Phenotype Of Ovarian Cancer Cells Research Articles

PDCD4 inhibits the malignant phenotype of ovarian cancer cells

Programmed cell death 4 (PDCD4) is a newly identified tumor suppressor that can inhibit activator protein (AP)-1 activation and protein translation. Our previous studies indicate that lost or reduced PDCD4 expression is associated with the progression of ovarian carcinoma. However, direct evidence that PDCD4 inhibits malignant phenotype of human cancer cells is limited. In the present study, we found that PDCD4 expression in ovarian cancer cell lines (SKOV3, 3AO, and CAOV3) inhibited significantly their proliferation and cell cycle progression, and induced apoptosis. More importantly, up-regulation of PDCD4 expression decreased the colony-forming capacity of ovarian cancer cells in vitro and tumorigenic capacity in mice. These results demonstrate that PDCD4 can suppress the malignant phenotype of ovarian cancer cells, and may represent a novel therapeutic target for the treatment of ovarian cancer.

Snail and Slug Mediate Radioresistance and Chemoresistance by Antagonizing p53-Mediated Apoptosis and Acquiring a Stem-Like Phenotype in Ovarian Cancer Cells

The transcriptional repressors Snail and Slug contribute to cancer progression by mediating epithelial-mesenchymal transition (EMT), which results in tumor cell invasion and metastases. We extend this current understanding to demonstrate their involvement in the development of resistance to radiation and paclitaxel. The process is orchestrated through the acquisition of a novel subset of gene targets that is repressed under conditions of stress, effectively inactivating p53-mediated apoptosis, while another subset of targets continues to mediate EMT. Repressive activities are complemented by a concurrent derepression of specific genes resulting in the acquisition of stem cell-like characteristics. Such cells are bestowed with three critical capabilities, namely EMT, resistance to p53-mediated apoptosis, and a self-renewal program, that together define the functionality and survival of metastatic cancer stem cells. EMT provides a mechanism of escape to a new, less adverse niche; resistance to apoptosis ensures cell survival in conditions of stress in the primary tumor; whereas acquisition of "stemness" ensures generation of the critical tumor mass required for progression of micrometastases to macrometastases. Our findings, besides achieving considerable expansion of the inventory of direct genes targets, more importantly demonstrate that such elegant cooperative modulation of gene regulation mediated by Snail and Slug is critical for a cancer cell to acquire stem cell characteristics toward resisting radiotherapy- or chemotherapy-mediated cellular stress, and this may be a determinative aspect of aggressive cancer metastases.

Roles of clusterin in progression, chemoresistance and metastasis of human ovarian cancer

Clusterin (CLU) is a multivalent glycoprotein with ubiquitous tissue distribution. To address the possible differential functional roles assumed by different isoforms of CLU in the progression of human ovarian cancer, we constructed 2 human ovarian cancer cell models that represent examples of contradistinctive CLU expression levels. One is constitutively overexpressing different clusterin isoforms in SKOV3 cells by transfection of the 3 different expression vectors, another is silencing the intrinsically expressing clusterin in cisplatin-resistant human A2780-cis(CP70) tumor cells with the usage of shRNA-mediated CLU gene silencing. Then, the different cellular localization, biological effects, and functional roles played in tumor progression and drug resistances were studied. We found that (i) in the distinct cellular contexts of human ovarian carcinoma SKOV3 and CP70 cells assayed, sCLU is a central molecule in cell homeostasis that functions as a cytoprotective protein, whereas nCLU is proapoptotic; (ii) In SKOV3 cells, nuclear localization of the truncated CLU is NLS dependent, without which the pnCLU protein was sequestrated in cytoplasm to prevent cytotoxicity. (iii) sCLU plays a significant role in the development of the chemoresistance phenotype in ovarian cancer cells. Moreover, with the CLU-specific shRNA oligonucleotides, we successfully sensitized cells for chemotherapy, and inhibited cells' proliferation, migration and invasion. Collectively, our results reveal that, CLU gene expression might play a crucial role in ovarian cancer progression, adaptation and eventual resistance to chemotherapy through differential processing of CLU isoforms. Specifically, sCLU as an antiapoptotic protein, upregulated in an adaptive cell-survival manner by chemotherapy, confers resistance to various cell-death triggers.

