Kepeng Wang

Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.

Approximately 2% of colorectal cancer is linked to pre-existing inflammation known as colitis-associated cancer, but most develops in patients without underlying inflammatory bowel disease. Colorectal cancer often follows a genetic pathway whereby loss of the adenomatous polyposis coli (APC) tumour suppressor and activation of β-catenin are followed by mutations in K-Ras, PIK3CA and TP53, as the tumour emerges and progresses. Curiously, however, ‘inflammatory signature’ genes characteristic of colitis-associated cancer are also upregulated in colorectal cancer. Further, like most solid tumours, colorectal cancer exhibits immune/inflammatory infiltrates, referred to as ‘tumour-elicited inflammation’. Although infiltrating CD4+ TH1 cells and CD8+ cytotoxic T cells constitute a positive prognostic sign in colorectal cancer, myeloid cells and T-helper interleukin (IL)-17-producing (TH17) cells promote tumorigenesis, and a ‘TH17 expression signature’ in stage I/II colorectal cancer is associated with a drastic decrease in disease-free survival. Despite its pathogenic importance, the mechanisms responsible for the appearance of tumour-elicited inflammation are poorly understood. Many epithelial cancers develop proximally to microbial communities, which are physically separated from immune cells by an epithelial barrier. We investigated mechanisms responsible for tumour-elicited inflammation in a mouse model of colorectal tumorigenesis, which, like human colorectal cancer, exhibits upregulation of IL-23 and IL-17. Here we show that IL-23 signalling promotes tumour growth and progression, and development of a tumoural IL-17 response. IL-23 is mainly produced by tumour-associated myeloid cells that are likely to be activated by microbial products, which penetrate the tumours but not adjacent tissue. Both early and late colorectal neoplasms exhibit defective expression of several barrier proteins. We propose that barrier deterioration induced by colorectal-cancer-initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth.

A gp130-Src-YAP module links inflammation to epithelial regeneration

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

JAK1–STAT1–STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts

Skeletal muscle stem cell–derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced muscle regeneration. However, the cellular signaling pathways controlling the proliferation and differentiation of myoblasts are not fully understood. We demonstrate that Janus kinase 1 (JAK1) is required for myoblast proliferation and that it also functions as a checkpoint to prevent myoblasts from premature differentiation. Deliberate knockdown of JAK1 in both primary and immortalized myoblasts induces precocious myogenic differentiation with a concomitant reduction in cell proliferation. This is caused, in part, by an accelerated induction of MyoD, myocyte enhancer–binding factor 2 (MEF2), p21Cip1, and p27Kip1, a faster down-regulation of Id1, and an increase in MEF2-dependent gene transcription. Downstream of JAK1, of all the signal transducer and activator of transcriptions (STATs) present in myoblasts, we find that only STAT1 knockdown promotes myogenic differentiation in both primary and immortalized myoblasts. Leukemia inhibitory factor stimulates myoblast proliferation and represses differentiation via JAK1–STAT1–STAT3. Thus, JAK1–STAT1–STAT3 constitutes a signaling pathway that promotes myoblast proliferation and prevents premature myoblast differentiation.

Signal-dependent incorporation of MyoD–BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex

Tissue‐specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin‐remodelling complex. However, the molecular mechanism that regulates SWI/SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD‐target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal‐dependent recruitment of the SWI/SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation‐activated p38α kinase is the signal that promotes incorporation of MyoD–BAF60c into a Brg1‐based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD‐target genes. Our data support an unprecedented two‐step model by which pre‐assembled BAF60c–MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues.

Interleukin-17 receptor A signaling in transformed enterocytes promotes early colorectal tumorigenesis

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine linked to rapid malignant progression of colorectal cancer (CRC) and therapy resistance. IL-17A exerts its pro-tumorigenic activity through its type A receptor (IL-17RA). However, IL-17RA is expressed in many cell types, including hematopoietic, fibroblastoid, and epithelial cells, in the tumor microenvironment, and how IL-17RA engagement promotes colonic tumorigenesis is unknown. Here we show that IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK, and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls the production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. These findings establish IL-17A and IL-17RA as therapeutic targets in colorectal cancer.

JAK2/STAT2/STAT3 are required for myogenic differentiation.

Skeletal muscle satellite cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced regeneration. However, the cellular signaling pathways that control proliferation and differentiation of myoblasts remain poorly defined. Recently, we found that JAK1/STAT1/STAT3 not only participate in myoblast proliferation but also actively prevent them from premature differentiation. Unexpectedly, we found that a related pathway consisting of JAK2, STAT2, and STAT3 is required for early myogenic differentiation. Interference of this pathway by either a small molecule inhibitor or small interfering RNA inhibits myogenic differentiation. Consistently, all three molecules are activated upon differentiation. The pro-differentiation effect of JAK2/STAT2/STAT3 is partially mediated by MyoD and MEF2. Interestingly, the expression of the IGF2 gene and the HGF gene is also regulated by JAK2/STAT2/STAT3, suggesting that this pathway could also promote differentiation by regulating signaling molecules known to be involved in myogenic differentiation. In summary, our current study reveals a novel role for the JAK2/STAT2/STAT3 pathway in myogenic differentiation.

