Karin Sundfeldt

E-cadherin expression in human epithelial ovarian cancer and normal ovary

The ovarian surface epithelium (OSE) is the origin of the majority of human ovarian cancers. These adenocarcinomas are characterized by initial local growth followed by spreading into the peritoneal cavity at later stages of tumor progression. The cell‐adhesion molecule E‐cadherin (E‐cad) plays an important role in maintaining tissue integrity. Disappearance or impaired function of E‐cad have often been associated with tumor formation and invasion in vivo and in vitro. The cell‐specific expression of E‐cad was investigated in normal human ovaries (n = 12), in benign (n = 5) and borderline (n = 4) ovarian epithelial tumors and in adenocarcinomas of different stages and histological grades (n = 18), by immunohistochemistry and immunoblotting. An ovarian cancer cell line (NIH‐OVCAR3) was used as a reference. The epithelial origin of the cells was confirmed with cytokeratin (AE1/AE3) staining. In normal ovaries, the expression of E‐cad was limited to inclusion cysts or deep clefts lined with OSE, whereas no staining of the OSE could be demonstrated at the surface of the ovary. In contrast, benign and borderline tumors uniformly expressed E‐cad. This was observed in malignant tumors of all stages despite their degree of differentiation. E‐cad was also present in metastasis from such tumors. The cell‐specific expression of E‐cad in inclusion cysts of normal ovaries and in epithelial layers of borderline tumors indicates a role for E‐cad in the early events of the progression to a malignant phenotype. E‐cad was not downregulated in later stages of ovarian cancer progression. Int. J. Cancer 74:275‐280, 1997.

Wnt-signalling pathway in ovarian epithelial tumours: increased expression of β-catenin and GSK3β

Beta-catenin is involved in both cell–cell adhesion and in transcriptional regulation by the Wingless/Wnt signalling pathway. Alterations of components of this pathway have been suggested to play a central role in tumorigenesis. The present study investigated, by immunohistochemistry and immunoblotting, the protein expression and localisation of β-catenin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and lymphocyte enhancer factor-1 (Lef-1) in normal human ovaries and in epithelial ovarian tumours in vivo and in vitro. Immortalised human ovarian surface epithelium and ovarian cancer cell cells (OVCAR-3) expressed β-catenin, APC, GSK3β and Lef-1. Nuclear staining of β-catenin and Lef-1 were demonstrated only in OVCAR-3 cells. There were significant increases of β-catenin and GSK3β, while APC was reduced in ovarian cancer compared to the normal ovary. Beta-catenin and Lef-1 were coimmunoprecipitated in ovarian tumours, but not in the normal ovary. Nuclear localisation of β-catenin or Lef-1 could not be demonstrated in the normal ovary or in the ovarian tumours. The absence of nuclear localisation of β-catenin could be due to an increased binding to the cadherin–α-catenin cell adhesion complex. In fact, we have earlier reported an increased expression of E-cadherin in ovarian adenocarcinomas. In summary, this study demonstrates an increase in the expression of components of the Wingless/Wnt pathway in malignant ovarian tumours. The increase suggests a role for this signalling pathway in cell transformation and in tumour progression. However, it remains to be demonstrated whether it is an increased participation of β-catenin in transcriptional regulation, or in the stabilisation of cellular integrity, or both, that is the crucial event in ovarian tumorigenesis.

Cell-cell adhesion in the normal ovary and ovarian tumors of epithelial origin; an exception to the rule

Cells are held together either by direct cell-cell contact or adhesion to extra-cellular matrix. Cell-cell adhesion in epithelial cell sheets consists of junctions, i.e. tight-, adherens- and gap-junctions. The adherens junctions, which are build up by the cadherin/catenin complex, are the main topic of this review, especially the aspect of its role in ovarian tumor biology. The ovarian surface epithelium is the origin for approximately 90% of the malignant ovarian tumors. The tumors arise from the inclusion cysts, localized in the ovarian stroma and grow solid, cystic or in mixed formations. Intra-abdominal spread of the ovarian cancer is common and this is a process that theoretically could be closely connected with impaired cell-cell adhesion. However, as we stand today, descriptive and functional studies on the cadherin-catenin complex and its cell signaling role in ovarian tumorigenesis reveals data that suggests a conversion of the mesothelial-like cells of the ovarian surface to a more epithelial phenotype with normal cell-cell adhesion prior to tumor differentiation. In later stages, invasive ovarian tumors still strongly express several cadherins, which are contrary to many other tumors, i.e. prostate and thyroid adenocarcinomas.

