Carcinogenesis, Vol. 23, No. 11, 1791-1796,
November 2002
© 2002 Oxford University Press
CANCER BIOLOGY |
Changes in expression of the human homologue of the Drosophila discs large tumour suppressor protein in high-grade premalignant cervical neoplasias
1 Cancer Research UK Institute for Cancer Studies, The Medical School, University of Birmingham, Birmingham B15 2TT,
2 Department of Pathology, Birmingham Women’s Hospital, Birmingham,
3 Department of Pathology, The Medical School, University of Birmingham, Birmingham B15 2TT, UK,
4 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy
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Abstract |
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The Drosophila tumour suppressor discs large (Dlg) is a cell-junction localized protein that is required for the maintenance of epithelial cyto-architecture and the negative control of cell proliferation. The mammalian homologue is likely to have a similar mode of action, and therefore functional perturbation of this protein may be linked to the development of epithelial-derived cancers. The finding that several unrelated viral oncoproteins, including the E6 protein of oncogenic human papillomaviruses, bind to the human homologue of Dlg (hDlg) supports this proposition. Employing immunohistochemistry, we show that in uterine cervical squamous epithelia, prominent localization of hDlg at sites of intercellular contact occurs in cells that have left the proliferating basal cell layers and begun maturation. The presence of hDlg at sites of cell:cell contact diminishes, whilst intracellular cytoplasmic levels increase significantly in high-grade, but not low-grade, cervical neoplasias. In invasive squamous cell carcinomas, total cellular hDlg levels are greatly reduced. Our data suggest that loss of hDlg at sites of intercellular contact may be an important step in the development of epithelial cancers.
Abbreviations: APC, adenomatous polyposis coli; CIN, cervical intraepithelial neoplasia; Dlg, Drosophila tumour suppressor discs large; hDlg, human homologue of Dlg; HPV, human papilloma virus; MAGUK, membrane-associated guanylate kinase; SIL, squamous intraepithelial lesions
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Introduction |
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Recent studies in Drosophila have shown that significant cyto-architectural changes of epithelial tissue, such as loss of cell polarity, aberrant cell adhesion and changes in cell shape, occur when the structure of the discs large (Dlg) gene is perturbed (1,2). These changes are accompanied by increased epithelial cell proliferation and neoplastic growth, indicating that Dlg functions in the maintenance of epithelial cell structure and as a negative regulator of cell growth; thus it is nominated as a tumour suppressor (2,3). Dlg is the founding member of the membrane-associated guanylate kinase (MAGUK) superfamily whose members are defined by a basic core of three different protein-interaction domains: a PDZ domain, an SH3 domain and a domain with homology to the enzyme guanylate kinase. MAGUKs are thought to act as scaffold proteins linking components of signalling pathways and structural proteins at specific domains at peripheral membranes of cells (4). In Drosophila imaginal disc epithelial cells, Dlg localizes to the cytoplasmic face of septate junctions (1) (equivalent to vertebrate tight junctions) whilst in Caenorhabditis elegans it resides at adherens junctions (5,6). Less is known about the human homologue of Dlg (hDlg), although it also localizes at intercellular contact sites in epithelial cells and has been shown to bind to members of the protein 4.1/ezrin, radixin, moesin (ERM) family; interactions that may be important for targeting of hDlg to the cell periphery (7–10). HDlg binds to several cellular factors that have roles in cell growth and proliferation (11,12). For example, it interacts with the tumour suppressor adenomatous polyposis coli (APC) protein and formation of an APC:hDlg complex is important in inducing a cell cycle suppression signal (11,13). It would seem likely therefore, that the human homologue of Dlg has similar functions as its Drosophila counterpart, regulating the structure and growth of epithelial tissue in response to cell:cell contact. Interestingly, hDlg has recently been described as a target of several viral oncoproteins; the E6 protein of high-risk types of human papillomaviruses (HPVs), E4-ORF1 of adenovirus 9 and Tax of human T-cell leukaemia virus (14–16). E6 has also been shown to target hDlg for ubiquitin-mediated degradation via the proteasome (17). The physiological effects of binding hDlg by these viral oncoproteins is not clear, however it has been demonstrated that Tax is able to suppress the growth inhibitory properties of hDlg and that E6 mutants unable to bind hDlg also lose their transforming activity in rodent cells (15,16). Therefore, given that binding to hDlg is a function conserved between unrelated viral oncoproteins, and the potential role of hDlg as a tumour suppressor, it has been proposed that inactivation of this MAGUK may contribute to the development of virus-associated cancers (14,15).
