium sulphate (DSS). This model is a reproducible, affordable model and most importantly it recapitulates the sequence of aberrant crypt foci to adenoma reaching carcinoma similar to human CRC [20,21]. Several studies used the AOM/DSS model to study the activation of NF-κB and/ or STAT3 signaling pathways in epithelial cells and the mechanism through which this activation contributed to tumor initiation and promotion during colorectal tumor development [22,23]. Our results revealed that the expression of the selected miRNAs seems to change in a stepwise pattern as colonic tissue transforms from normal to actively inflamed to neoplastic state in accordance with the gradual activation of NF-κB and STAT3, suggesting that the aberrant expression of these miRNAs represent important part in the feedback
2. Material and methods 2.1. Ethics statement The in vivo experiments in the present study were conducted with the approval of the Research Ethical Committee of the National Research Centre (Ethical approval No#16/300 NRC). All regulations concerning the animal model were carefully applied and strictly adhered to the rules of the animal ethics committee of the National Research Centre, Cairo, Egypt. 2.2. Establishment of AOM/DSS colitis associated carcinoma model AOM/DSS mice model was conducted following previous protocols with well established and reproducible procedures [20,24]. In our study, colitis associated carcinoma was induced in male CD-1 mice (25–30 g, 6-week old) via a single intraperitoneal injection of the carcinogen AOM (10 mg/kg) and then maintained for 7 days with a regular diet and autoclaved drinking water. One week later, mice were subjected to different cycles of DSS in drinking water. In each DSS cycle, mice received 2.5% DSS in autoclaved drinking water for 7 days followed by 14 days of recovery period. According to the number of DSS cycles, mice were separated into 4 groups (n = 12) where each group represented a phase of induction during the CAC development process (Fig. 1). Mice that have not received AOM or DSS and only received autoclaved drinking water during the whole experiment represented the vehicle group. During the animal experiment, the physical signs like change in body weight, stool consistency, bloody diarrhea and anorectal prolapse, were regularly tracked and monitored. Mice of each group were sacrificed at the determined collection point as shown in (Fig. 1). Blood was collected and colons were dissected from the ileocecal junction to anal verge, then immediately opened longitudinally and rinsed in cold PBS to remove excess feces. For each dissected colon section, part was preserved in RNALater and stored at −80°C for RNA extraction and the other part fixed in 10% phosphate-buffered formalin for histopathological and immunohistochemical analysis. 2.3. Histopathological detection of aberrant crypt foci (AFC) Changes in the morphology of colon crypts are detected by methylene blue staining that positively stain AFC [25]. Formalin-fixed tissue section (5–7 μm thick) stained with 0.2% methylene blue solution for 10 min were visualized using a light Zeiss Axiostar plus microscope (Zeiss Inc., Goettingen, Germany) equipped with digital camera (PowerShot A20, canon, USA). ACF characterization was conducted by evaluating the enlarged and morphologically abnormal crypts relative to normal mucosa and the increased pericryptal space and irregular lumen. The histologic characterization of ACFs varied from nearly normal morphology to those with severe epithelial dysplasia and invasion. 2.4. Immunohistochemical (IHC) analysis for NF-κB and STAT3 Formalin-fixed tissue slides from all groups were deparaffinized, rehydrated, and then pre-treated with 1% H2O2/PBS for 15–20 min to quench the activity of endogenous peroxidases. After blocking with bovine serum albumin, slides were incubated with primary antibodies (1:100) in PBS containing 1% BSA and incubated overnight at 4 °C. For detection of NF-κB expression we used NF-κB/p65 polyclonal antibody (Thermo Fischer Scientific, Catalog # 51–0500), and for evaluating the active form of STAT3 we used STAT3 (phosphor Tyr705) antibody (Gene Tex #GTX118000) to detect only the phosphorylated form of STAT3. Following overnight incubation, slides were washed with PBS 2
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Fig. 1. Schematic overview of the AOM/DSS colitis-associated carcinoma model. Each rectangle represents one week. AOM (10 mg/kg) is injected on week 1. At the beginning of the second week different cycles of DSS are received, where each cycle consists of 2.5% DSS administered to mice in their drinking water followed by two weeks of autoclaved water. The number of groups represented the number of DSS cycles.
