Loss of SMAD4 alters BMP signaling to promote colorectal cancer cell metastasis via activation of Rho and ROCK.

BACKGROUND & AIMS: SMAD4 frequently is lost from colorectal cancers (CRCs), which is associated with the development of metastases and a poor prognosis. SMAD4 loss is believed to alter transforming growth factor ß signaling to promote tumor progression. However, SMAD4 is also a central component of the bone morphogenetic protein (BMP) signaling pathway, implicated in CRC pathogenesis by human genetic studies. We investigated the effects of alterations in BMP signaling on the invasive and metastatic abilities of CRC cells and changes in members in this pathway in human tumor samples. METHODS: We activated BMP signaling in SMAD4-positive and SMAD4-negative CRC cells (HCT116, HT-29, SW480, and LS174T); SMAD4 was stably expressed or knocked down using lentiviral vectors. We investigated the effects on markers of epithelial-mesenchymal transition and on cell migration, invasion, and formation of invadopodia. We performed kinase activity assays to characterize SMAD4-independent BMP signaling and used an inhibitor screen to identify pathways that regulate CRC cell migration. We investigated the effects of the ROCK inhibitor Y-27632 in immunocompromised (CD-1 Nu) mice with orthotopic metastatic tumors. Immunohistochemistry was used to detect BMPR1a, BMPR1b, BMPR2, and SMAD4 in human colorectal tumors; these were related to patient survival times. RESULTS: Activation of BMP signaling in SMAD4-negative cells altered protein and messenger RNA levels of markers of epithelial-mesenchymal transition and increased cell migration, invasion, and formation of invadopodia. Knockdown of the BMP receptor in SMAD4-negative cells reduced their invasive activity in vitro. SMAD4-independent BMP signaling activated Rho signaling via ROCK and LIM domain kinase (LIMK). Pharmacologic inhibition of ROCK reduced metastasis of colorectal xenograft tumors in mice. Loss of SMAD4 from colorectal tumors has been associated with reduced survival time; we found that this association is dependent on the expression of BMP receptors but not transforming growth factor ß receptors. CONCLUSIONS: Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.

Oestrogens promote tumorigenesis in a mouse model for colitis-associated cancer.

BACKGROUND: Hormone replacement therapy increases the risk of developing ulcerative colitis in postmenopausal women. Chronic intestinal inflammation predisposes to colon cancer development, but effects of female hormones on colitis-associated cancer development have not been examined. AIM: To investigate the role of female hormones in the dextran sodium sulfate (DSS)-azoxymethane (AOM) mouse model for colitis-associated cancer. DESIGN: We performed ovariectomies, or sham operations, on mice, and supplemented these animals with indicated hormones. Additionally, we used oestrogen receptor α or ß (Erα or Erß) mutant mice. To study colitis or colitis-associated cancer, we used DSS only, or DSS and AOM, respectively. RESULTS: Ovariectomy protects female mice against colitis-associated tumour development. Hormone replacement in ovariectomised mice with either oestradiol (E2), medroxyprogesterone acetate or a combination of both suggests that oestrogens are the ovary-derived factor that promotes tumour development in the context of inflammatory damage. E2-treated animals showed increased clinical symptoms and Il-6 production upon DSS-induced colitis and enhanced epithelial proliferation. Treatment with E2 markedly increased the numbers of polyps in ovariectomised mice and also strongly promoted tumour progression with all E2-treated animals developing at least one invasive adenocarcinoma, whereas, placebo-treated animals developed adenomas only. Using Er mutant mice, we find that the protumorigenic effect of oestrogen depends on both Erα and Erß. CONCLUSIONS: Our results suggest that oestrogens promote inflammation-associated cancer development by impairing the mucosal response to inflammatory damage.

Correction: Intestinal Tumorigenesis Is Not Affected by Progesterone Signaling in Rodent Models.

[This corrects the article DOI: 10.1371/journal.pone.0022620.].

Blimp1 regulates the transition of neonatal to adult intestinal epithelium.

In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mother's milk to the adult diet at the end of the suckling period. These complex developmental changes are the result of a genetic programme intrinsic to the gut tube, but its regulators have not been identified. Here we show that transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) is highly expressed in the developing and postnatal intestinal epithelium until the suckling to weaning transition. Intestine-specific deletion of Blimp1 results in growth retardation and excessive neonatal mortality. Mutant mice lack all of the typical epithelial features of the suckling period and are born with features of an adult-like intestine. We conclude that the suckling to weaning transition is regulated by a single transcriptional repressor that delays epithelial maturation.

