Metastatic Ovarian Cancer Tissues Research Articles

Mitochondrial fission enhances IL-6-induced metastatic potential in ovarian cancer via ERK1/2 activation.

Ovarian cancer is a lethal gynecologic cancer mostly diagnosed in an advanced stage with an accumulation of ascites. Interleukin-6 (IL-6), a pro-inflammatory cytokine is highly elevated in malignant ascites and plays a pleiotropic role in cancer progression. Mitochondria are dynamic organelles that undergo fission and fusion in response to external stimuli and dysregulation in their dynamics has been implicated in cancer progression and metastasis. Here, we investigate the effect of IL-6 on mitochondrial dynamics in ovarian cancer cells (OVCs) and its impact on metastatic potential. Treatment with IL-6 on ovarian cancer cell lines (SKOV3 and PA-1) led to an elevation in the metastatic potential of OVCs. Interestingly, a positive association was observed between dynamin-related protein 1 (Drp1), a regulator of mitochondrial fission, and IL-6R in metastatic ovarian cancer tissues. Additionally, IL-6 treatment on OVCs was linked to the activation of Drp1, with a notable increase in the ratio of the inhibitory form p-Drp1(S637) to the active form p-Drp1(S616), indicating enhanced mitochondrial fission. Moreover, IL-6 treatment triggered the activation of ERK1/2, and inhibiting ERK1/2 mitigated IL-6-induced mitochondrial fission. Suppressing mitochondrial fission through siRNA transfection and a pharmacological inhibitor reduced the IL-6-induced migration and invasion of OVCs. This was further supported by 3D invasion assays using patient-derived spheroids. Altogether, our study suggests the role of mitochondrial fission in the metastatic potential of OVCs induced by IL-6. The inhibition of mitochondrial fission could be a potential therapeutic approach to suppress the metastasis of ovarian cancer.

Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer

Although the pro-tumorigenic functions of hyaluronan (HA) are well documented there is limited information on the effects and targets of different molecular weight HA. Here, we investigated the effects of 27 kDa, 183 kDa and 1000 kDa HA on ES-2 ovarian cancer cells overexpressing the stem cell associated protein, Notch3. 1000 kDA HA promoted spheroid formation in ES-2 cells mixed with ES-2 overexpressing Notch3 (1:3). We report disabled-2 (DAB2) as a novel protein regulated by 1000 kDa HA and further investigated its role in ovarian cancer. DAB2 was downregulated in ovarian cancer compared to normal tissues but increased in metastatic ovarian tumors compared to primary tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with HA synthesis enzyme HAS2, HA receptor CD44 and EMT and macrophage markers. Stromal DAB2 immunostaining was significantly increased in matched ovarian cancer tissues at relapse compared to diagnosis and associated with reduced survival. The proportion of DAB2 positive macrophages was significantly increased in metastatic ovarian cancer tissues compared to primary cancers. However, DAB2 overexpression significantly reduced invasion by both A2780 and OVCAR3 cells in vivo. Our research identifies a novel relationship between HA signalling, Notch3 and DAB2. We highlight a complex relationship of both pro-tumorigenic and tumor suppressive functions of DAB2 in ovarian cancer. Our findings highlight that DAB2 has a direct tumor suppressive role on ovarian cancer cells. The pro-tumorigenic role of DAB2 may be mediated by tumour associated macrophages and requires further investigation.

Metastatic pattern of ovarian cancer delineated by tracing the evolution of mitochondrial DNA mutations

Ovarian cancer (OC) is the most lethal gynecologic tumor and is characterized by a high rate of metastasis. Challenges in accurately delineating the metastatic pattern have greatly restricted the improvement of treatment in OC patients. An increasing number of studies have leveraged mitochondrial DNA (mtDNA) mutations as efficient lineage-tracing markers of tumor clonality. We applied multiregional sampling and high-depth mtDNA sequencing to determine the metastatic patterns in advanced-stage OC patients. Somatic mtDNA mutations were profiled from a total of 195 primary and 200 metastatic tumor tissue samples from 35 OC patients. Our results revealed remarkable sample-level and patient-level heterogeneity. In addition, distinct mtDNA mutational patterns were observed between primary and metastatic OC tissues. Further analysis identified the different mutational spectra between shared and private mutations among primary and metastatic OC tissues. Analysis of the clonality index calculated based on mtDNA mutations supported a monoclonal tumor origin in 14 of 16 patients with bilateral ovarian cancers. Notably, mtDNA-based spatial phylogenetic analysis revealed distinct patterns of OC metastasis, in which a linear metastatic pattern exhibited a low degree of mtDNA mutation heterogeneity and a short evolutionary distance, whereas a parallel metastatic pattern showed the opposite trend. Moreover, a mtDNA-based tumor evolutionary score (MTEs) related to different metastatic patterns was defined. Our data showed that patients with different MTESs responded differently to combined debulking surgery and chemotherapy. Finally, we observed that tumor-derived mtDNA mutations were more likely to be detected in ascitic fluid than in plasma samples. Our study presents an explicit view of the OC metastatic pattern, which sheds light on efficient treatment for OC patients.

