Machine learning assisted raman spectroscopy for the classification of ovarian cancer cells.

Raman spectroscopy and machine learning for the classification of breast cancers

DNA Methylation in Ovarian Cancer: II. Expression of DNA Methyltransferases in Ovarian Cancer Cell Lines and Normal Ovarian Epithelial Cells

Regulation of growth of normal ovarian epithelial cells andovarian cancer cell lines by transforming growth factor-ß

As important cell to cell communicator, exosomes carry a range of bioactive molecules which can significantly influence phenotype of recipient cells. Inhibiting or removing cancer cell-derived exosomes are of therapeutic interest. However, regulation of secretion and release mechanism of exosomes is still unclear. To explore the regulation of exosomes released from normal ovarian epithelial cells and ovarian cancer cells, a normal ovarian epithelial cell line and three ovarian epithelial cancer cell lines were utilized to investigate their exosomes' release and regulation. A cervical cancer cell SiHa was used for identifying tissue specificity. NanoSight NS500 was used to quantify exosome numbers. Exosomes were labeled and observed by confocal microscopy to investigate their interaction with different ovarian cell lines. Exosomes released from normal or ovarian cancer cells were regulated by the extracellular exosomes. Exosome release was inhibited with the extracellular exosome concentration increase. Exosomes from normal ovarian cell and cervical cancer cell also inhibited ovarian cancer cell-derived exosome release, and there was no tissue specificity. PKH26-labeled exosomes from normal ovarian cell and cervical cancer cell were uptaken by ovarian cancer cells. Release of exosomes from ovarian cancer cell is regulated by a feedback mechanism without tissue specificity. This may provide a therapeutic approach to control the release of exosomes from ovarian cancer cells.

OBJECTIVES: Prior studies have identified antibodies to casein kinase I epsilon (CKI-ε) in the serum of patients with ovarian cancer. We investigated the expression pattern, functional roles, and clinical correlation of CKI-ε in ovarian cancer. METHODS: The expression of CKI-ε in 8 healthy ovaries and 68 ovarian tumor samples was determined by IHC and scored on intensity of antibody expression (scale 0-3) and the percentage of the area stained (scale 0-3). The average from both scores was assigned to each patient. Clinical characteristics for each patient were retrieved from electronic medical records. Statistical significance was determined using the Fisher exact test. Univariate analysis was performed by constructing probability curves according to the Kaplan-Meier method. Immortalized normal human ovarian surface epithelial (HOSE) cell lines with ectopic expression of CKI-ε were established by transfection with a CKI-ε expression construct. CKI-ε-targeting shRNAs were used to generate ovarian cancer cell lines that have stably suppressed CKI-ε expression. Protein expression was determined by Western blot. Growth rate was determined by cell counting. Three-dimensional spheroid culture was performed on Matrigel. The growth of ovarian cancer cell lines harboring either a control shRNA or a CKI-ε-shRNA was evaluated in a mouse xenograft model. The effects of IC261, an inhibitor of CKI-ε, on ovarian cancer cells were evaluated by MTT assay. RESULTS: CKI-ε was overexpressed in 67% of invasive ovarian tumors (P-value=0.001) and in 14 of 17 ovarian cancer cell lines. Normal HOSE cells that have ectopic expression of CKI-ε have accelerated growth rate and formed spheroids in Matrigel that were two to four times larger than those formed by control HOSE. In contrast, cancer cell lines harboring specific CKI-ε shRNA had reduced growth rate and 6-fold reduced tumor weight in the xenograft model (P-value=0.007). The cancer cell lines also showed CKI-ε-dependent sensitivity to IC261. The median CKI-ε expression level among the ovarian cancer tissue samples was 3. For patients whose score was ≤ 3 and > 3, the mean overall survival was 91 months and 48 months, respectively. Similarly, the mean disease free survival interval was 43 months and 22 months for patients whose score was ≤ 3 and > 3, respectively. Patients who scored > 3 tended to have late stage disease (61%) compared to patients who scored ≤ 3 (39%), (p=0.06). CONCLUSIONS: CKI-ε is highly expressed in ovarian tumors and cancer cell lines. Studies of cell lines with alterations in CKI-ε expression showed that this signaling protein regulates cell growth and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4789.