Intestinal tumorigenesis is not affected by progesterone signaling in rodent models.

Clinical data suggest that progestins have chemopreventive properties in the development of colorectal cancer. We set out to examine a potential protective effect of progestins and progesterone signaling on colon cancer development. In normal and neoplastic intestinal tissue, we found that the progesterone receptor (PR) is not expressed. Expression was confined to sporadic mesenchymal cells. To analyze the influence of systemic progesterone receptor signaling, we crossed mice that lacked the progesterone receptor (PRKO) to the Apc(Min/+) mouse, a model for spontaneous intestinal polyposis. PRKO-Apc(Min/+) mice exhibited no change in polyp number, size or localization compared to Apc(Min/+). To examine effects of progestins on the intestinal epithelium that are independent of the PR, we treated mice with MPA. We found no effects of either progesterone or MPA on gross intestinal morphology or epithelial proliferation. Also, in rats treated with MPA, injection with the carcinogen azoxymethane did not result in a difference in the number or size of aberrant crypt foci, a surrogate end-point for adenoma development. We conclude that expression of the progesterone receptor is limited to cells in the intestinal mesenchyme. We did not observe any effect of progesterone receptor signaling or of progestin treatment in rodent models of intestinal tumorigenesis.

Angiogenic markers endoglin and vascular endothelial growth factor in gastroenteropancreatic neuroendocrine tumors.

AIM: To investigate the expression and potential prognostic role of vascular endothelial growth factor (VEGF) and endoglin in gastroenteropancreatic neuroendocrine tumors (GEP-NETs). METHODS: Microvessel density (MVD) in GEP-NETs was evaluated using endoglin and CD31 immunohistochemistry. In addition, tissue levels of endoglin and VEGF were determined in homogenates by ELISA. RESULTS: Endoglin was highly expressed on tumor endothelial cells. CD31 MVD in GEP-NETs was significantly higher compared to endoglin MVD (P < 0.01). Two- to four-fold higher tissue levels of endoglin and VEGF were seen in tumors compared to associated normal tissue. This increased endoglin tissue expression in tumors was significantly related to tumor size (P < 0.01), presence of metastases (P = 0.04), and a more advanced tumor stage (P = 0.02), whereas expression of VEGF was not. CONCLUSION: We suggest that endoglin is a potential marker to indicate and predict metastases, which might be useful in the post-resection therapeutic approach of patients with GEP-NETs.

Pathological incidence of duodenopancreatic neuroendocrine tumors in the Netherlands: a Pathologisch Anatomisch Landelijk Geautomatiseerd Archief study.

OBJECTIVES: Duodenopancreatic neuroendocrine tumors are rare, although current epidemiological studies worldwide suggest an incidence rate increase. We assessed the pathological incidence of duodenopancreatic neuroendocrine tumors for 18 years in The Netherlands. METHODS: Standardized excerpts from pathological reports of all patients who had a diagnosis of duodenopancreatic neuroendocrine tumors from 1991 until 2009 were collected from the Pathologisch Anatomisch Landelijk Geautomatiseerd Archief and reviewed. This nationwide network and registry of histopathological and cytopathological data covers 100% of the pathological reports in The Netherlands. RESULTS: We identified 905 patients with pancreatic (n = 692) or duodenal (n = 213) neuroendocrine tumors. Most of these patients (69.4%) had a nonfunctional tumor. Functional tumors were diagnosed at a younger age compared with nonfunctional tumors (mean [SD] age, 52.3 [17.7] years vs 60.0 [14.6] years, respectively; P < 0.0001). The mean annual incidence rates per 1,000,000 persons over 1991 to 2009 were 2.54 for pancreatic and 0.81 for duodenal neuroendocrine tumors. The highest incidence was found in patients 65 to 79 years of age. The incidence of nonfunctional neuroendocrine tumors had increased significantly for 2 decades (P < 0.0001). CONCLUSIONS: The incidence of duodenopancreatic nonfunctional neuroendocrine tumors in The Netherlands increased over 1991 to 2009. The etiology for this change includes improved diagnostic techniques and clinical awareness, as discussed.

Loss of Indian Hedgehog activates multiple aspects of a wound healing response in the mouse intestine.

