Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.

Chir99021 and Valproic acid reduce the proliferative advantage of Apc mutant cells.

More than 90% of colorectal cancers carry mutations in Apc that drive tumourigenesis. A 'just-right' signalling model proposes that Apc mutations stimulate optimal, but not excessive Wnt signalling, resulting in a growth advantage of Apc mutant over wild-type cells. Reversal of this growth advantage constitutes a potential therapeutic approach. We utilised intestinal organoids to compare the growth of Apc mutant and wild-type cells. Organoids derived from Apc Min/+ mice recapitulate stages of intestinal polyposis in culture. They eventually form spherical cysts that reflect the competitive growth advantage of cells that have undergone loss of heterozygosity (LOH). We discovered that this emergence of cysts was inhibited by Chiron99021 and Valproic acid, which potentiates Wnt signalling. Chiron99021 and Valproic acid restrict the growth advantage of Apc mutant cells while stimulating that of wild-type cells, suggesting that excessive Wnt signalling reduces the relative fitness of Apc mutant cells. As a proof of concept, we demonstrated that Chiron99021-treated Apc mutant organoids were rendered susceptible to TSA-induced apoptosis, while wild-type cells were protected.

Morphologic characterization of hamartomatous gastrointestinal polyps in Cowden syndrome, Peutz-Jeghers syndrome, and juvenile polyposis syndrome.

The morphologic features of the gastrointestinal polyps in hamartomatous polyposis syndromes are poorly defined. Our aim was to better characterize the gastrointestinal hamartomas in these syndromes. A blinded review was performed regarding many histologic features for every polyp. The study included 15 Cowden syndrome, 13 Peutz-Jeghers (PJS), 12 juvenile polyposis (JuvPS) patients, and 32 cases of sporadic hamartomatous polyps. A total of 375 polyps were examined. Cowden syndrome polyps were characteristically colonic, sessile, small, without surface erosion, and showing mildly inflamed fibrotic lamina propria with smooth muscle proliferation and lymphoid follicles. They showed the least degree of cystic glands and had no thick mucin. Uncommon but specific features were ganglion cells and nerve fibers within the lamina propria and mucosal fat. PJS polyps were typically of small or large bowel origin, often exophytic, seldom eroded, with inflamed edematous and fibrotic lamina propria and dilated cystic glands filled with often thick mucin. All PJS polyps showed smooth muscle proliferation, frequently widespread. The polyps of JuvPS were typically colonic, large, exophytic, eroded, with strikingly edematous, fibrotic markedly inflamed lamina propria, cystic glands filled with frequently thick mucin, and the least degree of smooth muscle proliferation. Nonsyndromic hamartomatous polyps were similar to JuvPS polyps; however, they were more often colonic, were smaller, showed more widespread smooth muscle proliferation, and were less likely to contain thick mucin. In conclusion, we were able to define the characteristic hamartomatous polyp for each hamartomatous polyposis syndrome. Awareness to these features may aid in the diagnosis of these rare syndromes.

Endogenous conversion of ω-6 to ω-3 polyunsaturated fatty acids in fat-1 mice attenuated intestinal polyposis by either inhibiting COX-2/ß-catenin signaling or activating 15-PGDH/IL-18.

Omega-3 polyunsaturated fatty acids (ω-3PUFAs) have inhibitory effects in various preclinical cancer models, but their effects in intestinal polyposis have never been examined. As attempts have been made to use nutritional intervention to counteract colon cancer development, in this study we evaluated the effects of ω-3 PUFAs on intestinal polyposis in the Apc(Min/+) mouse model. The experimental groups included wild-type C56BL/6 mice, Apc(Min/+) mice, fat-1 transgenic mice expressing an n-3 desaturase to enable ω-3 PUFA synthesis, and Apc(Min/+) × fat-1 double-transgenic mice; all mice were 20 weeks of age. Small intestines were collected for gross and pathologic evaluation, including assessment of polyp number and size, followed by immunohistochemical staining and Western blotting. After administration of various concentrations of ω-3 PUFAs, PUFA levels were measured in small intestine tissue by GC/MS/MS analysis to compare with PUFA synthesis of between C57BL6 and fat-1mice. As a result, ω-3 PUFAs significantly attenuated Apc mutation-induced intestinal polyposis accompanied with significant inhibition of Wnt/ß-catenin signaling, COX-2 and PGE2, but induced significant levels of 15-PGDH. In addition, significant induction of the inflammasome-related substrates as IL-1ß and IL-18 and activation of caspase-1 was observed in Apc(Min/+) × fat-1 mice. Administration of at least 3 g/60 kg ω-3 PUFAs was equivalent to ω-3 PUFAs produced in fat-1 mice and resulted in significant increase in the expression of IL-1ß, caspase-3 and IL-18, as seen in Apc(Min/+) × fat-1 mice. We conclude that ω-3PUFAs can prevent intestinal polyp formation by inhibition of Wnt/ß-catenin signaling, but increased levels of 15-PGDH and IL-18.