Abstract 906: Targeting CD166 to overcome platinum resistance in ovarian cancer

Abstract Background: Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemoresistance and fatal tumor recurrence after primary treatment. The development of intra-peritoneal metastases is correlated with the formation of multicellular spheroids by OC cells disseminated in the peritoneal fluid. Within a spheroid, OC cells undergo an epithelial-to-mesenchymal transition (EMT), which involves changes in the expression of cell adhesion molecules (CAMs) and acquisition of cancer stem cell (CSC) characteristics, therefore protecting OC cells during chemotherapy. Cluster of differentiation 166 (CD166) is one of the CAMs involved in tumor progression and its modulation impacts cell-cell adhesion, leading to impaired ability of tumor cells to metastasize. However, limited number of studies are available on the functional role of CD166 in the initiation, progression, and chemoresistance of OC. Our goal was to investigate the contribution of CD166 in supporting chemoresistance and the tumorigenic OCSC phenotype, thus proposing this molecule as a potential new therapeutic target in OC. Methods: We compared CD166 expression in primary OC cells grown as spheroids and monolayers and in chemoresistant versus sensitive OC cell lines by q-PCR and Western Blot (WB). CD166 blockade by shRNA knockdown (KD) or anti-CD166 inhibitory antibody (clone AZN-L50) was evaluated on spheroid assay and colony formation. Combinatorial carboplatin and AZN-L50 treatment was assessed on spheroid assays and by WB of pro-apoptotic proteins. Chromatin immunoprecipitation (ChIP) assay detected the interaction between the β-catenin and the CD166 promoter. Results: OC cells grown as spheroids showed a significant increase in CD166 expression compared to monolayers. Our results also demonstrated an increase in CD166 expression levels in chemoresistant OC cell lines when compared to the chemosensitive counterpart. A comparison of normal adjacent ovarian epithelium, localized, and metastatic OC tissues on a multitissue array revealed that CD166 expression coincided with an advanced promigratory phenotype (n=88, P<0.001), correlating with poor outcome. CD166 blockade by either shRNA-mediated KD or AZN-L50 treatment in combination with carboplatin led to a significant decrease in the expression levels of stemness-related genes, spheroid and colony formation. The combination of carboplatin and CD166 blockade increased cleaved-PARP and -caspase-3 levels compared to single agent alone, indicating sustained DNA damage. Mechanistically, β-catenin-KD decreased CD166 expression levels and β-catenin coimmunoprecipitated with the CD166 promoter, suggesting that CD166 is a direct β-catenin target. Conclusions: CD166 is strongly linked to chemoresistance and is regulated by the oncogenic β-catenin pathway. Our data suggest that inhibition of CD166 along with carboplatin treatment could be a potential combination therapy in OC. Citation Format: Rula Atwani, Mayuri Prasad, George E Sandusky, Salvatore Condello. Targeting CD166 to overcome platinum resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 906.

Knockdown of Hypoxia-Inducible Factor 1α (HIF-1α) Promotes Autophagy and Inhibits Phosphatidylinositol 3-Kinase (PI3K)/AKT/Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Ovarian Cancer Cells

BackgroundOvarian cancer has the highest mortality rate among all female genital tumors because of its insidious onset and drug resistance. Hypoxia-inducible factor 1α (HIF-1α), one of the best-studied oncogenes, plays an important part in tumor adaptation to microenvironmental hypoxia and was found to be overexpressed in several malignancies, including ovarian cancer. Previous studies found that the effect of HIF-1α on cancers may be correlated with autophagy and some signaling pathways, such as PI3K/AKT/mTOR, in several tumors. However, the function and potential mechanism have not been clearly defined.Material/MethodsThe expression of HIF-1α in ovarian cancer tissues were detected by immunohistochemistry. HIF-1α was knocked down by siRNA transfection. Cell viability was examined by CCK8 and colony formation assay. Apoptosis and autophagy were detected with flow cytometry, transmission electron microscopy, and laser scanning confocal microscopy, respectively. The proteins related to autophagy and PI3K/AKT/mTOR were detected through Western blot analysis.ResultsHIF-1α was expressed at higher levels in epithelial or metastatic ovarian cancer tissue than in normal fallopian tube tissue. When HIF-1α was knocked down by siRNA in A2780 and SKOV3 cells, the viability of ovarian cancer cells was weakened, but the apoptosis and autophagy were strengthened. Accordingly, autophagosome formation increased and the expression of autophagy-related proteins LC3 and P62 increased in HIF-1α knockdown cells. The PI3K/Akt/mTOR signaling pathway was also found to be inactivated in HIF-1α knockdown cells.ConclusionsThese findings show that knockdown of HIF-1α promoted autophagy and inhibited the PI3K/AKT/mTOR signaling pathway in ovarian cancer cells.