BACKGROUND & AIMS: Indian Hedgehog (Ihh) is expressed by the differentiated epithelial cells of the small intestine and signals to the mesenchyme where it induces unidentified factors that negatively regulate intestinal epithelial precursor cell fate. Recently, genetic variants in the Hh pathway have been linked to the development of inflammatory bowel disease. METHODS: We deleted Ihh from the small intestinal epithelium in adult mice using Cyp1a1-CreIhh(fl/fl) conditional Ihh mutant mice. Intestines were examined by immunohistochemistry, in situ hybridization, and real-time polymerase chain reaction. RESULTS: Deletion of Ihh from the intestinal epithelium initially resulted in a proliferative response of the intestinal epithelium with lengthening and fissioning of crypts and increased Wnt signaling. The epithelial proliferative response was associated with loss of bone morphogenetic protein and Activin signaling from the epithelium of the villus and crypts, respectively. At the same stage we observed a substantial influx of fibroblasts and macrophages into the villus core with increased mesenchymal transforming growth factor-ß signaling and deposition of extracellular matrix proteins. Prolonged loss of Ihh resulted in progressive leukocyte infiltration of the crypt area, blunting and loss of villi, and the development of intestinal fibrosis. CONCLUSIONS: Loss of Ihh initiates several events that are characteristic of an intestinal wound repair response. Prolonged loss resulted in progressive inflammation, mucosal damage, and the development of intestinal fibrosis. Ihh is a signal derived from the superficial epithelial cells that may act as a critical indicator of epithelial integrity.

Diagnostic efficacy of the secretin stimulation test for the Zollinger-Ellison syndrome: an intra-individual comparison using different dosages in patients and controls.

BACKGROUND/AIMS: The secretin stimulation test is the principal diagnostic tool to identify Zollinger-Ellison syndrome (ZES). We investigated, by intra-individual comparison, which dose of secretin results in the highest diagnostic efficacy to identify the ZES. METHODS: Fifty-seven paired secretin stimulation tests, using both 0.26 microg/kg and 0.78 microg/kg secretin, performed in 13 ZES patients and 12 controls, were analyzed and the findings confirmed in a validation cohort. RESULTS: A gastrin increase of >100 ng/l was found to be the most sensitive and specific criterion for a positive test. Higher gastrin increases after 0.78 microg/kg compared to 0.26 microg/kg secretin contributed to a slightly more sensitive (82.9 vs. 80.5%) but less specific (68.8 vs. 81.3%) test. A validation cohort, with 98 tests using 0.26 microg/kg secretin in 21 ZES patients and 39 controls, provided similar results. In ZES patients with normal fasting serum gastrin levels (<100 ng/l), there was no diagnostic benefit from the use of a higher secretin dose. CONCLUSIONS: The 0.26 microg/kg secretin stimulation test has the best diagnostic efficacy for the ZES. and IAP.

A case of recurrent gastrinoma in the liver with a review of "primary" hepatic gastrinomas.

The present report concerns a patient with a malignant gastrin-producing neuroendocrine tumour (ie, Zollinger-Ellison syndrome) with recurrent hepatic gastrinomas, in whom no gastrinoma in the duodenum, pancreas or other extrahepatic sites could be identified despite the use of multiple, repeatedly performed imaging and exploration techniques over the past 20 years. A short review on primary liver gastrinomas published since 1981 is also given. Interestingly, our patient is the only case with documented recurrent gastrinoma in the liver. None of the cases in the literature had liver gastrinomas as part of the multiple endocrine neoplasia type I syndrome. The interpretation of hepatic gastrinomas as primary lesions is questionable unless comprehensive investigation and well documented long-term follow-up is performed.

Motilin receptor expression in smooth muscle, myenteric plexus, and mucosa of human inflamed and noninflamed intestine.

BACKGROUND: Besides regulation of upper gastrointestinal motility, motilin seems to play a role in the inflammatory response. Motilin receptor expression in human intestine has not been studied thoroughly. This study aimed to describe the intestinal distribution of motilin receptors in inflammatory bowel disease (IBD) and control patients. METHODS: Quantitative autoradiography, immunohistochemistry, and reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect motilin receptors in tissue of 25 IBD patients (13 Crohn's disease [CD], 12 ulcerative colitis [UC]) and 19 patients with a neoplasm (controls). RESULTS: Median muscular motilin binding was 3 and 8 fmol/g tissue in colon and ileum, respectively. In the gastroduodenal region the median was higher (93 fmol/g). In UC colonic muscular motilin binding was significantly increased compared to controls (7 vs. 3 fmol/g, P < or = 0.05). Expression in CD was similar to controls. Besides the binding found in the muscular compartment, motilin binding was also found in the mucosa, which was even higher than in the muscle (3 versus 11 and 8 versus 27 fmol/g for colon and ileum (P < or = 0.06), respectively). RT-PCR and immunohistochemistry confirmed the mucosal motilin receptor expression. The mucosal motilin receptors were located in the epithelial cells. In the muscular compartment receptors were strongly present in the myenteric plexus and weakly in the smooth muscle cells. In IBD tissue the expression pattern was not different. CONCLUSIONS: The motilin receptor is expressed in human colonic and ileal smooth muscle. Further, motilin receptor expression was also shown in the mucosa. Muscular binding in UC patients is increased but no different expression pattern was found.

