Invasion Of Ovarian Cancer Cells Research Articles

BPA exposure enhances the metastatic aggression of ovarian cancer through the ERα/AKT/mTOR/HIF-1α signaling axis.

Long-term exposure to bisphenol A (BPA) in humans may promote ovarian cancer development. In present study, the mechanisms by which BPA mediates the aggression metastatic behavior of ovarian cancer were investigated in vitro/in vivo. The results showed that BPA (10μM) significantly promoted the proliferation, migration and invasion of human ovarian cancer cells (ES-2 and OVCAR-3cells); moreover, it promoted ES-2 and OVCAR-3cell glucose uptake, lactic acid release and intracellular ATP synthesis. After administration of 5μg/kg/day BPA, tumor volume was increased compared with that in control group. KEGG and GO enrichment analyses showed that the genes from ES-2cell in 10μM BPA-treated group were enriched mainly in central carbon metabolism and PI3K-AKT signaling pathway. Then, qRT‒PCR and western blotting results showed that BPA (10μM) increased the mRNA and protein expression levels of glycolysis-related genes and mTOR, p-AKT HIF-1α and ERα in vitro/vivo; whereas this effect was reduced after treatment with the ERα inhibitor methyl-piperidino-pyrazole. Furthermore, coimmunoprecipitation and mass spectrometry showed that BPA promoted the direct interaction of ERα with lactate dehydrogenase A. These results show that BPA directly promoted the proliferation, migration and invasion of ovarian cancer cells through the ERα/AKT/mTOR/HIF-1α signaling axis to enhance glycolysis.

Esomeprazole Alleviates Cisplatin Resistance by Inhibiting the AKT/mTOR Pathway in Ovarian Cancer Cells.

Ovarian cancer is the most lethal malignancy in gynecology. Due to limited treatment strategies and platinum resistance, newer drugs and therapeutic options are needed. Esomeprazole (ESO) has been reported to have multiple anticancer activities in preclinical and clinical research. Therefore, this study aimed to explore the anticancer effects of esomeprazole on ovarian cancer and its underlying molecular mechanisms. CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect cell viability and proliferation. The Transwell assay was used to evaluate cell migration and invasion capacity. Flow cytometry was used to detect cell apoptosis. Western blotting and immunofluorescence were used to detect protein expression. ESO effectively inhibited the cell viability, proliferation, invasion, migration, and induced apoptosis of ovarian cancer cells in a concentration-dependent manner. Treatment with ESO decreased the expression of c-MYC, SKP2, E2F1, N-cadherin, vimentin, and matrix metalloproteinase 2 (MMP2), while it increased E-cadherin, caspase3, p53, BAX, and cleaved poly (ADP-ribose) polymerase (PARP) expression, and downregulated the PI3K/AKT/mTOR signaling pathway. Furthermore, ESO combined with cisplatin showed synergistic effects in inhibiting proliferation, invasion, and migration of cisplatin-resistant ovarian cancer cells. The mechanism may be related to the increased inhibition of c-MYC, epithelial-mesenchymal transition (EMT), and the AKT/mTOR signaling pathway and enhanced the upregulation of the pro-apoptotic protein BAX and cleaved PARP levels. Moreover, ESO combined with cisplatin synergistically upregulated the expression of the DNA damage marker γH2A.X. ESO exerts multiple anticancer activities and has a synergistic effect in combination with cisplatin on cisplatin-resistant ovarian cancer cells. This study provides a promising strategy to improve chemosensitivity and overcome resistance to cisplatin in ovarian cancer.

LncRNA gadd7 promotes mitochondrial membrane potential decrease and apoptosis of alveolar type II epithelial cells by positively regulating MFN1 in an in vitro model of hyperoxia-induced acute lung injury.

