Alterations in mucin expression in common malignancies

Walsh, Michael (2011). Alterations in mucin expression in common malignancies PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Walsh, Michael
Thesis Title Alterations in mucin expression in common malignancies
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2011-06
Thesis type PhD Thesis
Supervisor Michael A. McGuckin
Total pages 394
Total colour pages 39
Total black and white pages 355
Language eng
Subjects 111202 Cancer Diagnosis
110307 Gastroenterology and Hepatology
110316 Pathology (excl. Oral Pathology)
Formatted abstract Colorectal cancer and breast cancer are both complex diseases. Recent advances in our understanding of both malignancies have demonstrated that there are distinctive differences in phenotype and behaviour between cancers attributable to a range of genetic and environmental risk factors.

For both diseases, there is an unmistakable familial or hereditary component in about 15-25%, and over the past two decades, germline mutations in several genes such as BRCA1, BRCA2, PALB2, CHEK2, and p53 have been linked to increased susceptibility to breast cancers. Similarly, syndromic colorectal cancers are seen in individuals harbouring mutations in genes as diverse as APC (giving rise to familial adenomatous polyposis – FAP), the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 (leading to Lynch syndrome or HNPCC) and MUTYH (MYH Associated Polyposis). There are other syndromes such as hyperplastic (or serrated) polyposis for which the causative gene(s) remain elusive but which have a marked increased risk for the development of cancers.

The family of proteins known as mucins is primarily characterized by the presence of one or more tandem repeat (VNTR) sequences of amino acids rich in serine and threonine such that the mature protein is highly glycosylated. The two main categories of mucin are the gel-forming or secreted mucins which are encoded by a gene cluster on chromosome 11, and the transmembrane mucins.

Alterations in mucin gene expression have been demonstrated to have significant consequences in terms of tumour biology. Mucinous tumours of the breast (those over-expressing gel mucins) have a better prognosis than conventional invasive carcinomas whereas the converse is true in the colorectum where mucin lakes tend to dissect the muscularis propria facilitating tumour dissemination. Increased expression of transmembrane mucins has also been linked to more aggressive tumour behaviour and poorer patient outcome with evidence mounting that these glycoproteins have functions as diverse as providing a physicochemical barrier for the cells interfering with cell-cell and cell-matrix adhesion, acting as ligands for growth factor receptors stimulating cell growth, and modulating signal transduction pathways by binding various proteins in a number of cascades.

Expression of MUC1 mucin was assessed immunohistochemically in an unselected series of breast cancer patients, and expression patterns were related to clinicopathological features of the tumours. The prognostic significance of MUC1 expression was determined for both disease-free interval and overall patient survival. MUC1 expression in breast cancer was further explored in a panel of 14 BC cell lines utilizing flow cytometry, immunocytochemistry and Western blotting for protein expression, and Northern blotting for mRNA levels. These same cell lines were grown as xenografts in SCID and nude mice, and MUC1 expression asses using immunohistochemistry in both primary implants and resultant pulmonary metastases. The patterns of expression of another transmembrane mucin, MUC13, were characterized in an unselected series of colorectal cancers, and expression related to patient outcome and DNA microsatellite instability status.

The gel-forming mucins, MUC2, MUC5AC, and MUC6 were assessed in a large series of colorectal polyps and colorectal cancers arsing in patients with the hyperplastic polyposis syndrome. Patients were recruited through an international collaboration with Northern Regional Genetics Service, Auckland, Ohio State University, Columbus, and the Australasian Colorectal Cancer Family Registry (ACCFR) (Melbourne/Brisbane). Expression of the mucins was determined immunohistochemically, and all lesions were subject to a full review by the candidate and a consultant specialist histopathologist. Somatic V600E BRAF and codons 12/13 KRAS mutations were determined using high resolution melt PCR and direct sequencing, and methylation-induced silencing of MLH1 determined using IHC for MLH1 and PMS2. The relationship between DNA methylation, somatic BRAF mutation and chromosome 11 mucin expression was further explored in a population-based series of colorectal cancers occurring in participants recruited into the Health2020 – Melbourne Collaborative Cohort Study.