Cross talk of signals between EGFR and IL-6R through JAK2/STAT3 mediate epithelial–mesenchymal transition in ovarian carcinomas

Epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas, with direct or indirect activation of EGFR able to trigger tumour growth. We demonstrate significant activation of both signal transducer and activator of transcription (STAT)3 and its upstream activator Janus kinase (JAK)2, in high-grade ovarian carcinomas compared with normal ovaries and benign tumours. The association between STAT3 activation and migratory phenotype of ovarian cancer cells was investigated by EGF-induced epithelial–mesenchymal transition (EMT) in OVCA 433 and SKOV3 ovarian cancer cell lines. Ligand activation of EGFR induced a fibroblast-like morphology and migratory phenotype, consistent with the upregulation of mesenchyme-associated N-cadherin, vimentin and nuclear translocation of β-catenin. This occurred concomitantly with activation of the downstream JAK2/STAT3 pathway. Both cell lines expressed interleukin-6 receptor (IL-6R), and treatment with EGF within 1 h resulted in a several-fold enhancement of mRNA expression of IL-6. Consistent with that, EGF treatment of both OVCA 433 and SKOV3 cell lines resulted in enhanced IL-6 production in the serum-free medium. Exogenous addition of IL-6 to OVCA 433 cells stimulated STAT3 activation and enhanced migration. Blocking antibodies against IL-6R inhibited IL-6 production and EGF- and IL-6-induced migration. Specific inhibition of STAT3 activation by JAK2-specific inhibitor AG490 blocked STAT3 phosphorylation, cell motility, induction of N-cadherin and vimentin expression and IL6 production. These data suggest that the activated status of STAT3 in high-grade ovarian carcinomas may occur directly through activation of EGFR or IL-6R or indirectly through induction of IL-6R signalling. Such activation of STAT3 suggests a rationale for a combination of anti-STAT3 and EGFR/IL-6R therapy to suppress the peritoneal spread of ovarian cancer.

Reversal of the malignant phenotype of ovarian cancer A2780 cells through transfection with wild-type PTEN gene

Objective PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene identified on human chromosome 10q23. Substantial studies have demonstrated that PTEN can inhibit cell proliferation, migration and invasion of many cancer cells. The purpose of this study was to determine whether upregulation of PTEN gene by transfection wild-type PTEN gene to ovarian cancer cells can inhibit growth and migration and to explore the potential for PTEN gene therapy of ovarian cancers. Method Wild-type and phosphatase-inactive (C124A) PTEN plasmids were transfected into ovarian epithelial cancer A2780 cells, and their effects on cell apoptosis, cell proliferation, cell migration and cell invasion were analyzed by flow cytometry analysis, TUNEL assay, MTT assay, wound-healing assay and transwell assay. Results Both wild-type and mutant PTEN can upregulate the expression of PTEN gene dramatically; however, it is wild-type PTEN not phosphatase-inactive PTEN that can induce apoptosis and decrease cell migration, invasion and proliferation in ovarian cancer cells. Conclusion These results demonstrated that PTEN had played an important role in the cell proliferation, cell migration and invasion dependent on its phosphatase activity. Enhanced expression of PTEN by gene transfer is sufficient to reverse the malignant phenotype of ovarian cancer cells and transfection of ovarian cancer cells with wild-type PTEN gene might be another novel approach for therapeutic intervention in ovarian cancer.