Comparison between serology and histology in the diagnosis of advanced gastric body atrophy: a study in a Dutch primary community.

GOALS: To assess serologically diagnosed gastric body atrophy (GBA) by histology in a sample of the general population. BACKGROUND: GBA is a precursor lesion in gastric cancer. Data on GBA in a primary health care community in the Netherlands have not been reported. STUDY: Thirty-four subjects of 997 consecutive adults from a Dutch family practice had serologic GBA, according to hypergastrinemia (>100 ng/L), hypopepsinogenemia A (<17 microg/L), and a low pepsinogen A/C ratio (<1.6). Two years later, 25 subjects of this group, agreed in serologic retesting and gastroscopy with biopsies for histologic assessment according to the Sydney system. RESULTS: At serologic retesting, 20 of 25 subjects again fulfilled the serologic criteria of GBA. Histologic examination of the corpus biopsies showed advanced GBA in 18 subjects (75%) of 24 (1 subject had no corpus biopsies) and 17 of 19 (89%) subjects with repeated positive serology. After disclosure of serology results, reexamination of the biopsies revealed GBA also in the 2 patients with initially insufficient evidence of GBA, giving a concordance of 100% (19/19). One subject with normal serum gastrin at retesting had both antral and body atrophy giving a concordance between serologic and histologic GBA of 95% (19/20). No adenomatous polyps, tumors, or dysplastic alterations were found. CONCLUSIONS: Identification by serology of asymptomatic patients with advanced GBA in primary care is adequately possible and useful in selecting for endoscopy.

Role of Helicobacter pylori and autoimmunity in serological atrophic corpus gastritis in a Dutch primary care community.

BACKGROUND: Atrophic corpus gastritis predisposes to vitamin B12 deficiency and gastric cancer. Little is known about the seroprevalence of atrophic corpus gastritis in the general population of Western Europe. AIM: To investigate the seroprevalence of atrophic corpus gastritis in a West-European primary care community in relation to Helicobacter pylori infection and autoimmunity. METHODS: Nine hundred and ninety-seven consecutive persons attending one general practice were asked to participate in the study by completing a questionnaire and donating fasting blood. Gastrin, pepsinogen A and C, and antibodies to H. pylori and parietal cells were measured by well-validated immunological methods. Criteria for serological atrophic corpus gastritis were pepsinogen A < 17 microg/l, pepsinogen A/C ratio <1.6, and gastrin >100 ng/l. RESULTS: Thirty-four participants (3.4%) fulfilled the serological criteria of atrophic corpus gastritis. Twenty-one of them (62%) and 17 of 34 (50%) age-matched and sex-matched nested controls were H. pylori positive [NS; odds ratio, 1.62 (0.62-4.24)], while 15 of them (44%) and one of 34 controls had antibodies to parietal cells [P < 0.005; odds ratio, 24.0 (3.00-201)]. CONCLUSIONS: The seroprevalence of atrophic corpus gastritis in this primary care community is 3.4%. When compared with controls, the approximate relative risk of having atrophic corpus gastritis was significantly higher (P < 0.025) for antibodies to parietal cells (24.0) than to H. pylori (1.62). In view of the decreasing risk of H. pylori infection in the western world, it is likely that the impact of H. pylori on the development of atrophic corpus gastritis will further diminish.

Proximal and distal gut hormone secretion in irritable bowel syndrome.

