Rutger J. Jacobs

Statins augment the chemosensitivity of colorectal cancer cells inducing epigenetic reprogramming and reducing colorectal cancer cell ‘stemness’ via the bone morphogenetic protein pathway

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.

Key elements of the BMP/SMAD pathway co-localize with CDX2 in intestinal metaplasia and regulate CDX2 expression in human gastric cell lines

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. Copyright

The role of EZH2 and DNA methylation in the silencing of the tumour suppressor RUNX3 in colorectal cancer

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.

The activities of Smad and Gli mediated signalling pathways in high-grade conventional osteosarcoma

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 tumours aggressive clinical course, however targeted pathway modulation in vitro did not affect cell proliferation.

Cholesterol metabolism and colorectal cancers.

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.

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC

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 β-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the β-catenin paradox.Methods:We analysed the expression patterns of SMAD4, p53 and β-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity.Results:Eighty-four percent of CRCs with high nuclear β-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner.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.

Reduced expression of bone morphogenetic protein receptor IA in pancreatic cancer is associated with a poor prognosis

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.

A Meta-Analysis of SMAD4 Immunohistochemistry as a Prognostic Marker in Colorectal Cancer

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.

Evaluation of the prognostic value of pSMAD immunohistochemistry in colorectal cancer.

SMAD4 mutations and recent genome-wide association studies (GWAS) show the importance of bone morphogenetic protein (BMP) and transforming growth factor-&bgr; (TGF-&bgr;) 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-&bgr; 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.

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

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