The mortality and morbidity rates of ovarian cancer (OC) are high, but the underlying mechanisms of OC have not been characterized. In this study, we determined the role of Rho GTPase Activating Protein 30 (ARHGAP30) in OC progression. We measured ARHGAP30 abundance in OC tissue samples and cells using immunohistochemistry (IHC) and RT-qPCR. EdU, transwell, and annexin V/PI apoptosis assays were used to evaluate proliferation, invasiveness, and apoptosis of OC cells, respectively. The results showed that ARHGAP30 was overexpressed in OC tissue samples and cells. Inhibition of ARHGAP30 suppressed growth and metastasis of OC cells, and enhanced apoptosis. Knockdown of ARHGAP30 in OC cells significantly inhibited the PI3K/AKT/mTOR pathway. Treatment with the PI3K/AKT/mTOR pathway inhibitor buparlisib simulated the effects of ARHGAP30 knockdown on growth, invasiveness, and apoptosis of OC cells. Following buparlisib treatment, the expression levels of p-PI3K, p-AKT, and p-mTOR were significantly decreased. Furthermore, buparlisib inhibited the effects of ARHGAP30 upregulation on OC cell growth and invasiveness. In conclusion, ARHGAP30 regulated the PI3K/AKT/mTOR pathway to promote progression of OC.

Autophagy-related gene PXN as a prognostic marker: Promotion of ovarian cancer progression by suppressing the p110β/Vps34/Beclin1 pathway.

Among gynecological malignancies, ovarian cancer has the highest mortality rate and has sparked widespread interest in studying the mechanisms underlying ovarian cancer development. Based on TCGA and GEO databases, we investigated the highly expressed autophagy-related genes that determine patient prognosis using limma differential expression and Kaplan-Meier survival analyses. The biological processes associated with these genes were also predicted using GO/KEGG functional enrichment analysis. CCK-8, cell scratch, and transwell assays were used to investigate the effects of PXN on the proliferation, migration, and invasion abilities of ovarian cancer cells. Transmission electron microscopy was used to observe the autophagosomes. The expression of autophagy proteins and the PI3K/Akt/mTOR and p110β/Vps34/Beclin1 pathway proteins in ovarian cancer cells was detected using western blot; autophagy protein expression was further detected and localized using cellular immunofluorescence. A total of 724 autophagy-related genes were found to be overexpressed in ovarian -cancer tissues, with high expression of PEX3, PXN, and RB1 associated with poor prognosis in patients (p < .05). PXN activates and regulates signaling pathways related to cellular autophagy, ubiquitination, lysosomes, PI3K-Akt, and mTOR. Autophagosomes were observed in all cell groups. The increase in PXN gene expression promoted the proliferation, migration, and invasion of ovarian cancer cells, increased the expression of SQSTM1/p62 protein, decreased LC3II/LC3Ⅰ, inhibited the phosphorylation of Akt and mTOR proteins, and suppressed the expression of PI3K(p110β) and Beclin1 proteins. The decrease in PXN expression also confirmed these changes. Thus, PXN is highly expressed during ovarian cancer and is associated with poor patient prognosis. It may promote ovarian cancer cell proliferation, migration, and invasion by inhibiting cellular autophagy via suppression of the p110β/Vps34/Beclin1 pathway.

The overexpression of actin related protein 2/3 complex subunit 1B(ARPC1B) promotes the ovarian cancer progression via activation of the Wnt/β-catenin signaling pathway.

Ovarian cancer is one of the most fatal malignancies of the female reproductive system. The purpose of this study is to explore the mechanism of Actin Related Protein 2/3 Complex Subunit 1B(ARPC1B) in the progression of ovarian cancer. The expressions and prognostic value of ARPC1B in ovarian cancer were identified using the GEPIA database and the Kaplan-Meier Plotter database. The expression of ARPC1B was manipulated to evaluate its impact on the malignant phenotypes of ovarian cancer. The cell proliferation ability was analyzed through CCK-8 assay and clone formation assay. The cell migration and invasion capacity was evaluated through wound healing assay and trans well assay. Mice xenografts were conducted to measure the effects of ARPC1B on tumor development in vivo. Our data suggested that ARPC1B was overexpressed in ovarian cancer, which was correlated with a poorer survival compared to low mRNA expression of ARPC1B in ovarian cancer patients. The overexpression of ARPC1B promoted cell proliferation, migration, and invasion of ovarian cancer cells. Conversely, the knockdown of ARPC1B resulted in the opposite effect. Additionally, ARPC1B expression could activate Wnt/β-catenin signaling pathway. The administration of the β-catenin inhibitor XAV-939 abolished the promotion of cell proliferation, migration, and invasion activities induced by ARPC1B overexpression in vitro. ARPC1B was overexpressed in ovarian cancer and was correlated with poor prognosis. ARPC1B promoted ovarian cancer progression through activation of Wnt/β-catenin Signaling Pathway.