Therefore, in this study we have examined hDlg expression in the normal ectocervical and endocervical epithelium of the uterine cervix – a common site of infection of carcinogenic HPV types. Development of invasive cervical cancer is preceded by cervical intraepithelial neoplasia [CIN, also termed squamous intraepithelial lesions (SIL), low and high-grade] that is graded by reference to the level of epithelial involvement by dedifferentiated cells, together with several additional morphological features. Therefore, to determine whether hDlg expression is altered during the transformation of normal cervical epithelium to neoplastic epithelium, immunohistochemistry was used to examine the distribution of hDlg in archival paraffin-embedded tissues of normal cervix, low and high-grade CINs and invasive squamous carcinoma.
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Tissue samples
Archival formalin-fixed, paraffin-embedded cervical tissue sections were obtained from Good Hope Hospital, Sutton Coldfield, Birmingham, UK. A total of 52 samples were analyzed. Six samples had no squamous epithelium and were removed from the study. The remaining 46 samples ranged from histologically normal through all grades of CIN. A majority of biopsies also contained endocervical epithelium and, a few cases, squamous mature metaplasia and immature metaplasia, as well as regions showing morphological evidence of HPV infection in non-CIN areas. The numbers for each lesion type were normal cervical epithelium, n = 14; CIN1, n = 11 CIN2, n = 10; CIN3, n = 11. Because some of the high-grade lesions also contained regions of a lower grade the numbers for each lesion type were therefore CIN1 =13, CIN2 = 15 and CIN3 = 11. In addition, four cases of invasive cervical cancer were studied, although only two of these had adjacent haematoxylin/eosin (H&E)-stained sections, kindly provided by Dr G.Teal, Good Hope Hospital, Birmingham, UK. For all cases, both the H&E-stained and hDlg-stained sections were re-reviewed by a single pathologist (T.P.R.). Sections were photographed using a Leica DC200 camera or an Olympus digital camera (invasive carcinomas) and images were assembled in Adobe Photoshop, San Jose, CA.
Antibodies, cell culture, western blotting and immunofluorescence staining
Monoclonal antibodies (mAbs) against hDlg (cl. 2D11) and E-cadherin (cl. 36) were purchased from Santa Cruz Biotechnology, California, CA and Transduction Laboratories Lexington, KY respectively. Epithelial lines examined were Madine-Darby canine kidney (MDCK), an SV40-immortalized human skin keratinocyte line, and two spontaneously immortalized keratinocyte lines, HaCaT and SCC-12F. All except HaCaTs, were grown in DME medium supplemented with 10% foetal calf serum, 4 mM glutamine and antibiotics. HaCaT cells were grown in the media described with 0.4 µg/ml hydrocortisone added. Cells were lysed in 8 M urea, 50 mM Tris pH 8.0, 0.15 M ß-mercaptoethanol and equal amounts of protein electrophoresed on 10% SDS-polyacrylamide gels, western-blotted using 2D11 (1 : 400 dilution) and developed using ECL (Amersham International). Immunofluorescence microscopy was performed on confluent cultures of cells as described previously (18). 2D11 was used at 1 in 30 and immune-complexes were detected using goat anti-mouse IgG-Alexa 488 antibody (Molecular Probes, Eugene, OR).