were purchased from Qiagen as following: miR-181b-1-3p (Mm_miR181b-1*_1 Cat# MS00032368), miR-34a (Mm_miR-34a_1 Cat# MS00001428), miR-221 (Mm_miR-221_2 Cat# MS00032585), miR146b (Mm_miR-146b_1 Cat#MS00001645), miR-155 (Mm_miR-155_1 Cat# MS00001701), miR-31 (Mm_miR-31_1 Cat# MS00001407), and RNU6-2_11 (Cat# MS00033740). For each miRNA, RT-PCR was performed in triplicate for each sample and the Ct values were calculated using automatic threshold setting. The average expression levels (ΔCt) were calculated following normalization to the internal control RNU6. Relative expression levels of each miRNA in tissue samples were evaluated by normalization to mean ΔCt value of control group and then represented as 2- ΔΔCt or relative expression level.
and then biotinylated secondary antibodies suitable for each primary antibody were added and incubated at room temperature for 2 h. Signals were developed and detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich). Slides were examined and images were captured using a light Zeiss Axiostar plus microscope (Zeiss Inc., Goettingen, Germany) equipped with digital camera (PowerShot A20, canon, USA). A manual semi-quantitative scoring based on staining intensity was applied; where (0) is no staining, (1) is weak staining representing 10% of cells are stained, (2) is moderate staining representing 20–50% of cells are stained, and (3) is strong staining where more than 50% of cells are stained. 2.5. Evaluating level of serum interlukin-6 (IL-6)
2.8. Statistical analysis The level of the pro-Inflammatory cytokine IL-6 in the serum of mice from all groups were meausred using mouse ELISA kit (BioVision, Catalog #K4144) following the manufacturer's protocol.
Data of the present study were analyzed using GraphPad Prism version 6.03. The expression levels of miRNAs were represented as Boxand-whisker plots. One-way ANOVA and a Dunnett multiple comparison tests were used to evaluate the significant differences in miRNAs expression between the different groups of the stepwise tumorigenesis process. p value of < 0.05 was considered statistically significant.
2.6. RNA isolation and reverse transcription Total RNA enriched with small sequence RNAs (miRNAs) was extracted from colonic tissue samples preserved in RNAlater. Disruption and homogenization of colon tissues were conducted by Qiagen TissueLyser (cat#85300) using stainless steel beads 5 mm (cat# 69989). Following homogenization, RNA was extracted from the homogenate using miRNeasy™ RNA isolation kit (Qiagen, Germany) following the manufacturer's protocol. The concentration and purity of eluted RNA was evaluated using a Nanodrop1000 spectrophotometer (Thermo Scientific, Wilmington, USA). All extracted RNA samples from all tissue samples of all groups were reverse-transcribed to first-strand complementary DNA (cDNA) using Qiagen miScript II RT Kit (Qiagen, Cat# 218161).