TMTC1 promotes invasiveness of ovarian cancer cells through integrins β1 and β4

Ovarian cancer is the most lethal gynecological malignancy and is characterized by peritoneal disseminated metastasis. Although O-mannosyltransferase TMTC1 is highly expressed by ovarian cancer, its pathophysiological role in ovarian cancer remains unclear. Here, immunohistochemistry showed that TMTC1 was overexpressed in ovarian cancer tissues compared with adjacent normal ovarian tissues, and high TMTC1 expression was associated with poor prognosis in patients with ovarian cancer. Silencing TMTC1 reduced ovarian cancer cell viability, migration, and invasion in vitro, as well as suppressed peritoneal tumor growth and metastasis in vivo. Moreover, TMTC1 knockdown reduced cell-laminin adhesion, which was associated with the decreased phosphorylation of FAK at pY397. Conversely, TMTC1 overexpression promoted these malignant properties in ovarian cancer cells. Glycoproteomic analysis and Concanavalin A (ConA) pull-down assays showed that integrins β1 and β4 were novel O-mannosylated protein substrates of TMTC1. Furthermore, TMTC1-mediated cell migration and invasion were significantly reversed by siRNA-mediated knockdown of integrin β1 or β4. Collectively, these results suggest that TMTC1-mediated invasive behaviors are primarily through integrins β1 and β4 and that TMTC1 is a potential therapeutic target for ovarian cancer.

MiR-2053 inhibits the growth of ovarian cancer cells by downregulating SOX4.

Ovarian cancer is one of the major gynaecological malignancies and a leading cause of cancer-related deaths worldwide. Dysregulation of miR-2053 has been reported in numerous types of cancer; however, its function in ovarian cancer remains largely unknown. In our study, the roles of miR-2053 during the development of ovarian cancer were investigated. miR-2053 expression was examined in ovarian cancer specimens and cells. Furthermore, the detailed functions and downstream targets of miR-2053 were identified. Briefly, the levels of miR-2053 were assessed in ovarian cancer tissues and paired non-cancerous samples, as well as in ovarian cancer cells using reverse transcription-quantitative polymerase chain reaction. The proliferation of cells was determined by cell counting kit-8 kit, and the levels of PCNA were also examined using immunostaining. Cell migration and invasion were evaluated using Transwell assay, and E-cad expression was assessed by immunostaining. In addition, cell apoptosis was determined by flow cytometry, and the expression of cleaved caspase-3 was examined using western blotting. The results revealed the downregulation of miR-2053 in ovarian cancer tissues and cells. Moreover, miR-2053 mimics suppressed the proliferation, migration, and invasion of ovarian cancer cells, while cell apoptosis was promoted. In addition, SOX4 was a putative downstream molecule of miR-2053 in ovarian cancer. Furthermore, SOX4 is involved in miR-2053-regulated growth and metastasis of ovarian cancer cells. In summary, miR-2053 and its novel target SOX4 could serve essential roles during tumour development of ovarian cancer, more importantly, miR-2053/SOX4 axis may be novel candidate for targeted therapy for patients with ovarian cancer.

Pharmacological inhibition of EZH2 using a covalent inhibitor suppresses human ovarian cancer cell migration and invasion.

Metastasis is the cause of poor prognosis in ovarian cancer (OC). Enhancer of Zeste homolog 2 (EZH2), a histone-lysine N-methyltransferase enzyme, promotes OC cell migration and invasion by regulating the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and matrix metalloproteinases-9 (MMP9). Hence, we speculated that EZH2-targeting therapy might suppress OC migration and invasion. In this study, the expression of EZH2, TIMP2, and MMP9 in OC tissues and cell lines was analyzed using The Cancer Genome Atlas (TCGA) database and western blotting, respectively. The effects of SKLB-03220, an EZH2 covalent inhibitor, on OC cell migration and invasion were investigated using wound-healing assays, Transwell assays, and immunohistochemistry. TCGA database analysis confirmed that the EZH2 and MMP9 mRNA expression was significantly higher in OC tissues, whereas TIMP2 expression was significantly lower than that in normal ovarian tissues. Moreover, EZH2 negatively correlated with TIMP2 and positively correlated with MMP9 expression. In addition to the anti-tumor activity of SKLB-03220 in a PA-1 xenograft model, immunohistochemistry results showed that SKLB-03220 markedly increased the expression of TIMP2 and decreased the expression of MMP9. Additionally, wound-healing and Transwell assays showed that SKLB-03220 significantly inhibited the migration and invasion of both A2780 and PA-1 cells in a concentration-dependent manner. SKLB-03220 inhibited H3K27me3 and MMP9 expression and increased TIMP2 expression in PA-1 cells. Taken together, these results indicate that the EZH2 covalent inhibitor SKLB-03220 inhibits metastasis of OC cells by upregulating TIMP2 and downregulating MMP9, and could thus serve as a therapeutic agent for OC.

Downregulation of HHLA2 inhibits ovarian cancer progression via the NF-κB signaling pathway and suppresses the expression of CA9

HHLA2 has been recently demonstrated to play multifaceted roles in several types of cancers. However, its underlying mechanism in the progression of human ovarian cancer (OC) remains largely unexplored. In the present study, we aimed to determine whether downregulation of HHLA2 inhibited malignant phenotypes of human OC cells and explore its specific mechanism. Our results revealed that downregulation of HHLA2 by transfection with a lentiviral vector significantly suppressed the viability, invasion, and migration of OC cells. Interaction study showed that downregulation of HHLA2 in OC cells reduced the expression of CA9 and increased the expressions of p-IKKβ and p-RelA. Conversely, the viability, invasion, and migration of HHLA2-depleted OC cells were increased when CA9 was upregulated. In vivo, we found that downregulation of HHLA2 significantly inhibited tumor growth, which was reversed by CA9 overexpression. In addition, downregulation of HHLA2 inhibited the OC progression via activating the NF-κB signaling pathway and decreasing the expression of CA9. Collectively, our data suggested a link between HHLA2 and NF-κB axis in the pathogenesis of OC, and these findings might provide valuable insights into the development of novel potential therapeutic targets for OC.

Development of the nonreceptor tyrosine kinase FER-targeting PROTACs as a potential strategy for antagonizing ovarian cancer cell motility and invasiveness

Aberrant overexpression of nonreceptor tyrosine kinase FER (Fps/Fes Related) has been reported in various ovarian carcinoma-derived tumor cells and is a poor prognosis factor for patient survival. It plays an essential role in tumor cell migration and invasion, acting concurrently in both kinase-dependent and -independent manners, which is not easily suppressed by conventional enzymatic inhibitors. Nevertheless, the PROteolysis-TArgeting Chimera (PROTAC) technology offers superior efficacy over traditional activity-based inhibitors by simultaneously targeting enzymatic and scaffold functions. Hence in this study, we report the development of two PROTAC compounds that promote robust FER degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a Food and Drug Administration-approved drug, brigatinib, in ovarian cancer cell motility suppression. Importantly, these PROTAC compounds also degrade multiple oncogenic FER fusion proteins identified in human tumor samples. These results lay an experimental foundation to apply the PROTAC strategy to antagonize cell motility and invasiveness in ovarian and other types of cancers with aberrant expression of FER kinase and highlight PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions.

PDLIM2 can inactivate the TGF-β/Smad pathway to inhibit the malignant behavior of ovarian cancer cells.

PDZ-LIM domain-containing Protein 2 (PDLIM2) has been reported to be downregulated in ovarian cancer. However, its exact function and mechanism in regulating ovarian cancer progression have not been elucidated. This work researched the exert effect and mechanism of PDLIM2 on ovarian cancer progression. Briefly, PDLIM2 expression in clinical tissues of ovarian cancer patients and cells was investigated by qRT-PCR and Western blot. The function of PDLIM2 on the proliferation, colony formation, migration and invasion of ovarian cancer cells was explored via cell counting kit-8, colony formation and Transwell assays. To verify whether PDLIM2 regulates ovarian cancer progression via regulating the transforming growth factor-β (TGF-β)/Smad pathway, exogenous TGF-β (10 ng/mL) treatment was performed on the PDLIM2-overexpressed ovarian cancer cells. PDLIM2 effect on the in vivo growth of ovarian cancer cells was researched by establishing a xenograft tumor model. Immunohistochemistry and Western blot were performed to protein expression in cells and tissues. As a result, PDLIM2 was low-expressed in ovarian cancer tissues/cells. PDLIM2 upregulation attenuated the proliferation, colony formation, migration, invasion and epithelial-mesenchymal transition (EMT) of ovarian cancer cells, and inactivated the TGF-β/Smad pathway. The opposite results were found in the PDLIM2-silenced ovarian cancer cells. Exogenous TGF-β treatment abrogated the inhibition of PDLIM2 on the malignant behavior of ovarian cancer cells. PDLIM2 upregulation attenuated the in vivo growth and EMT of ovarian cancer cells. Thus, PDLIM2 attenuates the proliferation, migration, invasion and EMT of ovarian cancer cells via inactivating the TGF-β/Smad pathway. PDLIM2 may be a usefully target for ovarian cancer treatment.