6541 POSTER Genetic polymorphism of the epidermal growth factor gene – value for the treatment of non-small cell lung cancer (NSCLC)

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene identified on human chromosome 10q23. Substantial studies have demonstrated that PTEN can inhibit cell proliferation, migration and invasion of many cancer cells. The purpose of this study was to determine whether upregulation of PTEN gene by transfection wild-type PTEN gene to ovarian cancer cells can inhibit growth and migration and to explore the potential for PTEN gene therapy of ovarian cancers.Wild-type and phosphatase-inactive (C124A) PTEN plasmids were transfected into ovarian epithelial cancer A2780 cells, and their effects on cell apoptosis, cell proliferation, cell migration and cell invasion were analyzed by flow cytometry analysis, TUNEL assay, MTT assay, wound-healing assay and transwell assay.Both wild-type and mutant PTEN can upregulate the expression of PTEN gene dramatically; however, it is wild-type PTEN not phosphatase-inactive PTEN that can induce apoptosis and decrease cell migration, invasion and proliferation in ovarian cancer cells.These results demonstrated that PTEN had played an important role in the cell proliferation, cell migration and invasion dependent on its phosphatase activity. Enhanced expression of PTEN by gene transfer is sufficient to reverse the malignant phenotype of ovarian cancer cells and transfection of ovarian cancer cells with wild-type PTEN gene might be another novel approach for therapeutic intervention in ovarian cancer.

The activity of the synthetic progestin medroxyprogesterone acetate on the invasive phenotype of ovarian cancer cells

16003 Background: Androgens play an integral role in the physiologic and pathologic processes of the ovary. Yet it has been difficult to study the role of the androgen recptors (AR) separately from the other steroid receptors such as the progesterone receptor (PR) in ovarian cancer. This has been made more complicated because most synthetic progestins such as Medroxyprogesterone acetate (MPA) bind both PR and AR. The objectives of our study were: 1. To create an ovarian cancer cell line constitutively expressing only AR. 2. To compare the role of AR activated by the synthetic progestin MPA vs. the pure androgen dihydrotestosterone (DHT) on the invasiveness of human breast and ovarian cancer cells. 3. To investigate the role of matrix metalloproteases (MMP's) associated with invasion. Methods: ER- and PR- human breast (T47D-Y) and ovarian (OvCa 429) cancer cells were engineered to stably express AR. Immunocytochemistry and western blot analyses confirmed that these breast and ovarian cancer cell lines (called Y-AR and OvCa-AR respectively) are PR-, but AR+. Boyden chamber invasion assays were performed using Y-AR and OvCa-AR cells treated with either vehicle, MPA or DHT. The MMP's associated with invasion were further investigated using zymographic assays. Results: AR activation by either MPA or DHT increased the invasive potential of both breast (p<0.05) and ovarian cancer cells with MPA being significantly more effective than DHT at stimulating invasion. However, regardless of the ligand, activation of AR increases tumor cell invasion. To elucidate the MMP's associated with this activation in OvCa-AR cells, we used zymographic analysis. Interestingly, we found that MPA activation of AR decreases both the total level and activation of MMP-9 compared to DHT and vehicle control. Conclusions: Using our model system we are able to study the role of AR independent of PR on the biology of breast and ovarian cancer cells. Our studies suggest that the use of pharmacological doses of synthetic progestins may actually increase the invasive potential of ovarian cancer cells through AR. We hypothesize that blockade of downstream AR targets or the use of selective AR modulators (SARMS) may be of therapeutic value in the treatment of ovarian cancer. No significant financial relationships to disclose.

0739: Exploiting the Glycolytic Phenotype in Ovarian Cancer Cells: Novel Target for Therapy

0739: Exploiting the Glycolytic Phenotype in Ovarian Cancer Cells: Novel Target for Therapy

ID: 119 Effects of overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 on the malignant phenotype of ovarian cancer cells

ID: 119 Effects of overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 on the malignant phenotype of ovarian cancer cells

Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells

The human tissue kallikrein family of serine proteases (hK1-hK15 encoded by the genes KLK1-KLK15) is involved in several cancer-related processes. Accumulating evidence suggests that certain tissue kallikreins are part of an enzymatic cascade pathway that is activated in ovarian cancer and other malignant diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells displayed similar proliferative capacity as the vector-transfected control cells (which do not express any of the four tissue kallikreins), but showed significantly increased invasive behavior in an in vitro Matrigel invasion assay (p<0.01; Mann-Whitney U-test). For in vivo analysis, the cancer cells were inoculated into the peritoneum of nude mice. Simultaneous expression of hK4, hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden compared to the vector-control cell line. Five out of 14 mice in the 'tissue kallikrein overexpressing' group displayed a tumor/situs ratio greater than 0.198, while this weight limit was not exceeded at all in the vector control group consisting of 13 mice (p=0.017; chi2 test). Our results strongly support the view that tumor-associated overexpression of tissue kallikreins contributes to ovarian cancer progression.

