MAL2 and tumor protein D52 (TPD52) are frequently overexpressed in ovarian carcinoma, but differentially associated with histological subtype and patient outcome

Epithelial ovarian carcinoma is a disease often characterised by poor outcome, despite intensive efforts to improve early disease detection, and to understand the causes of frequent treatment failure [1, 2]. To improve our understanding of the underlying molecular basis of this histologically heterogeneous group of tumours, large numbers of cytogenetic and comparative expression studies have been undertaken. Cytogenetic analyses have consistently identified chromosome 8q gain as a common event in ovarian carcinoma [summarised in 3], and in other cancer types [4, 5], and recent studies continue to highlight the fact that several distinct regions along chromosome 8q are increased in copy number [6–8]. One such region occurs at chromosome 8q24.12, and includes the gene encoding the four-transmembrane protein MAL2 [9], which is increased in copy number and/or overexpressed in breast and other cancers [10–18]. MAL2 is a 176 amino acid protein that contains a MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain commonly identified in proteins associated with membrane apposition events [19], and is an essential component of the basolateral-to-apical transcytotic machinery [20]. Increased MAL2 expression in ovarian cancer has been repeatedly identified by independent expression microarray studies [21–24], with two meta-analyses highlighting the same finding [22, 25]. Increased MAL2 expression has been validated in other cancer types using RT-PCR [26, 27], and demonstrated at the protein level in renal cell [18, 28] and breast carcinomas [12]. However, no study to date has examined whether MAL2 expression is increased in ovarian carcinoma, or its potential clinical significance.

MAL2 is known to bind the product of another gene on chromosome 8q, tumor protein D52 (TPD52) [9, 29], which is a member of the similarly-named gene and protein family [30]. The TPD52 gene maps to chromosome 8q21.13, and demonstrates copy number increases and overexpression in a variety of cancers [reviewed in 31]. The TPD52 protein is 184 amino acids in length and contains a coiled-coil domain, but does not show significant levels of sequence identity to proteins beyond the TPD52-like family [30]. Its expression in normal secretory epithelia has been implicated in regulating exocytotic secretion [32], whereas exogenous TPD52 expression in cultured cell lines results in increased proliferation and anchorage-independent growth [12, 33, 34], and in vivo metastasis in immunocompetent hosts [34]. In ovarian cancer, TPD52 overexpression has been identified in all histological subtypes of ovarian carcinoma relative to normal ovarian epithelium, with a significant positive correlation between TPD52 expression and gene copy number being found in an independent serous carcinoma cohort [3]. Other studies have similarly reported increased TPD52 expression in ovarian cancer using expression microarray [23, 24, 35, 36] and proteomic approaches [37]. While high TPD52 expression in breast cancer has been reported to be an adverse prognostic factor [12], the clinical significance of increased TPD52 expression in ovarian cancer has not been directly investigated.

The aim of the present study was therefore to define MAL2 and TPD52 expression in a large cohort of ovarian carcinomas, relative to other clinical parameters. Immunohistochemical staining using previously described polyclonal antisera [3, 12, 29] was assessed both visually and digitally, as previously described in breast carcinoma [12].

The present study has confirmed MAL2 overexpression in ovarian cancer, as predicted by previous expression microarray analyses [21–25], and has shown this to be a frequent event. MAL2 was significantly overexpressed in high-grade serous carcinomas compared with borderline tumours, and in high-grade serous carcinomas relative to the remaining histological subtypes combined. High-level MAL2 expression was also noted in mucinous, clear cell and endometrioid ovarian carcinomas, albeit less frequently than in serous carcinomas. The frequent overexpression of MAL2 in ovarian carcinoma, coupled with its low expression in benign and borderline lesions, may indicate a potential role for MAL2 in disease detection and/or monitoring, particularly in high-grade serous carcinoma. This is consistent with the previous inclusion of MAL2 in marker panels to discriminate pancreatic cancer from pancreatitis [26] and metastatic from non-affected lymph nodes in colorectal cancer patients [27].