Knockdown of ARHGAP30 inhibits ovarian cancer cell proliferation, migration, and invasiveness by suppressing the PI3K/AKT/mTOR signaling pathway

The mortality and morbidity rates of ovarian cancer (OC) are high, but the underlying mechanisms of OC have not been characterized. In this study, we determined the role of Rho GTPase Activating Protein 30 (ARHGAP30) in OC progression. We measured ARHGAP30 abundance in OC tissue samples and cells using immunohistochemistry (IHC) and RT-qPCR. EdU, transwell, and annexin V/PI apoptosis assays were used to evaluate proliferation, invasiveness, and apoptosis of OC cells, respectively. The results showed that ARHGAP30 was overexpressed in OC tissue samples and cells. Inhibition of ARHGAP30 suppressed growth and metastasis of OC cells, and enhanced apoptosis. Knockdown of ARHGAP30 in OC cells significantly inhibited the PI3K/AKT/mTOR pathway. Treatment with the PI3K/AKT/mTOR pathway inhibitor buparlisib simulated the effects of ARHGAP30 knockdown on growth, invasiveness, and apoptosis of OC cells. Following buparlisib treatment, the expression levels of p-PI3K, p-AKT, and p-mTOR were significantly decreased. Furthermore, buparlisib inhibited the effects of ARHGAP30 upregulation on OC cell growth and invasiveness. In conclusion, ARHGAP30 regulated the PI3K/AKT/mTOR pathway to promote progression of OC.

LncRNA AC005224.4/miR-140-3p/SNAI2 regulating axis facilitates the invasion and metastasis of ovarian cancer through epithelial-mesenchymal transition.

Ovarian cancer is one of the most widespread malignant diseases of the female reproductive system worldwide. The plurality of ovarian cancer is diagnosed with metastasis in the abdominal cavity. Epithelial-mesenchymal transition (EMT) exerts a vital role in tumor cell metastasis. However, it remains unclear whether long non-coding RNA (lncRNA) are implicated in EMT and influence ovarian cancer cell invasion and metastasis. This study was designed to investigate the impacts of lncRNA AC005224.4 on ovarian cancer. LncRNA AC005224.4, miR-140-3p, and snail family transcriptional repressor 2 ( SNAI2 ) expression levels in ovarian cancer and normal ovarian tissues were determined using real-time quantitative polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) and Transwell (migration and invasion) assays were conducted to measure SKOV3 and CAOV-3 cell proliferation and metastasis. E-cadherin, N-cadherin, Snail, and Vimentin contents were detected using Western blot. Nude mouse xenograft assay was utilized to validate AC005224.4 effects in vivo . Dual-luciferase reporter gene assay confirmed the targeted relationship between miR-140-3p and AC005224.4 or SNAI2 . AC005224.4 and SNAI2 upregulation and miR-140-3p downregulation were observed in ovarian cancer tissues and cells. Silencing of AC005224.4 observably moderated SKOV3 and CAOV-3 cell proliferation, migration, invasion, and EMT process in vitro and impaired the tumorigenesis in vivo . miR-140-3p was a target of AC005224.4 and its reduced expression level was mediated by AC005224.4. miR-140-3p mimics decreased the proliferation, migration, and invasion of ovarian cancer cells. SNAI2 was identified as a novel target of miR-140-3p and its expression level was promoted by either AC005224.4 overexpression or miR-140-3p knockdown. Overexpression of SNAI2 also facilitated ovarian cancer cell viability and metastasis. AC005224.4 was confirmed as an oncogene via sponging miR-140-3p and promoted SNAI2 expression, contributing to better understanding of ovarian cancer pathogenesis and shedding light on exploiting the novel lncRNA-directed therapy against ovarian cancer.

