Jason Yongsheng Chan

Molecular pathology of RUNX3 in human carcinogenesis

A major goal of molecular biology is to elucidate the mechanisms underlying cancer development and progression in order to achieve early detection, better diagnosis and staging and novel preventive and therapeutic strategies. We feel that an understanding of Runt-related transcription factor 3 (RUNX3)-regulated biological pathways will directly impact our knowledge of these areas of human carcinogenesis. The RUNX3 transcription factor is a downstream effector of the transforming growth factor-beta (TGF-beta) signaling pathway, and has a critical role in the regulation of cell proliferation and cell death by apoptosis, and in angiogenesis, cell adhesion and invasion. We previously identified RUNX3 as a major gastric tumor suppressor by establishing a causal relationship between loss of function and gastric carcinogenesis. More recently, we showed that RUNX3 functions as a bona fide initiator of colonic carcinogenesis by linking the Wnt oncogenic and TGF-beta tumor suppressive pathways. Apart from gastric and colorectal cancers, a multitude of epithelial cancers exhibit inactivation of RUNX3, thereby making it a putative tumor suppressor in human neoplasia. This review highlights our current understanding of the molecular mechanisms of RUNX3 inactivation in the context of cancer development and progression.

Genomic landscapes of breast fibroepithelial tumors

Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors. Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors, the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA, SETD2 and KMT2D, suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.

Resveratrol displays converse dose-related effects on 5-fluorouracil-evoked colon cancer cell apoptosis: The roles of caspase-6 and p53

We have reported that resveratrol (RSV) evoked apoptosis through caspase-6 activation in HCT116 human colon cancer cells wild-type (p53+/+) or knockout (p53-/-) for p53. In this study, the role of caspase-6 activation in 5-fluorouracil (5-FU)-elicited apoptosis as well as the combination effects between RSV and 5-FU on their apoptosis induction was further investigated in the same colon cancer cell model. The combination effects were determined by calculation of combination indices (CI). We found that 5-FU triggered apoptosis and caspase-6 activation in the cancer cells, which were entirely abrogated by caspase-6 inhibitors. RSV (200 µM) increased 5-FU-triggered apoptosis and caspase-6 activation. Lower doses (25 or 50 µM) inhibited 5-FU-mediated apoptosis and caspase-6 activation only in p53+/+ cells. Moreover, G1-arrest of the p53+/+ cells was elicited by lower doses of RSV and 5-FU in combination, but not with either agent alone. RSV (200 µM) interacted with 5-FU in a synergistic manner (mean CI < 0.9). At lower doses (25 or 50 µM), it interacted with 5-FU in antagonistic (mean CI > 1.1) and additive manners (0.9 < mean CI

RUNX3 Inactivation in Colorectal Polyps Arising Through Different Pathways of Colonic Carcinogenesis

OBJECTIVES:We hypothesized that RUNX3 inactivation by promoter hypermethylation in colorectal polyps is an early molecular event in colorectal carcinogenesis.METHODS:RUNX3 protein expression was analyzed immunohistochemically in 50 sporadic colorectal polyps comprising 19 hyperplastic polyps (HPs), 14 traditional serrated adenomas (TSAs), and 17 sporadic traditional adenomas (sTAs) as well as in 19 familial adenomatous polyposis (FAP) samples from 10 patients showing aberrant crypt foci (ACF) (n=91), small adenomas (SmAds) (n=40), and large adenomas (LAds) (n=13). In addition, we assessed the frequency of promoter hypermethylation of RUNX3 by methylation-specific PCR (MSP) in all the 50 sporadic polyps as well as 38 microdissected FAP polyps comprising ACF, SmAds, and LAds obtained from 7 FAP samples. A total of 12 normal colon samples were also included for RUNX3 MSP analysis.RESULTS:Compared to normal colon (2 of 12, 16%) and sTAs (3 of 17, 18%), HPs (15 of 19, 79%) and TSAs (8 of 14, 57%) displayed significant inactivation of RUNX3 (P<0.05). In FAP, RUNX3 inactivation was more frequently seen in ACF (78 of 91, 86%), SmAds (25 of 40, 62%), and LAds (6 of 13, 46%) compared to normal mucosa (0 of 19, 0%) in the same samples (all P<0.05). Promoter hypermethylation of RUNX3 was significantly higher in colorectal polyps (64 of 87, 74%) compared to normal colon (2 of 12, 16%) (P=0.001). Serrated polyps such as HPs (17 of 19, 89%) and TSAs (12 of 14, 86%) were significantly more methylated than sTAs (7 of 17, 44%) (P=0.004). RUNX3 hypermethylation was observed in 28 of the total 38 (74%) FAP polyps. Overall, RUNX3 promoter methylation correlated with inactivation of RUNX3 expression in sporadic (27 of 36, 75%) (P=0.022) and FAP (21 of 28, 75%) (P=0.021) polyps.CONCLUSIONS:Our data suggest that RUNX3 inactivation due to promoter hypermethylation in colorectal polyps represents an early event in colorectal cancer (CRC) progression. In addition, epigenetic RUNX3 inactivation is a frequent event in the serrated colonic polyps as well as in the ACF of FAP polyps.