OBJECTIVE: Sensory and motor dysfunctions of the gut are both important characteristics of irritable bowel syndrome (IBS). Several gut peptides contribute to the regulation of gastrointestinal function but little is known about gut hormone secretion in IBS. MATERIAL AND METHODS: We evaluated perceptual thresholds and fasting and postprandial plasma levels of proximal (cholecystokinin (CCK), motilin) and distal (peptide YY) gut peptides up to 1 h after ingestion of a high caloric meal in 99 IBS patients and 40 age- and gender-matched healthy controls. RESULTS: Fasting plasma CCK levels were significantly elevated in patients (1.2+/-0.8 pM) compared with those in controls (0.8+/-0.7 pM, p=0.006), as was the incremental postprandial CCK response (72+/-73 versus 40+/-42 pM.60 min, respectively; p=0.003). No differences in fasting and postprandial motilin or PYY levels were found. The postprandial PYY response was significantly increased in hypersensitive compared to normosensitive patients (215+/-135 versus 162+/-169 pM, p=0.048). Patients with a diarrhoea predominant bowel habit had higher fasting motilin levels compared to constipated patients or alternating type IBS patients (82.1+/-36.5 versus 60.8+/-25.1 versus 57.5+/-23.9 pM, one-way ANOVA p=0.003). CONCLUSIONS: IBS patients have increased fasting and postprandial plasma levels of CCK. Changes in plasma levels of motilin and PYY may contribute to the clinical expression of IBS, such as the presence of visceral hypersensitivity or a predominant bowel habit.

Effect of peptide YY on pancreatico-biliary secretion in humans.

OBJECTIVE: The negative feedback on pancreatico-biliary secretion induced by ileal nutrients has been well documented but the role of the distal gut peptide YY (PYY) as mediator is less well defined. We determined the effect of PYY on basal, sham feeding and feeding-stimulated pancreatico-biliary secretion in humans. MATERIAL AND METHODS: Eight healthy volunteers participated in a placebo-controlled, double-blind, randomized study with intravenous infusion of a physiological dose of PYY. Subjects were intubated with a naso-jejunal tube. Duodenal samples were aspirated continuously while a recovery marker perfused the duodenum. Outputs were measured in the basal state, in response to modified sham feeding (MSF) and during jejunal perfusion with a liquid meal. Blood samples were drawn for plasma cholecystokinin (CCK) and PYY measurements. RESULTS: PYY infusion significantly (p<0.01) increased plasma PYY, from 16+/-2 to 55+/-6 pM. Basal outputs of amylase, lipase, trypsin and bilirubin were not affected by PYY. In the placebo experiment, MSF significantly increased amylase, lipase, trypsin and bilirubin outputs. However, during PYY infusion lipase, trypsin and bilirubin outputs did not significantly increase in response to MSF. Jejunal feeding, however, significantly (p<0.01) increased the outputs of bilirubin and enzymes equally during both the placebo and PYY infusions. CONCLUSIONS: Infusion of PYY to physiological plasma levels exerts an inhibitory effect on pancreatico-biliary secretion during the cephalic phase of digestion, but not in the basal state or during the intestinal phase. PYY suppresses pancreatico-biliary secretion during the cephalic phase but not during the intestinal phase of nutrient digestion.

Motilin effects on the proximal stomach in patients with functional dyspepsia and healthy volunteers.

This study investigates motilin effects on the proximal stomach in patients with functional dyspepsia (FD) and healthy volunteers. Eight healthy volunteers and 12 patients with FD were infused with synthetic motilin or placebo. Proximal gastric volume was measured with a barostat at constant pressure and during isobaric distensions. Abdominal symptoms were scored by visual analog scales. Plasma motilin concentrations were measured by radioimmunoassay. Motilin concentrations and baseline gastric volumes were similar for patients and healthy volunteers. Motilin, compared with placebo, reduced gastric volume by 112 ml [F(29,195); confidence interval (CI) 95%] in patients and by 96 ml [F(-7,200); CI 95%] in healthy volunteers. In patients, motilin decreased compliance by 76 ml/mmHg [F(9,143); CI 95%] compared with placebo, which was similar in volunteers [66 ml/mmHg; F(11,120); CI 95%]. Patients were more nauseous during motilin compared with placebo (P = 0.04), whereas healthy volunteers did not experience nausea. We conclude that in a fasted condition, FD patients have a similar proximal gastric motor response to motilin as healthy volunteers, but experience an exaggerated sensation of nausea.

Pancreatic Polypeptide Secretion in Patients with Chronic Pancreatitis and After Pancreatic Surgery.

Pancreatic polypeptide (PP) is a 36-amino acidpolypeptide with a molecular weight of 4200 (1). PP is mainly produced in the head of the pancreas in adistinct cell type both within the islets and scatteredamong the exocrine parenchyma (2,3). PP release isunder neural and hormonal control. Ingestion of nutrients,cholinergic neural activation and infusion ofgastro-intestinal peptides (especially CCK) stimulatePP release (1,4,5). It has been clearly shown that PPsecretion is under vagal cholinergic control since thePP response to a meal or CCK infusion is bluntedduring vagal cholinergic blockade with atropine orfollowing truncal vagotomy (1,6).