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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.

Purpose: Endoglin is a coreceptor for TGFβ ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody. Experimental Design: The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor–knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg). Results: In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcγ receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8+ cytotoxic T cells and CD4+ Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens. Conclusions: Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role.

Patients with the mesenchymal subtype colorectal cancer (CRC) have a poor prognosis, in particular patients with stroma-rich tumors and aberrant SMAD4 expression. We hypothesized that interactions between SMAD4-deficient CRC cells and cancer-associated fibroblasts provide a biological explanation. In transwell invasion assays, fibroblasts increased the invasive capacity of SMAD4-deficient HT29 CRC cells, but not isogenic SMAD4-proficient HT29 cells. A TGF-beta/BMP-specific array showed BMP2 upregulation by fibroblasts upon stimulation with conditioned medium from SMAD4-deficient CRC cells, while also stimulating their invasion. In a mouse model for experimental liver metastasis, the co-injection of fibroblasts increased metastasis formation of SMAD4-deficient CRC cells (p = 0.02) but not that of SMAD4-proficient CRC cells. Significantly less metastases were seen in mice co-injected with BMP2 knocked-down fibroblasts. Fibroblast BMP2 expression seemed to be regulated by TRAIL, a factor overexpressed in SMAD4-deficient CRC cells. In a cohort of 146 stage III CRC patients, we showed that patients with a combination of high stromal BMP2 expression and the loss of tumor SMAD4 expression had a significantly poorer overall survival (HR 2.88, p = 0.04). Our results suggest the existence of a reciprocal loop in which TRAIL from SMAD4-deficient CRC cells induces BMP2 in fibroblasts, which enhances CRC invasiveness and metastasis.

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.

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The introduction of a wide range of immunotherapies in clinical practice has revolutionized the treatment of cancer in the last decade. The majority of these therapeutic modalities are centered on reinvigorating a tumor-reactive cytotoxic T-cell response. While impressive clinical successes are obtained, the majority of cancer patients still fail to show a clinical response, despite the fact that their tumors express antigens that can be recognized by the immune system. This is due to a series of other cellular actors, present in or attracted towards the tumor microenvironment, including regulatory T-cells, myeloid-derived suppressor cells and cancer-associated fibroblasts (CAFs). As the main cellular constituent of the tumor-associated stroma, CAFs form a heterogeneous group of cells which can drive cancer cell invasion but can also impair the migration and activation of T-cells through direct and indirect mechanisms. This singles CAFs out as an important next target for further optimization of T-cell based immunotherapies. Here, we review the recent literature on the role of CAFs in orchestrating T-cell activation and migration within the tumor microenvironment and discuss potential avenues for targeting the interactions between fibroblasts and T-cells.

Purpose: Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis. Experimental Design: CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer–derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model. Results: CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9–induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver. Conclusions: Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations. See related commentary by Becker and LeBleu, p. 6110.

Cell contact inhibition (CCI) is deregulated in cancer. Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. We found that dual-specificity phosphatase 10 (DUSP10) is involved in CRC. DUSP10 overexpression increased the growth of CRC cell lines and mouse xenografts, while the opposite phenotype was observed by DUSP10 silencing. High cell density (HD) induced DUSP10 expression in CRC cell lines, particularly within the nucleus. Yes-associated protein 1 (YAP1) is activated by dephosphorylation, controlling organ growth and CCI, both processes being deregulated in CRC. Expression levels and localization of DUSP10 matched with YAP1 levels in CRC cell lines. DUSP10 and YAP1 co-immunoprecipitated and their interaction was dependent on YAP1 Ser397. The existence of DUSP10 and YAP1 pathway in vivo was confirmed by using a transgenic Drosophila model. Finally, in CRC patients&rsquo, samples, high levels of nuclear DUSP10 correlated with nuclear YAP1 in epithelial tumor tissue. Strong nuclear DUSP10 staining also correlated with high tumor stage and poor survival. Overall, these findings describe a DUSP10&ndash, YAP1 molecular link in CRC cell lines promoting cell growth in HD. We present evidence suggesting a pro-tumorigenic role of nuclear DUSP10 expression in CRC patients.

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.

