Role played by MDSC in colitis-associated colorectal cancer and potential therapeutic strategies.

Colitis-associated colorectal cancer has been a hot topic in public health issues worldwide. Numerous studies have demonstrated the significance of myeloid-derived suppressor cells (MDSCs) in the progression of this ailment, but the specific mechanism of their role in the transformation of inflammation to cancer is unclear, and potential therapies targeting MDSC are also unclear. This paper outlines the possible involvement of MDSC to the development of colitis-associated colorectal cancer. It also explores the immune and other relevant roles played by MDSC, and collates relevant targeted therapies against MDSC. In addition, current targeted therapies for colorectal cancer are analyzed and summarized.

Therapeutic Effect of Proteinase-Activated Receptor-1 Antagonist on Colitis-Associated Carcinogenesis.

BACKGROUND & AIMS: Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR1) increases in inflammatory bowel disease. The present study investigated the therapeutic effects of PAR1 antagonism on colitis-associated carcinogenesis. METHODS: A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR1 antagonist E5555 was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblasts of the Crohn's disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR1 agonist proteinases. RESULTS: AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The ß-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota ß-diversity in AOM/DSS mice. The thrombin expression was up-regulated in tumor and non-tumor areas, whereas PAR1 mRNA expression was higher in tumor areas compared with non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca2+ concentration and ERK1/2 phosphorylation, as well as IL6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned medium contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR1 at the thrombin cleavage site. CONCLUSIONS: PAR1 antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and its associated carcinogenesis.

Tropomyosin 2 Regulates Tumor Cell Proliferation, Immune Suppression, and Activation of the JNK Signaling Pathway in Colitis-Associated Cancer (CAC).

BACKGROUND: Tropomyosin 2 (TPM2) has been linked to the advancement of various tumor types, exhibiting distinct impacts on tumor progression. In our investigation, the primary objective was to identify the potential involvement of TPM2 in the development of colitis-associated cancer (CAC) using a mice model. METHODS: This study used lentiviral vector complex for TPM2 knockdown (sh-TPM2) and the corresponding negative control lentiviral vector complex (sh-NC) for genetic interference in mice. CAC was induced in mice using azoxymethane (AOM) and dextran sulfate sodium salt (DSS). This study included 6 groups of mice models: Control, Control+sh-NC, Control+sh-TPM2, CAC, CAC+sh-NC, and CAC+sh-TPM2. Subsequently, colon tissues were collected and assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for TPM2 mRNA levels and flow cytometry for infiltrating immune cells. Tumor number, size, and weight within colon tissues from CAC mice were measured and recorded. The hematoxylin-eosin staining was used for observing tissue pathology changes. The intestinal epithelial cells (IECs) were isolated and analyzed for cell proliferation. This analysis included examining the levels of 5-bromo-2-deoxyuridine (BrdU) and Ki-67 using immunohistochemistry. Additionally, the mRNA levels of proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by qRT-PCR. This study also investigated the activation of the c-Jun N-terminal kinase (JNK) pathway using western blot analysis. Immunogenicity analyses were conducted using immunohistochemistry for F4/80 and flow cytometry. RESULTS: In 8-week-old mice, AOM injections and three cycles of DSS treatment induced TPM2 upregulation in tumor tissues compared to normal tissues (p < 0.05). Fluorescence-activated cell sorting (FACS)-isolated lamina CAC adenomas revealed macrophages and dendritic cells as primary TPM2 contributors (p < 0.001). Lentiviral TPM2 gene knockdown significantly reduced tumor numbers and sizes in CAC mice (p < 0.01, and p < 0.001), without invasive cancer cells. TPM2 suppression resulted in decreased IEC proliferation (p < 0.001) and reduced PCNA and Ki-67 expression (p < 0.05). Western blot analysis indicated reduced JNK pathway activation in TPM2-knockdown CAC mice (p < 0.05, p < 0.001). TPM2 knockdown decreased tumor-associated macrophage infiltration (p < 0.01) and increased CD3+ and CD8+ T cells (p < 0.01, and p < 0.001), with increased levels of regulator of inflammatory cytokines (CD44+, CD107a+) (p < 0.01, and p < 0.001), decreased levels of PD-1+ and anti-inflammatory factor (IL10+) (p < 0.01, and p < 0.001). CONCLUSIONS: Our results demonstrated that TPM2 knockdown suppressed the proliferation of CAC IECs, enhanced immune suppression on CAC IECs, and inhibited the JNK signaling pathway within the framework of CAC. These findings suggest TPM2 can serve as a potential therapeutic target for CAC treatment.

