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Background Epidemiological studies and meta-analyses show an association between statin use and a reduced incidence of colorectal cancer (CRC). We have shown that statins act on CRC through bone morphogenetic protein (BMP) signalling, but the exact cellular targets and underlying mechanism of statin action remain elusive. In this study, we set out to assess the influence of statins on global cancer cell signalling by performing an array-based kinase assay using immobilised kinase substrates spanning the entire human kinome. Methods CRC cells with or without Lovastatin treatment were used for kinome analysis. Findings on kinome arrays were further confirmed by immunoblotting with activity-specific antibodies. Experiments in different CRC cell lines using immunoblotting, siRNA-mediated knockdown and treatment with specific BMP inhibitor Noggin were performed. The relevance of in vitro findings was confirmed in xenografts and in CRC patients treated with Simvastatin. Results Kinome analysis can distinguish between non-specific, toxic effects caused by 10 mu M of Lovastatin and specific effects on cell signalling caused by 2 mu M Lovastatin. Statins induce upregulation of PTEN activity leading to downregulation of the PI3K/Akt/mTOR signalling. Treatment of cells with the specific BMP inhibitor Noggin as well as PTEN knockdown and transfection of cells with a constitutively active form of AKT abolishes the effect of Lovastatin on mTOR phosphorylation. Experiments in xenografts and in patients treated with Simvastatin confirm statin-mediated BMP pathway activation, activation of PTEN and downregulation of mTOR signalling. Conclusions Statins induce BMP-specific activation of PTEN and inhibition of PI3K/Akt/mTOR signalling in CRC.

BACKGROUND & AIMS: We aimed to perform geriatric assessment in older patients with inflammatory bowel disease (IBD) to evaluate which IBD characteristics associate with deficits in geriatric assessment and the impact of deficits on disease burden (health-related quality of life).METHODS: A prospective multicenter cohort study including 405 consecutive outpatient patients with IBD aged ≥65 years. Somatic domain (comorbidity, polypharmacy, malnutrition), impairments in (instrumental) activities of daily living, physical capacity (handgrip strength, gait speed), and mental (depressive symptoms, cognitive impairment) and social domain (life-partner) were assessed. Deficits in geriatric assessment were defined as ≥2 abnormal domains; 2-3 moderate deficits and 4-5 severe deficits. Clinical (Harvey Bradshaw Index >4/partial Mayo Score >2) and biochemical (C-reactive protein ≥10 mg/L and/or fecal calprotectin ≥250 μg/g) disease activity and disease burden (short Inflammatory Bowel Disease Questionnaire) were assessed.RESULTS: Somatic domain (51.6%) and activities of daily living (43.0%) were most frequently impaired. A total of 160 (39.5%) patients had moderate deficits in their geriatric assessment; 32 (7.9%) severe. Clinical and biochemical disease activity associated with deficits (clinical: adjusted odds ratio, 2.191; 95% confidence interval, 1.284-3.743; P = .004; biochemical: adjusted odds ratio, 3.358; 95% confidence interval, 1.936-5.825; P < .001). Deficits in geriatric assessment independently associate with lower health-related quality of life.CONCLUSION: Deficits in geriatric assessment are highly prevalent in older patients with IBD. Patients with active disease are more prone to deficits, and deficits associate with lower health-related quality of life, indicating higher disease burden. Prospective data validating impact of frailty and geriatric assessment on outcomes are warranted to further improve treatment strategies.

BACKGROUND: Long-term use of statins is associated with a small reduced risk of colorectal cancer but their mechanism of action is not well understood. While they are generally believed to act on KRAS, we have previously proposed that they act via influencing the BMP pathway. The objective of this study was to look for associations between statin use and the risk of developing colorectal cancer of a particular molecular subtype.METHODS: By linking two registries unique to the Netherlands, 69,272 statin users and 94,753 controls were identified and, if they developed colorectal cancer, their specimens traced. Colorectal cancers were molecularly subtyped according to the expression of SMAD4 and the mutation status of KRAS and BRAF.RESULTS: Statin use was associated with a reduction in the risk of developing colorectal cancer regardless of molecular subtype (HR 0.77; 95% CI 0.66-0.89) and a larger reduction in the risk of developing SMAD4-positive colorectal cancer (OR 0.64; 95% CI 0.42-0.82). There was no relationship between statin use and the risk of developing colorectal cancer with a mutation in KRAS and/or BRAF.CONCLUSIONS: Statin use is associated with a reduced risk of developing colorectal cancer with intact SMAD4 expression.