Role of CXCL12-CXCR4 axis in ovarian cancer metastasis and CXCL12-CXCR4 blockade with AMD3100 suppresses tumor cell migration and invasion in vitro.

Ovarian cancer (OC) is a lethal gynecologic tumor, which brings its mortality to the head. CXCL12 and its receptor chemokine receptor 4 ( CXCR4) have been found to be highly expressed in OC and contribute to the disease progression by affecting tumor cell proliferation and invasion. Here, in this study, we aim to explore whether the blockade of CXCL12-CXCR4 axis with AMD3100 (a selective CXCR4 antagonist) has effects on the progression of OC. On the basis of the gene expression omnibus database of OC gene expression chips, the OC differentially expressed genes were screened by microarray analysis. OC (nonmetastatic and metastatic) and normal ovarian tissues were collected to determine the expressions of CXCL12 and CXCR4. A series of AMD3100, shRNA against CXCR4, and pCNS-CXCR4 were introduced to treat CAOV3 cells with the highest CXCR4 was assessed. Cell viability, apoptosis, migration, and invasion were all evaluated. The microarray analysis screened out the differential expression of CXCL12-CXCR4 in OC. CXCL12 and CXCR4 expressions were increased in OC tissues, particularly in the metastatic OC tissues. Downregulation of CXCR4 by AMD3100 or shRNA was observed to have a critical role in inhibiting cell proliferation, migration, and invasion of the CAOV3 OC cell line while promoting cell apoptosis. Overexpressed CXCR4 brought significantly promoting effects on the proliferation and invasiveness of OC cells. These results reinforce that the blockade of CXCL12-CXCR4 axis with AMD3100 inhibits the growth of OC cells. The antitumor role of the inhibition of CXCL12-CXCR4 axis offers a preclinical validation of CXCL12-CXCR4 axis as a therapeutic target in OC.

Metabolomics of biomarker discovery in ovarian cancer: a systematic review of the current literature.

Metabolomics is the emerging member of "omics" sciences advancing the understanding, diagnosis and treatment of many cancers, including ovarian cancer (OC). To systematically identify the metabolomic abnormalities in OC detection, and the dominant metabolic pathways associated with the observed alterations. An electronic literature search was performed, up to and including January 15th 2016, for studies evaluating the metabolomic profile of patients with OC compared to controls. QUADOMICS tool was used to assess the quality of the twenty-three studies included in this systematic review. Biological samples utilized for metabolomic analysis include: serum/plasma (n = 13), urine (n = 4), cyst fluid (n = 3), tissue (n = 2) and ascitic fluid (n = 1). Metabolites related to cellular respiration, carbohydrate, lipid, protein and nucleotide metabolism were significantly altered in OC. Increased levels of tricarboxylic acid cycle intermediates and altered metabolites of the glycolytic pathway pointed to perturbations in cellular respiration. Alterations in lipid metabolism included enhanced fatty acid oxidation, abnormal levels of glycerolipids, sphingolipids and free fatty acids with common elevations of palmitate, oleate, and myristate. Increased levels of glutamine, glycine, cysteine and threonine were commonly reported while enhanced degradations of tryptophan, histidine and phenylalanine were found. N-acetylaspartate, a brain amino acid, was found elevated in primary and metastatic OC tissue and ovarian cyst fluid. Further, elevated levels of ketone bodies including 3-hydroxybutyrate were commonly reported. Increased levels of nucleotide metabolites and tocopherols were consistent through out the studies. Metabolomics presents significant new opportunities for diagnostic biomarker development, elucidating previously unknown mechanisms of OC pathogenesis.