Lynch syndrome (LS), one of the most frequent forms of hereditary colorectal cancer (CRC), is caused by a defect in one of the mismatch repair (MMR) genes. Carriers of MMR defects have a strongly increased risk of developing CRC and endometrial cancer. Over the last few years, value-based healthcare has been introduced as an approach to the cost-effective delivery of measurable patient value over complete cycles of care. This requires all involved stakeholders to formulate and validate ‘patient value’ for Lynch syndrome, as well as to identify targets and associated costs. The aim of this study was to develop a value-based care model for Lynch syndrome that can determine patient value and associated costs, and to design a coordinated care pathway from existing guidelines. All specialists in our hospital involved in the management of LS patients evaluated the care delivered to these patients at their department and formulated outcome measures relevant to patient value. Patients were then invited to complete a questionnaire that assessed the importance of these measures on a scale of 1–10. Six high-value outcomes were identified: (1) prevention of cancer or detection of early stage cancer (2) rapid results from MMR gene mutation testing (3) rapid investigation of the colon and uterus (4) no/little pain during colonoscopy and gynaecologic examination/biopsy (5) the offer of psychological help and (6) registration with the Dutch Lynch syndrome registry. A total of 38 (59 %) out of 62 patients completed the questionnaire. The relevance of all outcomes was confirmed by the patients and mean scores varied from 7.2 to 9.9. Patients underscored the relevance of both proper patient education and the efficiency of surveillance during their care cycle. Value-based care delivery for Lynch syndrome includes the implementation of six parameters related to prevention and early detection of cancer, a short cycle time and registration to ensure continuation of care. Estimated costs are € 3320 for the first cycle of care (€ 3550 including gynaecologic surveillance) and approximately 720 per subsequent annual cycle (€ 950 including gynaecologic surveillance).Keywords: Lynch syndrome, Colorectal cancer, Endometrial cancer, Prevention, Value-based health care, Patient value, Outcome measures, Lynch syndrome registry

Endoglin, a transforming growth factor-beta co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical trials for anti-angiogenic therapy. In this project, the redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis and the effects of targeting both pathways on breast cancer metastasis were explored. In patient samples, increased endoglin signaling after VEGF inhibition was observed. In vitro TRC105, an endoglin-neutralizing antibody, increased VEGF signaling in endothelial cells. Moreover, combined targeting of the endoglin and VEGF pathway, with the VEGF receptor kinase inhibitor SU5416, increased antiangiogenic effects in vitro and in a zebrafish angiogenesis model. Next, in a mouse model for invasive lobular breast cancer, the effects of TRC105 and SU5416 on tumor growth and metastasis were explored. Although TRC105 and SU5416 decreased tumor vascular density, tumor volume was unaffected. Strikingly, in mice treated with TRC105, or TRC105 and SU5416 combined, a strong inhibition in the number of metastases was seen. Moreover, upon resection of the primary tumor, strong inhibition of metastatic spread by TRC105 was observed in an adjuvant setting. To confirm these data, we assessed the effects of endoglin-Fc (an endoglin ligand trap) on metastasis formation. Similar to treatment with TRC105 in the resection model, endoglin-Fc-expressing tumors showed strong inhibition of distant metastases. These results show, for the first time, that targeting endoglin, either with neutralizing antibodies or a ligand trap, strongly inhibits metastatic spread of breast cancer in vivo.

Development of colon cancer is a multistep process that is regulated by intrinsic and extrinsic cellular signals. Extrinsic factors include molecular patterns that are derived from either pathogens (PAMPs) or cellular damage (DAMPs). These molecules can promote tumourigenesis by activation of the innate immune system, but the individual contribution of ligands and their receptors remains elusive. The receptor for advanced glycation end products (Rage) is a pattern recognition receptor that binds multiple ligands derived from a damaged cell environment such as Hmgb1 and S100 protein. Here we show that Rage signalling has a critical role in sporadic development of intestinal adenomas, as Apc(Min/+) Rage(-/-) mice are protected against tumourigenesis.

