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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 β 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 β 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., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Promoter hypermethylation is an important and potentially reversible mechanism of tumour suppressor gene silencing in cancer. Compounds that demethylate tumour suppressor genes and induce differentiation of cancer cells, but do not have toxic side effects, would represent an exciting option in cancer therapy. Statins are cholesterol-lowering drugs with an excellent safety profile and associated with a reduced incidence of various cancers including colorectal cancer (CRC). The authors have previously shown that statins act by activating tumour suppressive bone morphogenetic protein (BMP) signalling in CRC, increasing expression of BMP2. BMP2 is silenced by hypermethylation in gastric cancer., Aim: To investigate whether BMP2 is methylated in CRC, whether statins can reverse this, and what implications this has for the use of statins in CRC., Methods: Methylation-specific PCR, bisulphite sequencing, immunoblotting, reverse transcription PCR, quantitative PCR, fluorescence-activated cell sorting analysis, an in vitro DNA methyltransferase (DNMT) assay, and cell viability studies were performed on CRC cells. The effect of statins was confirmed in a xenograft mouse model. Results BMP2 is silenced by promoter hypermethylation in cell lines with the hypermethylator phenotype and in primary tumours. Treatment with lovastatin downregulates DNMT activity, leading to BMP2 promoter demethylation and to upregulation of expression of BMP2 as well as other genes methylated in CRC. Statins alter gene expression, indicating a shift from a stem-like state to a more differentiated state, thereby sensitising cells to the effects of 5-fluorouracil. In a xenograft mouse model, simvastatin treatment induces BMP2 expression, leading to differentiation and reduced proliferation of CRC cells., Conclusions: Statins act as DNMT inhibitors, demethylating the BMP2 promoter, activating BMP signalling, inducing differentiation of CRC cells, and reducing 'stemness'. This study indicates that statins may be able to be used as differentiating agents in combined or adjuvant therapy in CRC with the CpG island methylator phenotype.

Much of the current understanding of colorectal cancer stems from the study of rare, inherited colorectal cancer syndromes. Mutations in the bone morphogenetic protein (BMP) pathway have been found in juvenile polyposis, an inherited polyposis syndrome that predisposes to colorectal cancer. The hamartomas that develop in these patients and in BMP pathway mutant mice have a remarkable mesenchymal component. Further evidence in mice suggests a primary role for mesenchymal loss of BMP signalling in hamartoma development. Here, we examine this evidence and question its relevance to sporadic colorectal carcinogenesis.

Background: Transforming growth factor beta (TGFbeta) is important in colorectal cancer (CRC) progression. Bone morphogenetic proteins (BMPs), a subgroup within the TGFbeta superfamily, recently also have been implicated in CRC, but their precise role in CRC has yet to be investigated., Methods: The authors used a tissue microarray and immunohistochemistry of BMP receptors and signal transduction elements in adenomas and CRC specimens to elucidate the role of BMP signaling in CRC carcinogenesis., Results: The adenoma specimens expressed all 3 BMP receptors (BMPRs) (BMPR type 1a [BMPR1a], BMPR1b, and BMPR2) and expressed SMAD family member 4 (SMAD4); and 20 of 22 adenomas (90.9%) exhibited active BMP signaling, as determined by nuclear phosphorylated SMAD1,5,8 (pSMAD1,5,8) expression. In contrast, pSMAD1,5,8 nuclear staining was present in 5 CRC specimens (22.7%) but was lost in 17 CRC specimens (77.3%; cancer vs adenoma; P< .0001). The earliest loss of pSMAD1,5,8 nuclear staining was detected in regions of high-grade dysplasia/carcinoma in situ within adenomas. CRCs showed frequent loss of BMPR2 (P< .0001) and SMAD4 (P< .01) compared with adenomas. Negative expression of BMPR2 was observed more frequently in earlier stage cancers (Dukes stage B) than in advanced cancers (Dukes stage C; P< .05)., Conclusions: Taken together, the current results indicated that loss of BMP signaling correlates tightly with progression of adenomas to cancer and occurs relatively early during cancer progression.