Immunohistochemistry
Sections (4 µm) were cut, mounted on Vectabond (Vector Laboratories, Peterborough, UK)-treated Superfrost Plus slides and air-dried overnight at 37°C. One section of each case was stained with H&E. Sections to be stained with antibodies were de-paraffinized and re-hydrated by first immersing in xylene, followed by absolute ethanol and then water. To block endogenous peroxidase activity, sections were immersed in 3% hydrogen peroxide in methanol for 10 min. Antigen retrieval was performed using a modified low-temperature method (19). Briefly, sections were incubated in EDTA buffer (1 mM EDTA, pH 8.0, 0.01% Tween 20) at 65°C for 16 h with stirring (600 r.p.m.). When cool, sections were incubated in 0.2 M Tris-buffered saline (TBS), pH 7.6 for 30 min at 20°C. Primary antibody (2D11 diluted 1 in 25; E-cadherin mAb diluted 1 in 200) was applied for 60 min at room temperature and subsequently slides were washed in TBS containing 0.1% Tween 20. Negative controls were sections in which the primary antibody had been omitted. Primary antibody was localized by using a streptavidin–biotin–peroxidase kit (Universal ABC kit, The Binding Site, Birmingham, UK) according to the manufacturer’s instructions. The chromogenic reaction was performed using NovaRED substrate (Vector Laboratories, Peterborough, UK) for 10 min at 20°C. After counterstaining in Mayer’s haematoxylin, sections were dehydrated by immersion in absolute ethanol followed by xylene and then mounted in DPX. There was no evidence that endogenous biotin interfered with the staining patterns of hDlg or E-cadherin as identical staining patterns were obtained in sections in which endogenous biotin was blocked prior to staining.
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Testing the specificity of hDlg monoclonal antibody 2D11
The commercially available hDlg mAb 2D11 was raised against the N-terminus (amino acids 1–229) of hDlg, a region that is unique to hDlg between MAGUK family members. To confirm that 2D11 was specific for hDlg, various epithelial cell lines were examined by western blotting and immunofluorescence microscopy. On western blots, 2D11 identified between two and three bands around 120 kDa and no other bands were detected (Figure 1
, top panel). A similar molecular mass and multiple band pattern has been described for hDlg (predicted molecular mass is 103 kDa) and its rat homologue, synapse-associated protein 97 (SAP97) (7,10,20). In confluent cultures of the different epithelial cells stained with 2D11, hDlg was immunolabelled at the cell periphery at sites of cell:cell contact (Figure 1, bottom panel and data not shown). A similar localization of hDlg in epithelial cells has been previously reported (7,10). Also, 2D11 labelled small clusters of cytoplasmic granules near the cell boundary. These structures may represent vesicular structures in which hDlg is thought to reside prior to recruitment to the cell periphery (10). We conclude that 2D11 specifically recognizes hDlg.
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hDlg protein expression in normal cervix
The distribution and cellular localization of hDlg was examined in the different types of epithelium found in the uterine cervix. Twelve cases (12/14) contained both normal endocervical (glandular) and ectocervical epithelia, and hDlg staining was most often stronger in cells of the endocervix than in those of ectocervical epithelium. Furthermore, it was noticeable that intensity of hDlg staining in the ectocervix became significantly weaker at sites farthest from the transformation zone (data not shown). In all cases of normal ectocervical squamous epithelia, expression of hDlg was limited to the basal, parabasal and intermediate suprabasal layers, with the superficial layers being largely negative (Figure 2A
). This distribution pattern was similar to the adherens junction protein E-cadherin (Figure 2C). A diffuse cytoplasmic hDlg distribution was observed in basal cells and the immediate two to three layers of parabasal cells (Figure 2B). However, variations in basal hDlg staining occurred, with some samples showing a more intense cytoplasmic labelling than those of the parabasal compartment (data not shown). In addition, weak staining at cell:cell borders and cell junctions was observed in both compartments. In the intermediate suprabasal layers, hDlg levels increased at the cell periphery, particularly at structures resembling desmosomes, and decreased significantly in the cytoplasm (Figure 2B). In some cells of basal and suprabasal compartments, hDlg was detectable at the nuclear periphery (Figure 2B and inset). In columnar cells of the endocervix, hDlg was present at the baso-lateral cell borders and in many of these cells cytoplasmic staining was also detected (Figure 2D). Sub-columnar reserve cells showed intense cytoplasmic staining (data not shown). Significant cytoplasmic hDlg was evident in cells of immature squamous metaplasia although some cells also showed hDlg at cell borders (Figure 2E). The staining pattern in mature squamous metaplasia was similar to that in ectocervical tissue (data not shown). No staining was seen in controls.