Somatic alterations in juvenile polyps from BMPR1A and SMAD4 mutation carriers.

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant disorder predisposing to gastrointestinal hamartomatous polyps and cancer with a pathogenic SMAD4 or BMPR1A germline mutation (1st-hit) being identified in about 40-50% of patients. Little is known, however, about the occurrence and nature of somatic alterations (2nd-hit) in SMAD4-/BMPR1A-related juvenile polyps. In this study, we screened 25 polyps from three patients carrying either a pathogenic SMAD4 (c.1244-1247delACAG) or BMPR1A (c.583C>T; p.Gln195*) germline mutation for somatic alterations. The SMAD4-related polyps were also analyzed for SMAD4 protein expression by immunohistochemistry. Despite comprehensive screening for loss of heterozygosity (LOH), mutations in the coding sequence, chromosomal rearrangements, and promoter methylation, no somatic alterations could be identified in 14 SMAD4-related polyps. SMAD4 protein expression, however, was lost in 8 (57%) of 14 juvenile polyps with 6 showing concomitant loss in both, the epithelial and stromal, compartments. In the BMPR1A-related polyps, five out of nine (56%) displayed LOH. Further analysis of selected polyps revealed that LOH was gene copy number neutral and had occurred in the epithelial compartment. The heterogeneity of genetic mutations and protein expression levels indicates that different modes of gene inactivation can be operational in SMAD4- and BMPR1A-related polyp formation. Our observation, that about half of BMPR1A-related polyps displayed LOH, predominantly in the epithelial compartment, is compatible with BMPR1A acting as a tumour suppressor gene. Still, it remains to be determined whether juvenile polyp development generally requires loss of BMPR1A expression or, as observed in some SMAD4-related polyps, can occur despite normal protein expression.

IL-33 activates tumor stroma to promote intestinal polyposis.

Tumor epithelial cells develop within a microenvironment consisting of extracellular matrix, growth factors, and cytokines produced by nonepithelial stromal cells. In response to paracrine signals from tumor epithelia, stromal cells modify the microenvironment to promote tumor growth and metastasis. Here, we identify interleukin 33 (IL-33) as a regulator of tumor stromal cell activation and mediator of intestinal polyposis. In human colorectal cancer, IL-33 expression was induced in the tumor epithelium of adenomas and carcinomas, and expression of the IL-33 receptor, IL1RL1 (also referred to as IL1-R4 or ST2), localized predominantly to the stroma of adenoma and both the stroma and epithelium of carcinoma. Genetic and antibody abrogation of responsiveness to IL-33 in the Apc(Min/+) mouse model of intestinal tumorigenesis inhibited proliferation, induced apoptosis, and suppressed angiogenesis in adenomatous polyps, which reduced both tumor number and size. Similar to human adenomas, IL-33 expression localized to tumor epithelial cells and expression of IL1RL1 associated with two stromal cell types, subepithelial myofibroblasts and mast cells, in Apc(Min/+) polyps. In vitro, IL-33 stimulation of human subepithelial myofibroblasts induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression. IL-33 deficiency reduced mast cell accumulation in Apc(Min/+) polyps and suppressed the expression of mast cell-derived proteases and cytokines known to promote polyposis. Based on these findings, we propose that IL-33 derived from the tumor epithelium promotes polyposis through the coordinated activation of stromal cells and the formation of a protumorigenic microenvironment.

Esophageal cancer in a family with hamartomatous tumors and germline PTEN frameshift and SMAD7 missense mutations.