Increased expression of the transcription factors CCAAT-enhancer binding protein-β (C/EBβ) and C/EBPζ (CHOP) correlate with invasiveness of human colorectal cancer

Regulation of cell differentiation is most often impaired in malignant tumors and may represent a key mechanism for the progression of the disease. CCAAT‐enhancer binding protein (C/EBP) is a family of transcription factors involved in the regulation of embryonic gut development in rodents, which has also been detected in various malignancies, e.g., liposarcomas and breast and ovarian epithelial tumors. We studied the relationship between C/EBP and tumor histology (Dukes invasive stage and pathological grade) in colorectal cancer. Immunoblotting techniques were used on microdissected fresh frozen tumor specimens, and expression of C/EBPα, C/EBPβ and C/EBPζ (CHOP) was analyzed in addition to that of the cell‐cycle regulator p53 and the proliferation marker PCNA. Expression of C/EBPβ (LAP isoforms) was markedly increased in all tumors compared with normal colon mucosa. Although the inter‐patient variability was large, we found that LIP, the isoform of C/EBPβ known to inhibit transcription, was expressed at higher levels in Dukes stage B tumors compared with Dukes stage A, whereas Dukes C tumors had the lowest LIP expression. A similar relationship was seen for CHOP. The cell‐cycle regulator gene p53 was the only factor that clearly correlated with pathological grade: a decrease in p53 expression was demonstrated. Our data suggest that genetic and cellular events involving C/EBPβ and CHOP are important for tumor invasion and that these events do not appear to be related to the pathological grade of the tumor. Int. J. Cancer 86:337–343, 2000.

The expression of CCAAT/enhancer binding protein (C/EBP) in the human ovary in vivo: specific increase in C/EBPβ during epithelial tumour progression

SummaryThe CCAAT/enhancer binding protein (C/EBP) family of transcription factors is involved in metabolism and differentiation of cells, especially in rodent liver cells and adipocytes. Their roles in vivo and in particular during pathophysiological conditions in humans are largely unknown. We have investigated the presence of C/EBPα, -β, -δ and -ζ in normal ovaries and in epithelial ovarian tumours of different stages. Immunohistochemical experiments demonstrated that C/EBPα and C/EBPβ were preferentially expressed in epithelial/tumour cells irrespective of stage or grade of the tumour. C/EBPβ was located in the nuclei of the cells, in contrast to C/EBPα, which was present only in the cytoplasm of these cells. The nuclear localization of C/EBPβ indicates an active role of this transcription factor in tumour cells, whereas the cytoplasmic distribution suggests a more passive function of C/EBPα. C/EBPδ and -ζ demonstrated a more diverse distribution with predominant localization to epithelial cells, but stromal distribution was also noted. The intracellular distribution was confined to both the nucleus and the cytoplasm for C/EBPδ and -ζ. Western blotting demonstrated that C/EBPα, -β, -δ and -ζ were present in a majority of the samples. The amount of C/EBPβ increased markedly with malignancy, i.e. with degree of dedifferentiation, while the other members of the C/EBP family displayed a more constant expression level. These results demonstrate an association between the expression of members of the C/EBP family and the formation of epithelial ovarian tumours, with C/EBPβ as a potential marker for these tumours. As C/EBPβ is known to be expressed during proliferation of cells in vitro, it may participate in the proliferative process of ovarian epithelial tumour cells in vivo and play a central role in tumour progression.

Ovarian epithelial cancer: a role for PGE2-synthesis and signalling in malignant transformation and progression.

BackgroundThe involvement of the cyclooxygenases (COX), in particular COX-2, is well documented for many tumours, e.g. colon, breast and prostate cancer, by both experimental and clinical studies. There are epidemiological data from subjects using NSAIDs, and experimental evidence supporting the hypothesis of prostaglandins (PGs) as regulators of tumourigenesis in the ovary. One of the end products of PG-synthesis, PGE2, regulates several key-processes, which are characteristic for tumour growth, e.g. angiogenesis, proliferation and apoptosisis. The present study investigated the pathway for PGE2 – synthesis and signalling in ovarian tumourigenesis by analysing specimen from normal ovaries (n = 18), benign (B) (n = 8), borderline type (BL) (n = 6) and malignant tumours (AC) (n = 22). The expression and cell-specific localization of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1) and two of the receptors for PGE2, EP1 and EP2, were examined by immunoblotting (IB) and immunohistochemistry (IHC).ResultsThe results are in line with earlier studies demonstrating an increase of COX-2 in AC compared to the normal ovary, B and BL tumours. Increased expressions were also observed for COX-1, mPGES-1 and EP-1 which all were significantly (p < 0.05) augmented in less differentiated AC (grades: moderately-, poorly- and undifferentiated). The increase of COX-2 was also correlated to stage (FIGO classification) with significant elevations in stages II and III. EP1 was increased in stage III while no significant alterations were demonstrated for COX-1, mPGES-1 or EP2 for stage. IHC revealed staining of the tumour cells, but also increase of COX-1, COX-2, mPGES-1 and EP1–2 in the stromal compartment of AC (grades: moderately-, poorly- and undifferentiated). This observation suggests interactions between tumour cells and stromal cells (fibroblasts, immune cells), e.g. paracrine signalling mediated by growth factors, cytokines and possibly PGs.ConclusionThe increases of COX-1, COX-2, mPGES-1 and EP1–2 in epithelial ovarian cancer, supports the hypothesis that PGE2-synthesis and signalling are of importance for malignant transformation and progression. The observed augmentations of COX-1, COX-2 and mPGES-1 have implications for future therapeutic strategies.

Differences in expression patterns of the tight junction proteins,claudin 1, 3, 4 and 5, in human ovarian surface epithelium as compared to epithelia in inclusion cysts and epithelial ovarian tumours

Human ovarian surface epithelium (OSE), regarded as the precursor cell of epithelial ovarian adenocarcinoma, is not a fully developed epithelium when situated on the ovarian surface. It lacks epithelial characteristics such as the cell–cell adhesion factor epithelial (E)‐cadherin, but as we have shown earlier, this OSE can form functional tight junctions (TJs) in culture. Recent gene‐expression data on ovarian adenocarcinoma has pointed out a family of TJ proteins, the claudins, to be highly expressed in malignant compared to benign ovarian tumours. The purpose of this study was to investigate the distribution of claudin 1–5 proteins in cultured OSE (n = 4), normal ovarian (n = 11), benign (n = 8), borderline (n = 7) and ovarian cancer (n = 22) tissues. We found that a weak or absence of expression of claudin 3 and claudin 4 in surface OSE changed to typical cell‐border localisation in OSE of inclusion cysts in the normal ovarian stroma. Semiquantitative estimations of immunoblots showed that claudin 3 was significantly increased in ovarian adenocarcinomas compared to benign and borderline‐type tumours. Claudin 4 was significantly increased in both borderline‐type and ovarian adenocarcinomas compared to benign tumours, whereas no changes were found for claudin 1 or 5. Concerning relation to Federation for International Gynaecology and Obstetrics (FIGO) grade, claudin 3, but not claudin 4, was significantly increased in moderately, poorly and undifferentiated adenocarcinomas compared to well‐differentiated and borderline‐type tumours. No significant changes were noticed for any claudin with regard to FIGO stages. We conclude that both claudin 3 and 4, even though they differ in expression during ovarian malignant transformation, might be used as novel markers for ovarian tumours. The observations of lack of claudin 4 and low expression of claudin 3 in OSE strengthen our current knowledge about the biological nature of these cells as an undeveloped epithelium.