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hDlg in CIN and invasive carcinomas
No substantial differences were found with respect to hDlg expression or localization in epithelia showing morphological evidence of HPV (wart) infection (data not shown). Similarly, the pattern of hDlg localization and level of protein expression in low-grade dysplasias (12/13, CIN1) was not significantly different from that found in normal tissue (Figure 3A
). However, one case (1/13) showed strong hDlg staining at the periphery of cells in the mid-epithelial layers, but none (or very little) in cells of lower layers (Figure 3B).
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In contrast, hDlg expression was strikingly different in high-grade (CIN2 and CIN3) dysplastic lesions (Figure 3C–F
). The typical pattern of hDlg distribution (22/26 cases) was a strong diffuse cytoplasmic staining that extended through almost the entire thickness of epithelium (Figure 3C–E). The presence of hDlg at cell:cell boundaries was evident in some cases, but the intensity varied and was restricted either to the uppermost layers of the epithelium or to small foci of cells (Figure 3C and E). In more differentiated lesions, no hDlg staining was evident at the cell boundaries or in the cytoplasm in the most superficial cell layers (Figure 3D). As was noted in normal epithelium, there was some variation in the level of basal staining; in six out of the 22 typical cases basal hDlg levels were higher then elsewhere in the epithelium (data not shown). Although in general cytoplasmic hDlg levels were high, small areas or patches of weak staining were evident in a minority of lesions (data not shown). In the four cases showing atypical hDlg staining, three (two CIN2 and one CIN3) had relatively weak levels of hDlg in both the cytoplasm and at cell boundaries compared with areas of normal squamous and glandular epithelium, and to the levels in separate cases of high-grade CIN (Figure 3F). The other case, graded CIN3, had weak staining of hDlg both within the lesion and in normal epithelia (data not shown).
Four invasive squamous carcinomas were stained with 2D11 (Figure 3G–I). Two cases also contained high-grade (CIN3) dysplasia and in these regions hDlg protein expression was high (Figure 3G), in agreement with hDlg expression in high-grade CIN samples. In the invasive tumour islands, however, hDlg protein levels were very weak (Figure 3H) and, significantly, staining was absent at the leading edge of the invading tumours (Figure 3I).
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Discussion |
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The human homologue of Drosophila tumour suppressor Dlg is expressed in all the different types of epithelia present in the uterine cervix (Figure 2
). In glandular epithelium, it localizes primarily to the basal and lateral membranes of columnar cells, similar to hDlg distribution in cells of human colonic tissue (20). Peripheral hDlg staining is also prominent in suprabasal cell layers of ectocervical epithelium. These findings are consistent with a role for hDlg at sites of intercellular contacts. However, hDlg is also located in the cytoplasm and nucleus. Cytoplasmic hDlg staining was most pronounced in the proliferating compartments of the ectocervix and immature squamous metaplasia that have either no, or little, peripheral hDlg. This sub-cellular distribution of hDlg is reminiscent of that of SAP97 (the rat homologue of hDlg) in epithelial cells on intestinal villi (21). Cells of the proliferating compartment of the intestinal crypts have lower levels of peripheral SAP97 compared with the intense labelling of the lateral membranes of more mature cells in the distal segments of the villi (21). Localization of hDlg to the periphery may therefore be important for the negative regulation of proliferation. Nuclear hDlg was present in basal and suprabasal compartments of the ectocervix. In cultured epithelial cells, a nuclear form of hDlg is detected by an antibody that recognizes alternatively spliced forms of hDlg containing the I2 insertion, suggesting that nuclear localization may be regulated by post-transcriptional modification of hDlg transcripts (22). It remains to be seen however, whether the different sub-cellular localizations of hDlg reflect alternative functions of the protein.