MUC1-mediated Macrophage Activation Promotes Colitis-associated Colorectal Cancer via Activating the Interleukin-6/ Signal Transducer and Activator of Transcription 3 Axis.

BACKGROUND & AIMS: MUC1 is abnormally expressed in colorectal cancer, including colitis-associated colorectal cancer (CAC), but its role in tumorigenesis is unclear. This study investigated MUC1's effects in murine models of colitis and CAC and elucidated mechanisms of action. METHODS: Colitis and CAC were induced in mice by exposure to dextran sodium sulfate or azoxymethane plus dextran sodium sulphate. Clinical parameters, immune cell infiltration, and tumor development were monitored throughout disease progression. Experiments in knockout mice and bone marrow chimeras were combined with an exploration of immune cell abundance and function. RESULTS: Deficiency of Muc1 suppressed inflammation, inhibited tumor progression, increased abundance of CD8+ T lymphocytes, and reduced abundance of macrophages in colon tumors. Bone marrow chimeras showed promotion of CAC was primarily mediated by Muc1-expressing hematopoietic cells, and that MUC1 promoted a pro-tumoral immunosuppressive macrophage phenotype within tumors. Mechanistic studies revealed that Muc1 deficiency remarkably reduced interleukin-6 levels in the colonic tissues and tumors that was mainly produced by infiltrating macrophages at day 21, 42, and 85. In bone marrow-derived macrophages, MUC1 promoted responsiveness to chemoattractant and promoted activation into a phenotype with high Il6 and Ido1 expression, secreting factors which inhibited CD8+ T cell proliferation. MUC1 potently drives macrophages to produce interleukin-6, which in turn drives a pro-tumorigenic activation of signal transducer and activator of transcription 3 in colon epithelial tumor and stromal cells, ultimately increasing the occurrence and development of CAC. CONCLUSIONS: Our findings provide cellular and molecular mechanisms for the pro-tumorigenic functions of MUC1 in the inflamed colon. Therapeutic strategies to inhibit MUC1 signal transduction warrant consideration for the prevention or therapy of CAC.

T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model.

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.

Suppression of Transmembrane Tumor Necrosis Factor Alpha Processing by a Specific Antibody Protects Against Colitis-Associated Cancer.

Soluble tumor necrosis factor-α (sTNF-α) plays an important role in colitis-associated cancer (CAC); however, little is known about transmembrane TNF-α (tmTNF-α). Here, we observed an increase in sTNF-α mainly in colitis tissues from an azoxymethane/dextran sodium sulfate (DSS)-induced CAC mouse model whereas tmTNF-α levels were chiefly increased on epithelial cells at the tumor stage. The ratio of intracolonic tmTNF-α/sTNF-α was negatively correlated with the levels of pro-inflammatory mediators (IL-1ß, IL-6, and NO) and M1 macrophages but positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and the level of the anti-inflammatory cytokine IL-10, suggesting an anti-inflammatory effect of tmTNF-α. This effect of tmTNF-α was confirmed again by the induction of resistance to LPS in colonic epithelial cell lines NCM460 and HCoEpiC through the addition of exogenous tmTNF-α or transfection of the tmTNF-α leading sequence that lacks the extracellular segment but retains the intracellular domain of tmTNF-α. A tmTNF-α antibody was used to block tmTNF-α shedding after the first or second round of inflammation induction by DSS drinking to shift the time window of tmTNF-α expression ahead to the inflammation stage. Antibody treatment significantly alleviated inflammation and suppressed subsequent adenoma formation, accompanied by increased apoptosis. An antitumor effect was also observed when the antibody was administered at the malignant phase of CAC. Our results reveal tmTNF-α as a novel molecular marker for malignant transformation in CAC and provide a new insight into blocking the pathological process by targeting tmTNF-α processing.

