Heterogeneous changes in gut and tumor microbiota in patients with pancreatic cancer: insights from clinical evidence.

BACKGROUND: Pancreatic cancer is the foremost contributor to cancer-related deaths globally, and its prevalence continues to rise annually. Nevertheless, the underlying mechanisms behind its development remain unclear and necessitate comprehensive investigation. METHODS: In this study, a total of 29 fresh stool samples were collected from patients diagnosed with pancreatic cancer. The gut microbial data of healthy controls were obtained from the SRA database (SRA data number: SRP150089). Additionally, 28 serum samples and diseased tissues were collected from 14 patients with confirmed pancreatic cancer and 14 patients with chronic pancreatitis. Informed consent was obtained from both groups of patients. Microbial sequencing was performed using 16s rRNA. RESULTS: The results showed that compared with healthy controls, the species abundance index of intestinal flora in patients with pancreatic cancer was increased (P < 0.05), and the number of beneficial bacteria at the genus level was reduced (P < 0.05). Compared with patients with chronic pancreatitis, the expression levels of CA242 and CA199 in the serum of patients with pancreatic cancer were increased (P < 0.05). The bacterial richness index of tumor microorganisms in patients with pancreatic cancer increased, while the diversity index decreased(P < 0.05). Furthermore, there was a change in the species composition at the genus level. Additionally, the expression level of CA242 was found to be significantly positively correlated with the relative abundance of Acinetobacter(P < 0.05). CONCLUSION: Over all, the expression levels of serum tumor markers CA242 and CA19-9 in patients with pancreatic cancer are increased, while the beneficial bacteria in the intestine and tumor microenvironment are reduced and pathogenic bacteria are increased. Acinetobacter is a specific bacterial genus highly expressed in pancreatic cancer tissue.

Collagenous Colitis Is Associated With HLA Signature and Shares Genetic Risks With Other Immune-Mediated Diseases.

BACKGROUND & AIMS: Collagenous colitis (CC) is an inflammatory bowel disorder with unknown etiopathogenesis involving HLA-related immune-mediated responses and environmental and genetic risk factors. We carried out an array-based genetic association study in a cohort of patients with CC and investigated the common genetic basis between CC and Crohn's disease (CD), ulcerative colitis (UC), and celiac disease. METHODS: DNA from 804 CC formalin-fixed, paraffin-embedded tissue samples was genotyped with Illumina Immunochip. Matching genotype data on control samples and CD, UC, and celiac disease cases were provided by the respective consortia. A discovery association study followed by meta-analysis with an independent cohort, polygenic risk score calculation, and cross-phenotype analyses were performed. Enrichment of regulatory expression quantitative trait loci among the CC variants was assessed in hemopoietic and intestinal cells. RESULTS: Three HLA alleles (HLA-B∗08:01, HLA-DRB1∗03:01, and HLA-DQB1∗02:01), related to the ancestral haplotype 8.1, were significantly associated with increased CC risk. We also identified an independent protective effect of HLA-DRB1∗04:01 on CC risk. Polygenic risk score quantifying the risk across multiple susceptibility loci was strongly associated with CC risk. An enrichment of expression quantitative trait loci was detected among the CC-susceptibility variants in various cell types. The cross-phenotype analysis identified a complex pattern of polygenic pleiotropy between CC and other immune-mediated diseases. CONCLUSIONS: In this largest genetic study of CC to date with histologically confirmed diagnosis, we strongly implicated the HLA locus and proposed potential non-HLA mechanisms in disease pathogenesis. We also detected a shared genetic risk between CC, celiac disease, CD, and UC, which supports clinical observations of comorbidity.

Farnesoid X receptor expression is decreased in colonic mucosa of patients with primary sclerosing cholangitis and colitis-associated neoplasia.