Cancer is the most common disease to threaten human health. The ability to screen individuals with malignant tumours with only a blood sample would be greatly advantageous to early diagnosis and intervention. This study explores the possibility of discriminating between cancer patients and normal subjects with serum surface-enhanced Raman spectroscopy (SERS) and a support vector machine (SVM) through a peripheral blood sample. A total of 130 blood samples were obtained from patients with liver cancer, colonic cancer, esophageal cancer, nasopharyngeal cancer, gastric cancer, as well as 113 blood samples from normal volunteers. Several diagnostic models were built with the serum SERS spectra using SVM and principal component analysis (PCA) techniques. The results show that a diagnostic accuracy of 85.5% is acquired with a PCA algorithm, while a diagnostic accuracy of 95.8% is obtained using radial basis function (RBF), PCA–SVM methods. The results prove that a RBF kernel PCA–SVM technique is superior to PCA and conventional SVM (C-SVM) algorithms in classification serum SERS spectra. The study demonstrates that serum SERS, in combination with SVM techniques, has great potential for screening cancerous patients with any solid malignant tumour through a peripheral blood sample.

11063 Background: Ovarian cancer is one of the leading causes of death from gynecological tumors in women. Several lines of evidence suggest that estrogens may play an important role in ovarian carcinogenesis, through binding with their receptors, ERα and ERβ. Two novel estrogen receptor β (ERβ) isoforms that diverge in their 5’-untranslated regions, ERβ mRNA (0K-1) and ERβ mRNA (0N-1), have recently been identified. This finding indicates that transcription of the human ERβ gene occurs from at least two different promoters, i.e., promoter 0K and promoter 0N. The purpose of this study was to study the expression of ERβ isoforms in primary cultures of normal human ovarian surface epithelial cells (OSE), a panel of ovarian cancer cell lines and in normal human ovarian tissues and ovarian cancer tissues; and to examine whether methylation of the two ERβ promoters is involved in regulation of ERβ gene expression or not. Methods: The expression of ERβ isoforms and the methylation status was evaluated in 8 OSE cells, 15 ovarian cancer cell lines, 6 normal, and 57 ovarian cancer tissues. Using quantitative real-time PCR techniques, mRNA expression levels of ERβ1, ERβ2 (ERβcx), ERβ3, ERβ4 and ERβ5 were evaluated. The methylation status of two ERβ promoters were confirmed by direct bisulfate sequencing. Results: ERβ1, ERβ2 and ERβ4 were significantly lower in ovarian cancer cell lines than OSE cells (p<0.01). In contrast, ERβ5 were significantly higher in ovarian cancer cell lines than OSE cells (p<0.01). Promoter 0N was unmethylated in OSE, but was extensively methylated in ovarian cancer cell lines. In contrast, promoter 0K was unmethylated in both normal and malignant ovarian epithelial cells. Correlation was detected between promoter 0N hypermethylation and loss of ERβ mRNA expression in ovarian cancer cell lines. These findings were also detected in ovarian cancer tissues. Conclusions: Decreased level of ERβ mRNA is considered to be associated with ovarian tumorigenesis, and that DNA methylation is an important mechanism for ERβ gene silencing in ovarian cancer. No significant financial relationships to disclose.

The effect of interferon gamma on epidermal growth factor receptor expression in normal and malignant ovarian epithelial cells