Activation of p70S6KInduces Expression of Matrix Metalloproteinase 9 Associated with Hepatocyte Growth Factor-Mediated Invasion in Human Ovarian Cancer Cells

The expression of hepatocyte growth factor (HGF) receptor, encoded by the Met oncogene, is elevated in ovarian and a variety of cancers. Here we show that human ovarian cancer cells with high Met expression were more sensitive to the cell motility and invasion effect of HGF. Met down-regulation by small interfering RNAs or K252a resulted in reduced migration in response to HGF. The invasive/migratory phenotype activated by HGF can be blocked by specific inhibitors of the phosphatidylinositol-3-kinase (PI3K) cascade, inhibitor of p70(S6K), and also the expression of a dominant-negative Akt, demonstrating that HGF transmits the motogenic signal through PI3K and Akt to p70(S6K). A significant role for p70(S6K) in cell invasion is further supported by the observation that expression of constitutively active forms of p70(S6K) is sufficient to induce invasive and migratory phenotypes in ovarian cancer cells. Importantly, activation of p70(S6K) stimulated expression and proteolytic activity of matrix metalloproteinase (MMP)-9 and cellular invasion, whereas it had little effect on MMP-2, suggesting for the first time that MMP-9 up-regulation by p70(S6K) as a key step for HGF-induced invasion and migration. These data suggest that interfering p70(S6K) may provide a novel means of controlling tumor cell invasiveness.

Cyclooxygenase-2 Functions as a Downstream Mediator of Lysophosphatidic Acid to Promote Aggressive Behavior in Ovarian Carcinoma Cells

Elevated levels of the bioactive lipid lysophosphatidic acid (LPA) are detectable in the majority of patients with both early- and late-stage ovarian cancer, suggesting that LPA promotes early events in ovarian carcinoma dissemination. LPA contributes to the development, progression, and metastasis of ovarian cancer in part by inducing the expression of genes that contribute to proliferation, survival, or invasion, including cyclooxgenase-2 (COX-2) and matrix metalloproteinase-2 (MMP-2). We have previously shown that LPA promotes proMMP-2 activation and MMP-2-dependent migration and invasion in ovarian cancer cells. The purpose of the current study was to determine whether the effect of LPA on acquisition of the metastatic phenotype in ovarian cancer cells is mediated via a COX-2-dependent mechanism. Immunohistochemical analysis of 173 ovarian tumors showed strong COX-2 immunoreactivity in 63% of tumor specimens, including 50% of borderline tumors. LPA increased COX-2 protein expression in a time- and concentration-dependent manner in two of three immortalized borderline ovarian epithelial cells as well as in four of six ovarian cancer cell lines. This was accomplished by both activation of the Edg/LPA receptor and LPA-mediated transactivation of the epidermal growth factor receptor, which increased COX-2 expression via the Ras/mitogen-activated protein kinase pathway. COX-2 also played a role in LPA-induced invasion and migration, as treatment with the COX-2 specific inhibitor NS-398 reduced LPA-induced proMMP-2 protein expression and activation and blocked MMP-dependent motility and invasive activity. These data show that COX-2 functions as a downstream mediator of LPA to potentiate aggressive cellular behavior.

194 BARD1 catalyzes of p53 phosphorylation by DNA-damage response kinase and reverses malignant phenotype of ovarian cancer cells

194 BARD1 catalyzes of p53 phosphorylation by DNA-damage response kinase and reverses malignant phenotype of ovarian cancer cells

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

The alpha-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 alpha6beta4 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.

