Marina Bagnoli

Overexpression of folate binding protein in ovarian cancers

The high affinity folate binding protein (FBP) is overexpressed in ovarian cancers. However, its role in the pathogenesis and biological behaviour of these neoplasms is not clearly understood. Using the monoclonal antibody (MAb) MOv 18 and cytofluorimetric analysis, we investigated FBP expression in frozen neoplastic tissues from 136 patients diagnosed with epithelial ovarian cancer. FBP values were compared with clinico‐pathological characteristics (age, stage, histologic grade, histologic type, DNA ploidy, percentage of S‐phase cells, and previous chemotherapeutic treatment). Some amount of FBP overexpression was observed in 122 of the 136 tumours examined. The overall mean value of FBP fluorescence index (FBP FI) was 5.6 (median 2.7; min 0.8 − max, 78.9). By univariate analysis, FBP FI was overexpressed to a higher degree in ovarian neoplasms with high histologic grade, advanced stage, serous histology, aneuploid status, and high percentage of cells in S‐phase. Of the total number (136) of cases, 106 had all the parameters assessed and were thus selected for stepwise selection procedure. The only significant independent variable was the percentage of S‐phase cells, which accounted for about 31% of variance of FBP FI. Our results indicate that FBP is associated with parameters of biological aggressiveness in ovarian cancers. Int. J. Cancer 74:193‐198, 1997.

Activation of Phosphatidylcholine Cycle Enzymes in Human Epithelial Ovarian Cancer Cells

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation, (b) increased PC-specific phospholipase C (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)-mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKalpha (but not ChoKbeta) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKalpha mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC biosynthesis and degradation, respectively.

Subcellular Localization of Activated Leukocyte Cell Adhesion Molecule Is a Molecular Predictor of Survival in Ovarian Carcinoma Patients

Purpose: Currently available clinicopathologic prognostic factors are imperfect predictors of clinical course in advanced-stage epithelial ovarian cancer patients. New molecular predictors are needed to identify patients with higher risk of relapse or death from disease. In a retrospective study, we investigated the prognostic impact of activated leukocyte cell adhesion molecule (ALCAM) expression in epithelial ovarian cancer. Experimental Design: We analyzed the effect of cell-anchorage loss on ALCAM cellular localization in vitro and assessed ALCAM expression by immunohistochemistry in a series of 109 well-characterized epithelial ovarian cancer patient samples. Chi-square test, Kaplan-Meier method, and Cox proportional hazard analyses were used to relate ALCAM cellular localization to clinical-pathologic parameters and to overall survival (OS) rate. Results: Loss of epithelial ovarian cancer cell anchorage was associated both in vitro and in vivo with decreased ALCAM membrane expression. In vivo, ALCAM was localized to cell membrane in normal surface ovarian epithelium, whereas in 67% of the epithelial ovarian cancer samples, membrane localization was decreased or even lost, and the molecule was mainly expressed in cytoplasm. Median OS in this group of patients was 58 months, whereas a median OS was not yet reached in patients with ALCAM membrane localization (P = 0.036, hazard ratio [HR] = 2.0, 95% confidence interval [CI] 1.1 to 3.5). In a multivariate Cox regression model including all the available clinicopathologic variables, loss of ALCAM membrane expression was an independent factor of unfavorable prognosis (P = 0.042, HR = 2.15, 95% CI: 1.0 to 4.5). Conclusions: Decreased/lost ALCAM membrane expression is a marker of poorer outcome in epithelial ovarian cancer patients and might help to identify patients who could benefit from more frequent follow-up or alternative therapeutic modalities.

Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti folate receptor antibodies.