BACKGROUND: The expression and distribution of farnesoid X receptor (FXR) in colitis and colitis-associated neoplasia (CAN) is unknown. We investigated FXR expression in neoplastic and nonneoplastic tissue from ulcerative colitis (UC) patients, with or without primary sclerosing cholangitis (PSC), as well as the role of DNA methylation in FXR expression in colorectal cancer (CRC) cell lines. METHODS: Samples from the right (RC) and left (LC) colon of patients with UC, with and without PSC, and with or without CAN, were stained by immunohistochemistry and scored semiquantitatively for nuclear FXR expression. FXR expression was analyzed by western blot and polymerase chain reaction (PCR) in nine different CRC cell lines before and after demethylation with 5-azacytidine. RESULTS: In nondysplastic samples, FXR expression demonstrated a diminishing expression from proximal to distal colon (strong FXR expression: 39% RC samples vs. 14% LC samples; P = 0.007). With moderate-to-severe inflammation, FXR expression was almost always absent or weak in both UC and PSC-UC, regardless of location. With quiescent/mild inflammation, 56% of UC samples in the RC retained strong FXR expression versus 24% of PSC-UC samples (P= 0.017). FXR was absent in 72% of the neoplastic samples, with an inverse association with the grade of dysplasia. FXR expression was absent in all CRC cell lines, in some cases due to DNA methylation. CONCLUSIONS: FXR expression is inversely correlated with neoplastic progression and severity of inflammation in UC. Patients with PSC-UC have diminished FXR expression in the proximal colon compared to UC patients. This finding could contribute to the higher risk of proximal neoplasia in PSC patients.

The role of prostaglandin E2 (PGE 2) in toll-like receptor 4 (TLR4)-mediated colitis-associated neoplasia.

BACKGROUND: We have previously found that TLR4-deficient (TLR4-/-) mice demonstrate decreased expression of mucosal PGE 2 and are protected against colitis-associated neoplasia. However, it is still unclear whether PGE 2 is the central factor downstream of TLR4 signaling that promotes intestinal tumorigenesis. To further elucidate critical downstream pathways involving TLR4-mediated intestinal tumorigenesis, we examined the effects of exogenously administered PGE 2 in TLR4-/- mice to see if PGE 2 bypasses the protection from colitis-associated tumorigenesis. METHOD: Mouse colitis-associated neoplasia was induced by azoxymethane (AOM) injection followed by two cycles of dextran sodium sulfate (DSS) treatment. Two different doses of PGE 2 (high dose group, 200 microg, n = 8; and low dose group, 100 microg, n = 6) were administered daily during recovery period of colitis by gavage feeding. Another group was given PGE 2 during DSS treatment (200 microg, n = 5). Inflammation and dysplasia were assessed histologically. Mucosal Cox-2 and amphiregulin (AR) expression, prostanoid synthesis, and EGFR activation were analyzed. RESULTS: In control mice treated with PBS, the average number of tumors was greater in WT mice (n = 13) than in TLR4-/- mice (n = 7). High dose but not low dose PGE 2 treatment caused an increase in epithelial proliferation. 28.6% of PBS-treated TLR4-/- mice developed dysplasia (tumors/animal: 0.4 +/- 0.2). By contrast, 75.0% (tumors/animal: 1.5 +/- 1.2, P < 0.05) of the high dose group and 33.3% (tumors/animal: 0.3 +/- 0.5) of the low dose group developed dysplasia in TLR4-/- mice. Tumor size was also increased by high dose PGE 2 treatment. Endogenous prostanoid synthesis was differentially affected by PGE 2 treatment during acute and recovery phases of colitis. Exogenous administration of PGE 2 increased colitis-associated tumorigenesis but this only occurred during the recovery phase. Lastly, PGE 2 treatment increased mucosal expression of AR and Cox-2, thus inducing EGFR activation and forming a positive feedback mechanism to amplify mucosal Cox-2. CONCLUSIONS: These results highlight the importance of PGE 2 as a central downstream molecule involving TLR4-mediated intestinal tumorigenesis.

Toll-like receptor 4 differentially regulates epidermal growth factor-related growth factors in response to intestinal mucosal injury.