Germline mutations in the PTEN tumor-suppressor gene cause autosomal-dominant conditions such as Cowden and Bannayan-Riley-Ruvalcaba syndromes with variable presentations, including hamartomatous gastrointestinal tumors, dermatologic abnormalities, neurologic symptoms, and elevated cancer risk. We describe a father and son with extensive hamartomatous gastrointestinal polyposis who both developed early-onset esophageal cancer. Exome sequencing identified a novel germline PTEN frameshift mutation (c.568_569insC, p.V191Sfs*11). In addition, a missense mutation of SMAD7 (c.115G>A, p.G39R) with an allele frequency of 0.3% in the Exome Variant Server was detected in both affected individuals. Fluorescence in situ hybridization for PTEN in the resected esophageal cancer specimen demonstrated no PTEN copy loss in malignant cells; however, results of an immunohistochemical analysis demonstrated a loss of PTEN protein expression. While the risks of many cancers are elevated in the PTEN hamartoma tumor syndromes, association between esophageal adenocarcinoma and these syndromes has not been previously reported. Esophageal adenocarcinoma and extensive polyposis/ganglioneuromatosis could represent less common features of these syndromes, potentially correlating with this novel PTEN frameshift and early protein termination genotype. Alternatively, because simultaneous disruption of both the PTEN and TGF-ß/SMAD4 pathways is associated with development of esophageal cancer in a mouse model and because SMAD4 mutations cause gastrointestinal hamartomas in juvenile polyposis syndrome, the SMAD7 mutation may represent an additional modifier of these individuals' PTEN-mutant phenotype.

Pathogenesis and management of serrated polyps: current status and future directions.

Hyperplastic or serrated polyps were once believed to have little to no clinical significance. A subset of these polyps are now considered to be precursors to colorectal cancers (CRC) in the serrated pathway that may account for at least 15% of all tumors. The serrated pathway is distinct from the two other CRC pathways and involves an epigenetic hypermethylation mechanism of CpG islands within promoter regions of tumor suppressor genes. This process results in the formation of CpG island methylator phenotype tumors. Serrated polyps are divided into hyperplastic polyps, sessile serrated adenomas/polyps (SSA/Ps), and traditional serrated adenomas (TSAs). The SSA/P and the TSA have the potential for dysplasia and subsequent malignant transformation. The SSA/Ps are more common and are more likely to be flat than TSAs. Their flat morphology may make them difficult to detect and thus explain the variation in detection rates among endoscopists. Challenges for endoscopists also include the difficulty in pathological interpretation as well surveillance of these lesions. Furthermore, serrated polyps may be inadequately resected by endoscopists. Thus, it is not surprising that the serrated pathway has been linked with interval cancers. This review will provide the physician or clinician with the knowledge to manage patients with serrated polyps.

¹8F-FDG PET/CT imaging for a gastrointestinal mantle cell lymphoma with multiple lymphomatous polyposis.

Multiple lymphomatous polyposis (MLP) is an uncommon type of gastrointestinal lymphoma characterized by the presence of multiple polyps along the gastrointestinal tract. Most of this entity is in fact considered the counterpart of gastrointestinal tract involvement for mantle cell lymphoma (MCL). To our knowledge, there have been no reports on [fluorine-18]-fluorodeoxy-glucose ((18)F-FDG)-positron emission tomography (PET)/computed tomography (CT) imaging for gastrointestinal MCL with MLP. We present the results of (18)F-FDG PET/CT imaging in a patient with gastrointestinal tract involvement of MCL showing continuous MLP from the stomach to the rectum and intestinal intussusception. FDG-PET/CT findings were false negative in typical MLP spreading widely over the gastrointestinal tract, but uptake was noted in large lesions with deep infiltration considered atypical as MLP. On FDG-PET/CT imaging, the Ki-67 proliferative index, which is a cell proliferation marker, showed neither correlation with the presence of uptake nor the maximum standardized uptake value.

Gastrin promotes intestinal polyposis through cholecystokinin-B receptor-mediated proliferative signaling and fostering tumor microenvironment.