Background & Aims: Epidemiological evidence suggests that statins prevent colorectal cancer (CRC), but the biological mechanism remains obscure. Statins induce bone morphogenetic protein (BMP) expression in bone cells. We have previously shown that BMPs act as tumor suppressors in CRC. We hypothesized that the action of statins in CRC involves the induction of BMPs., Methods: We investigated the effects of statins on CRC cell lines using immunoblotting, measurements of apoptosis and cell proliferation, and luciferase reporter assays. The effect of statins was confirmed in a xenograft mouse model., Results: CRC cell lines show widely differing sensitivities to statin treatment. Sensitive cell lines show induction of BMP2 protein levels and a BMP2 reporter construct, activation of the BMP pathway, and induction of the BMP target gene ID-2, whereas resistant cell lines do not. The addition of the specific inhibitor of BMPs, noggin, completely prevents lovastatin-induced apoptosis in sensitive cells. Sensitive cell lines express the central BMP pathway element SMAD4, whereas the resistant cell lines do not. Targeted knockout of SMAD4 leads to the loss of statin sensitivity and reconstitution with SMAD4, to the restoration of statin sensitivity. In a xenograft mouse model, tumors from sensitive and insensitive cell lines were treated with oral simvastatin. Significant inhibition of tumor growth using sensitive cells but increased tumor growth when using insensitive cells was observed., Conclusions: Statins induce apoptosis in CRC cells through induction of BMP2. Statin therapy may only be effective in SMAD4-expressing CRCs and may have adverse effects in SMAD4-negative tumors.

Nonsteroidal anti-inflammatory drugs show chemopreventive efficacy in colon cancer, but the mechanism behind this remains unclear. Elucidating this mechanism is seen as vital to the development of new chemopreventive agents. We studied the effects of aspirin on the oncogenic Wnt/beta-catenin pathway activity in colorectal cancer cell lines and observed that aspirin dose-dependently decreased the activity of this pathway, as judged by TCF-driven luciferase activity, reduced Wnt target gene expression and increased phosphorylation of beta-catenin by immunoblotting. Furthermore, the ubiquitination and cytoplasmic levels of beta-catenin were assessed by immunoblotting, and also the localization of beta-catenin was shown by green fluorescent protein-tagged beta-catenin and time-lapse fluorescent imaging. Importantly, aspirin treatment caused increased phosphorylation of protein phosphatase 2A (PP2A), an event associated with inhibition of PP2A enzymatic activity, which was confirmed by a reduction in enzymatic PP2A activity. Moreover, this inhibition of PP2A enzymatic activity was essential for the effects of aspirin on the Wnt/beta-catenin pathway as shown by transient transfection with PP2A constructs. The findings in this article provide a molecular explanation for the efficacy of aspirin in chemoprevention of colorectal cancer and shows biochemical evidence that PP2A is an important regulator of Wnt/beta-catenin pathway activity in these cells.

Morphogens regulate epithelial cell fate decisions in the adult gastrointestinal tract. The authors hypothesized that influx of inflammatory cells into the lamina propria may disturb the normal expression gradients of morphogens (morphogenetic landscape) in gastrointestinal epithelia. Changes in the activity of the bone morphogenetic protein (BMP) pathway in normal and Helicobacter pylori-infected gastric mucosa were therefore examined. It is shown that BMP receptors, the activated (phosphorylated) form of the intracellular BMP signal transduction protein SMAD1, and BMP target ID2 all localize to gastric epithelial cells that are at the end of the axis of epithelial renewal in normal mucosa. Colonization of human gastric mucosa with H. pylori was associated with an increase in BMP2 expression due to influx of inflammatory cells that produce BMP2. Furthermore, whereas no BMP4 was detected in the normal antrum, focal infiltrates of BMP4-expressing cells were found in the H. pylori-infected stomach. This influx of BMP-expressing cells was associated with an increase in epithelial BMP signalling. Interestingly, a shift in activity of the BMP pathway was observed towards the precursor cell compartment (isthmus) of the gastric units. Thus, H. pylori infection results in an influx of inflammatory cells that disturb the normal activity gradient of a morphogenetic pathway with an established role in epithelial cell fate regulation. The data suggest that morphological changes in epithelial histology may result from alterations in the morphogenetic landscape secondary to changes in the cellular composition of the lamina propria., (Copyright (c) 2006 Pathological Society of Great Britain and Ireland.)

BackgroundConstitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the β-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the β-catenin paradox.MethodsWe analysed the expression patterns of SMAD4, p53 and β-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity.ResultsEighty-four percent of CRCs with high nuclear β-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner.ConclusionsThe BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.