Epiregulin (EPI) and amphiregulin (AR) are epidermal growth factor receptor (EGFR) ligands implicated in mucosal repair and tumorigenesis. We have shown that Toll-like receptor 4 (TLR4) induces intestinal epithelial cell (IEC) proliferation by activating EGFR through AR expression. We examined whether TLR4 differentially regulates expression of EGFR ligands in response to mucosal injury. The human IEC line SW480 was examined expression of EGFR ligands, EGFR phosphorylation, and proliferation in response to lipopolysaccharide (LPS). Small-interfering RNA (siRNA) was used to block TLR4. Neutralizing antibodies to EGFR ligands were used to examine inhibition of LPS-dependent EGFR activation. Acute colitis and recovery were examined in the mice given 2.5% dextran sodium sulfate (DSS). Colonic secretion of EPI and AR was analyzed by enzyme-linked immunosorbent assay. LPS selectively induces EPI and AR but not other EGFR ligands. LPS induced early EPI mRNA expression between 30 min and 24 h. The neutralizing antibodies to EPI and AR prevented activation of EGFR by LPS. LPS induces IEC proliferation (200%, P=0.01) in 24 h but blocking EPI and AR significantly decreased proliferation. In vivo, mucosal EPI and AR expression are significantly decreased in TLR4(-/-) mice (P=0.02) compared to wild-type mice during acute colitis. EPI and AR exhibit different kinetics in response to mucosal damage: EPI expression is upregulated acutely at day 7 of DSS, but falls during recovery at day 14. By contrast, a sustained upregulation of AR expression is seen during mucosal injury and repair. We show that TLR4 regulates EPI and AR expression and that both these EGFR ligands are necessary for optimal proliferation of IEC. The diverse kinetics of EPI and AR expression suggest that they function in distinct roles with respect to acute injury vs repair. Our results highlight the role of bacterial sensing for IEC homeostasis and may lead to targeted therapy for mucosal healing and prevention of tumorigenesis.

A novel Toll-like receptor 4 antagonist antibody ameliorates inflammation but impairs mucosal healing in murine colitis.

Dysregulated innate immune responses to commensal bacteria contribute to the development of inflammatory bowel disease (IBD). TLR4 is overexpressed in the intestinal mucosa of IBD patients and may contribute to uncontrolled inflammation. However, TLR4 is also an important mediator of intestinal repair. The aim of this study is to examine the effect of a TLR4 antagonist on inflammation and intestinal repair in two murine models of IBD. Colitis was induced in C57BL/6J mice with dextran sodium sulfate (DSS) or by transferring CD45Rb(hi) T cells into RAG1-/- mice. An antibody (Ab) against the TLR4/MD-2 complex or isotype control Ab was administered intraperitoneally during DSS treatment, recovery from DSS colitis, or induction of colitis in RAG1-/- mice. Colitis severity was assessed by disease activity index (DAI) and histology. The effect of the Ab on the inflammatory infiltrate was determined by cell isolation and immunohistochemistry. Mucosal expression of inflammatory mediators was analyzed by real-time PCR and ELISA. Blocking TLR4 at the beginning of DSS administration delayed the development of colitis with significantly lower DAI scores. Anti-TLR4 Ab treatment decreased macrophage and dendritic cell infiltrate and reduced mucosal expression of CCL2, CCL20, TNF-alpha, and IL-6. Anti-TLR4 Ab treatment during recovery from DSS colitis resulted in defective mucosal healing with lower expression of COX-2, PGE(2), and amphiregulin. In contrast, TLR4 blockade had minimal efficacy in ameliorating inflammation in the adoptive transfer model of chronic colitis. Our findings suggest that anti-TLR4 therapy may decrease inflammation in IBD but may also interfere with colonic mucosal healing.

Innate immune signaling by Toll-like receptor-4 (TLR4) shapes the inflammatory microenvironment in colitis-associated tumors.

BACKGROUND: Patients with ulcerative colitis are at increased risk for developing colorectal cancer. We have shown that Toll-like receptor-4 (TLR4) is overexpressed in human colitis-associated cancer (CAC) and that mice deficient in TLR4 are markedly protected against colitis-associated neoplasia. We wished to elucidate the specific contributions of TLR4 signaling by myeloid cells and colonic epithelial cells (CEC) in colitis-associated tumorigenesis. METHODS: TLR4-deficient mice or wildtype littermates (WT) were transplanted with bone marrow (BM) cells: TLR4(-/-) BM-->WT mice (TLR4-expressing CEC) and WT BM-->TLR4(-/-) mice (TLR4-expressing myeloid cells). Colitis-associated neoplasia was induced by azoxymethane (AOM 7.3 mg/kg) injection and 2 cycles of dextran sodium sulfate (DSS) treatment. RESULTS: The number and size of dysplastic lesions were greater in TLR4(-/-) BM-->WT mice than in WT BM-->TLR4(-/-) mice (P < 0.005). Histologically, TLR4(-/-) BM-->WT mice had greater numbers of mucosal neutrophils and macrophages compared to WT BM-->TLR4(-/-) mice. The chemokines KC and CCL2, important in recruitment of neutrophils and macrophages, respectively, were induced in mice expressing TLR4 in CEC rather than the myeloid compartment. The lamina propria infiltrate of mice expressing TLR4 in CEC was characterized by macrophages expressing Cox-2. Moreover, mice expressing TLR4 in CEC rather than the myeloid compartment had increased production of amphiregulin and EGFR activation. CONCLUSIONS: These findings indicate that TLR4 signaling on CEC is necessary for recruitment and activation of Cox-2-expressing macrophages and increasing the number and size of dysplastic lesions. Our results implicate innate immune signaling on CEC as a key regulator of a tumor-promoting microenvironment.