Increased serum gastrin concentrations in patients with colorectal cancer suggested the tumorigenic trophic effect of gastrin. Detailed and global molecular mechanisms explaining trophic effect of gastrin had not been revealed. In the current study, intestinal polyposis of APC(Min/⁺) mice was compared between phosphate buffered saline (PBS) injected and gastrin (10 µg/kg, thrice per week) injected group. Total number of intestinal polyposis was counted and immunohistochemical staining with F4/80 and CD3 was done. MTT assay, cell cycle analysis, and Western blot for cyclin D1, CDK4, and ß-catenin were performed in Raw 264.7 and HCT116 cells before and after gastrin administration. Experiments were repeated with YM022 or transfection with si-cholecystokinin-B receptor (CCK-B-R). Intraperitoneal gastrin significantly increased intestinal polyposis in APC(Min/⁺) mice (P<0.005), in which significant increases in macrophage were noted on F4/80 immunohistochemical staining (Plt;0.05) as well as Ki-67 staining (Plt;0.05) after gastrin. On comparative cytokine array, gastrin increased interleukin-1ß (IL-1ß), interleukin 3Rß (IL-3Rß), stromal cell-derived factor-1α (SDF-1α), thymus and activation-regulated chemokine (TARC), and thymus-derived chemotactic agent 3 (TCA-3) in macrophage cells, which was further confirmed with real time polymerase chain reaction (RT-PCR) analysis (P<0.05). In addition to increased inflammatory cytokines, gastrin increased macrophage proliferation accompanied with increased cyclin D1 and CDK4. Targeted for HCT116 cells, gastrin significantly increased proliferation as well as increases in synthetic phase of cell cycle. YM022 as gastrin antagonist significantly abolished the trophic actions of gastrin (P<0.05). HCT116 cells transfected with siCCK-B-R, gastrin did not increase either cell cycle or ß-catenin in spite of gastrin administration. Conclusively, gastrin promoted intestinal polyposis through either direct gastrin receptor-mediated proliferative signaling or fostering tumor microenvironment such as macrophage activation.

Grape antioxidant dietary fiber inhibits intestinal polyposis in ApcMin/+ mice: relation to cell cycle and immune response.

Epidemiological and experimental studies suggest that fiber and phenolic compounds might have a protective effect on the development of colon cancer in humans. Accordingly, we assessed the chemopreventive efficacy and associated mechanisms of action of a lyophilized red grape pomace containing proanthocyanidin (PA)-rich dietary fiber [grape antioxidant dietary fiber (GADF)] on spontaneous intestinal tumorigenesis in the Apc(Min/+) mouse model. Mice were fed a standard diet (control group) or a 1% (w/w) GADF-supplemented diet (GADF group) for 6 weeks. GADF supplementation greatly reduced intestinal tumorigenesis, significantly decreasing the total number of polyps by 76%. Moreover, size distribution analysis showed a considerable reduction in all polyp size categories [diameter <1mm (65%), 1-2mm (67%) and >2mm (87%)]. In terms of polyp formation in the proximal, middle and distal portions of the small intestine, a decrease of 76, 81 and 73% was observed, respectively. Putative molecular mechanisms underlying the inhibition of intestinal tumorigenesis were investigated by comparison of microarray expression profiles of GADF-treated and non-treated mice. We observed that the effects of GADF are mainly associated with the induction of a G1 cell cycle arrest and the downregulation of genes related to the immune response and inflammation. Our findings show for the first time the efficacy and associated mechanisms of action of GADF against intestinal tumorigenesis in Apc(Min/+) mice, suggesting its potential for the prevention of colorectal cancer.

BMPR1A mutations in juvenile polyposis affect cellular localization.

BACKGROUND: Juvenile polyposis (JP) is characterized by the development of hamartomatous polyps of the gastrointestinal tract that collectively carry a significant risk of malignant transformation. Mutations in the bone morphogenetic protein receptor type 1A (BMPR1A) are known to predispose to JP. We set out to study the effect of such missense mutations on BMPR1A cellular localization. METHODS: We chose eight distinct mutations for analysis. We tagged a BMPR1A wild-type (WT) expression plasmid with green fluorescent protein on its C-terminus. Site-directed mutagenesis was used to recreate JP patient mutations from the WT-green fluorescent protein BMPR1A plasmid. We verified mutant expression vector sequences by direct sequencing. First, we transfected BMPR1A expression vectors into HEK-293T cells; then, we performed confocal microscopy to determine cellular localization. Four independent observers used a scoring system from 1 to 3 to categorize the degree of membrane versus cellular localization. RESULTS: Of the eight selected mutations, one was within the signaling peptide, four were within the extracellular domain, and three were within the intracellular domain. The WT BMPR1A vector had strong membrane staining, whereas all eight mutations had much less membrane and much more intracellular localization. Enzyme-linked immunosorbent assays for BMPR1A demonstrated no significant differences in protein quantities between constructs, except for one affecting the start codon. CONCLUSIONS: Bone morphogenetic protein receptor type 1A missense mutations occurring in patients with JP affected cellular localization in an in vitro model. These findings suggest a mechanism by which such mutations can lead to disease by altering downstream signaling through the bone morphogenetic protein pathway.