Background & aims: It is currently unclear whether reported changes in the gut microbiome are cause or consequence of inflammatory bowel disease (IBD). Therefore, we studied the gut microbiome of IBD-discordant and -concordant twin pairs, which offers the unique opportunity to assess individuals at increased risk of developing IBD, namely healthy cotwins from IBD-discordant twin pairs. Methods: Fecal samples were obtained from 99 twins (belonging to 51 twin pairs), 495 healthy age-, sex-, and body mass index–matched controls, and 99 unrelated patients with IBD. Whole-genome metagenomic shotgun sequencing was performed. Taxonomic and functional (pathways) composition was compared among healthy cotwins, IBD-twins, unrelated patients with IBD, and healthy controls with multivariable (ie, adjusted for potential confounding) generalized linear models. Results: No significant differences were observed in the relative abundance of species and pathways between healthy cotwins and their IBD-twins (false discovery rate

Background Frailty could be useful for risk stratification in older patients with Inflammatory Bowel Diseases (IBD). However, no prospective evidence is yet available. We aimed to assess how frailty screening associates with hospital admissions and functional and (health related) quality of life (HR)QoL decline. Methods This is a prospective multicenter cohort study. Consecutive IBD patients aged ≥65 years were included at outpatient departments and infusion centers in six hospitals. Clinical disease activity was assessed through Harvey Bradshaw Index (HBI) or partial Mayo Score (pMS) (remission: HBI Results 405 patients were included, median age 70 (IQR 67–74), 85 (21%) clinical disease activity, 93 (23%) biochemical disease activity and 196 (48%) abnormal G8. Until February 2021, 293 (72%) patients were eligible for follow-up: 255 participated in follow-up visits, 38 patients did not (15 not reachable, 16 not willing, 7 died). 74 all-cause and 33 IBD-related hospital admissions occurred. Abnormal G8 was associated with occurrence of both all-cause (adjusted HR 3.9, 95%CI 1.9–7.8, p Conclusion Abnormal frailty screening independently associates with both all-cause and IBD-related hospital admissions and with functional and QoL decline. Therefore, we provide the first prospective evidence for frailty screening as a useful risk stratification tool in IBD.

AIM: SMAD4 immunohistochemistry is considered a valuable prognostic marker in colorectal cancer, but individual studies have often been small and the results variable. A meta-analysis could potentially clarify these findings. METHODS: In September 2014, a Pubmed and Google Scholar search was conducted to find publications that reported the prognostic value of SMAD4 expression. A meta-analysis was performed to clarify the association between SMAD4 expression and survival outcomes. RESULTS: 137 studies were found, of which 13 were considered eligible. The studies consisted of a total of 3800 patients. Three different endpoints were taken into account, namely, overall survival (OS), disease-free survival (DFS), and cancer-specific survival (CSS). In addition, the studies were divided into univariate and multivariate analyses. The pooled hazard ratios were given as follows: univariate CSS = 1.75 [95% confidence interval (CI): 0.93-3.32; z= 1.69; P= .09]; multivariate CSS = 2.17 (95% CI: 1.56-3.01; z= 4.65; P= .000); univariate DFS = 2.11 (95% CI: 1.36-3.28; z= 3.32; P= .001); multivariate DFS = 2.15 (95% CI: 1.56-3.01; z= 4.65; P= .000); univariate OS and DFS = 2.30 (95% CI: 1.41-3.73; z= 3.36; P= .001); univariate OS = 2.28 (95% CI: 1.30-4.00; z= 2.89; P= .004). CONCLUSION: The results of the presented meta-analyses indicate that SMAD4 expression status using immunohistochemistry is a prognostic marker for patient survival.

Colorectal cancer is a leading cause of death by cancer in the western world. Despite major progress, even new chemotherapeutic regimens have had relatively little impact on long term survival in the approximately 50% of patients with advanced disease at presentation meaning that prevention is the only realistic way to reduce the burden of this disease. Many countries have implemented population-based screening methods to prevent colorectal cancer by the physical removal of its precursor lesion the adenoma, or to detect cancer at an earlier stage when it is amenable to surgical cure. However these programs have only been shown to reduce colorectal cancer deaths by 30% in those screened and therefore new or complimentary approaches are needed. One such approach is chemoprevention. A number of compounds have shown potential in reducing the incidence of colorectal cancer. Most widely known are NSAIDs but recently inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, also known as statins, commonly prescribed medications that lower serum cholesterol, have been shown to reduce colorectal cancer incidence. A critical issue in chemoprevention is the weighing of benefits against risks. In chemoprevention this balance is likely to be unfavourable when used in a wide unselected population even for the safest of compounds. Therapy should therefore be tailored to the individual patient. The balance will be more favourable in high risk groups such as individuals especially susceptible to neoplasia because of environmental risk factors, patients with inflammatory bowel disease, those with a hereditary predisposition and patients with a previous history of colorectal cancer or polyps. Furthermore colorectal cancer is not one disease but a heterogeneous group of diseases with different underlying molecular mechanisms. It is likely that both prevention and therapy will need to be tailored to the molecular subtype of the cancer in question. This may explain why studies of colorectal cancer in statin users do not show consistent protective effects. Evidence in vitro has shown a dichotomous effect of statins with either a cancer inhibiting or cancer promoting effect depending on their molecular subtype. Further studies are needed to determine in which patient groups statins can be used to prevent colorectal cancer and whether in other patients groups they should be avoided.

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.

In gastric cancer, a new epigenetic mechanism of tumour suppressor loss has been suggested where the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is responsible for loss of expression of RUNX3. This is consistent with EZH2 upregulation in multiple cancer types being associated with poor prognosis. We investigated whether EZH2 influences the expression of RUNX3 in colorectal cancer (CRC) and whether this is independent of methylation. We determined protein and messenger RNA (mRNA) levels of EZH2 and RUNX3 and assessed RUNX3 methylation with methylation-specific polymerase chain reaction using 72 human CRCs and 8 CRC cell lines. We assessed the effect of efficient RNA interference-mediated knockdown of EZH2 on RUNX3 levels, cell viability and H3K27 trimethylation of the RUNX3 promoter using chromatin immunoprecipitation. Despite higher levels of EZH2 and lower levels of RUNX3 in CRC specimens in general, no inverse correlation between EZH2 and RUNX3 in paired samples was found arguing against a major role for histone methylation in silencing RUNX3 in CRC. Conversely, downregulation of RUNX3 mRNA in the same tumours was associated with RUNX3 DNA methylation (P < 0.05). In cell lines, knockdown of EZH2 removed the repressive chromatin marks from RUNX3 but did not result in RUNX3 re-expression. However, it prevented the re-silencing of RUNX3 after the removal of demethylating agents. In conclusion, DNA methylation is primarily responsible for the transcriptional silencing of RUNX3 in CRC, but EZH2 and histone methylation are necessary for its methylation-dependent re-silencing after the removal of demethylating agents. These results would predict that inhibitors of EZH2 and histone methylation would enhance the effects of demethylating agents in cancer therapy.

BACKGROUND & AIMS: Statin use has been associated with a reduced incidence of colorectal cancer and might also affect survival of patients diagnosed with colon cancer. Statins are believed to inhibit Ras signaling and may also activate the bone morphogenetic protein (BMP) signaling pathway in colorectal cancer cells. We investigated the effects of statins on overall survival of patients with a diagnosis of colon cancer, and whether their effects were associated with changes in KRAS or the BMP signaling pathways.METHODS: Data were derived from the PHARMO database network (Netherlands) and linked to patients diagnosed with colon cancer from 2002 through 2007, listed in the Eindhoven Cancer Registry. We obtained information on causes of death from statistics Netherlands. We constructed a tissue microarray of 999 colon cancer specimens from patients who underwent surgical resection from 2002 through 2008. Survival was analyzed with statin user status after diagnosis as a time-dependent covariate. Multivariable Poisson regression survival models and Cox analyses were used to study the effect of statins on survival. Tumor tissues were analyzed by immunohistochemistry for levels of SMAD4, BMPR1A, BMPR1B, and BMPR2 proteins. Tumor tissues were considered to have intact BMP signaling if they contained SMAD4 plus BMPR1A, BMPR1B, or BMPR2. DNA was isolated from tumor tissues and analyzed by quantitative polymerase chain reaction to detect mutations in KRAS. The primary outcome measures were overall mortality and cancer-specific mortality.RESULTS: In this cohort, 21.0% of the patients (210/999) were defined as statin users after diagnosis of colon cancer. Statin use after diagnosis was significantly associated with reduced risk of death from any cause (adjusted relative risk [RR], 0.67; 95% confidence interval [CI], 0.51-0.87; P = .003) and death from cancer (adjusted RR, 0.66; 95% CI, 0.49-0.89; P = .007). Statin use after diagnosis was associated with reduced risk of death from any cause or from cancer for patients whose tumors had intact BMP signaling (adjusted RR, 0.39; 95% CI, 0.22-0.68; P = .001), but not for patients whose tumors did not have BMP signaling (adjusted RR, 0.81; 95% CI, 0.55-1.21; P = .106; P < .0001 for the interaction). Statin use after diagnosis was not associated with reduced risk of death from any cause or from cancer for patients whose tumors did not contain KRAS mutations (adjusted RR, 0.81; 95% CI, 0.56-1.18; P = .273) or whose tumors did have KRAS mutations (adjusted RR, 0.59; 95% CI 0.35-1.03; P = .062; P = .90 for the interaction).CONCLUSIONS: In an analysis of 999 patients with a diagnosis of colon cancer, we associated statin with reduced risk of death from any cause or from cancer. The benefit of statin use is greater for patients whose tumors have intact BMP signaling, independent of KRAS mutation status. Randomized controlled trials are required to confirm these results.

Background: Constitutive 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 beta-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 beta-catenin paradox. Methods: We analysed the expression patterns of SMAD4, p53 and beta-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. Results: Eighty-four percent of CRCs with high nuclear beta-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. Conclusions: The 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.

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 beta 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 beta 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.

Background: The expression of SMAD4, the central component of the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathways, is lost in 50% of pancreatic cancers and is associated with a poor survival. Although the TGF-β pathway has been extensively studied and characterised in pancreatic cancer, there is very limited data on BMP signalling, a well-known tumour-suppressor pathway. BMP signalling can be lost not only at the level of SMAD4 but also at the level of BMP receptors (BMPRs), as has been described in colorectal cancer. Methods: We performed immunohistochemical analysis of the expression levels of BMP signalling components in pancreatic cancer and correlated these with survival. We also manipulated the activity of BMP signalling in vitro. Results: Reduced expression of BMPRIA is associated with a significantly worse survival, primarily in a subset of SMAD4-positive cancers. In vitro inactivation of SMAD4-dependent BMP signalling increases proliferation and invasion of pancreatic cancer cells, whereas inactivation of BMP signalling in SMAD4-negative cells does not change the proliferation and invasion or leads to an opposite effect. Conclusion: Our data suggest that BMPRIA expression is a good prognostic marker and that the BMP pathway is a potential target for future therapeutic interventions in pancreatic cancer.

SMAD4 mutations and recent genome-wide association studies (GWAS) show the importance of bone morphogenetic protein (BMP) and transforming growth factor-β (TGF-β) signalling in the development of colorectal cancer (CRC). Loss of SMAD4 has been implicated as a predictive marker in CRC. As activation of the BMP and TGF-β pathways leads to phosphorylation of SMAD1,5,8 and SMAD2,3, respectively, and both need SMAD4 for translocation to the nucleus, we aimed to investigate whether nuclear staining of pSMAD1,5,8 and pSMAD2, 3 can be used as predictive markers in CRC. A tissue microarray (TMA) was constructed using tissue from 209 patients diagnosed with CRC. TMA was stained and scored for the nuclear presence of SMAD4, pSMAD2,3 and pSMAD1,5,8. Loss of SMAD4, pSMAD2,3 and pSMAD1,5,8 was observed in 40, 38 and 73% of the cases, respectively. The incidence of SMAD4 loss was significantly higher in the advanced stages. There was a correlation between loss of SMAD4 and loss of pSMAD1,5,8, but not between loss of SMAD4 and loss of pSMAD2,3. Loss of SMAD4 correlated with a poorer survival. Loss of one of the pSMADs did not correlate with a poorer outcome. Combining different SMAD stainings did not improve the prediction. SMAD4 expression is a prognostic marker in CRC. Nuclear expressions of pSMAD1,5,8 and pSMAD2,3 are not useful prognostic markers in CRC.

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

Colorectal cancer (CRC) is primarily a lifestyle disease of the western world. As such it can be likened to cardiovascular disease and indeed it shares many of the same risk factors. It is therefore perhaps unsurprising that cholesterol metabolism and colorectal cancer are also intricately linked. Many of the initial studies suggesting a link between dietary cholesterol, blood cholesterol levels and cholesterol lowering drugs were performed more than a decade ago. The most recent insights in this field are the result of meta-analyses, advances in pharmacogenetics and the new field of molecular pathological epidemiology. This review summarises the current evidence linking cholesterol metabolism with colorectal cancer including the suggested underlying molecular causes and the implications for colorectal cancer prevention.

High-grade conventional osteosarcoma is a malignant tumour predominantly affecting adolescents and, despite multimodal intensive therapy, lethal for one third of the patients. Although there is currently detailed knowledge of normal skeletal development, this has not been integrated into research on the genesis of osteosarcoma. Recently we showed that the canonical Wnt pathway is not active in osteosarcoma and that its reactivation is disadvantageous to osteosarcoma cells. Since Wnt is regulating normal skeletogenesis together with other pathways, here we report on the activities of the bone morphogenic protein (BMP), the transforming growth factor beta (TGFβ) and the hedgehog (Hh) pathways in osteosarcoma. Human osteosarcoma samples (n=210), benign bone tumours of osteoblastic lineage called osteoblastoma (n=25) and osteosarcoma cell lines (n=19) were examined. For pathway activity luciferase transcriptional reporter assays and gene and protein expression analyses were performed. Immunohistochemical analysis of phosphorylated Smad1 and Smad2, the intracellular effectors of BMP and TGFβ, respectively, showed nuclear expression of both proteins in 70% of the osteosarcoma samples at levels comparable to osteoblastoma. Interestingly cases with lower expression showed significantly worse disease free survival. This may imply that drugs restoring impaired signalling pathways in osteosarcoma might change the tumour’s aggressive clinical course, however targeted pathway modulation in vitro did not affect cell proliferation.

Helicobacter pylori infection induces intestinal metaplasia of the stomach, a preneoplastic lesion associated with an increased risk for gastric cancer development. Intestinal metaplasia is induced by the intestine-specific transcription factor CDX2 but the mechanisms responsible for this ectopic expression have never been described. We hypothesized that the BMP/SMAD pathway has a role in CDX2 regulation, in this context, for the following reasons: (1) the BMP pathway is crucial for normal intestinal differentiation and (2) there is an influx of BMP2 and BMP4-producing cells to the stomach upon Helicobacter pylori infection. We evaluated the expression of key elements of the BMP pathway in human stomach specimens with IM. Growth factor treatments, with BMP2 and BMP4, were performed in cultured cells and a knock-down experiment of SMAD4 was done using RNAi. We showed overexpression in IM of BMP2/4, BMPR1A, and SMAD4 in 56% of IM foci, and pSMAD1/5/8 in 100% of IM foci as compared to adjacent mucosa. In vitro, treatment of AGS cells with BMP2 and BMP4 increased endogenous CDX2 expression as well as the intestinal differentiation markers MUC2 and LI-cadherin. On the other hand, SMAD4 knock-down led to decreased endogenous CDX2, MUC2, and LI-cadherin in AGS. Treatment of the SMAD4 knock-down cells had no influence on CDX2 expression as opposed to wild-type cells. A 9.3 kb CDX2 promoter could be transactivated by SMAD4 and SMAD1 in a cell-dependent manner. In conclusion, we identified for the first time that the BMP pathway is active in intestinal metaplasia and that BMP2 and BMP4 regulate CDX2 expression and promote intestinal differentiation through the canonical signal transducers.