MiR-124 Suppresses Growth of Human Colorectal Cancer by Inhibiting STAT3

Emerging evidence indicate that microRNAs (miRNAs) may play important roles in cancer. Aberrant expression of miRNAs has been frequently identified in different human malignancies, including colorectal cancer (CRC). However, the mechanism by which deregulated miRNAs impact the development of CRC remains largely elusive. In this study, we show that miR-124 is significantly down-regulated in CRC compared to adjacent non-tumor colorectal tissues. MiR-124 suppresses the expression of STAT3 by directly binding to its 3′-untranslated region (3′-UTR). Overexpression of miR-124 led to increased apoptosis of CRC cells and reduced tumor growth in vitro and in vivo. Knocking down STAT3 expression by specific siRNA suppressed the growth of CRC cells in vitro and in vivo, resembling that of miR-124 overexpression. Moreover, overexpression of STAT3 in miR-124-transfected CRC cells effectively rescued the inhibition of cell proliferation caused by miR-124. These data suggest that miR-124 serves as a tumor suppressor by targeting STAT3, and call for the use of miR-124 as a potential therapeutic tool for CRC, where STAT3 is often hyper-activated.

Intestinal glucuronidation protects against chemotherapy-induced toxicity by irinotecan (CPT-11)

Significance Camptothecin (CPT)-11 (irinotecan) is an antitumor agent used in cancer chemotherapy primarily for the treatment of solid tumors. CPT-11 is a prodrug that is metabolized by carboxylesterases to the DNA topoisomerase 1 inhibitor, called SN-38. Detoxification of SN-38 occurs by UDP-glucuronosyltransferase 1A1 (UGT1A1)-dependent glucuronidation. A serious side effect of CPT-11 chemotherapy is SN-38–induced intestinal toxicity, which is believed to result in part from the delivery of SN-38 into intestinal tissue through enterohepatic circulation. By selectively targeting the deletion of the Ugt1 locus in either liver or intestinal tissue, we have confirmed that intestinal UGT1A-specific glucuronidation of SN-38 is essential in preventing SN-38–induced toxicity. Being able to induce intestinal UGT1A1 may be effective in reducing CPT-11 toxicity. Camptothecin (CPT)-11 (irinotecan) has been used widely for cancer treatment, particularly metastatic colorectal cancer. However, up to 40% of treated patients suffer from severe late diarrhea, which prevents CPT-11 dose intensification and efficacy. CPT-11 is a prodrug that is hydrolyzed by hepatic and intestinal carboxylesterase to form SN-38, which in turn is detoxified primarily through UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed glucuronidation. To better understand the mechanism associated with toxicity, we generated tissue-specific Ugt1 locus conditional knockout mouse models and examined the role of glucuronidation in protecting against irinotecan-induced toxicity. We targeted the deletion of the Ugt1 locus and the Ugt1a1 gene specifically in the liver (Ugt1ΔHep) and the intestine (Ugt1ΔGI). Control (Ugt1F/F), Ugt1ΔHep, and Ugt1ΔGI adult male mice were treated with different concentrations of CPT-11 daily for four consecutive days. Toxicities were evaluated with regard to tissue glucuronidation potential. CPT-11–treated Ugt1ΔHep mice showed a similar lethality rate to the CPT-11–treated Ugt1F/F mice. However, Ugt1ΔGI mice were highly susceptible to CPT-11–induced diarrhea, developing severe and lethal mucositis at much lower CPT-11 doses, a result of the proliferative cell loss and inflammation in the intestinal tract. Comparative expression levels of UGT1A1 in intestinal tumors and normal surrounding tissue are dramatically different, providing for the opportunity to improve therapy by differential gene regulation. Intestinal expression of the UGT1A proteins is critical toward the detoxification of SN-38, whereas induction of the UGT1A1 gene may serve to limit toxicity and improve the efficacy associated with CPT-11 treatment.

Implications of anti-cytokine therapy in colorectal cancer and autoimmune diseases

Up to 20% of all cancers have been linked to chronic inflammation and persistent infections. However, almost all solid tumours contain immune infiltrates, and tumour-associated inflammatory cells play broad roles in different stages of tumour development and malignant progression. Cytokines are important mediators of the inflammatory effect on tumorigenesis both in inflammation-induced cancer and in the inflammation that follows tumour development. We have shown interleukin (IL)-6 to be an important tumour promoter in early colitis-associated cancer (CAC). IL-6 is mainly produced by tumour-infiltrating myeloid cells under the control of NF-κB. IL-6 promotes proliferation of tumour-initiating cells derived from the intestinal epithelium and protects them from apoptotic elimination. These pro-survival and proliferative effects of IL-6 are mainly mediated by STAT3, whose ablation in intestinal epithelial cells significantly reduces CAC tumorigenesis. More recently, we found a critical role for IL-23 and its downstream cytokines IL-17 and IL-22 in the development of CAC. These findings suggest that such cytokines or the cells that produce them may provide new therapeutic or preventive targets in forms of colorectal cancer that are linked to inflammation.

Sequential Recruitment of PCAF and BRG1 Contributes to Myogenin Activation in 12-O-Tetradecanoylphorbol-13-acetate-induced Early Differentiation of Rhabdomyosarcoma-derived Cells

Myogenin and its upstream regulator MyoD are known to be required for myogenic cell differentiation. Although both of them can be expressed in rhabdomyosarcoma-derived RD cells, the cells are unable to undergo full-scale terminal myogenic differentiation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) has been found to be functional in the induction of RD cell differentiation, whereas its mechanism is not fully understood. By using quantitative real-time-based chromatin immunoprecipitation and real-time reverse transcription-PCR-based promoter activity assays, we examined the activation mechanism of the myogenin gene during TPA-induced differentiation of the RD cells. We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene. Both PCAF and BRG1 are also involved in the activation of the myogenin gene. In addition, we have found that the p38 mitogen-activated protein kinase is required for BRG1 recruitment in TPA-mediated myogenin induction. We propose that there are two distinct activation steps for the induction of myogenin in TPA-induced early differentiation of RD cells: 1) an early step that requires PCAF activity to acetylate core histones and MyoD to initiate myogenin gene expression, and 2) a later step that requires p38-dependent activity of the SWI/SNF remodeling complex to provide an open conformation for the induction of myogenin. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma.