Elsevier

Gynecologic Oncology

Volume 104, Issue 2, February 2007, Pages 461-469
Gynecologic Oncology

L1-CAM in a membrane-bound or soluble form augments protection from apoptosis in ovarian carcinoma cells

https://doi.org/10.1016/j.ygyno.2006.08.038Get rights and content

Abstract

Objective.

Apoptosis resistance is a hallmark of cancer progression, a phenomenon frequently observed in ovarian carcinoma. We reported previously, that L1 adhesion molecule (CD171) is overexpressed in ovarian and endometrial carcinomas and that L1 expression is a predictor of poor outcome. We investigated a possible role of L1 in apoptosis resistance.

Methods.

We used L1 transfectants and ovarian carcinoma cell lines and induced apoptosis by different stimuli such as C2-ceramide, staurosporine, cisplatin or hypoxia.

Results.

We found that cells expressing L1 are more resistant against apoptosis. In HEK293 cells, L1-expresssion leads to a sustained ERK, FAK and PAK phosphorylation. Soluble L1 only partially rescued HEK293 cells from apoptosis. Treatment with apoptotic stimuli upregulated the anti-apoptotic molecule Bcl-2 to a greater extend in HEK293 cells expressing L1. In the ovarian carcinoma cell line OVMz, the depletion of L1 by RNA interference sensitized cells for apoptosis induction. No changes in activation of ERK or FAK were observed after L1 knockdown. The selection of m130 ovarian carcinoma or SW707 colon carcinoma cells with cisplatin leads to upregulated expression of L1.

Conclusions.

Our results suggest a link between L1 expression and chemoresistance of ovarian carcinomas. Upregulation of L1 after cisplatin treatment might indicate a more malignant tumor phenotype given the established role of L1 in cell motility and invasion.

Introduction

The acquisition of apoptosis resistance is a hallmark of cancer progression. In ovarian carcinoma, this is frequently observed. Chemotherapy is important in controlling residual disease following cyto-reductive surgery and as neo-adjuvant therapy in patients with advanced disease [1]. The standard chemotherapy for advanced ovarian cancer is currently paclitaxel–carboplatin or paclitaxel–cisplatin which is routinely given together with dexamethasone, a synthetic corticoid [2]. Despite initial response to therapy, ovarian carcinomas often acquire resistance to chemotherapeutic drugs leading to tumor recurrence and frequent death of the patients [1], [2]. A better understanding of molecular mechanisms underlying chemoresistance is urgently needed.

L1 is a type I membrane glycoprotein of 200–220 kDa structurally belonging to the Ig superfamily [3]. L1 plays a crucial role in axon guidance and cell migration in the developing nervous system [4], [5]. Recent studies have also implicated L1 expression in the progression of human carcinomas. L1 expression was found on different tumors including lung cancer [6], gliomas [7], melanomas [8], [9], renal carcinoma [10], [11], and colon carcinoma [12]. We reported before that L1 is overexpressed in ovarian and endometrial carcinomas in a stage-dependent manner and that L1 expression was a predictor of poor outcome [13]. A clear mechanism by which L1 expression could contribute to the progression of human tumors is still missing. However, several recent studies have shown that overexpression of L1 can augment cell motility of carcinoma cells on extracellular matrix proteins [14], [15], [16], and invasiveness in matrigel invasion assays [12], [17], [18]. L1 expression was also found to enhance tumor growth in NOD/SCID mice [12], [19] and was found to induce L1-dependent gene expression [16], [18].

We demonstrated before that L1 is released from the cell membrane by the metalloproteases ADAM10 [14], [20] and ADAM17 [21], [22]. The soluble L1 ectodomain, as a product of L1 cleavage, is detectable in serum and ascites from ovarian carcinoma patients [13]. Soluble L1 from ascites is a potent inducer of cell migration [23]. Other functions, for instance in apoptosis protection, have not been investigated. In the present communication, we have addressed the question if expression of L1 and/or soluble L1 has an influence on apoptosis of ovarian carcinoma cells. We used cell lines stably transfected with L1 and ovarian carcinoma cell lines to study the influence of L1 expression on apoptosis induced through different apoptotic stimuli including C2-ceramide, staurosporine, cisplatin or hypoxic conditions. Our results show that expression of L1 affected apoptosis sensitivity and suggests a link between L1 expression and chemoresistance of ovarian carcinomas.

Section snippets

Cells

The ovarian carcinoma cell lines OVMz and m130 have been described before [19], [20]. The human epithelial kidney cell line HEK293 and the Chinese hamster ovary (CHO) cell line stably expressing human L1 (hL1) were established by transfection with superfect (Stratagene, Heidelberg, Germany) and selection for L1 expression with mAb L1-11A and magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) or sorting by FACS as described before [19], [20]. All cells were cultivated in DMEM

Expression of L1 enhances apoptosis resistance in HEK293 cells

We initially analyzed the role of L1 in apoptosis resistance using stably transfected cell lines. HEK293 and HEK293-hL1 cells were treated with C2-ceramide or staurosporine under serum-free conditions and apoptosis was analyzed by Nicoletti staining. L1-expressing cells were more resistant against apoptosis induced through both stimuli (Figs. 1B and C). 93% of the cells were still viable after 24-h treatment with C2-ceramide, compared to only 63% of wild-type HEK293 cells. Similar differences

Discussion

High-grade ovarian carcinoma is a life-threatening disease with a low 5-year survival rate. Currently, the preferred treatment regimen after surgery is combined chemotherapy comprising usually a platinum-based drug, such as cisplatin or carboplatin, coupled with paclitaxel. While this treatment course shows promising effects in a high percentage of cases, the development of chemoresistance is a hurdle that significantly reduces successful treatment outcomes. We previously reported that the

Acknowledgments

This work was supported by a grant from Deutsche Krebshilfe (Schwerpunktprogramm Migration and Invasion) and the European Community (EC-Strep Signalling & Traffic) to P.A. We thank A. Strecker and C. Math for the excellent technical assistance and Prof. Ingrid Herr for the helpful comments.

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