Overproduction of Gastrointestinal 5-HT Promotes Colitis-Associated Colorectal Cancer Progression via Enhancing NLRP3 Inflammasome Activation.

Chronic inflammation is a key driver for colitis-associated colorectal cancer. 5-hydroxytryptamine (5-HT), a neurotransmitter, has been reported to promote inflammation in the gastrointestinal tract. However, the mechanism behind this remains unclear. In this study, we found that 5-HT levels, as well as the expression of tryptophan hydroxylase 1 (TPH1), the 5-HT biosynthesis rate-limiting enzyme, were significantly upregulated in colorectal tumor tissues from patients with colorectal cancer, colorectal cancer mouse models, and colorectal cancer cell lines when compared with normal colorectal tissues or epithelial cell lines. Colorectal cancer cell-originated 5-HT enhanced NLRP3 inflammasome activation in THP-1 cells and immortalized bone marrow-derived macrophages (iBMDM) via its ion channel receptor, HTR3A. Mechanistically, HTR3A activation led to Ca2+ influx, followed by CaMKIIα phosphorylation (Thr286) and activation, which then induced NLRP3 phosphorylation at Ser198 (mouse: Ser194) and inflammasome assembling. The NLRP3 inflammasome mediated IL1ß maturation, and release upregulated 5-HT biosynthesis in colorectal cancer cells by inducing TPH1 transcription, revealing a positive feedback loop between 5-HT and NLRP3 signaling. Silencing TPH1 or HTR3A by short hairpin RNA slowed down tumor growth in an established CT26 and iBMDM coimplanted subcutaneous allograft colorectal cancer mouse model, whereas treatment with TPH1 inhibitor 4-chloro-DL-phenylalanine or HTR3A antagonist tropisetron alleviated tumor progression in an azoxymethane/dextran sodium sulfate-induced colorectal cancer mouse model. Addressing the positive feedback loop between 5-HT and NLRP3 signaling could provide potential therapeutic targets for colorectal cancer.

Xanthine oxidoreductase promotes the progression of colitis-associated colorectal cancer by causing DNA damage and mediating macrophage M1 polarization.

In addition to its pivotal role in purine metabolism, xanthine oxidoreductase (XOR) is one of the key enzymes involved in superoxide radical generation. Oxidative stress has been implicated in the etiology of colorectal cancer, but the contribution of XOR remains unclear. Here we investigated the role of XOR in colitis-associated colorectal cancer (CAC) and the underlying mechanisms. Using clinical samples, we demonstrated that XOR up-regulation was an early event in colonic carcinogenesis. Pharmacological inhibition of XOR effectively delayed the progression of CAC. Moreover, XOR activity positively correlated with tumor necrosis factor-alpha (TNFα) protein levels. Mechanistically, TNFα may activate XOR transcription via activator protein-1 and, thus, promote endogenous hydrogen peroxide generation, resulting in oxidative DNA damage in colon cancer cells. On the other hand, XOR may regulate the TNFα mRNA transcripts by mediating LPS-induced macrophage M1 polarization. Collectively, XOR promotes tumor development by programming the tumor microenvironment and stimulates CAC progression via DNA damage-induced genetic instability.

Scutellarin ameliorates colitis-associated colorectal cancer by suppressing Wnt/ß-catenin signaling cascade.