Cisplatin-centered chemotherapy is the first-line treatment for human ovarian cancer. However, chemoresistance remains a major obstacle to successful treatment. Evidence has indicated that signal transducer and activator of transcription-3 (STAT3) is a determinant of chemoresistance; it was related to tumor recurrence in a large number of solid malignancies. Unfortunately, none of the compounds currently developed to block STAT3 signaling has been considered a serious clinical candidate because of toxicity or limited bioavailability. In this study, we clarified the significance of STAT3 activation in chemoresistant ovarian cancer and assessed the suitability of a novel oncolytic adenovirus (M4) designed to specifically deplete STAT3 and reverse cisplatin resistance in ovarian cancer. We showed that aberrant expression and constitutive activation of STAT3 was instrumental in cisplatin resistance in ovarian cancer cell lines and in ovarian cancer tissue samples. The M4 adenovirus could specifically deplete constitutive and inducible STAT3 and phosphorylated STAT3 proteins in ovarian cancer cells. This significantly inhibited cell survival and enhanced cisplatin-induced apoptosis. In contrast, normal human umbilical vein endothelial cells and human ovarian surface epithelial cells appeared to be unaffected by M4 treatment. Furthermore, a combined cisplatin plus M4 therapy substantially eliminated populations enriched in tumor-initiating cells. In mice, systemic intraperitoneal administration of M4 significantly potentiated the antitumor effect of cisplatin. These results suggest that M4 has great potential as a therapy against cisplatin resistance in human ovarian cancer. Thus, it warrants further clinical investigation.

Ovarian cancer (OC) is a common malignancy characterized by disseminated peritoneal metastases. Smad ubiquitin regulatory factor 2 (SMURF2) is involved in OC progression by stabilizing receptor for activated C kinase 1 (RACK1). However, the functions and mechanisms of action of SMURF2 in OC remain unclear. This biological function of SMURF2 in OC and its potential mechanisms of action were investigated in this study. The expression of SMURF2 in ovarian tumor tissues, patient serum, and OC cell lines was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and/or western blotting. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, flow cytometry, and terminal deoxynucleotidyl transferase dUTPnick-endlabeling(TUNEL) were used for detecting cell proliferation and apoptosis. Autophagosomes in SKOV3 cells were observed using transmission electron microscopy. Immunohistochemistry and RT-qPCR were performed to evaluate SMURF2 expression. The levels of proteins related to autophagy and RACK1 were measured using western blotting and RT-qPCR, respectively. Western blotting was performed to assess the expression of AKT/mTOR pathway-related proteins. SMURF2 was underexpressed in OC tissues and cell lines compared with that in adjacent normal tissues or normal ovarian epithelial cells. RT-qPCR results suggested that SMURF2 was downregulated in the serum of patients with OC. SMURF2 overexpression inhibited SKOV3 cell growth and autophagy, and induced apoptosis both in vitro and in vivo. Moreover, SMURF2 overexpression suppressed RACK1 expression in SKOV3 cells. The AKT/mTOR pathway was activated by SMURF2 overexpression in SKOV3, and OC cells and tissues. SMURF2 plays a key role in OC by inhibiting cell autophagy and growth via activation of the RACK1/AKT/mTOR pathway, which might potentially be a new biomarker for OC diagnosis and therapy.

ARHI, an imprinted putative tumor suppressor gene, is expressed in normal ovarian epithelial cells, but its expression is down-regulated or lost in most ovarian cancer cell lines. Reexpression of ARHI in cancer cells induces p21(WAF1/CIP1), down-regulates cyclin D1 promoter activity and inhibits growth in cell culture and in heterografts. To determine the relevance of these observations to clinical cancer, we have now measured ARHI expression in normal, benign and malignant ovarian tissues using immunohistochemistry and in situ hybridization. Paraffin embedded tissues from 7 normal ovaries, 22 cystadenomas and 42 borderline lesions were analyzed using standard immunoperoxidase and in situ hybridization techniques to assess ARHI expression. In addition, immunohistochemistry against ARHI was performed on a tissue microarray containing 441 consecutive cases of ovarian carcinoma. Strong ARHI expression was found in normal ovarian surface epithelial cells, cysts and follicles using immunohistochemistry and in situ hybridization. Reduced ARHI expression was observed in tumors of low malignant potential as well as in invasive cancers. ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47%. When immunohistochemistry and in situ hybridization were compared, ARHI protein expression could be down-regulated in the presence of ARHI mRNA. ARHI expression was correlated with expression of p21(WAF1/CIP1) (P = 0.0074) but not with cyclin D1 and associated with prolonged disease free survival (P = 0.001). On multivariate analysis, ARHI expression, grade and stage were independent prognostic factors. ARHI expression did not correlate with overall survival. Persistence of ARHI expression in epithelial ovarian cancers correlated with prolonged disease free survival and expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