Critical role of SEMA5A expression in invasion and metastasis of ovarian cancer cell

Semaphorins are a large family of genes involved in the development and morphogenesis of the nervous system. SEMA5A has been reported as a bi-functional molecule, acting as both oncogene and tumor suppressor in different types of cancer. High expression levels of SEMA5A and its receptor, Plexin-B3, were associated with aggressiveness in pancreatic and prostate cancers. Our previous study in ovarian cancer metastasis indicates that FAK knock-down can suppress ovarian cancer cells migration and invasion. We hypothesized that SEMA5A expression promotes ovarian cancer invasion and metastasis. We investigated the expression of SEMA5A in patients with metastatic ovarian cancer (n = 43), localized tumor (n = 37) and normal ovarian tissue (n = 12) from non-malignant diseases as control with different histopathological characteristics. For Silencing of SEMA5A in vitro, we treated human ovarian cancer cells (OVCAR-3, A2780/CP70) with miR-27a and miR-27b. We observed significantly higher expression of SEMA5A protein (P= 0.001) in metastatic ovarian cancer tissue associated with poor overall survival outcomes compared to localized ovarian cancer and control. In vitro silencing of SEMA5A reduced migration and invasion of ovarian cancer cell. Our data offer opportunities for the therapeutic modulation and biomarker of metastatic ovarian cancer.

Method for Recovery and Immunoaffinity Enrichment of Membrane Proteins Illustrated with Metastatic Ovarian Cancer Tissues

Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss. In this paper, we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new (ProteoSolve-TD) buffer system. This new extraction buffer is compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots). We demonstrate near quantitative recovery of membrane proteins EDG2, EDG4, FASLG, KDR, and LAMP-3 by western blots. We have also adapted commercial ELISAs for serum-soluble membrane protein fragments (e.g., sVEGFR2) to measure the tissue titers of their transmembrane progenitors. Finally, we demonstrate the compatibility of the new buffers with immunoaffinity enrichment/mass spectrometric characterization of tissue proteins.

Tiam1, negatively regulated by miR-22, miR-183 and miR-31, is involved in migration, invasion and viability of ovarian cancer cells

Tiam1 has been implicated in the invasive phenotype of various carcinomas. However, its role in ovarian cancer remains to be elucidated, including its upstream regulatory mechanisms. In the present study, we examined the differential expression of Tiam1 in 10 normal ovarian tissues and 17 paired primary and corresponding metastatic ovarian cancer tissues by semi-quantitative immunohistochemistry. It was found that Tiam1 expression was remarkably increased in both primary and metastatic ovarian cancer tissues relative to normal ovarian tissues. Loss-of-function study revealed that downregulation of Tiam1 in SKOV-3ip and HO-8910PM cells lead to reduced cell migration and invasion, and growth inhibition without significantly affecting cell apoptosis. Subsequent regulatory study further confirmed the negative regulatory effects of miR-22, miR-183 and miR-31 on Tiam1 expression. Taken together, our data suggested that Tiam1 may be involved in the aggressive behavior of ovarian cancer, and differential expression profiles of microRNA (miRNA) may contribute to the dysregulation of Tiam1 abundance, which contributes to the invasive, migratory and viability properties of ovarian cancer cells.

Ovarian cancer immuno-reactive antigen domain containing 1 (OCIAD1), a key player in ovarian cancer cell adhesion

Objectives To identify proteins unique to metastatic ovarian cancer and test their potential involvement in cell adhesion. Methods We purified plasma membrane from paired metastatic and primary tumor tissues from patients with stage IIIC ovarian cancer. Membrane proteins unique to metastases were identified by liquid chromatographic mass spectrometry (LC-MS). The role of one of the identified proteins, ovarian cancer immuno-reactive antigen domain containing 1 (OCIAD1) in cell adhesion was determined in the presence of LPA using both over-expression and down regulation approaches. Results We identified a differentially expressed 29 kDa protein as OCIAD1 over-expressed in metastatic tissues, when compared to primary tumor tissues. OCIAD1 over-expression in HEY ovarian cancer cells increased LPA-induced, but not basal level cell adhesion to extracellular matrix proteins collagen I and laminin 10/11. This enhancement was not blocked by LY294002 and GF109203X, suggesting that OCIAD1 does not use PKC and PI3K signaling pathways to exert its effect on adhesion. In addition, LPA induced cell adhesion to collagen I was unaffected by paclitaxel (5 µM) in OCIAD1 overexpressing cells. Conclusions This is the first report that OCIAD1 is over-expressed in metastatic ovarian cancer tissues. The effect of OCIAD1 on cell adhesion may be related to its function in ovarian cancer. Failure of paclitaxel to affect ovarian cancer cell adhesion in presence of OCIAD1 raises the possibility of OCIAD1's role in tumor metastasis. Ongoing studies using a mouse orthotopic LPA-dependent ovarian cancer metastasis model are focused on strategies to inhibit the potential role of OCIAD1 in tumor metastasis.