Ovarian epithelial neoplasia after hormonal infertility treatment: long-term follow-up of a historical cohort in Sweden

OBJECTIVE To study the association between hormonal infertility treatment and ovarian neoplasia. DESIGN Historical cohort study. SETTING Three university hospitals in Sweden. PATIENT(S) A total of 2,768 women assessed and treated for infertility and infertility-associated disorders between 1961 and 1975. INTERVENTION(S) Exposed women received clomiphene citrate and/or gonadotropins. MAIN OUTCOME MEASURE(S) Incidence of ovarian neoplasia. RESULT(S) No overall excess risk of invasive ovarian cancer emerged compared with the general population. In women with gonadotropin treatment for non-ovulatory disorders, the risk was elevated (standardized incidence ratio [SIR] = 5.89; 95% confidence interval [CI] 1.91-13.75); four of the five cases reported hCG treatment only, rendering the biological plausibility uncertain. Multivariate analysis within the cohort indicated that treatment with gonadotropins only was associated with an increased risk of invasive cancer (relative risk = 5.28; 95% CI 1.70-16.47). For borderline tumors, a more than threefold overall increase of tumors (SIR = 3.61; 95% CI 1.45-7.44) was noted; women exposed to clomiphene because of ovulatory disorders showed the highest risk (SIR = 7.47; 95% CI 1.54-21.83). CONCLUSION(S) Our findings of increased risk of ovarian cancer after gonadotropins and of borderline tumors after clomiphene treatment need to be interpreted with caution. However, concern is raised, and further research on the long-term safety particularly of modern hormonal infertility treatment in IVF programs is warranted.

Four potential biomarkers as prognostic factors in stage III serous ovarian adenocarcinomas.

The mortality rate for patients with ovarian carcinomas is high and the available prognostic factors are insufficient. The use of biomarkers may contribute to better prediction and survival for these patients. We aimed to study the gene and protein expressions for 7 potential biomarkers, to determine if it is possible to use them as prognostic factors. Genes selected from our previous microarray analysis (2006), CLU, ITGB3, TACC1, MUC5B, CAPG, PRAME and TROAP, were analyzed in 19 of the tumors with quantitative real‐time polymerase chain reaction (QPCR). We found that CLU and ITGB3 were more expressed in tumors from survivors and PRAME and CAPG were more expressed in tumors from deceased patients. None of the other 3 genes were significantly differently expressed. The protein expressions of CLU, ITGB3, PRAME and CAPG were analyzed in 43 of the tumors with western blot for semiquantitative analysis. We established that the mRNA and protein expressions correlated and that all 4 proteins were significantly differently expressed. Further, immunohistochemistry (IHC) was used to localize the expression of the proteins in the tumor samples. According to our results, the 4 biomarkers CLU, ITGB3, PRAME and CAPG may be used as prognostic factors for patients with stage III serous ovarian adenocarcinomas.

The transcription factor C/EBP-beta and its role in ovarian function; evidence for direct involvement in the ovulatory process.

Gonadotropins are responsible for maturation of the ovarian follicle and the oocyte. Ovulation is the ultimate step in this process and involves disintegration of the follicular wall and subsequent release of an oocyte into the oviduct. These events are triggered by a surge of luteinizing hormone (LH). Genes expressed in the ovary, that respond to LH, are likely to be involved in the biochemical pathways that regulate ovulation. The transcription factor C/EBP‐β is induced promptly in the ovary, as a response to an ovulatory dose of gonadotropins. We used an ex vivo perfusion system to demonstrate that a specific reduction in ovarian C/EBP‐β expression inhibits ovulation. In such ovaries the oocytes appeared to be entrapped within the follicle. We have found a correlation between the expression level of the activating isoform of C/EBP‐β and the number of oocytes ovulated in response to gonadotropins. Since a reduction in C/EBP‐β expression does not affect the level of the ovulatory mediator prostaglandin endoperoxide synthase‐2 (PGS‐2), these findings support the view of C/EBP‐β as an important factor in the ovulatory process and highlight a C/EBP‐β‐dependent and PGS‐2‐independent pathway that takes part in regulation of ovulation.