3. Results 3.1. Identification and characterization of aberrant crypt foci (ACF) The histopathological manifestation of aberrant crypt foci (ACF) is used to evaluate the earliest alterations in crypt morphology in the adenoma-carcinoma sequence [25–27]. In the present study, the colon tissue sections of control vehicle group showed crypts with uniform size and shape and round lumen with no sign of ACF (Fig. 2). However, the alteration in the crypts morphology started to be detected as early as Group I that received AOM and one cycle of DSS. In this group crypts showed slight altered morphology and ACF appeared sporadically distributed in few regions along normal crypts. As the number of DSS cycles increase the progressive alterations in the colonic crypts were observed. The morphology of crypts in mice received AOM and 2–3 cycles of DSS (Group II and III) appeared hyperplastic and heavily stained, crypts were round shaped with thick walls and narrow lumen. Colon sections of mice received AOM and 4 cycles of DSS (Group IV)
2.7. Analysis of miRNAs expression by real time-PCR Tissue levels of the selected panel of miRNAs were evaluated by Real-time PCR using miScript SYBR green PCR kit (Qiagen, Cat#218075). RT-PCR was performed in duplicate with cycling conditions; 95 °C for 15 min, followed by 40 cycles of 94 °C for 15s, 55 °C for 30s, and 70 °C for 30s. RT-PCR primers (miScript Primer Assay) 3
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Fig. 2. Morphological characterization of ACF in distal colon of mice treated with AOM followed by different cycles of DSS for monitoring tumor development process. A) Colonic tissue sections of vehicle group stained with methylene blue showing uniform size and shape of colonic crypts with regular colonic architecture. B) Colonic tissue sections of Group I received one cycle of DSS showing slight altered morphology of the colonic crypts. C) Colonic tissue sections of Group II received two cycles of DSS showing moderate altered morphology and staining of aberrant crypts. D) Colonic tissue sections of Group III where mice received three cycles of DSS showing aberrant crypts heavily stained with methylene blue and the presence of large focal areas of abnormal crypts were detected. E) Colonic tissue sections of Group IV that received four cycles of DSS showing many focal areas of abnormal crypts that intensely stained blue, individual crypts have thickened epithelium accompanied with nuclear elongation and stratification indicating signs of dysplasia. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
characterized by many dark staining aberrant crypts assembled together in cluster as foci, and several individual aberrant crypts show signs of dysplasia with nuclear stratification and elongation (Fig. 2). 3.2. Elevation of Interleukin-6 level in serum Interleukin-6 (IL-6) is a pro-inflammatory cytokine and is considered as a key regulator in the development of colitis-associated cancer. In our study the level of Il-6 significantly increased in all groups that received AOM and DSS and represent different stages of colon carcinogenesis (Group I - IV) in comparison to the vehicle group. However, no significant differences were detected in the level of IL-6 between these four groups (Fig. 3). 3.3. Activation of NF-κB and STAT3 during the stepwise progression of colitis-associated colorectal cancer Fig. 3. Serum levels of IL‐6 measured by ELISA in the different groups of the AOM/DSS‐induced colon tumorigenesis. Statistically significant differences between the different cycle groups and the vehicle group were evaluated using one-way ANOVA followed by Dunnett multiple comparison test. *P value ˂0.05, **P value ˂0.001.
Activation of NF-κB and STAT3 during the stepwise colon tumorigenesis were evaluated in our study at the protein level by immunohistochemistry using polyclonal antibodies against the NF-κB -subunit p65 (Fig. 4) and p-STAT3 (Fig. 5). In our study, colon tissue sections revealed faint staining of NF-κB in normal epithelial cells (scoring between 0 and 1). Meanwhile, the expression of NF-κB was slightly activated in Group I that received AOM followed by one cycle of 4
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Fig. 4. Immunohistochemical staining for the NF-κB subunit p65 in colon tissue sections. (A) Colonic tissue sections of vehicle group showing faint immunostaining of NF-κB (scoring 0–1). (B) Colonic tissue sections of Group I where mice received one cycle of DSS showing mild immunopositive staining of NF-κB (scoring 1). (C) and (D) Colonic tissue sections of Group II and Group III that received two and three cycles of DSS, respectively, showing increased immunopositive staining (scoring 2). (E) Colonic tissue sections of Group IV that received four cycle of DSS showed strong immunopositive staining in the nuclei of the aberrant crypts (scoring 3).
κB/p-STAT3 staining and the malignant transformation of epithelial cells, indicating that these two markers are implicated in the stepwise tumorigenesis process of CRC.