Dysregulated Wnt/ß-catenin signaling pathway plays a critical role in the pathogenesis of colorectal cancer (CRC). Scutellarin, a flavonoid compound in Scutellaria barbata, has been reported to suppress CRC, with the action mechanism elusive. In this study, Scutellarin was found to inhibit the carcinogenesis of colitis-associated cancer (CAC) in mice caused by azoxymethane/dextran sulfate sodium, with alleviation of pathologic symptoms. Besides, Scutellarin attenuated mouse serum concentrations of TNF-α and IL-6, heightened Bax expression and diminished B-cell lymphoma-2 (Bcl-2) level in CAC tissues of mice, through down-regulating Wnt/ß-catenin signaling cascade. In CRC HT-29 cells, Scutellarin retarded the proliferation and migration, induced apoptosis, with boosted Bax expression and decreased Bcl-2 level, which may be attributed to its repression of Wnt/ß-catenin signals in HT-29 cells. Our findings demonstrate that Scutellarin may ameliorate colitis-associated colorectal cancer by weakening Wnt/ß-catenin signaling cascade.

Colorectal cancer cells utilize autophagy to maintain mitochondrial metabolism for cell proliferation under nutrient stress.

Cancer cells reprogram cellular metabolism to maintain adequate nutrient pools to sustain proliferation. Moreover, autophagy is a regulated mechanism to break down dysfunctional cellular components and recycle cellular nutrients. However, the requirement for autophagy and the integration in cancer cell metabolism is not clear in colon cancer. Here, we show a cell-autonomous dependency of autophagy for cell growth in colorectal cancer. Loss of epithelial autophagy inhibits tumor growth in both sporadic and colitis-associated cancer models. Genetic and pharmacological inhibition of autophagy inhibits cell growth in colon cancer-derived cell lines and patient-derived enteroid models. Importantly, normal colon epithelium and patient-derived normal enteroid growth were not decreased following autophagy inhibition. To couple the role of autophagy to cellular metabolism, a cell culture screen in conjunction with metabolomic analysis was performed. We identified a critical role of autophagy to maintain mitochondrial metabolites for growth. Loss of mitochondrial recycling through inhibition of mitophagy hinders colon cancer cell growth. These findings have revealed a cell-autonomous role of autophagy that plays a critical role in regulating nutrient pools in vivo and in cell models, and it provides therapeutic targets for colon cancer.

Transcriptome analysis of potential candidate genes and molecular pathways in colitis-associated colorectal cancer of Mkp-1-deficient mice.

BACKGROUND: The nuclear phosphatase mitogen-activate protein kinase phosphatase-1 (MKP-1) is a key negative regulator of the innate immune response through the regulation of the biosynthesis of proinflammatory cytokines. In colorectal cancer (CRC), which is induced mainly by chronic inflammation, Mkp-1 overexpression was found in addition to disturbances in Mkp-1 functions, which may play a role in cancer development in different types of tumors. However, the potential molecular mechanisms by which Mkp-1 influences CRC development is not clear. Here, we performed global gene expression profiling of Mkp-1 KO mice using RNA sequencing (RNA-seq) to explore the role of Mkp-1 in CRC progression using transcriptome analysis. METHODS: Azoxymethane/dextran sodium sulfate (AOM/DSS) mouse models were used to examine the most dramatic molecular and signaling changes that occur during different phases of CRC development in wild-type mice and Mkp-1 KO mice. Comprehensive bioinformatics analyses were used to elucidate the molecular processes regulated by Mkp-1. Differentially expressed genes (DEGs) were identified and functionally analyzed by Gene Ontology (GO), Kyoto Enrichment of Genes and Genomes (KEGG). Then, protein-protein interaction (PPI) network analysis was conducted using the STRING database and Cytoscape software. RESULTS: Persistent DEGs were different in adenoma and carcinoma stage (238 & 251, respectively) and in WT and MKp-1 KO mice (221& 196, respectively). Mkp-1 KO modulated key molecular processes typically activated in cancer, in particular, cell adhesion, ion transport, extracellular matrix organization, response to drug, response to hypoxia, and response to toxic substance. It was obvious that these pathways are closely associated with cancer development and metastasis. From the PPI network analyses, nine hub genes associated with CRC were identified. CONCLUSION: These findings suggest that MKp-1 and its hub genes may play a critical role in cancer development, prognosis, and determining treatment outcomes. We provide clues to build a potential link between Mkp-1 and colitis-associated tumorigenesis and identify areas requiring further investigation.