Background: The use of statins has been associated with a reduced incidence of colorectal cancer. Potentially, statins may also have an influence on survival of colon cancer patients when used after diagnosis. The primary aim of this observational study was to evaluate the influence of statin use after diagnosis of colon cancer on overall survival. The secondary aim was to investigate the molecular background in which statins are effective adjuvant therapeutic agents, specifically KRAS mutational status and Bone Morphogenetic Protein pathway functionality, as these are the two most promising and frequently mentioned potential molecular mechanisms. Methods: Data was derived from the PHARMO database network (PHARMO, Netherlands) and was linked to a colon cancer cohort (Eindhoven Cancer Registry). A Tissue Micro Array (TMA) consisting of 999 colon cancer specimens was constructed from patients who had a surgical resection between 2002 and 2008. Survival was analyzed with statin user status after diagnosis as a time-dependent covariate. Multivariate Poisson regression survival models were used to study the effect of statins on overall survival (OS). The tumors were analyzed for SMAD4, BMPR1a, BMPR1b and BMPR2 expression, and KRAS mutations to perform stratified analyses for intact BMP signaling status and KRAS mutation status. Results: In this cohort 21.0% (210/999) of the patients were defined as statin users after colon cancer diagnosis. Statin use after diagnosis was significantly associated with an improved OS with an adjusted Rate Ratio (RR) of 0.67 (95% CI 0.51-0.87, P= 0.003). When stratified for intact BMP signaling status, survival benefit of statin use after diagnosis was stronger in tumors with intact BMP signaling (adjusted RR 0.46 (95% CI 0.29-0.74, P=0.001) than in tumors with a non-intact BMP signaling pathway (adjusted RR 0.75, 95% CI 0.53-1.06, P=0.106). Statin use after diagnosis was not associated with an improved OS in KRAS wild-type tumors (Adjusted RR 0.81, 95% CI 0.56-1.18, P=0.273) or KRAS mutated-tumors (Adjusted RR 0.59, 95% CI 0.35-1.03, P=0.062). Conclusion: Statin use after diagnosis is associated with an improved overall survival in colon cancer patients. This survival benefit is more prominent in tumors with intact BMP signaling. The survival benefit is independent of KRAS mutation status.

Introduction:Notch1-mediated signaling influences cell fate decisions, cell cycle and carcinogenesis via a transcription activation complex consisting of ICN1 (the activated form of Notch1), the co-activatorMAML and the transcription factor CSL. However, the consequences of Notch activation are complex and context dependent. The role of Notch signaling in esophageal epithelial biology andmalignant transformation remain elusive. Methods:Human esophageal keratinocytes immortalized by telomerase (EPC1-hTERT and EPC2-hTERT) as well as non-transformed and transformed derivatives including EPC2T overexpressing EGFR, cyclin D1 and p53R175H; transformed human esophageal keratinocytes carrying a human papilloma virus (HPV)-E6E7 fusion oncogene (EN60) and esophageal squamous cell carcinoma (ESCC) cell lines were genetically engineered to express tetracycline-inducible ICN1; dominant-negative MAML1 (DNMAML1), a genetic pan-Notch inhibitor; or short hairpin RNA (shRNA) directed against CSL, HPV-E7 or cyclin-dependent kinase inhibitor p16INK4A. Notch activity was determined by transfection assays of an 8xCSL-luciferase reporter construct. Notch target genes were determined by quantitative RT-PCR. Western blotting determined retinoblastoma (RB) protein phosphorylation status. Cell proliferation was determined by colorimetric assays. Anchorage-independent colony formation was evaluated in soft agar. Tumor formation was assessed by xenograft transplantation in immunodeficient mice. Senescence associated β-galactosidase (SABG) activity was determined. Results: ICN1 activated CSL-dependent transcription in all cell lines tested and this activation was antagonized by either DNMAML1 expression or CSL knockdown. In response to ICN1 induction, nontransformed cells underwent growth arrest displaying flat and enlarged cell morphology with SABG positive staining (40-80%), suggesting senescence. ICN1-mediated senescence was found to be CSL-dependent and was associated with decreased RB protein phosphorylation and p16INK4A upregulation. ICN1-mediated senescence was inhibited by p16INK4A knockdown, but not the presence of p53R175H. ICN1 failed to induce senescence in EN60 cells expressing EPV-E7 protein or ESCC cell lines lacking the INK4A locus. ICN1 stimulated colony formation and tumor growth in these transformed cell lines. HPV E7 knockdown in EN60 cells resulted in senescence with activation of endogenous Notch1. Conclusions: These novel data indicate that loss of the RB protein-dependent senescence checkpoint function may be permissive for Notch1 to facilitate malignant transformation in human esophageal epithelial cells.