DSS, where a moderate immunoreactivity for NF-κB subunit p65 was detected (scoring 1). With the increase in the number of DSS cycles given to mice and the subsequent transformation of epithelial cells from well-to poorly-differentiated adenoma, the staining of NF-κB gradually increased reaching its maximum expression in Group IV (scoring 3) where a significant immunoreactivity and nuclear translocation of NFκB was detected (Fig. 4). Our data suggest that NF-κB is constitutively activated during the gradual transformation of epithelial cells. Analysis of STAT3 activation in intestinal epithelial cells during the tumorigenesis process was evaluated by immunohistochemical staining of p-STAT3. The same pattern of gradual increase in the staining intensity that detected for NF-κB was also observed for p-STAT3 immunostaining where the scoring ranged from 0 to 3 as the staining intensity gradually increased (Fig. 5). Colonic tissue sections of vehicle group showed no staining of p-STAT3 (scoring 0) however, transformed epithelial cells in colonic tissue sections of the groups received AOM and DSS cycles showed positive immunostaining for p-STAT3 in both cytoplasm and nucleus where the staining intensity increased as more DSS cycles were received (scoring ranged from 1 to 3). In our study, there was a detected positive correlation between the intensity of NF-
3.4. Differential expression of miRNAs during the stepwise tumorigenesis process Using RT-PCR, we verified the differential expression levels of the six selected miRNAs in the different groups that represent stages of tumor development, in comparison to vehicle group. Our results revealed a significant dynamic change in the expression of miRNAs during the stepwise tumor development process (Fig. 6). The miRNAs expression profiles dynamically changed either by overexpression (miR181b, miR-31, miR-146b, miR-221, miR-155) or by suppression (miR34a) during the four stages of colitis associated tumorigenesis and there was a dependent increase/decrease in the expression of this panel of miRNAs associated with the tumor development stage. Worth to note, the significance in the aberrant expression was mostly observed in the late stages of tumorigenesis (Group III and IV) in comparison with vehicle control. 5
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Fig. 5. Immunohistochemical images of pSTAT3 in colon tissue sections of mice treated with AOM followed by different cycles of DSS. A) Vehicle control group showing immunonegative staining of pSTAT3 (scoring 0). (B) Colonic tissue sections of Group I where mice received one cycle of DSS showing moderate immunopositive brown staining of p-STAT3 (scoring 2). (C), (D), and (E) Colonic tissue sections of Group II, III and IV that received two, three and four cycles of DSS, respectively, showing strong immunopositive staining of p-STAT3 as indicated by the brown granular deposits in the cytoplasms and nuclei (scoring 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4. Discussion
In our endeavor to clarify and understand the molecular markers controlling the activity of IL-6/NF-κB/STAT3 cooperative signaling pathway during the inflammatory tumorigenesis process, we evaluated the differential expression of a panel of miRNAs that directly or indirectly target NF-κB and/or STAT3 (Fig. 7). Our results revealed that the expression of the selected miRNAs seems to change in a stepwise pattern as colonic tissue transforms from normal to actively inflamed to neoplastic state in accordance with the gradual activation of NF-κB and STAT3. The gradual and significant up-regulation of miR-181b that is detected in our study during the different stages of colorectal tumorigenesis process was in accordance with several studies that considered miR-181b as a critical link between inflammation and cancer [19,30]. The clinical significance of miR-181b was investigated in several cancer types and it was shown to be inversely correlated with the survival rate of colon and gastric cancer patients [31,32]. In colorectal cancer, STAT3 was reported to up-regulate miR-181b through directly binding to multiple sites in the miR-181b promoter region, which in turn enhanced the expression of this miRNA [19]. The elevation in the level of miR-181b in turn enhances NF-κB activity through directly targeting and inhibiting the tumor suppressor cylindromatosis (CYLD) which is responsible for negatively modulating NF-κB activity [19,33] (Fig. 7). Therefore, the STAT3-miR-181b- CYLD- NF-κB link is an important part of the positive feedback loop that underlies the epigenetic switch