Differential expression of the 67-kD laminin receptor and 31-kD human laminin-binding protein in human ovarian carcinomas

The expression of the 67-kD laminin receptor (67LR) and the 31-kD human laminin-binding protein (HLBP31), two proteins involved in cancer cell laminin interaction, was evaluated on 30 ovarian cancer specimens. Expression of the 67LR was increased (up to 2.5-fold, in 87% of the patients), while HLBP31 expression was downregulated in cancer cells compared with the normal tissue, as detected by northern blotting and immunohistochemistry. The immunohistochemical study demonstrated that the 67LR was significantly overexpressed ( P < 0.05) in the group of patients whose cytoreductive surgery was suboptimal, and those with poor clinical outcome. No correlation was observed between HLBP31 expression and clinicopathological features. Increased expression of the 67LR appears to correlate with the invasive phenotype of ovarian cancer cells and suggests a role of the latter in ovarian cancer invasion.

The effects of dimethyl sulfoxide and retinoic acid on the cell growth and the phenotype of ovarian cancer cells

We have compared the in vitro effects of the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA) on a polyclonal human ovarian cancer cell line (HOC-7). Density gradient fractionation of untreated cells reveals that a proportion of rapidly growing, polygonal cells with medium density is capable of spontaneous reversion into a slowly growing low-density phenotype with flattened morphology similar to non-transformed human ovarian surface epithelial cells. Clonal expansion of these low-density cells proves that the observed characteristics are stable for prolonged culture periods. Exposure of HOC-7 cells to DMSO and RA or removal of the serum from the medium is effective in enhancing the proportion of these low-density cells. Application of DMSO causes the cells to become flattened and elongated, and to develop rod-like protrusions. In these cytoplasmic extensions thick filament bundles are dominant. Immunofluorescence studies demonstrate that both untreated low-density subclones and DMSO-treated polyclonal cells are much more reactive for cytokeratin than medium-density subclones or untreated parental cells. Furthermore, immunocytochemistry and fixed-cell ELISA reveal 2- to 5-fold greater amounts of desmoplakins I and II and of fibronectin in low-density subclones and in DMSO-treated cells as compared to medium-density subclones and control cultures. RA exerts weaker effects on the phenotype of the cells. Both inducers reduce DNA synthesis and inhibit the anchorage-dependent and the anchorage-independent cell growth in a dose- and time-dependent manner. The restoration of the original morphology and growth rate after removal of the differentiation-inducing agents proves that the observed changes are reversible; this indicates that the cells do not become terminally differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of clonogenic and differentiated cell phenotypes of ovarian cancer cells (HOC-7) by discontinuous density gradient centrifugation

We isolated clonogenic cells from differentiated HOC-7 ovarian cancer cells. Both cell subsets were characterised in respect to morphology, growth behaviour, DNA content and expression of tumour-associated antigens and nuclear oncogenes. Ten cell fractions (Fr) were separated by centrifugation in a discontinuous density gradient (Fr 1 < 1.037 g/ml to Fr 10 > 1.069 g/ml, steps 0.004 g/ml). Large adenoid cells containing vacuoles filled with neutral polysaccharides were concentrated in Fr 1–4. These cells were non-clonogenic in soft agar. The growth on solid substrate was highest in Fr 6 and 7, intermediate in Fr 2–5 and Fr 8–10 and lowest in Fr 1. The mean cloning efficiencies of the fractions in soft agar were highest in Fr 6 (8.1%) and lowest in Fr 2 and 3 (0.1%). Diploid and near tetraploid cell subsets were found with similar frequency in all fractions. Immunocytochemistry revealed 4–7% Ki-67 positive cells in Fr 1–6 and 12–20% in Fr 7–10. In Fr 3–10 ≥ 79% of the cells expressed CA 125. Positivity for c-myc, c-myb and c-fos (≥74%) was not correlated with clonogenicity. In conclusion, differentiated cells (Fr 1–4) were separated from cells with higher growth rates (Fr 5–10). Clonogenic cells were enriched in Fr 6. These data indicate that discontinuous density gradient fractionation represents a useful method for separation of cells with different degrees of differentiation, growth potential and clonogenicity.