Loss of CSMD1 expression is associated with high tumour grade and poor survival in invasive ductal breast carcinoma Mohamed Kamal, Abeer M. Shaaban, Liqun Zhang, Clare Walker, Sally Gray, Nalin Thakker, Carmel Toomes, Valerie Speirs, Sandra M. Bell To cite this version: Mohamed Kamal, Abeer M. Shaaban, Liqun Zhang, Clare Walker, Sally Gray, et al.. Loss of CSMD1 expression is associated with high tumour grade and poor survival in invasive ductal breast carcinoma. Breast Cancer Research and Treatment, Springer Verlag, 2009, 121 (3), pp.555-563. 10.1007/s10549- 009-0500-4. hal-00612973 HAL Id: hal-00612973 https://hal.archives-ouvertes.fr/hal-00612973 Submitted on 2 Aug 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Breast Cancer Res Treat (2010) 121:555–563 DOI 10.1007/s10549-009-0500-4 PRECLINICAL STUDY Loss of CSMD1 expression is associated with high tumour grade and poor survival in invasive ductal breast carcinoma Mohamed Kamal Æ Abeer M. Shaaban Æ Liqun Zhang Æ Clare Walker Æ Sally Gray Æ Nalin Thakker Æ Carmel Toomes Æ Valerie Speirs Æ Sandra M. Bell Received: 22 July 2009 / Accepted: 25 July 2009 / Published online: 8 August 2009 Ó Springer Science+Business Media, LLC. 2009 Abstract CUB and SUSHI multiple domain protein 1 samples was investigated by immunohistochemistry in 275 (CSMD1) is a candidate tumour suppressor gene that maps to patients. Univariate and multivariate Cox regression analy- chromosome 8p23, a region deleted in many tumour types ses were performed. In normal breast duct epithelial cells, including 50% of breast cancers. CSMD1 has homologies to luminal, membranous and cytoplasmic CSMD1 staining proteins implicated in carcinogenesis. We aimed to study the was identified. Reduced expression of CSMD1 was detected expression pattern of the CSMD1 protein and evaluate its in 79/275 (28.7%) of IDC cases. Low CSMD1 expression prognostic importance in invasive ductal carcinoma (IDC). was significantly associated with high tumour grade An anti-CSMD1 antibody was developed and validated. The (P = 0.003). CSMD1 expression was associated with expression pattern of CSMD1 in normal breast and IDC overall survival (OS; HR = 0.607, 95%CI: 0.4–0.91, P = 0.018) but not with disease-free survival (DFS; HR = 0.81, 95%CI: 0.46–1.43, P = 0.48). Multivariate analysis showed that CSMD1, together with Nottingham Prognostic Index, was considered an independent predictor of OS (HR = 0.607, 95%CI: 0.4–0.91, P = 0.018) but not Electronic supplementary material The online version of this DFS (HR = 0.84, 95%CI: 0.46–1.5, P = 0.573). Reduction article (doi:10.1007/s10549-009-0500-4) contains supplementary of CSMD1 expression was significantly associated with high material, which is available to authorized users. tumour grade and decreased OS. Therefore, our results M. Kamal Á C. Walker Á C. Toomes Á S. M. Bell (&) support the idea that CSMD1 is a tumour suppressor gene Leeds Institute of Molecular Medicine, University of Leeds, and suggest its possible use as a new prognostic biomarker. Wellcome Trust Brenner Building, Level 8, St. James’s The membrane expression pattern of CSMD1 suggests that it University Hospital, Leeds LS9 7TF, UK may be a receptor or co-receptor involved in the process of e-mail: [email protected] signal transduction. M. Kamal Department of Zoology, Faculty of Science, University Keywords CSMD1 Á Breast cancer Á Prognosis Á of Benha, Benha, Egypt Differentiation Á Immunohistochemistry A. M. Shaaban Á C. Walker Á S. Gray St James’s Institute of Oncology, St James’s University Hospital, Leeds LS9 7TF, UK Introduction L. Zhang Á N. Thakker Department of Medical Genetics, University of Manchester, Manchester M13 0JH, UK Breast cancer is one of the leading causes of death in women [1, 2]. It is a complex and heterogeneous disease at V. Speirs the molecular level. Abnormal expression of growth factor Leeds Institute of Molecular Medicine, University of Leeds, Wellcome Trust Brenner Building, Level 4, St. James’s receptors, cell-cycle regulatory molecules and cell-adhe- University Hospital, Leeds LS9 7TF, UK sion proteins frequently occurs [3]. Investigating the 123 556 Breast Cancer Res Treat (2010) 121:555–563 expression and functions of these molecules, and their Materials and methods association with patient outcome, is extremely important to aid the understanding of the disease process and assist in Patients clinical decision-making [4]. CUB and SUSHI multiple domains-1 (CSMD1) encodes All patients gave informed consent to participate in this multiple mRNA transcripts with the largest being 14.3 Kb study, and ethical approval was obtained from the Local long. The gene spans over 2 Mb of genomic DNA and Research Ethics Committee of the