Over the past few years, several studies reported the vital role of miRNAs in the molecular pathogenesis of colorectal cancer, it was even suggested that the expression profiles of miRNAs can classify tumor types more accurately than gene expression profiles [28,29]. While transcription factors like NF-κB and STAT3 are associated with cancer progression, the network crosstalk connecting and regulating these transcription factors with miRNAs are still under investigation. In this study we provide strong evidence that the differential expression of miRNAs that control the crosstalk of NF-κB and STAT3 is important for the inflammatory process responsible for colorectal tumorigenesis. Using the AOM/DSS murine model of colitis associated tumorigenesis we were able to recapitulate the different stages of tumor development. According to our results, the growth and morphological features of ACF evaluated in colon tissue sections of the different groups support the sequential tumorigenesis process that originates from normal epithelial cells to aberrant crypts and preneoplastic lesions and eventually the traditional sequence of colitis development. This sequential transformation of epithelial cells was accompanied by a significant elevation in the level of serum IL-6 as detected from ELISA measurement (Fig. 3) and gradual consecutive activation of NF-κB and STAT-3 as can be detected from colonic tissue immunostaining (Figs. 4 and 5). 6
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Fig. 6. Box-and-whisker plots of the relative expression levels of the six selected miRNAs in the 4 groups representing the stepwise development of colitis associated tumorigenesis. The relative expression was calculated using the 2−ΔΔCt method. Statistically significant differences between the different cycle groups and the vehicle group were evaluated using one-way ANOVA followed by Dunnett multiple comparison test. *P value ˂0.05, **P value ˂0.001.
metastatic colorectal cancer. The authors of this study proved that repression of miR-34a by STAT3 is required for tumor progression. The miR-34a deficient mice with colitis-associated intestinal tumors displayed significant up-regulation of p-STAT3 and IL-6 receptor and progressed efficiently to invasive carcinomas more than wild type mice. This data indicates that reduction of miR-34a level, which is common in different cancer types, is essential for the IL-6/STAT3 activation
between inflammation and colorectal cancer. Another miRNA that has is reported in our study to be differentially expressed during the tumorigenesis process is the tumor suppressor miR-34a. The link between suppression of miR-34a and colorectal cancer development was represented by IL-6 receptor/STAT3/miR-34a feedback loop which was first reported by Rokavec et al., 2014 [34] and considered as a vital feedback that occurs in both primary tumor and 7
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221. The mechanism that is suggested through which up-regulation of miR-221 contributes to the constitutive activation of NF-κB and STAT3, is through the direct targeting and inhibition of miR-221 to the PDZLIM domain protein (PDLIM2) [47]. PDLIM2 is a gene that is known to be essential for restraint NF-κB activity through intranuclear sequestration and degradation of the p65 subunit of the NF-κB [48]. At the same time PDLIM2 is responsible for the polyubiquitination and/or proteasomal degradation of STAT3 [49]. Therefore inhibiting the expression of this gene by miR-221 enhances the NF-κB/STAT3 activity. To prove the efficiency of this positive feedback loop in tumor progression, delivering miR-221 antagonism to CRC cell line or to colitisassociated cancer mice significantly suppressed the activity of NFκB and STAT3 and subsequently reduced inflammation, cell proliferation and increased apoptosis as proved previously [47], this suggested mechanism can delineate the link between our detected stepwise elevation in miR-221 and the increase in the activity of NF-κB-STAT3 crosstalk. miR-155 is one of the well-studied oncomiRs with elevated expression in several solid and hematologic malignancies where it is involved in proliferation and cell survival. It is even documented that the overexpression of miR-155 could be sufficient to trigger and promote malignant transformation [50,51]. miR-155 plays an essential role in the activation of immune cells (B- and T-cells) and therefore it acts as a link between inflammation and cancer [52,53]. In the study by Gerloff et al., 2015 [54] NF-κB is found to be a potential regulator and inducer of miR-155 via direct promoter binding and this regulation is even enhanced by activated STAT5 in acute myeloid leukemia [54]. A relation between miR-155 and STAT3 has also been found where the overexpression of miR-155 caused a constitutive activation of STAT3 as a result of the direct targeting and inhibition of the Suppressor of Cytokine Signaling 1 gene (SOCS1) [55–57]. SOCS1 is a tumor suppressor that functions as a negative feedback regulator of STAT3 signaling by preventing the JAK-STAT3 binding activity [58,59]. These direct and indirect links between the expression of miR-155 and NF-κB-STAT3 activation is linked in our study with the different stages of CAC. In conclusion, in our study we were able to provide strong evidence that the differential expression of miRNAs that control the crosstalk of NF-κB and STAT3 is important for the inflammatory process responsible for colorectal tumorigenesis. Identifying the molecular regulators of the NF-κB and STAT3 crosstalk could improve the understanding of the molecular pathology of colorectal tumorigenesis and provide novel strategies to target these signaling pathways.