Background: Upregulation of the matrix metalloproteinases MMP-2 and MMP-9 in various cancers has been associated with worse survival of the patients. Methods: We assessed MMP-2 and MMP-9 levels in normal colorectal mucosa from colorectal cancer patients in relation to the course of the disease. Results: A high protein expression of MMP-2 as well as MMP-9 in normal mucosa was found to be correlated with worse 5-year survival. The combination of both parameters was an even stronger prognostic factor. These protein levels were found not to be related to the corresponding single nucleotide polymorphisms of MMP-2 (−1306C>T) and MMP-9 (−1562C>T). Multivariate analyses indicated that the MMP-2 and MMP-9 levels in normal mucosa are prognostic for survival, independent of TNM classification. Conclusion: MMP-2 and MMP-9 levels in normal mucosa are indicative of the course of disease in colorectal cancer patients.

Background 5-aminosalicylic acid (5-ASA) may protect against the development of inflammation-associated colorectal cancer. In vitro data suggest that, in colorectal cancer cells, 5-ASA induces cell cycle arrest, but the molecular mechanism leading to this arrest remains to be determined. Aim To dissect the signal transduction events that lead to 5-ASA mediated inhibition of proliferation of colorectal cancer cells, focusing on mammalian target of rapamycin (mTOR), a regulator of cell cycle progression. Methods The influence of 5-ASA on mTOR signalling was examined in a panel of colorectal cancer cell lines. The effects of 5-ASA on the pathways that control mTOR activity were studied in detail in two different colorectal cancer cell lines, using western blot, siRNA, a phospholipase D (PLD) activity assay, proliferation assays and cell cycle analysis. The phosphorylation status of mTOR and its downstream target, ribosomal protein S6, was studied in colorectal cancers before and after topical 5-ASA treatment. Results Treatment of colorectal cancer with 5-ASA inhibited mTOR signalling in vitro and in vivo. 5-ASA had no effect on any of the pathways that regulate the activity of the tuberous sclerosis complex in colorectal cancer cells. Both proliferation and mTOR activity depended on PLD, an enzyme that generates phosphatidic acid (PA). 5-ASA treatment inhibited PLD activity and proliferation; these effects could be rescued with exogenous PA. Conclusion 5-ASA interferes with proliferation of colorectal cancer cells via inhibition of PLD-dependent generation of PA and loss of mTOR signalling.

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.

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 β (TGFβ) is important in colorectal cancer (CRC) progression. Bone morphogenetic proteins (BMPs), a subgroup within the TGFβ 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. Cancer 2008. © 2007 American Cancer Society.

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 fines 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.

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.

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 b-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.

Despite recent additions to the armory of chemotherapeutic agents for colorectal cancer (CRC) treatment, the results of chemotherapy remain unsatisfactory. 5-Fluorouracil (5-FU) still represents the cornerstone of treatment and resistance to its actions is a major obstacle to successful chemotherapy. Therefore, new active agents in CRC and agents that increase the chemosensitivity of cancer cells to 5-FU are still urgently required. Violacein, a pigment isolated from Chromobacterium violaceum in the Amazon river, has a diverse spectrum of biological activities, and represents a novel cytotoxic drug with known antileukemic properties. To assess the suitability of violacein as a chemotherapeutic agent in CRC its cytotoxic effects were evaluated both as a single agent and in combination with 5-FU. Its underlying mechanisms of action were further investigated by studying its effects on the cell cycle, apoptosis and cell survival pathways [phosphatidylinositol-3-kinase/Akt, p44/42 mitogen activated protein kinase and nuclear factor kappa B (NF-kappa B)] in colon cancer cell lines. Violacein inhibits the growth of all four colon cancer cell lines tested. It induces apoptosis, and potentiates the cytotoxic effect of 5-FU in a poorly differentiated microsatellite unstable cell line (HCT116). Violacein causes cell cycle block at G(1), upregulates p53, p27 and p21 levels and decreases the expression of cyclin D1. Violacein leads to dephosphorylation of retinoblastoma protein and activation of caspases and a pancaspase inhibitor abrogates its biological activity. Our data provide evidence that violacein acts through the inhibition of Akt phosphorylation with subsequent activation of the apoptotic pathway and downregulation of NF-kappa B signaling. This leads to the increase in chemosensitivity to 5-FU in HCT116 colon cancer cells. Taken together, our findings suggest that violacein will be active in the treatment of colorectal tumors and offers new prospects for overcoming 5-FU resistance.

Phosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influences the phosphorylation of signaling pathway mediators involved in cancer and is thus postulated to be a tumor-promoting enzyme, but neither unequivocal clinical evidence nor convincing mechanistic actions for a role of LMWPTP have been identified. In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia. Chemical inhibition of LMWPTP significantly reduces CRC growth. Furthermore, downregulation of LMWPTP in CRC leads to a reduced migration ability in both 2D- and 3D-migration assays, and sensitizes tumor cells to the chemotherapeutic agent 5-FU. In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.

Wnt signaling defines the colonic epithelial progenitor cell phenotype(1), and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers(2). The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4-beta-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal

Aspirin and other non-steroidal anti-inflammatory drugs show efficacy in the prevention of colon cancer. The mechanism by which they do this is unclear. We used a commercially available DNA microarray to study changes in gene expression in 1176 cancer related genes in the HT29 colon cancer cell line induced by aspirin. Overall we find more genes that are significantly induced than are repressed. The pattern of gene expression changes is different at high concentrations of aspirin (5 mM) than at lower levels (500 and 50 microM). Genes involved in DNA damage signaling, nucleotide metabolism and the stress response are induced, and cell cycle related genes repressed. The small GTPase Rac1 is highly induced and this was confirmed by immunoblotting. We show using immunohistochemistry that Rac1 is expressed in mature colonocytes at the intercrypt table in human and mouse colon tissue. These results support the previous findings that aspirin has different actions at high concentrations than at low concentrations and further show the use of DNA array technology in the investigation of drug mechanisms of action. Furthermore, they point towards a role for Rac1 in the action of aspirin in colon cancer.

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.

The factors that govern the progression from colonic adenomatous polyp to colon cancer are poorly understood. The observation that NSAIDs act as chemopreventative agents and reduce the size of colonic polyps suggests the involvement of inflammatory signalling, but inflammatory signalling in colonic polyps has not been studied. We investigated the expression of the active forms of NF-kappaB, JNK and p38 MAPK using immunohistochemistry with activation specific antibodies in human colonic adenomas. We show that active NF-kappaB is seen in stromal macrophages that also express COX-2 and TNF-alpha, active JNK is seen in stromal and intraepithelial T-lymphocytes and periendothelial cells of new blood vessels, and active p38 MAPK is most highly expressed in macrophages and other stromal cells. These results demonstrate the presence of active inflammatory signal transduction in colonic polyps and that these are predominantly in the stroma. In the case of NF-kappaB this coincides with the cellular localisation of COX-2. These results support evidence that NSAIDs may act through effects on stromal cells rather than epithelial cells.

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.].

Bone morphogenetic protein (BMP) signaling is known to suppress oncogenesis in the small and large intestine of mice and humans. We examined the role of Bmpr1a signaling in the stomach. On conditional inactivation of Bmpr1a, mice developed neoplastic lesions specifically in the squamocolumnar and gastrointestinal transition zones. We hypothesized that the regulation of epithelial cell fate may be less well defined in these junctional zones than in the adjacent epithelium and found that the mucosa at the squamocolumnar junction in mice shows a lack of differentiated fundic gland cell types and that foveolar cells at the gastrointestinal junctional zone lack expression of the foveolar cell marker Muc5ac. Precursor cell proliferation in both transition zones was higher than in the surrounding epithelium. Our data show that BMP signaling through Bmpr1a suppresses tumorigenesis at gastric epithelial junctional zones that are distinct from the adjacent gastric epithelium in both cellular differentiation and proliferation. [Cancer Res 2007;67(17):8149–55]

The rising incidence and poor prognosis of colorectal cancer have aroused substantial interest in novel chemopreventive strategies. Interestingly, treatment of ulcerative colitis with mesalazine, which displays few side effects during long-term treatment, is associated with a reduced incidence of colorectal cancer, but its molecular mechanism is not known. The effect of mesalazine on the Wnt/beta-catenin pathway was studied in colorectal cancer cell lines to find a molecular basis underlying its chemopreventive features. Mesalazine affects the Wnt/beta-catenin pathway in adenomatous polyposis coli mutated cells with intact beta-catenin, judged by luciferase reporter assays. Furthermore, mesalazine treatment reduced expression of nuclear beta-catenin and Wnt/beta-catenin target genes, and increased beta-catenin phosphorylation. This effect on the Wnt/beta-catenin pathway is mediated via protein phosphatase 2A (PP2A): increased phosphorylation of PP2A after mesalazine treatment is observed, which coincides with decreased PP2A enzymatic activity. The inhibition of PP2A enzymatic activity by mesalazine is essential for its effect on the Wnt/beta-catenin pathway, as shown by transient transfection with siPP2A and mutant PP2A. This study shows, using concentrations of mesalazine identical to concentrations seen in patients with inflammatory bowel disease, that mesalazine inhibits the Wnt/beta-catenin pathway via inhibition of PP2A.