STAT6 Is Critical for the Induction of Regulatory T Cells In Vivo Controlling the Initial Steps of Colitis-Associated Cancer.

Inflammation is the main driver of the tumor initiation and progression in colitis-associated colorectal cancer (CAC). Recent findings have indicated that the signal transducer and activator of transcription 6 (STAT6) plays a fundamental role in the early stages of CAC, and STAT6 knockout (STAT6-/-) mice are highly resistant to CAC development. Regulatory T (Treg) cells play a major role in coordinating immunomodulation in cancer; however, the role of STAT6 in the induction and function of Treg cells is poorly understood. To clarify the contribution of STAT6 to CAC, STAT6-/- and wild type (WT) mice were subjected to an AOM/DSS regimen, and the frequency of peripheral and local Treg cells was determined during the progression of CAC. When STAT6 was lacking, a remarkable reduction in tumor growth was observed, which was associated with decreased inflammation and an increased number of CD4+CD25+Foxp3+ cells in the colon, circulation, and spleen, including an over-expression of TGF-beta, IL-10, and Foxp3, compared to WT mice, during the early stages of CAC development. Conversely, WT mice showed an inverse frequency of Treg cells compared with STAT6-/- mice, which was followed by intestinal tumor formation. Increased mucosal inflammation, histological damage, and tumorigenesis were restored to levels observed in WT mice when an early inhibition/depletion of Treg cells was performed in STAT6-/- mice. Thus, with STAT6 deficiency, an increased number of Treg cells induce resistance against tumorigenesis, arresting tumor-promoting inflammation. We reported a direct role of STAT6 in the induction and function of Treg cells during CAC development and suggest that STAT6 is a potential target for the modulation of immune response in colitis and CAC.

Genetic and Epigenetic Characteristics of Inflammatory Bowel Disease-Associated Colorectal Cancer.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder associated with an elevated risk of colorectal cancer (CRC). IBD-associated CRC (IBD-CRC) may represent a distinct pathway of tumorigenesis compared to sporadic CRC (sCRC). Our aim was to comprehensively characterize IBD-associated tumorigenesis integrating multiple high-throughput approaches, and to compare the results with in-house data sets from sCRCs. METHODS: Whole-genome sequencing, single nucleotide polymorphism arrays, RNA sequencing, genome-wide methylation analysis, and immunohistochemistry were performed using fresh-frozen and formalin-fixed tissue samples of tumor and corresponding normal tissues from 31 patients with IBD-CRC. RESULTS: Transcriptome-based tumor subtyping revealed the complete absence of canonical epithelial tumor subtype associated with WNT signaling in IBD-CRCs, dominated instead by mesenchymal stroma-rich subtype. Negative WNT regulators AXIN2 and RNF43 were strongly down-regulated in IBD-CRCs and chromosomal gains at HNF4A, a negative regulator of WNT-induced epithelial-mesenchymal transition (EMT), were less frequent compared to sCRCs. Enrichment of hypomethylation at HNF4α binding sites was detected solely in sCRC genomes. PIGR and OSMR involved in mucosal immunity were dysregulated via epigenetic modifications in IBD-CRCs. Genome-wide analysis showed significant enrichment of noncoding mutations to 5'untranslated region of TP53 in IBD-CRCs. As reported previously, somatic mutations in APC and KRAS were less frequent in IBD-CRCs compared to sCRCs. CONCLUSIONS: Distinct mechanisms of WNT pathway dysregulation skew IBD-CRCs toward mesenchymal tumor subtype, which may affect prognosis and treatment options. Increased OSMR signaling may favor the establishment of mesenchymal tumors in patients with IBD.

MondoA-Thioredoxin-Interacting Protein Axis Maintains Regulatory T-Cell Identity and Function in Colorectal Cancer Microenvironment.