The α-folate receptor (FR) is selectively overexpressed in 90% of nonmucinous ovarian carcinomas, whereas no expression is detectable in normal ovarian surface epithelium (OSE). Indirect evidence suggests that FR expression is associated with tumor progression and affects cell proliferation. To evaluate better the role of FR, we developed an approach based on intracellular expression of single-chain (sc) antibodies (intrabody) to downmodulate membrane expression of FR in ovary cancer cells. IGROV-1 and SKOV3 ovarian carcinoma cell lines were transfected with an anti-FR intrabody. Transfectants and parental cells were tested for FR, integrins and anti-FR intrabody expression by fluorescence-activated cell sorting (FACS), reverse transcription and polymerase chain reaction (RT-PCR) and/or immunoblotting. Cell growth characteristics and adhesion properties were evaluated in liquid, semisolid and organotypic cultures. The anti-FR scFv inhibited FR expression from 60 to 99%. At physiological concentrations of folate, proliferation varied directly as a function of FR expression. FR downmodulation was accompanied by reduced colony-forming ability in soft agar, morphological change of the cells, significant enhanced adhesion to laminin or Matrigel, a two- to three-fold increase in α6β4 integrin expression, and a marked reduction in laminin production. In three-dimensional organotypic cultures, anti-FR intrabody-transfected IGROV1 cells grew as a single-ordered layer, reminiscent of normal OSE growth in vivo. In conclusion, the anti-FR intrabody reverses the transformed phenotype in ovary cancer cells and may provide an efficient means to inhibit selectively the growth of these cells.

Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to α-folate receptor overexpression

Caveolin (cav-1) and the GPI-anchored α-folate receptor (αFR) are membrane proteins both found associated to caveolar structures. Several studies in tumor cells independently reported cav-1 downregulation and αFR overexpression. Here we analysed the expression of the two molecules in normal and tumor ovarian samples derived from fresh specimens and from cultured cell lines. Whereas normal ovary surface epithelial cells displayed only cav-1 expression, ovarian tumor surgical samples and cell lines (COR, IGROV1, OVCAR3 and OVCA432) displayed high αFR and low-level or no cav-1 expression, except those cell lines (SKOV3 and SW626) with the lower αFR expression. SKOV3, but not two αFR-negative non-ovarian cell lines, exhibited downregulation of cav-1 expression following stable αFR cDNA transfection. Conversely, cav-1 transfection in IGROV1 cells led to downregulated αFR expression, together with formation of caveolar structures and reduction of growth capability. Moreover, cav-1 expression was induced in IGROV1 cells by transfection with intracellular anti-αFR antibodies to downmodulate αFR expression. In cav-1 transfected cells, transcriptional activity of the αFR-specific promoter P1 was reduced by 70% and an additional specific DNA-protein complex was identified by gel-shift assay, indicating that cav-1 expression influences αFR gene transcription. Together these results support the notion that αFR and cav-1 protein expression is reciprocally regulated in ovary cancer cells.

Role of microRNAs in ovarian cancer pathogenesis and potential clinical implications.

Despite important improvements over the past two decades, the overall cure rate of epithelial ovarian cancer (EOC) remains only approximately 30%. Although much has been learned about the proteins and pathways involved in early events of malignant transformation and drug resistance, a major challenge still remaining is the identification of markers for early diagnosis and prediction of response to chemotherapy. Recently, it has become clear that alterations in the expression of microRNAs (miRNAs) contribute to the pathogenesis and progression of several human malignancies. In this review we discuss current data concerning the accumulating evidence of the role of miRNAs in EOC pathogenesis and tumor characterization; their dysregulated expression in EOC; and their still undefined role in diagnosis, prognosis and prediction of response to therapy. The most frequently deregulated miRNAs are members of the let-7 and miR-200 families, the latter involved in epithelial-to-mesenchymal transition (EMT). EMT is part of normal ovarian surface epithelium physiology, being the key regulator of the post-ovulatory repair process, and failure to undergo EMT may be one of the events leading to transformation. A general down-modulation of miRNA expression is observed in EOC compared to normal tissue. However, a clear consensus on the miRNA signatures associated with prognosis or prediction of response to therapy has not yet been reached.