Background & Aims: The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon. Methods: We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients. Results: BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients. Conclusions: These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.

Background: c9orf140 is a novel gene that has been recently isolated and indentified by the mRNA differential display (mRNAD), which is associated with cell proliferation and tumorigenesis in Gastric caner. Moreover, it was reported that the expression of c9orf140 is significantly elevated in colorectal caner (CRC) tissues when compared with normal tissues. However, the role of c9orf140 in the invasion of CRC and the mechanism of c9orf140 overexpression are still unclear. Method: c9orf140, EZH2, beta-catanin, STAT5 and pSTAT5 expressions were measured by Western Blot and immunohistochemical staining. To establish stable cells, CRC cells were infected with c9orf140 shRNA, c9orf140 overexpression or control lentivirus. Stable cell lines with different protein or shRNA expressions were introduced into nude mice respectively via tail veins injection. The photos were taken by the NightOWL II LB 983 in vivo Imaging system. Chromatin Immunoprecipitation (ChIP) and Luciferase reporter gene assay were carried out according to the protocol of manufactures. Result: there is almost no expression of C9orf140 in a normal human colon epithelial cell line, CRL1790, but the expression of C9orf140 is significantly increased in all CRC cell line especially in those highly invasive CRC cells. Immunofluorence analysis showed that C9orf140 is mainly detected in the cytoplasm of CRC HCT116 and HT29 cells. Immunohistochemical analysis of 150 patients' tissue sections showed that the staining of C9orf140 was found at higher expression levels in CRC samples than normal samples. Furthermore, the expression of C9orf140 was gradually increased from well differentiated to poor differentiated in CRC tissues. Knockdown of C9orf140 dramatically increased E-cadherin expression and decreased N-cadherin expression. Knockdown of C9orf140 significantly reduced the CRC cell invasion. More lung metastasis and shorter overall survival were detected after overexpression of C9orf140 compared with control in vivo. Knockdown of C9orf140 dramatically increased overall survival and decreased metastasis of lung in vivo. Western Blot data showed that C9orf140 expressions may be regulated by EZH2, STAT5 and β-catanin. Moreover, Immunoprecipitation, ChIP and Luciferase reporter gene assay showed that EZH2, STAT5 and βcatanin may colocalized together and directly bind to the promoter of C9orf140 gene to upregulate this gene expression in CRC cells. Conclusion: we firstly revealed the role of C9orf140 in the metastatic progression of CRC in vitro and in vivo. C9orf140-induced CRC cell migration and invasion may depend on promote the progression of EMT. Furthermore, we firstly identified that c9orf140 expression may be directly regulated by EZH2, STAT5 and β-catanin. Our data may provide potential targets to prevent and/or treat aggressive CRC.

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.

Background & Aims: The finding of bone morphogenetic protein (BMP) receptor la mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC. Methods: We investigated BMP receptor (BMPR) expression using immuno-blotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc. Results: BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P

Early detection and treatment are paramount to the clinical outcome of patients with colorectal cancer (CRC). Deciphering the dynamic interactions that occur between epithelial cells and stromal cells during tumorigenesis requires in-depth analyses of early-stage CRC lesions in spatial context. Here we employed spatially-resolved gene expression profiling to dissect molecular processes that associate with malignant transformation in CRC. We provide the transcriptional landscapes of colorectal cancer tumorigenesis from healthy mucosa, through different degrees of dysplasia, to cancer. The complementary examination of epithelial and stromal fractions allowed us to define whether specific oncogenic processes involved cancer cells, stromal cells, or the tumor microenvironment as a whole. We identified several genes that were consistently deregulated during CRC onset that could serve as clinical biomarkers for early-stage CRC. Furthermore, we uncovered an essential role for the innate immune system during CRC tumorigenesis.Abstract Figure