Trefoil factor-3 expression in human colon cancer liver metastasis.

Deaths from colorectal cancer are often due to liver metastasis. Trefoil factor-3 (TFF3) is expressed by normal intestinal epithelial cells and its expression is maintained throughout the colon adenoma-carcinoma sequence. Our previous work demonstrated a correlation between TFF3 expression and metastatic potential in an animal model of colon cancer. The aim of this study was to determine whether TFF3 is expressed in human colon cancer liver metastasis (CCLM) and whether inhibiting TFF3 expression in colon cancer cells would alter their invasive potential in vitro. Human CCLMs were analyzed at the mRNA and protein level for TFF3 expression. Two highly metastatic rat colon cancer cell lines that either natively express TFF3 (LN cells) or were transfected with TFF3 (LPCRI-2 cells), were treated with two rat TFF3 siRNA constructs (si78 and si365), and analyzed in an in vitro invasion assay. At the mRNA and protein level, TFF3 was expressed in 17/17 (100%) CCLMs and 10/11 (91%) primary colon cancers, but not in normal liver tissue. By real time PCR, TFF3 expression was markedly inhibited by both siRNA constructs in LN and LPCRI-2 cells. The si365 and si78 constructs inhibited invasion by 44% and 53%, respectively, in LN cells, and by 74% and 50%, respectively, in LPCRI-2 cells. These results provide further evidence that TFF3 contributes to the malignant behavior of colon cancer cells. These observations may have relevance for designing new diagnostic and treatment approaches to colorectal cancer.

The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease.

Abnormal T cell responses to commensal bacteria are involved in the pathogenesis of inflammatory bowel disease. MyD88 is an essential signal transducer for TLRs in response to the microflora. We hypothesized that TLR signaling via MyD88 was important for effector T cell responses in the intestine. TLR expression on murine T cells was examined by flow cytometry. CD4(+)CD45Rb(high) T cells and/or CD4(+)CD45Rb(low)CD25(+) regulatory T cells were isolated and adoptively transferred to RAG1(-/-) mice. Colitis was assessed by changes in body weight and histology score. Cytokine production was assessed by ELISA. In vitro proliferation of T cells was assessed by [(3)H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling. CD4(+)CD45Rb(high) T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules. MyD88(-/-) CD4(+) T cells showed decreased proliferation compared with WT CD4(+) T cells both in vivo and in vitro. CD4(+)CD45Rb(high) T cells from MyD88(-/-) mice did not induce wasting disease when transferred into RAG1(-/-) recipients. Lamina propria CD4(+) T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice receiving MyD88(-/-) cells than mice receiving WT cells. In vitro, MyD88(-/-) T cells were blunted in their ability to secrete IL-17 but not IFN-gamma. Absence of MyD88 in CD4(+)CD45Rb(high) cells results in defective T cell function, especially Th17 differentiation. These results suggest a role for TLR signaling by T cells in the development of inflammatory bowel disease.

Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors.