Leeds Teaching Hospitals contains 71 exons, which encode a 3,565 amino acid pro- (LTH), NHS Trust, Leeds, UK. Paraffin blocks from two tein consisting of 14 CUB domains and 28 SUSHI independent cohorts of breast cancer cases were retrieved domains. CSMD1’s function is unknown; however, CUB from the archives of the pathology departments of the LTH, and SUSHI domains are thought to be involved in receptor- NHS Trust. The first cohort containing 50 cases was desig- ligand interactions. The full-length CSMD1 protein is nated as the training set, and the second cohort containing predicted to be a membrane protein with an extracellular 446 cases was used as the validation set. These cases were region containing the CUB and SUSHI domains, a single selected from patients presenting between 1987 and 2005. transmembrane domain and a short cytoplasmic domain Only patients with IDC of no special type, NST (40 in the that contains a tyrosine phosphorylation site. From these training set and 331 in the validation set) were included in the features, one could predict that CSMD1 is a receptor or co- study due to the small numbers of patients with other tumour receptor for unknown ligand(s) and is involved in signal subtypes. Immunohistochemical data were available on all transduction [5, 6]. the 40 patients of the training set. While in the validation set, Many lines of evidence indicate CSMD1 could be a from 331 cases, immunohistochemical data were available tumour suppressor gene. Firstly, CSMD1 spans chromo- on a maximum of 275 patients due to loss of some tissue somal band 8p23, which is known to exhibit frequent microarray (TMA) cores during antigen retrieval and stain- deletions in many cancers, including breast cancer [7–15]. ing, a well-recognised phenomenon associated with TMA Secondly, loss of CSMD1 has been reported in many immunohistochemistry. Clinical history and tumour char- cancers. At the DNA level, an array-based comparative acteristics (age, tumour type, size, histological grade, lymph genomic hybridization (CGH) study detected the loss of node status and Nottingham prognostic index (NPI)) were CSMD1 in a large proportion of head and neck squamous available. DFS was defined as the interval (months) from cell carcinoma (HNSCC), lung squamous cell carcinoma primary surgical treatment to the first loco regional or distant (SCC) and breast cancers [16]. At the RNA level, quanti- recurrence. OS was taken as time (months) from primary tative real-time PCR (qPCR) detected the loss of CSMD1 surgical treatment to time of death from breast cancer. expression in HNSCCs and lung SCCs [16], liver [17], colorectal [18] and prostate cancer [19, 20]. Thirdly, Generation of anti-CSMD1 antibody CSMD1 shares homologies with many other proteins playing roles in cancer progression by the virtue of its Two peptide sequences HFTSDSNHRRKGFN and QRVTE CUB and SUSHI domains such as SEZ6L and DMBT1. TLAAWSDHR from the N-terminus of human CSMD1 SEZ6L is homozygously deleted in small cell lung cancer were selected as targets for generating a polyclonal anti- cell lines [5, 21, 22], and DMBT1 expression is reduced in body. In vitro synthesis of the peptides and immunisation of malignant brain tumours [23], lung cancer [24] and breast chickens with keyhole limpet hemocyanine-conjugated cancer [25]. A variety of developmental proteins have been CSMD1 peptides, followed by Ig purification from the egg implicated in carcinogenesis, and CUB and SUSHI yolks, were performed by Abcam (Cambridge, UK). domains are also found in many of these, including bone morphogenetic proteins, tolloid, neuropilin and spermad- Anti-CSMD1 antibody validation hesins [26–29]. The genomic localisation, expression pattern and protein Initially, the binding specificity of the anti-CSMD1 antibody homology of CSMD1 suggest that it is a tumour suppressor was tested by an enzyme-linked immunosorbent assay gene. In this study, we investigate its role in breast cancer (ELISA) against the BSA-conjugated peptides stated earlier progression. We have generated an anti-CSMD1 antibody [30]. Antibody specificity was also confirmed on histological and identified the expression pattern of CSMD1 in 275 IDC tissue sections by pre-absorption with the peptides. The patients with a follow-up period of 152 months. Univariate antibody was diluted to its working concentration and and multivariate Cox regression analyses have been per- incubated with a tenfold excess of relevant peptide overnight formed to correlate CSMD1 expression with OS and DFS. at 4°C before application to the tissue. Loss of CSMD1 expression was associated with high For further validation of the specificity and sensitivity of tumour grade and poor survival outcome.