The myo-inositol biosynthesis rate-limiting enzyme ISYNA1 suppresses the stemness of ovarian cancer via Notch1 pathway

Cancer stem cells (CSCs) play a central role in ovarian cancer (OC), understanding regulatory mechanisms governing their stemness is critical. Here, we report ISYNA1, the rate-limiting enzyme in myo-inositol biosynthesis, as a suppressor of OC regulating cancer stemness. We identified ISYNA1 as a differentially expressed gene in normal ovary and ovarian cancer tissues, as well as OC cells and OCSCs. Low ISYNA1 expression correlated with poor prognosis in OC patients. In addition, ISYNA1 was negatively correlated with cancer stem cell (CSC) markers, and ISYNA1-related pathways were enriched in Wnt, Notch, and other critical cancer pathways. ISYNA1 deficiency promoted OC cell growth, migration, and invasion ability in vitro and in vivo. Knockdown of ISYNA1 increased stemness of OC cells, including self-renewal, CSC markers expression, ALDH activity, and proportion of CD44+/CD117+ CSCs. Conversely, ectopic overexpression of ISYNA1 suppresses cell proliferation, migration, invasion and stemness of OC cells. Mechanistically, ISYNA1 inhibits OC stemness by regulating myo-inositol to suppress Notch1 signaling. In summary, these data provide evidence that ISYNA1 act as a tumor suppressor in OC and a regulator of stemness, providing insight into potentially targetable pathways for ovarian cancer therapy.

SPTBN2 regulates endometroid ovarian cancer cell proliferation, invasion and migration via ITGB4‑mediated focal adhesion and ECM receptor signalling pathway.

Ovarian cancer is as a major contributor to gynaecologic death globally. The present study aimed to investigate the regulatory role of spectrin β non-erythrocytic 2 gene (SPTBN2) in endometroid ovarian cancer and its mechanism of action. According to the Gene Expression Profiling Interactive Analysis (GEPIA) database, SPTBN2 expression is elevated in ovarian cancer tissues and higher SPTBN2 expression indicated a worse prognosis. The present study assessed SPTBN2 mRNA and protein expression levels by reverse transcription-quantitative PCR and western blotting, respectively. Cell viability, proliferation, migration and invasion were assessed with Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine incorporation, wound healing and Transwell assays, respectively. SPTBN2 expression was notably enhanced in ovarian cancer cell lines, especially in A2780 cells compared with HOSEPiC cells (P<0.001). Following transfection with small interfering (si)RNA targeting SPTBN2, the viability, proliferation, migration and invasion of A2780 cells were decreased compared with those of A2780 cells transfected with siRNA-NC (P<0.001). Gene Set Enrichment Analysis database revealed that SPTBN2 was primarily enriched in 'focal adhesion' and 'extracellular matrix (ECM)-receptor interaction', whereas SPTBN2 was significantly associated with integrin β4 (ITGB4) in the GEPIA database. In addition, rescue experiments were performed to determine the mechanism of SPTBN2 in endometroid ovarian cancer. ITGB4 overexpression reversed the inhibitory effects of the SPTBN2 knockdown on viability, proliferation, migration and invasion of A2780 cells (P<0.05). The impacts of SPTBN2 on the expression of focal adhesion and downstream ECM receptor signalling-related proteins, including Src and p-FAK/FAK, were significantly reversed by ITGB4 overexpression (P<0.01). Collectively, SPTBN2 may regulate endometroid ovarian cancer cell proliferation, invasion and migration through the ITGB4-mediated focal adhesion and ECM receptor signalling pathway.

Silencing of FAM111B inhibits tumor growth and promotes apoptosis by decreasing AKT activity in ovarian cancer.