Frequent reports of MAL2 overexpression in numerous cancer types, in association with increases in gene copy number, argue against MAL2 overexpression being an epiphenomenon. MAL2 has been recently shown to bind the MUC1 oncoprotein [29], and MAL2 transcripts are predicted to be co-expressed with EpCAM [38]. However, it is not yet clear how MAL2 overexpression promotes tumourigenesis or progression. Increased Myc-tagged MAL2 expression in MCF-10A breast epithelial cells produced reduced proliferation rates and an elongated cell phenotype [29]. Furthermore, other studies examining gene expression consequences of oncogene overexpression have reported reduced MAL2 expression in these models [39, 40]. MAL2 overexpression may therefore contribute to tumourigenesis through mechanisms that do not emerge from the study of homogenous in vitro systems. These could include modulating cell-cell interactions or signalling between cancer and/or other cell types. It was interesting to note that cytoplasmic expression of MAL2 was rare in benign and borderline tumours, but common in ovarian carcinomas. MAL2 also showed a broad cytoplasmic distribution in renal cell carcinomas [18, 28] and in breast carcinomas and cell lines [12, 29]. This might indicate that MAL2 plays an oncogenic function in some cell types when distributed throughout the cytoplasm.

Like MAL2, TPD52 was overexpressed in high-grade serous carcinomas relative to borderline tumours, but was more equivalently expressed in high-grade serous tumours relative to the combined cohort of other histological subtypes. This agrees with our previous finding that TPD52 was broadly overexpressed in an independent ovarian carcinoma cohort [3]. The present study identified high TPD52 staining as particularly characteristic of clear cell carcinomas, as this was detected in all cases examined. Frequent high-level TPD52 expression in clear cell carcinoma is consistent with the results of array-based comparative genomic hybridisation analyses of ovarian clear cell carcinoma cell lines, where increased TPD52 copy number was noted in 6/12 cell lines examined [8].

Despite MAL2 overexpression being more frequent in high-grade serous carcinoma, MAL2 expression was not significantly associated with overall patient survival in this study. In contrast, increased TPD52 staining was noted to be a favourable prognostic marker in ovarian carcinoma. While this finding derives from the analysis of immunohistochemical staining as a single technique, a recent expression microarray study also identified TPD52 overexpression as being associated with improved progression-free and overall survival in patients with serous and endometrioid tumours [24]. It may also be relevant that chromosome 8q21 gain has been previously associated with improved survival in clear cell carcinoma patients [41]. However, the finding that TPD52 is a favourable prognostic indicator in ovarian carcinoma patients contrasts with findings obtained in breast cancer [12]. Here, high TPD52 expression was an unfavourable indicator, both within the cohort overall, and patient subgroups already characterised by poorer survival [12]. This is also consistent with reports of TPD52 being included in gene signatures associated with unfavourable prognosis [42, 43], and with the clinical significance of chromosome 8q21 gain in breast cancer [44–47]. Thus, whereas increased TPD52 expression appears to be a common event in both breast and ovarian carcinoma, the contrasting clinical significance of both TPD52 overexpression and chromosome 8q21 gain suggests different roles for TPD52 in these cancer types. There is evidence that TPD52 may promote invasion through solid tissues, as suggested by the finding that Tpd52-expressing 3T3 cells injected into the mammary fat pad subsequently formed lung metastases in immunocompetent hosts [34]. In contrast, the pattern of metastatic spread is very different in ovarian carcinoma, as there is no anatomical barrier to widespread tumour dissemination and spread within the peritoneal cavity [1]. Such differences in tumour dissemination patterns could partially explain the opposing prognostic significance of increased TPD52 expression or copy number reported in breast [12, 42–44] and ovarian cancer [24]. Alternatively, a comparative lack of TPD52 expression could segregate with adverse prognostic markers in ovarian carcinoma, and thus the association between increased TPD52 expression and improved overall survival may be indirect.

The present study has highlighted frequent MAL2 overexpression in ovarian carcinoma, particularly in serous tumours. The frequent overexpression of MAL2 in ovarian carcinomas, coupled with its low expression in benign and borderline lesions, suggest that MAL2 may be a useful marker component to assist in disease detection and/or monitoring. High TPD52 staining was associated with significantly improved overall patient survival in ovarian carcinoma. The differential association of high TPD52 staining and overall patient survival in breast versus ovarian carcinoma may indicate different roles for TPD52 overexpression in ovarian versus breast tumour progression.