Metastasis and drug resistance remain significant contributors to cancer-related mortality globally. In ovarian cancer, a substantial 70% of cases develop resistance to the front-line therapy, cisplatin. Despite numerous proposed mechanisms for cisplatin resistance, the complete molecular events leading to this phenomenon in ovarian cancer remain incompletely understood. Mounting evidence suggests that dysregulation of microRNAs (miRNAs) plays a pivotal role in carcinogenesis, including the development of drug resistance. While various miRNAs have been identified in ovarian cancer samples and cell lines, few studies have compared miRNA expression in human paraffin ovarian samples and cisplatin-resistant high-grade serous ovarian cancer (HGSOC), the most prevalent and malignant of gynecological malignancies. This study aimed to identify key miRNAs implicated in cisplatin resistance. We performed miRNA expression profiles in RNA isolated from human paraffin ovarian tumors and ovarian cancers cell lines. Eight miRNAs were commonly increased in paraffin samples and cisplatin resistant ovarian cancer cells. Targeting these miRNAs with inhibitors significantly reduced cell proliferation by more than 50% for miR-203a, miR-96-5p, miR-10a-5p, miR-141-3p, miR-200c-3p, and miR-182-5p, and to a lesser extent, miR-296-5p and miR-1206. Inhibitors demonstrated a reduction in cell migration for all targets except miR-200c-3p and miR-10a-5p. Analysis of online databases, literature reports, and molecular pathway construction revealed that these eight miRNAs regulate key molecular pathways associated with cell proliferation, primarily through PTEN regulation in cisplatin-resistant HGSOC cells. Interrogation of the KM plotter patient database revealed that high expression of miR-1206, miR-10a, miR-141, and miR-96 correlates with a poor prognosis in ovarian cancer patients. These findings position these eight miRNAs as potential therapeutic targets and markers for cisplatin response in ovarian cancer therapy. Citation Format: Mariela Rivera-Serrano, Marienid Flores, Jose Rolon, Victor Reyes, Fatma Valiyeva, Pablo E. Vivas-Mejia. MicroRNA profiling in human samples and cell lines uncovers eight key players in the cisplain resistance of HGSOC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2989.

Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. We previously identified bone marrow stromal antigen 2 (BST2) as an endometrial cancer antigen using iTRAQ-based quantitative proteomic technology focused on cell surface membrane proteins, and also demonstrated the usefulness of an anti-BST2 monoclonal antibody (mAb) for endometrial cancer. In this study, we aimed to identify a new ovarian cancer antigen. We also aimed to develop a novel monoclonal antibody (mAb) and evaluate its preclinical efficacy against ovarian cancer. To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. As the new therapeutic target for ovarian cancer, we identified lipolysis-stimulated lipoprotein receptor (LSR) which had one of the largest significant differences in protein level between normal ovarian epithelial and ovarian cancer cell lines. Immunohistochemical analysis showed that the overall survival of ovarian serous carcinoma patients with high LSR expression was significantly shorter than those with low LSR (p = 0.0293). We newly developed anti-LSR mAb and investigated its preclinical efficacy. Anti-LSR mAb showed significant in vivo inhibition of tumor growth against a xenograft model of hLSR-positive ovarian cancer in an antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) independent manner (p = 0.0001). And anti-LSR mAb also induced G0/G1 cell cycle arrest by regulation of MEK and p44/42 MAPK activities and expression levels of cell cycle related proteins in vitro. Furthermore, anti-hLSR mAb, which crossreacts with mouse LSR, had little detectable toxicity in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition in ADCC and CDC independent manner in vivo. Anti-human LSR mAb also inhibited LSR function and showed direct tumor growth inhibition inducing G0/G1 cell cycle arrest in vitro. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Satoshi Nakagawa, Akiko Morimoto, Minoru Fujimoto, Takuhei Yokoyama, Yusuke Takahashi, Yutaka Ueda, Kiyoshi Yoshino, Eiichi Morii, Tadashi Kimura, Tetsuji Naka. Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2015-4382