BACKGROUND & AIMS: Chronic inflammation is a risk factor for colon cancer in patients with ulcerative colitis (UC). The molecular mechanisms linking inflammation and colon carcinogenesis are incompletely understood. We tested the hypothesis that Toll-like receptor 4 (TLR4) is involved in tumorigenesis in the setting of chronic inflammation. METHODS: Tissues from UC patients with cancer were examined for TLR4 expression. Colitis-associated neoplasia was induced using azoxymethane injection followed by dextran sodium sulfate treatment in TLR4-deficient or wild-type mice. Inflammation, polyps, and microscopic dysplasia were scored. Cyclooxygenase (Cox)-2 and prostaglandin E(2) production were analyzed by real-time polymerase chain reaction, immunohistochemistry, or enzyme immunoassay. Epidermal growth factor receptor (EGFR) phosphorylation and amphiregulin production were examined by Western blot analysis and enzyme-linked immunosorbent assay, respectively. RESULTS: We show that TLR4 is overexpressed in human and murine inflammation-associated colorectal neoplasia. TLR4-deficient mice were protected markedly from colon carcinogenesis. Mechanistically, we show that TLR4 is responsible for induction of Cox-2, increased prostaglandin E(2) production, and activation of EGFR signaling in chronic colitis. Amphiregulin, an EGFR ligand, was induced in a TLR4, Cox-2-dependent fashion and contributes to activation of EGFR phosphorylation in colonic epithelial cells. CONCLUSIONS: TLR4 signaling is critical for colon carcinogenesis in chronic colitis. TLR4 activation appears to promote the development of colitis-associated cancer by mechanisms including enhanced Cox-2 expression and increased EGFR signaling. Inhibiting TLR4 signaling may be useful in the prevention or treatment of colitis-associated cancer.

Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: Role in proliferation and apoptosis in the intestine.

BACKGROUND & AIMS: We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair. METHODS: Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice. RESULTS: Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor. CONCLUSIONS: We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.

Trefoil factor family-3 is associated with aggressive behavior of colon cancer cells.

BACKGROUND AND AIM: Trefoil factor family 3 (TFF3) is expressed by intestinal epithelial cells and it mainly functions to protect the mucosa from injury. Expression of TFF3 has been correlated with a poor prognosis in patients with cancer, but little is known about whether TFF3 directly contributes to the malignant behavior of cancer cells. The present study was conducted to determine whether TFF3 expression contributes to the malignant behavior of cancer cells in vitro and in vivo. METHODS: Two subclones of a metastatic rat colorectal cancer cell line, demonstrated previously to manifest aggressive (LN cells) and non-aggressive (LP cells) growth in vivo, were analyzed for expression of TFF3 and tested in assays of cancer cell migration, invasion, and apoptosis in vitro, and mortality in vivo. RESULTS: The aggressive LN cell line endogenously expressed TFF3 and supported the transcription of a TFF3 promoter-driven reporter construct, whereas the non-aggressive LP cell line did not express TFF3. LN cells demonstrated enhanced migration, invasion, and less apoptosis compared to LP cells. Transfecting TFF3 into LP cells enhanced their ability to migrate, invade, block apoptosis, and behave more aggressively in vivo, thereby resembling the phenotype of LN cells. CONCLUSIONS: In rat colon cancer cells, both endogenous and constitutive expression of TFF3 correlates with an aggressive phenotype. These data provide direct evidence that TFF3 contributes to the malignant behavior of cancer cells.

Intestinal trefoil factor: a marker of poor prognosis in gastric carcinoma.

PURPOSE: Intestinal trefoil factor (ITF) is a marker of intestinal differentiation that may also play a role in cancer cell biology by inhibiting cell adhesion, promoting cell invasion, and blocking apoptosis. Gastric adenocarcinomas can arise through a process of intestinalization, but no study has yet comprehensively examined the expression of ITF in gastric cancer or correlated ITF expression with clinical outcome in any cancer type. EXPERIMENTAL DESIGN: Patients (209) with primary gastric adenocarcinoma were evaluated for ITF expression by immunohistochemistry. Results of immunostaining were correlated with clinicopathological variables and overall survival. RESULTS: In normal gastric mucosa, ITF expression was absent, whereas areas of intestinal metaplasia revealed strong ITF expression by goblet cells. A portion of gastric cancers (55%) demonstrated ITF expression. Women were more likely than men to express ITF in gastric cancers. However, in men, the expression of ITF correlated with aggressive phenotype of tumors (advanced stage, infiltrative growth pattern, and positive lymph nodes). Multivariate analysis revealed that expression of ITF was associated with a poor prognosis, independent of tumor stage. CONCLUSIONS: This is the first study to correlate ITF expression with clinicopathological features or outcome in any cancer type. ITF expression in gastric cancer exhibited a curious gender-associated relationship, being more frequently expressed in tumors of women, but associated with more aggressive pathological features in men. The poor prognosis of patients with ITF-positive gastric cancers further implicates ITF in cancer cell biology.