Ovarian cancer is the most lethal gynecological tumor in women worldwide. FAM111B (family with sequence similarity 111 member B) is an oncoprotein associated with multiple cancers, but its biological functions in ovarian cancer remain elusive. In this study, FAM111B was overexpressed in ovarian cancer tissues and cell lines. Functional studies in vitro revealed that silencing of FAM111B inhibited ovarian cancer cell proliferation, invasion, and migration, as well as increased cell apoptosis. Furthermore, FAM111B silencing arrested the ovarian cancer cell cycle at the G1/S phase. Furthermore, western blot assays demonstrated that silencing of FAM111B resulted in downregulation of phospho-AKT (p-AKT) protein expression, as well as upregulation of p53 and caspase-1 protein expression. The xenograft animal model of ovarian cancer demonstrated that FAM111B silencing inhibited tumor growth, enhanced cell apoptosis, and inhibited Ki-67 and proliferating cell nuclear antigen (PCNA) protein expression in vivo. Conversely, the overexpression of FAM111B exhibited opposite effects on the ovarian cancer xenograft. It was previously established that inactivating AKT inhibited ovarian cancer progression. This study found that silencing of FAM111B inhibits tumor growth and promotes apoptosis by decreasing AKT activity in ovarian cancer. Caspase-1 and p53 signaling also influenced the function of FAM111B in SKOV3 cells. Collectively, our results demonstrate that silencing of FAM111B is a potential therapeutic strategy against ovarian cancer.

Retraction: Knockdown of TMEM14A expression by RNAi inhibits the proliferation and invasion of human ovarian cancer cells.

Retraction: Knockdown of TMEM14A expression by RNAi inhibits the proliferation and invasion of human ovarian cancer cells.

Single-cell transcriptomics in ovarian cancer identify a metastasis-associated cell cluster overexpressed RAB13

BackgroundMetastasis, the leading cause of cancer-related death in patients diagnosed with ovarian cancer (OC), is a complex process that involves multiple biological effects. With the continuous development of sequencing technology, single-cell sequence has emerged as a promising strategy to understand the pathogenesis of ovarian cancer.MethodsThrough integrating 10 × single-cell data from 12 samples, we developed a single-cell map of primary and metastatic OC. By copy-number variations analysis, pseudotime analysis, enrichment analysis, and cell–cell communication analysis, we explored the heterogeneity among OC cells. We performed differential expression analysis and high dimensional weighted gene co-expression network analysis to identify the hub genes of C4. The effects of RAB13 on OC cell lines were validated in vitro.ResultsWe discovered a cell subcluster, referred to as C4, that is closely associated with metastasis and poor prognosis in OC. This subcluster correlated with an epithelial–mesenchymal transition (EMT) and angiogenesis signature and RAB13 was identified as the key marker of it. Downregulation of RAB13 resulted in a reduction of OC cells migration and invasion. Additionally, we predicted several potential drugs that might inhibit RAB13.ConclusionsOur study has identified a cell subcluster that is closely linked to metastasis in OC, and we have also identified RAB13 as its hub gene that has great potential to become a new therapeutic target for OC.

Abstract 2499: Complement activation at the interface between cancer cells and adipocytes

Abstract Introduction: Ovarian cancer (OC) preferred site of metastasis is the fat-rich omentum. The interaction between cancer cells and adipocytes induces genotypical and phenotypical changes favoring increased invasiveness and metastatic potential of OC cells. In this study we stablish a novel in-vitro direct co-culture model by using immortalized human visceral pre-adipocytes and OC cells to understand the genotypical and phenotypical changes that adipocytes coculture generates over OC cells. Materials and Methods: OVCAR5 or OVCAR8 OC cells expressing either GFP or RFP were co-cultured with matured primary immortalized human visceral pre-adipocyte (VNPAD). The cells were seeded together in direct monolayer co-culture. After co-culture, OC cells were sorted by FACS and used to evaluate cell proliferation, resistance to cisplatin and transcriptomic changes. Results: Direct co-culture (3 days) of adipocytes and OC cells increased proliferation (p &amp;lt; 0.001), invasiveness (p &amp;lt; 0.005), and induced epithelial to mesenchymal transition (EMT) of OC cells relative to cells cultured alone. Co-culture with adipocytes also increased resistance to platinum (cisplatin IC50 from 3.90 µM to 7.07 µM for OVCAR5, and from 7.54 µM to 9.35 µM for OVCAR8 cells). RNAseq of co-cultured OVCAR5 cells detected 7783 differentially expressed genes (FDR&amp;lt;0.05, fold change &amp;gt;2). The most affected pathways included the PI3K/AKT pathway, inflammatory response, complement activation, and lipid localization. Activation of the complement (C) system in cells co-cultured with adipocytes was associated with upregulation of both C3 and C5 which was confirmed by quantitative RT-PCR, western blot and ELISA. Treatment of primary OC cells with media conditioned by adipocytes increased the expression of C3 and C5 (P&amp;lt;0.05). Similarly, treatment of OVCAR5 cells with a lipid mixture increased expression of C3 and C5 (mRNA and protein). The co-culture of OC cells with adipocytes and the treatment with lipids activated the PI3K pathway and the integrated stress response (ISR) pathway mediated by ATF4. shRNA knockdown of C3 and a chemical inhibitor for the C3 and C5 receptor (sb290157) reverted the increased proliferative phenotype and activation of PI3K in cocultured OC cells. Stable knockdown of C3 with shRNA in OVCAR5 cells reduced tumor volume and tumor weight in an intraperitoneal OC xenograft model (p&amp;lt;0.05). IHC of paired primary tumors and metastatic implants in the omentum showed increased C3 expression adjacent to fat (p&amp;lt;0.05). Conclusion: Co-culture of adipocytes and cancer cells drives changes in proliferation and cisplatin responsiveness in OC cells. These changes are induced by transfer of lipids from adipocytes to cancer cells and subsequent activation of the complement proteins C3 and C5 through the ISR pathway and ATF4. Citation Format: Andres Felipe Valdivia, Hao Huang, Horacio Cardenas, Aloysius Klingelhutz, Daniela Matei. Complement activation at the interface between cancer cells and adipocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2499.