Breast cancers associated with Lynch syndrome were identified from pedigrees of families enrolled in the ACCFR and individuals were classified as carrier, non-carrier or non-Lynch syndrome on the basis of germline mutation testing. Breast tumours were tested for expression of the four major mismatch repair proteins, MLH1, PMS2, MSH2 and MSH6 by immunohistochemistry, as was steroid hormone receptor status. MSI and BRAF testing was performed on DNA extracted from tumours. The tumours were subjected to an extensive histopathological re-review. These breast cancers, as well as an unselected series of BCs were then examined for a panel of mucin genes.

MUC1 expression in breast cancers was found to be prognostically significant. Patients whose tumours showed cytoplasmic staining in >75% tumour cells had a poorer disease-free interval and overall survival. High levels of MUC1 staining were also associated with oestrogen receptor expression and the presence of regional lymph node metastases, but were not related to other clinicopathological features. In a panel of BC cell lines, expression of MUC1 was found to be variable. Cell lines which expressed little to no MUC1 were found to be those which had undergone a mesenchymal transformation, expressing vimentin and with reduced cytokeratin 8/18 expression. There was generally good correlation between mRNA levels and protein levels as determined by flow cytometry and Western blotting. MUC1 expression remained relatively consistent between primary tumour xenografts and the resultant pulmonary metastases for cell lines grown in immunocompromised mice.

A similar association between high levels of MUC13 and clinicopathological features associated with poorer patient outcome in a series of colorectal tumours. Nearly all colorectal cancers examined expressed significant quantities of the MUC13 protein with highest levels in more poorly differentiated tumours. A redistribution of protein from the apical to basolateral membranes was associated with poor differentiation and tumour budding. Expression levels were higher in left-sided (distal) cancers than more proximal tumours. Basolateral expression was more commonly seen in patients with poorer overall survival.

Breast cancers occurring in a series of 104 individuals with a personal or family history of early-onset colorectal carcinoma were examined and evidence of MMR deficiency and MSI were found in 18 tumours from proven germline mutation carriers. There was no association between MMR loss and tumour type although these tumours were of poorer grade, lack steroid hormone receptors, have vesicular nuclei, growth in solid sheets and have tumour infiltrating lymphocytes. MMR deficiency was identified in approximately 60% of breast tumours from mutation carriers, and strikingly, the BC was the only tumour or first tumour diagnosed in the majority of these women.

Unlike MSI colorectal cancers with MSI, there was no association between MMR deficiency and de novo expression of MUC2, MUC5AC or MUC6 in breast cancers when compared with non-mutation carriers or a series of breast cancers from individuals without a family history of cancer. Breast cancers showing overt mucinous phenotype were not a feature of MMR deficiency.

Conclusions
Expression of many mucin genes is perturbed in neoplasia in both the breast and colorectum. The MUC1 mucin is frequently up regulated in breast carcinoma and increased expression is related to more aggressive phenotypic characteristics and poorer disease-free and overall patient survival. Similarly, MUC13 is expressed at higher levels in colorectal cancers of unfavourable phenotype. In vivo studies of MUC1 expression by a panel of breast carcinoma cell lines suggest that pulmonary metastases maintain expression levels comparable to the corresponding primary xenografts.

In the largest series of Lynch-syndrome associated breast cancers studied to date, carriage of a germline MMR gene mutation coincides with a high frequency of microsatellite instability and tumour histopathogical features such as poor differentiation and tumour lymphocytic infiltration similar to those reported for such colorectal cancers. Most importantly, the observation that, for many of these individuals, the breast cancer is the only malignancy diagnosed has particular clinical and diagnostic ramifications. The commonly described relationship between MSI and mucinous differentiation in the colorectum with over-expression of the chromosome 11 gel-forming mucin genes was not a characteristic of Lynch-syndrome associated breast cancers despite the fact that a proportion of breast cancers over-express these genes.
Keyword Mucin
Expression
Lynch syndrome
Carcinoma
Colorectal
Breast
Microsatellite Instability
Sporadic
Hereditary
Additional Notes Colour pages: 39,49,62,80,106,123,134,157,159,19-206,210,211,227,228,230-234,241,271-276,279-284 Landscape pages: 147-150,166-167,170-171,227-230,235,268,269,271-275,283,284

 
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Created: Wed, 04 Jul 2012, 05:30:04 EST by Mr Michael Walsh on behalf of Scholarly Publishing and Digitisation Service