Spontaneous and 5-fluorouracil-induced centrosome amplification lowers the threshold to resveratrol-evoked apoptosis in colon cancer cells.

We recently reported that caspase-6 activation is a major molecular event underlying resveratrol-triggered apoptosis in HCT116 colon cancer cells with or without p53. In the present study, we investigated the relationship between centrosome amplification and apoptosis sensitivity in cancer cells. We found that centrosome amplification, which occurs spontaneously in cancer cells, could be induced by a subtoxic concentration of 5-fluorouracil. Cancer cells with centrosome amplification, either spontaneous or 5-fluorouracil-induced, were more sensitive to apoptosis induction by resveratrol. In cancer cells, a subtoxic concentration of 5-fluorouracil also enhanced resveratrol-evoked caspase-6 activation. Functional loss of p53 promoted spontaneous, not 5-fluorouracil-induced, centrosome amplification. We conclude that centrosome amplification, either spontaneous or 5-fluorouracil-induced, confers higher apoptosis sensitivity to cancer cells. Drug induction of centrosome amplification might be a novel chemosensitization approach to cancer therapy.

MED12 protein expression in breast fibroepithelial lesions: correlation with mutation status and oestrogen receptor expression

Aims Recent reports have identified recurrent MED12 somatic mutations in fibroadenomas and phyllodes tumours. The frequency and type of somatic mutations were noted to be similar to those of uterine leiomyomas. We aimed to investigate protein expression of MED12, correlating it to MED12 mutational status and expression of oestrogen receptors (ER). Methods Immunohistochemistry was performed on a total of 232 fibroepithelial lesions (100 fibroadenomas, 132 phyllodes tumours) diagnosed at the Department of Pathology, Singapore General Hospital using MED12, ERα and ERβ antibodies. Expressions were evaluated in both stroma and epithelium, and correlated with MED12 mutational status. Results MED12 mutation was significantly associated with high MED12 protein expression (H-score >150) in the stroma (p=0.029), but not in the epithelium. It was not associated with ERα and ERβ protein expression in both stroma and epithelium. MED12 protein expression was significantly correlated with ERα in epithelial (p=0.007) and ERβ in stromal (p=0.049) components. MED12 was not significantly different between fibroadenomas and phyllodes tumours. Epithelial expression of ERα was significantly higher in fibroadenomas (p<0.001) than in phyllodes tumours. Conversely, both epithelial and stromal expression of ERβ was significantly higher in phyllodes tumours (p<0.001). Conclusions Positive associations observed between MED12 and ERα, ERβ immunohistochemical expression suggest a biological interplay between the proteins. The lack of significant association of MED12 mutation with ER protein expression indicates a need to further explore the functional impact of MED12 mutations on the ER signalling pathway in breast fibroepithelial lesions.

Opinion: molecular gestalt and modern pathology.

Since the early postmortem work by Morgagni or Rokitansky, the macroscopic evaluation of whole organs and systems has provided invaluable insights to the understanding of diseases. Virchow and Cohnheim pioneered the basic concept that diseases are correlated with specific histologic and cytologic appearances that lead to the description of microscopic physiopathologic processes. Both exercises are based on the link between appearances and disease which, translated into a diagnostic opinion, forms the backbone of traditional pathology, arguably one of the most common diagnostic strategies routinely used in modern medicine. Further extensions of contemporary pathology follow the same rationale but on a diminishing scale— immunohistochemistry, in situ hybridization, and electron microscopy are, for the practicing diagnostic pathologist, simply an intellectualization of pattern and color. Thus, one could argue that the history of Modern Pathology is primarily characterized by a reductionistic approach towards the interpretation and integration of morphologic information, defined as a method of understanding physiopathologic processes at progressively lower levels of physical organization—from the order of organ systems, tissues, cells, to organelles. The success of such a diagnostic approach may be largely attributed to its time and cost efficiency, as well as, perhaps more importantly, its tolerance to incomplete information. This confidence in the pathologic opinion comes from a learning process that, we believe, is very close to morphologic