Abstract 293: Non-metabolic regulatory functions of nuclear pyruvate kinase M2 induce a MAPK mediated phenotypic switch in invasive cancers

Abstract Accumulating evidence suggests that PKM2, the differentially spliced pyruvate kinase isoform that is expressed in most cancers and embryonic tissue, is linked with epithelial-mesenchymal transition (EMT). However, how PKM2 may drive this process of tumor invasiveness remains unclear. Here we demonstrate that in metastatic hormone-sensitive and ovarian cancer cells PKM2 undergoes modification by phosphorylation on its Serine 37 residue (pS37) and that this modified protein displays distinct localization patterns in response to pro-migratory cues emanating from an artificial nanopatterned substrate mimicking the tumor associated extracellular matrix. We find that in epithelial-mesenchymal (EM) hybrid cohorts, migrating cells undergo phenotypic switching from followers to leaders and the PKM2-pS37 signals correlate with different cell phenotypes (less/more invasive and leaders vs followers). This phenotypic switching occurs as a function of cell distance from the leading edge in an expanding epithelial sheet. We found that the putative novel non-metabolic functions of PKM2 are attributed to its autoregulation that results in not only a PKM2-pS37 upregulation but also an induction in PKM2 expression via ERK signaling. Cues emanating from either the extracellular matrix (mediated via Integrin/FAK) or through growth factor signaling (EGFR signaling), culminating in ERK activation induce PKM2 phosphorylation at S37. Thus, our data suggest that an increase in PKM2pS37 is accompanied by a switch to less enzymatically active molecular complexes which can translocate to the nucleus and increase PKM2 expression through an auto-positive feedback loop resulting in increased total PKM2 concentration and function in the leader cells. To further confirm the existence of this positive feedback loop we developed a mathematical model for PKM2 regulation. This model allows for the evaluation of inputs emerging from the extracellular matrix, such as the integrin/FAK signaling, and growth factor receptor signaling both of which regulate PKM2 expression through ERK activation in a switch-like fashion. The mathematical model includes non-linearity in the phosphorylation of S37 on PKM2 and the non-linearity in the nuclear localization and upregulation of PKM2. Further experimental analysis supports the functional role of the switch, as the observed PKM2 expression states correlate with the expression of characteristic EMT markers and invasion-promoting pseudo-hypoxic stress in the leader cells. Importantly, the small molecule activator of PKM2, TEPP 46, can restore a more epithelial-like state, decrease invasion, and increase glucose uptake in these EM hybrid ovarian cancer cells. Taken together, our results highlight a link between PKM2 and cancer aggressiveness driven by novel regulatory mechanisms that support both metabolic rewiring and invasive spread of tumor cells. Citation Format: Kiran Vanaja, Maria Apostolidi, Andre Levchenko, Jesse Rinehart. Non-metabolic regulatory functions of nuclear pyruvate kinase M2 induce a MAPK mediated phenotypic switch in invasive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 293.