In invertebrates, Dlg is required for the maintenance of epithelial cell:cell junctions (1,2,5,6,23), and in Drosophila this is important for the organization of epithelial tissues and the prevention of their neoplastic overgrowth (1,2,23). The changes in sub-cellular expression of hDlg as cells move up through the stratifying layers of uterine ectocervical epithelium could therefore indicate that hDlg may have a role in the proper growth and formation of differentiated epithelium. This role could be dependent on hDlg’s localization to the cell periphery, where it may participate in aspects of assembly and/or function of intercellular junctions. It is therefore of interest that the normal pattern of hDlg expression is significantly disturbed in high-grade dysplasias of cervical epithelium (see Figure 3). In these lesions, there was a loss of hDlg localization at the cell periphery concomitant with an increase in cytoplasmic staining. Also, in contrast to normal epithelia and low-grade lesions, cytoplasmic hDlg was expressed in almost all layers of the epithelium. This is consistent with the replacement of differentiated layers with more ‘basal-like’ proliferating cells. Furthermore, in invasive tumours total cellular hDlg levels were low and even absent in some regions (Figure 3). Thus, loss of hDlg from the cell periphery may negate its function as an inhibitor of neoplastic transformation. This is supported by recent work in cervical tumour cell lines that shows that cells that exhibit a more fully transformed phenotype have lower levels of hDlg protein at sites of cell:cell contact compared to less transformed cells (24; R.W. and S.R., unpublished observations). Our results therefore suggest that loss of hDlg at sites of intercellular contact may be a crucial step in the progression of cervical neoplasia.
HPV is a recognized human carcinogen and it is well established that HPV is present in nearly all cases of CIN and carcinomas of the cervix (25). The HPV E6 oncogene may primarily contribute to the late stages of progression to malignancy (26). Although inactivation of p53 is important in E6’s biological function, p53-independent activities may also be necessary. For instance, E6-induction of epithelia hyperplasia in transgenic mice is p53-independent (27). It is tempting to speculate that a loss of a functional hDlg through E6-mediated degradation may contribute to such changes in epithelial growth and that the changes in hDlg expression observed in high-grade dysplasias and cancers may be mediated by the activity of an oncogenic E6 molecule. However, it has been demonstrated that in cervical tumour cells a reduction in hDlg protein levels can occur in the absence of HPV genomes suggesting that regulation of hDlg expression in cervical tissue could be controlled through HPV-independent cellular mechanisms (24). For example, in HPV-negative C33I cervical tumour cells, the loss of hDlg expression is largely at the level of transcription, and therefore this may be one of the means by which hDlg is lost in the absence of E6 (24).
Studies of the invertebrate MAGUK proteins have shown that they act in pathways important in maintaining growth control and epithelial structure. It is proposed that the modes of action of these proteins are likely to be conserved between invertebrates and vertebrates. Because the loss of cell growth control and cyto-architecture disruption are hallmarks of oncogenic transformation, then perturbation of the function of mammalian MAGUKs may contribute to tumourigenesis. Interestingly, cellular levels of the MAGUK protein ZO-1, a component of tight junctions, are significantly reduced in breast cancers (28). We have shown that hDlg localization and expression is severely disrupted during the gradual transformation of cervical epithelia, indicating that like its Drosophila counterpart, hDlg may function in cellular pathways that inhibit cell proliferation and maintain tissue structure.
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5 To whom correspondence should be addressed Email: s.roberts{at}bham.ac.uk
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We thank Steven Hay for help in the collection of tissue samples and Jo Flavell for sectioning the paraffin blocks. RAW was supported by a Ph.D. studentship provided by the E.B. Jones Bequest, University of Birmingham’s Medical School. L.B. is supported by the Associazione Italiana per la Ricerca sul Cancro. This work was supported by Cancer Research UK.
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