MT110: A novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors

We have developed a novel single-chain Ep-CAM-/CD3-bispecific single-chain antibody construct designated MT110. MT110 redirected unstimulated human peripheral T cells to induce the specific lysis of every Ep-CAM-expressing tumor cell line tested. MT110 induced a costimulation independent polyclonal activation of CD4- and CD8-positive T cells as seen by de novo expression of CD69 and CD25, and secretion of interferon gamma, tumor necrosis factor alpha, and interleukins 2, 4 and 10. CD8-positive T cells made the major contribution to redirected tumor cell lysis by MT110. With a delay, CD4-positive cells could also contribute presumably as consequence of a dramatic upregulation of granzyme B expression. MT110 was highly efficacious in a NOD/SCID mouse model with subcutaneously growing SW480 human colon cancer cells. Five daily doses of 1 μg MT110 on days 0–4 completely prevented tumor outgrowth in all mice treated. The bispecific antibody construct also led to a durable eradication of established tumors in all mice treated with 1 μg doses of MT110 on days 8–12 after tumor inoculation. Finally, MT110 could eradicate patient-derived metastatic ovarian cancer tissue growing under the skin of NOD/SCID mice. MT110 appears as an attractive bispecific antibody candidate for treatment of human Ep-CAM-overexpressing carcinomas.

Absence of estrogen receptor-β expression in metastatic ovarian cancer

Objective: To evaluate the expression of estrogen receptor (ER)α and ERβ mRNA and protein in normal ovarian tissue and primary and metastatic tumors. Methods: Estrogen receptor α and ERβ expression was studied in normal ovarian biopsies ( n = 9) and primary ( n = 8) and metastatic ovarian epithelial cancers ( n = 8). Ovarian tissue was collected from surgical samples. Estrogen receptor α and ERβ mRNA expression was compared by coamplification of the mRNA of the ERs. Expression was confirmed at the protein level by Western blot analysis using antibodies specific for each receptor. Results: Among eight primary ovarian cancer samples, three had only ERα, two had only ERβ, and three had both. All eight metastatic ovarian cancer tissues expressed only ERα mRNA and protein. Biopsies from normal ovaries had ERα and ERβ mRNA and protein. Two of the ovarian epithelial cancer samples were paired and showed the same results. Conclusion: We found varying amounts of ERα and ERβ in normal ovaries, lower levels of ERβ expression in ovarian epithelial cancer primary tumors, and only ERα in metastatic tumors. Our findings indicate that a fundamental difference might exist between primary and metastatic cells, which could be caused by intrinsic or extrinsic factors that regulate ER gene expression.

Expression of the macrophage colony-stimulating factor and its receptor in gynecologic malignancies

Recently, hematopoietic growth factors have been implicated in protean nonhematopoietic processes. In the current study, expression of macrophage colony-stimulating factor (M-CSF) and its receptor (the c-fms proto-oncogene) was investigated in 42 samples of gynecologic tissues. There were 15 samples of normal ovarian and uterine tissue or benign conditions of these organs; 11 samples of primary ovarian cancer tissue; seven samples of metastatic ovarian cancer tissue; and nine samples of primary endometrial cancer tissue. Steady state transcript levels were assessed by Northern Blot analysis. Macrophage colony-stimulating factor (M-CSF) expression was not observed in any of the specimens of benign abnormalities or of normal organs; c-fms expression was detected in two of 15 (13%) of these specimens, albeit at very low levels. In contrast, 14 (78%) of 18 ovarian tumor specimens, and five (55%) of nine endometrial tumor specimens expressed M-CSF. Similarly, 16 (89%) of 18 ovarian tumor specimens and six (67%) of nine endometrial tumor specimens expressed c-fms. Most positive malignant tissues (19 [86%] of 22) showed coexpression of M-CSF and c-fms. Of interest, M-CSF and c-fms mRNA were detected in tumor, but not in adjacent normal tissue. Furthermore, M-CSF and c-fms transcripts were produced by all metastatic tumors, including two cases in which the corresponding primary tumor from the same patient was negative. Because M-CSF mediates its effects by binding to its receptor, the increased levels of both these gene products in gynecologic malignancies suggest that an interaction between M-CSF and c-fms may participate in the development of ovarian and endometrial carcinomas and especially in progression to the metastatic state.