Deficiency of phospholipase A2 group 7 decreases intestinal polyposis and colon tumorigenesis in Apc(Min/+) mice.

Platelet-activating factor (PAF) is a naturally occurring phospholipid that mediates diverse effects such as physiological and pathological inflammation, immunosuppression, and cancer. Several lines of evidence support both positive and negative roles for PAF in carcinogenesis. PAF stimulates cell growth, oncogenic transformation, and metastasis, but can also limit proliferation and induce apoptosis. The biological context and microenvironment seem to define whether PAF has pro- or anticarcinogenic effects. To investigate the role of exacerbated PAF signaling in colon cancer, we conducted cell-based and in vivo studies using genetically engineered mice lacking expression of phospholipase A2 group 7 (PLA2G7), an enzyme that specifically metabolizes PAF and structurally related glycerophospholipids. Absence of Pla2g7 robustly decreased intestinal polyposis and colon tumor formation in Apc(Min)(/+) mice, suggesting an antitumorigenic role for PAF in settings characterized by aberrant function of the tumor suppressor Adenomatous polyposis coli (Apc). In colonic epithelial cells, exposure to a PAF analog led to dephosphorylation of Akt at serine-473 and induction of apoptosis. The mechanism of this response involved formation of a complex between ß-arrestin 1 and the Akt phosphatase PHLPP2, and activation of the intrinsic pathway of apoptosis. Our results suggest that strategies based on inhibiting PLA2G7 activity or increasing PAF-mediated signaling hold promise for the treatment of intestinal malignancies that harbor mutations in APC.

A unifying working hypothesis for juvenile polyposis syndrome and Ménétrier's disease: specific localization or concomitant occurrence of a separate entity?

BACKGROUND: Juvenile polyposis syndrome with gastric involvement may mimic Ménétrier's disease, which is correlated to transforming growth factor (TGF)α overproduction and PDX1 upregulation in the gastric fundus. AIM: We report a family with juvenile polyposis syndrome where one member showed typical features of Ménétrier's disease and concomitant Helicobacter pylori infection. METHODS: We studied a 31-year-old woman belonging to a family with juvenile polyposis syndrome, who exhibited a particular form of hyperplastic gastropathy diagnosed as Ménétrier's disease with Helicobacter pylori infection. RESULTS: TGFα overexpression and undetectable PDX1 expression were demonstrated in the fundic gastric biopsy specimens. In all affected members of the family we identified a 4-bp deletion in exon 9 of SMAD4 gene, a mutation usually associated with a more virulent form of juvenile polyposis syndrome with a higher incidence of gastric and colonic polyposis. CONCLUSION: To explain the association of juvenile polyposis syndrome with Ménétrier's disease we hypothesized a new mechanism that involves TGFß-SMAD4 pathway inactivation and TGFα overexpression related to Helicobacter pylori infection.

Role of Gadd45a in Wip1-dependent regulation of intestinal tumorigenesis.

Conversion of intestinal stem cells into tumor-initiating cells is an early step in Apc(Min)-induced polyposis. Wild-type p53-induced phosphatase 1 (Wip1)-dependent activation of a DNA damage response and p53 has a permanent role in suppression of stem cell conversion, and deletion of Wip1 lowers the tumor burden in Apc(Min) mice. Here we show that cyclin-dependent kinase inhibitor 2a, checkpoint kinase 2, and growth arrest and DNA damage gene 45a (Gadd45a) exert critical functions in the tumor-resistant phenotype of Wip1-deficient mice. We further identified Gadd45a as a haploinsufficient gene in the regulation of Wip1-dependent tumor resistance in mice. Gadd45a appears to function through its ability to activate the Jnk-dependent signaling pathway that in turn is a necessary mediator of the proapoptotic functions of p53 that respond to activation of the ß-catenin signaling pathway. We propose that silencing of Gadd45a is sufficient to override p53 activation in the presence of active ß-catenin under conditions of an enhanced DNA damage response.

Two sides of the story? Smad4 loss in pancreatic cancer versus head-and-neck cancer.

TGFß signaling Smads (Smad2, 3, and 4) were suspected tumor suppressors soon after their discovery. Nearly two decades of research confirmed this role and revealed other divergent and cancer-specific functions including paradoxical tumor promotion effects. Although Smad4 is the most potent tumor suppressor, its functions are highly context-specific as exemplified by pancreatic cancer and head-and-neck cancer: in pancreatic cancer, Smad4 loss cannot initiate tumor formation but promotes metastases while in head-and-neck cancer Smad4 loss promotes cancer progression but also initiates tumor formation, likely through effects on genomic instability. The differing consequences of impaired Smad signaling in human cancers and the molecular mechanisms that underpin these differences will have important implications for the design and application of novel targeted therapies.