Fig. 7. Schematic view of the six selected miRNAs regulating the crosstalk of NF-κB/IL-6/STAT3 signaling pathway. (NF-κB) Nuclear Factor-Kappa B; (STAT3) Signal Transducer and Activator of Transcription; (IL-6) Interlukin-6; (SOCS1) Suppressor of Cytokine Signaling 1; (CYLD) Cylindromatosis; (PDLIM2) PDZ-LIM domain protein; (JAK) The Janus kinase; (TRAF6) TNF Receptor Associated Factor 6; (IRAK1) interleukin 1 receptor associated kinase 1.
involved in tumor progression, this link was confirmed in our study where the gradual significant reduction in miR-34a level was in accordance with the gradual NF-κB - STAT3 activation. Previous reports labeled miR-31 as an oncomiR in colorectal cancer with major clinical significance. The elevated expression of miR-31 was associated with advanced tumor stage, as its level at TNM stage III and IV was much higher than that detected at stage I and II in CRC patients [35–37]. Moreover, the overexpression of miR-31 was progressively increased during the transformation from normal to IBD-related neoplasia [38] which is similar to our detected results, revealing that miR31 expression is important for the development and progression of CAC. The up-regulation of miR-31 contributes to several pathways controlling CRC like the activation of Wnt, BMP and TGFb pathways. Additionally, a tight association between miR-31 induction and STAT3 pathway activation in intestinal tissues was also reported, where an active STAT3 signaling significantly elevated the expression of miR-31 in intestinal epithelial cells [39]. The elevated expression of miR-31 is reported to accompany an elevation in NF- κB activity in intestinal tissue of rats [40], at the same time an active state of NF-κB can enhance the expression of miR-31 through the binding of the p65 subunit of NF-κB to the putative binding sites in the promoter element of miR31, offering a positive feedback loope [41]. Aberrant expression of miR-146b-5p is documented in several cancer types however, the molecular mechanism of miR-146b-5p differs in each type. In human glioma miR-146b functions as a tumor suppressor [42] meanwhile, the expression of miR-146b-5p is significantly elevated in breast, lung, thyroid and colorectal cancer. The elevated levels of miR-146b is positively associated with tumor stage progression [43–45] and this can be observed in our study by the gradual elevation of miR-146b expression level during each stage of tumor development. miR-146b was identified as a direct STAT3 target and its expression is induced by activated STAT3. Previously it was reported that during STAT3 induction of miR-146b a negative feedback circuit inhibiting the activity of NF-κB occurs [46]. However, in our study this negative feedback loop was not detected, suggesting that miR-146b regulatory circuit could be subverted in AOM/DSS induced model of inflammatory bowel disease allowing the growth and progression of tumor cells contingent on the NF-κB/IL-6/STAT3 pathway. Another miRNA that is detected in our study to be elevated during AOM/DSS tumorigenesis process and has oncogenic properties is miR-
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