Overexpression of neurone glial-related cell adhesion molecule is an independent predictor of poor prognosis in advanced colorectal cancer

A downstream target of the Wnt pathway, neurone glial‐related cell adhesion molecule (Nr‐CAM) has recently been implicated in human cancer development. However, its role in colorectal cancer (CRC) pathobiology and clinical relevance remains unknown. In this study, we examined the clinical significance of Nr‐CAM protein expression in a retrospective series of 428 CRCs using immunohistochemistry and tissue microarrays. Cox proportional hazards regression was used to calculate hazard ratios (HR) of mortality according to various clinicopathological features and molecular markers. All CRC samples were immunoreactive for Nr‐CAM protein expression, compared to 10/245 (4%) matched normal tissue (P < 0.0001). Of 428 CRC samples, 97 (23%) showed Nr‐CAM overexpression, which was significantly associated with nodal (P = 0.012) and distant (P = 0.039) metastasis, but not with extent of local invasion or tumor size. Additionally, Nr‐CAM overexpression was associated with vascular invasion (P = 0.0029), p53 expression (P = 0.036), and peritoneal metastasis at diagnosis (P = 0.013). In a multivariate model adjusted for other clinicopathological predictors of survival, Nr‐CAM overexpression correlated with a significant increase in disease‐specific (HR 1.66; 95% confidence interval 1.11–2.47; P = 0.014) and overall mortality (HR 1.57; 95% confidence interval 1.07–2.30; P = 0.023) in advanced but not early stage disease. Notably, 5‐fluorouracil‐based chemotherapy conferred significant survival benefit to patients with tumors negative for Nr‐CAM overexpression but not to those with Nr‐CAM overexpressed tumors. In conclusion, Nr‐CAM protein expression is upregulated in CRC tissues. Nr‐CAM overexpression is an independent marker of poor prognosis among advanced CRC patients, and is a possible predictive marker for non‐beneficence to 5‐fluorouracil‐based chemotherapy. (Cancer Sci 2011; 102: 1855–1861)

The Topography of DNA Methylation in the Non-Neoplastic Colonic Mucosa Surrounding Colorectal Cancers

The degree of gene hypermethylation in non‐neoplastic colonic mucosa (NNCM) is a potentially important event in the development of colorectal cancer (CRC), particularly for the subgroup with a CpG island methylator phenotype (CIMP). In this study, we aimed to use an unbiased and high‐throughput approach to evaluate the topography of DNA methylation in the non‐neoplastic colonic mucosa (NNCM) surrounding colorectal cancer (CRC). A total of 61 tissue samples comprising 53 NNCM and 8 tumor samples were obtained from hemicolectomy specimens of two CRC patients (Cases 1 and 2). NNCM was stripped from the underlying colonic wall and samples taken at varying distances from the tumor. The level of DNA methylation in NNCM and tumor tissues was assessed at 1,505 CpG sites in 807 cancer‐related genes using Illumina GoldenGate® methylation arrays. Case 1 tumor showed significantly higher levels of methylation compared to surrounding NNCM samples (P < 0.001). The average level of methylation in NNCM decreased with increasing distance from the tumor (r = −0.418; P = 0.017), however this was not continuous and “patches” with higher levels of methylation were observed. Case 2 tumor was less methylated than Case 1 tumor (average β‐value 0.181 vs. 0.415) and no significant difference in the level of methylation was observed in comparison to the surrounding NNCM. No evidence was found for a diminishing gradient of methylation in the NNCM surrounding CRC with a high level of methylation. Further work is required to determine whether CIMP+ CRC develop from within “patches” of NCCM that display high levels of methylation.

Lack of RUNX3 inactivation in columnar cell lesions of breast

Subramaniam M M, Chan J Y, Omar M F M, Ito K, Ito Y, Yeoh K G, Salto‐Tellez M & Putti T C
(2010) Histopathology57, 555–563
Lack of RUNX3 inactivation in columnar cell lesions of breast