BACKGROUND & AIMS: The metabolic features and function of intratumoral regulatory T cells (Tregs) are ambiguous in colorectal cancer. Tumor-infiltrating Tregs are reprogrammed to exhibit high glucose-depleting properties and adapt to the glucose-restricted microenvironment. The glucose-responsive transcription factor MondoA is highly expressed in Tregs. However, the role of MondoA in colorectal cancer-infiltrating Tregs in response to glucose limitation remains to be elucidated. METHODS: We performed studies using mice, in which MondoA was conditionally deleted in Tregs, and human colorectal cancer tissues. Seahorse and other metabolic assays were used to assess Treg metabolism. To study the role of Tregs in antitumor immunity, we used a subcutaneous MC38 colorectal cancer model and induced colitis-associated colorectal cancer in mice by azoxymethane and dextran sodium sulfate. RESULTS: Our analysis of single-cell RNA sequencing data of patients with colorectal cancer revealed that intratumoral Tregs featured low activity of the MondoA-thioredoxin-interacting protein (TXNIP) axis and increased glucose uptake. Although MondoA-deficient Tregs were less immune suppressive and selectively promoted T-helper (Th) cell type 1 (Th1) responses in a subcutaneous MC38 tumor model, Treg-specific MondoA knockout mice were more susceptible to azoxymethane-DSS-induced colorectal cancer. Mechanistically, suppression of the MondoA-TXNIP axis promoted glucose uptake and glycolysis, induced hyperglycolytic Th17-like Tregs, which facilitated Th17 inflammation, promoted interleukin 17A-induced of CD8+ T-cell exhaustion, and drove colorectal carcinogenesis. Blockade of interleukin 17A reduced tumor progression and minimized the susceptibility of MondoA-deficient mice to colorectal carcinogenesis. CONCLUSIONS: The MondoA-TXNIP axis is a critical metabolic regulator of Treg identity and function in the colorectal cancer microenvironment and a promising target for cancer therapy.

Neutrophils Alter DNA Repair Landscape to Impact Survival and Shape Distinct Therapeutic Phenotypes of Colorectal Cancer.

BACKGROUND & AIMS: Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. METHODS: Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. RESULTS: We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. CONCLUSIONS: Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.

Wnt-ß-catenin activation epigenetically reprograms Treg cells in inflammatory bowel disease and dysplastic progression.

The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

Natural Polyphenols as Targeted Modulators in Colon Cancer: Molecular Mechanisms and Applications.

Colon cancer commonly develops from long-term chronic inflammation in the intestine and seriously threatens human health. Natural polyphenols have been valued as a crucial regulator of nutrient metabolism and metabolic diseases, owing to their anti-inflammatory and antioxidant functions and the ability to maintain a balance between gut microbes and their hosts. Notably, experimental and clinical evidence has shown that natural polyphenols could act as a targeted modulator to play a key role in the prevention or treatment of colon cancer. Thus, in this review, we summarized recent advances in the possible regulatory mechanism and the potential application of natural polyphenols in colon cancer, which might be regarded as a novel platform for the colon cancer management.

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.

A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy.

BACKGROUND & AIMS: Chronic bowel inflammation increases the risk of colon cancer; colitis-associated cancer (CAC). Thiopurine treatments are associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/ß-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/ß-catenin signalling. Autophagy is implicated in colonic tumors, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms. METHODS: Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harboring intestinal epithelial cell-specific deletion of autophagy related 7 gene (Atg7ΔIEC). TG or vehicle was administered intrarectally, and the effect on tumor burden and ß-catenin activity was assessed. The mechanisms of action of TG were investigated in vitro and in vivo. RESULTS: TG ameliorated DSS colitis in wild-type but not Atg7ΔIEC mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both wild-type and Atg7ΔIEC mice. This was associated with decreased ß-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines, and the dependency on Rac1 GTPase was demonstrated by siRNA knockdown and overexpression of constitutively active Rac1. CONCLUSIONS: Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches.

A Novel PAK1-Notch1 Axis Regulates Crypt Homeostasis in Intestinal Inflammation.

BACKGROUND & AIMS: p21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/ß-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice. METHODS: IL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT. RESULTS: When compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein-coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased ß-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation. CONCLUSIONS: PAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.