Relationship between folate-binding protein expression and cisplatin sensitivity in ovarian carcinoma cell lines

It has been suggested that sensitivity of ovarian carcinomas to cisplatin is in part related to an endogenous folate deficiency. In this work, we investigated whether overexpression of the folate-binding protein (FBP), a receptor involved in folate transport, might be associated with cisplatin sensitivity. The results obtained on a panel of ten ovarian carcinoma cell lines that overexpress different levels of the FBP showed a statistically significant relationship between FBP overexpression and cisplatin responsiveness, with the most sensitive cell lines expressing higher FBP levels on their membrane than the less sensitive ones. The relationship was observed both in cells growing in standard medium-containing high-folate concentrations (2.3 microM) and in cells adapted to growth in low-folate (20 nM) medium. Analysis of two cisplatin-resistant cell lines derived from the cisplatin-sensitive IGROV1 ovarian carcinoma cell line indicated that resistance was associated with a significant decrease in FBP expression. However, the receptor does not appear to be directly responsible for drug sensitivity per se as different cell lines transfected with FBP cDNA did not become more sensitive to the drug. Together, the data suggest the possible predictive value of FBP in ovarian carcinoma, as higher levels of expression can be indirectly but significantly associated with increased drug sensitivity.

SIMULTANEOUS ACTIVITY OF TWO DIFFERENT MECHANISMS OF FOLATE TRANSPORT IN OVARIAN CARCINOMA CELL LINES

We investigated whether the folate receptor α‐isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5‐methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate‐depleted medium and internalization of 10 nM evaluated as acid‐resistant radioactivity at 0° and 37°C. The amount of 5‐methyl[3H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time‐course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5‐methyl[3H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5‐methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5‐methyl[3H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier‐mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5‐methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491.

CD95-Mediated Apoptosis Is Impaired at Receptor Level by Cellular FLICE-Inhibitory Protein (Long Form) in Wild-Type p53 Human Ovarian Carcinoma

Purpose: Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status. Experimental Design: CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays. Results: CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIPL) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIPL down-modulation at the DISC level. Down-regulation of c-FLIPL with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIPL in apoptosis resistance. Removal of c-FLIPL block at DISC level allowed full activation of the mitochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIPL involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIPL expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIPL and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status. Conclusion: The inhibitory protein c-FLIPL is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.

MiR-506 inhibits multiple targets in the epithelial-to-mesenchymal transition network and is associated with good prognosis in epithelial ovarian cancer.

Extensive investigations have shown that miRNAs are important regulators of epithelial‐to‐mesenchymal transition (EMT), mainly targeting the transcriptional repressors of E‐cadherin (E‐cad). Less is known about the post‐transcriptional regulation of vimentin or N‐cadherin (N‐cad) in EMT. Our previous study identified miR‐506 as a key EMT inhibitor through directly targeting the E‐cad transcriptional repressor SNAI2. In this study, we provide evidence that miR‐506 simultaneously suppresses vimentin and N‐cad. The knockdown of vimentin using siRNA reversed EMT, suppressed cell migration and invasion, and increased E‐cad expression on the cell membrane in epithelial ovarian cancer (EOC) cells. In a set of tissue microarrays that included 204 EOCs of all major subtypes (eg serous, endometrioid, clear cell, and mucinous), miR‐506 was positively correlated with E‐cad and negatively correlated with vimentin and N‐cad in all subtypes of EOC. A high level of miR‐506 was positively associated with early FIGO stage and longer survival in EOC. Introduction of miR‐506, mediated by nanoparticle delivery, in EOC orthotopic mouse models resulted in decreased vimentin, N‐cad, and SNAI2 expression and increased E‐cad expression; it also suppressed the dissemination of EOC cells. Thus, miR‐506 represents a new class of miRNA that regulates both E‐cad and vimentin/N‐cad in the suppression of EMT and metastasis. Copyright