Background: Colorectal cancer (CRC) is a major public health problem worldwide and incidence rates are increasing, especially in areas historically at low risk. Removal of cancer precursor lesions at colonoscopy is the foundation of CRC prevention, but offers far from perfect protection from CRC. CRC prevention using chemical or natural compounds (chemoprevention) represents a promising alternative or adjuvant preventative strategy. Epidemiological studies and meta-analyses have shown an association between the use of statins and a reduced incidence of CRC. Statins are generally well tolerated and with proven beneficial effects on cardiovascular outcomes improving any overall benefit/risk ratio, they are an attractive candidate for use as a chemopreventive agent in CRC. Their exact cellular targets and underlying mechanism of action have, however, remained elusive. We have shown that statins act on CRC through activation of Bone Morphogenetic Protein (BMP) signaling. However, statins have been shown by others to have a wide range of effects on many other signaling pathways. Aim & Methods: In this study, we set out to assess the influence of statins on global cell signaling in a hypothesis-free manner. We performed an array-based kinase assay using immobilized kinase substrates spanning the entire human kinome. We used HCT116 colon cancer cells with or without Statin treatment. We confirmed our findings from the array using immunoblotting with activity-specific antibodies. Subsequent experiments in different CRC cell lines using immunoblotting with activation-specific antibodies, RNAi and kinase inhibitors focused on the most consistent and pronounced changes. Experiments in xenografts and in patients treated with Statins were performed to confirm our In Vitro data In Vivo. Results: The clinically relevant concentration of 2μM Lovastatin proved to be optimal for kinome analysis. Treatment with statin led to widespread changes in cell signaling compatible with anti-tumor activity. The strongest effect was seen on the PI3K/Akt pathway. Statins induced upregulation of PTEN activity leading to downregulation of the PI3K/Akt pathway and downstream mTOR-mediated signaling. In Vitro experiments showed that the effect on PTEN is dependent on BMP signaling. Further experiments in xenografts and in patients treated with Statins confirmed the changes in BMP pathway and PTEN activity. Conclusion: Statin treatment rapidly leads to significant changes in signal transduction favoring anti-tumor activity. Statins induce downregulation of Akt/mTOR signaling through upregulation of PTEN activity in a BMP dependent manner; both In Vitro as In Vivo. These findings should ultimately facilitate more targeted use of statins in CRC either alone or in combination with other therapies.

INTRODUCTION: Colorectal cancer (CRC) is one of the leading causes of death by cancer in theWestern world. Unfortunately, successful treatment is dependent still on early diagnosis and surgical resection. Epidemiological studies suggest that Statins have powerful chemopreventive properties with respect to colon cancer, but the mechanism of action of Statins in colon cancer is unknown. Most research has focused on the post-translational prenylation of proteins, but several studies have shown that statins also have additional effects. A limited number of well characterized signal transduction pathways allow cells to react to their environment and are regulated by phosphorylation. By generating a comprehensive description of the phosphoproteome we aimed to investigate the mechanism of action of statins in colon cancer cells in an unbiased fashion. AIMS & METHODS: Aim: To elucidate the molecular mechanism of action of Statins in colorectal cancer. Methods: We used arrays containing 1024 different kinase substrates spanning the entire human kinome. The peptide arrays were incubated with cell lysates plus radioactive ATP, exposed to a phospho-imaging screen and scanned. The density of the spots was measured and analyzed with array software. Western blotting was used to independently validate the results. RESULTS: To analyze the changes in the phosphoproteome we determined the number of significantly phosphorylated substrates for each condition. Statin treatment for 24 hours leads to a substantial downregulation of cell signaling. Non-treated cells displayed phosphorylation of 222 substrates, and treated cells 151 substrates. While most other pathways remained unaffected the activity of multiple Receptor Tyrosine Kinases (RTKs) was significantly decreased with down regulation of their downstream targets. As RTKs play a well known role in cell cycle progression, growth and metabolism control, their inhibition by statins may provide a new insight into the chemopreventive properties of this drug. CONCLUSION: Treatment of colon cancer cells with Statins alters intracellular signaling by downregulation of the activity of multiple Receptor Tyrosine Kinases and its downstream pathways. This insight could help to identify a group of patients with CRC where Statins can be successfully used as chemotherapeutic and chemopreventive drugs.