Germline mutations in SMAD4 disrupt bone morphogenetic protein signaling.

INTRODUCTION: Juvenile polyposis (JP) is an autosomal dominant disease that predisposes to GI malignancies. Germline mutations in the tumor suppressor gene SMAD4 account for approximately 20% of JP cases. SMAD4 is the common intracellular mediator of the TGF-ß and bone morphogenetic protein (BMP) pathways. Since mutations in BMP receptor 1A also cause JP, we hypothesize that altered BMP signaling is the underlying defect in JP. We therefore set out to investigate the effect of SMAD4 mutations on BMP signaling. METHODS: SMAD4 mutations identified in JP patients were selected for analysis. These were created in SMAD4 pCMV expression vectors (EV) using a PCR-based, site-directed mutagenesis (SDM) approach. SDM clones were confirmed by direct sequencing, then co-transfected with an IdI-BMP Luciferase Responsive Element (BRE-Luc) vector and Renilla control vector into HEK-293T cells. Lysates were then collected after 48 hours, and luciferase activity was quantified using a luminometer. A pCMV empty vector was used as a negative control, and its luciferase activity was considered the baseline for cellular BMP signaling. Results obtained for each SDM clone were compared to those with the wild type (WT) vector. Statistical analysis was performed with the Student's t-test. RESULTS: Eleven distinct mutations from 16 JP patients were analyzed; seven mutations were nonsense, and four were missense. Both type of mutations resulted in reduction of BMP signaling; missense mutations produced an 8-30% reduction in luciferase activity, whereas nonsense mutations led to 30-60% reduction in luciferase activity when compared to the WT clone (Figure 1). All nonsense mutations led to significantly reduced activity relative to WT (P < 0.05), while the reduction in signaling seen in missense mutations was not statistically significant. CONCLUSION: SMAD4 germline mutations as seen in the JP patients appear to negatively impact downstream BMP signaling. Nonsense mutations resulted in significantly reduced luciferase activity when compared to missense mutations. These results support the hypothesis that disruption of the BMP signaling pathway is the likely etiology of JP in patients with SMAD4 mutations.

HMGA1 induces intestinal polyposis in transgenic mice and drives tumor progression and stem cell properties in colon cancer cells.

BACKGROUND: Although metastatic colon cancer is a leading cause of cancer death worldwide, the molecular mechanisms that enable colon cancer cells to metastasize remain unclear. Emerging evidence suggests that metastatic cells develop by usurping transcriptional networks from embryonic stem (ES) cells to facilitate an epithelial-mesenchymal transition (EMT), invasion, and metastatic progression. Previous studies identified HMGA1 as a key transcription factor enriched in ES cells, colon cancer, and other aggressive tumors, although its role in these settings is poorly understood. METHODS/PRINCIPAL FINDINGS: To determine how HMGA1 functions in metastatic colon cancer, we manipulated HMGA1 expression in transgenic mice and colon cancer cells. We discovered that HMGA1 drives proliferative changes, aberrant crypt formation, and intestinal polyposis in transgenic mice. In colon cancer cell lines from poorly differentiated, metastatic tumors, knock-down of HMGA1 blocks anchorage-independent cell growth, migration, invasion, xenograft tumorigenesis and three-dimensional colonosphere formation. Inhibiting HMGA1 expression blocks tumorigenesis at limiting dilutions, consistent with depletion of tumor-initiator cells in the knock-down cells. Knock-down of HMGA1 also inhibits metastatic progression to the liver in vivo. In metastatic colon cancer cells, HMGA1 induces expression of Twist1, a gene involved in embryogenesis, EMT, and tumor progression, while HMGA1 represses E-cadherin, a gene that is down-regulated during EMT and metastatic progression. In addition, HMGA1 is among the most enriched genes in colon cancer compared to normal mucosa. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 drives proliferative changes and polyp formation in the intestines of transgenic mice and induces metastatic progression and stem-like properties in colon cancer cells. These findings indicate that HMGA1 is a key regulator, both in metastatic progression and in the maintenance of a stem-like state. Our results also suggest that HMGA1 or downstream pathways could be rational therapeutic targets in metastatic, poorly differentiated colon cancer.