Uterine Serous Carcinoma Cell Research Articles

Sulindac exhibits anti-proliferative and anti-invasive effects in uterine serous carcinoma cells

PurposeUterine serous carcinoma (USC) is a highly aggressive and frequently recurring subtype of endometrial cancer with limited treatment options for advanced or recurrent stages. Sulindac, a classic non-steroidal anti-inflammatory drug, has demonstrated anti-tumor activity in several pre-clinical tumor models. This study aims to evaluate the effect of sulindac on cell proliferation and invasion in USC cells.MethodsHuman USC cell lines ARK-1 and SPEC2 were treated with different concentrations of sulindac. Cell proliferation was assessed using MTT and colony formation assays. ELISA assays measured cellular stress, cleaved caspase 3 activity, antioxidant ability, and adhesion. Cell cycle arrest was evaluated by Cellometer. The invasive capability was detected by wound healing assay. Western blotting was used to analyze the changes in protein expression induced by sulindac.ResultsExposure to sulindac decreased cellular viability in a dose-dependent manner in ARK-1 and SPEC2 cells. Sulindac effectively inhibited cell cycle progression, increased cellular stress, caused apoptosis, and reduced cell adhesion and invasion in USC cells. Additionally, sulindac decreased the expression of COX-2 and blocked phosphorylation of NF-κB induced by TNF-α.ConclusionSulindac is a potential therapeutic agent for USC that deserves further exploration in pre-clinical studies and potentially future clinical trials.

The PPP2R1A cancer hotspot mutant p.R183W increases clofarabine resistance in uterine serous carcinoma cells by a gain-of-function mechanism.

Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs. Here, we investigated the effect of the p.R183W PPP2R1A hotspot variant on treatment response to the nucleoside analogue clofarabine. USC cells stably expressing p.R183W Aα showed increased resistance to clofarabine treatment in vitro and, corroborated by decreased clofarabine-induced apoptosis, G1 phase arrest, DNA-damage (γH2AX) and activation of ATM and Chk1/2 kinases. Phenotypic rescue by pharmacologic PP2A inhibition or dicer-substrate siRNA (dsiRNA)-mediated B56δ subunit knockdown supported a gain-of-function mechanism of Aα p.R183W, promoting dephosphorylation and inactivation of deoxycytidine kinase (dCK), the cellular enzyme responsible for the conversion of clofarabine into its bioactive form. Therapeutic assessment of related nucleoside analogues (gemcitabine, cladribine) revealed similar effects, but in a cell line-dependent manner. Expression of two other PPP2R1A USC mutants (p.P179R or p.S256F) did not affect clofarabine response in our cell models, arguing for mutant-specific effects on treatment outcome as well. While our results call for PPP2R1A mutant and context-dependent effects upon clofarabine/nucleoside analogue monotherapy, combining clofarabine with a pharmacologic PP2A inhibitor proved synergistically in all tested conditions, highlighting a new generally applicable strategy to improve treatment outcome in USC.

High-dose ascorbate exerts anti-tumor activities and improves inhibitory effect of carboplatin through the pro-oxidant function pathway in uterine serous carcinoma cell lines

ObjectiveUterine serous carcinoma is a highly aggressive non-endometrioid subtype of endometrial cancer with poor survival rates overall, creating a strong need for new therapeutic strategies to improve outcomes. High-dose ascorbate (vitamin C) has been shown to inhibit cell proliferation and tumor growth in multiple preclinical models and has shown promising anti-tumor activity in combination with chemotherapy, with a favorable safety profile. We aimed to study the anti-tumor effects of ascorbate and its synergistic effect with carboplatin on uterine serous carcinoma cells. MethodsCell proliferation was evaluated by MTT and colony formation assays in ARK1, ARK2 and SPEC2 cells. Cellular stress, antioxidant ability, cleaved caspase 3 activity and adhesion were measured by ELISA assays. Cell cycle was detected by Cellometer. Invasion was measured using a wound healing assay. Changes in protein expression were determined by Western immunoblotting. ResultsHigh-dose ascorbate significantly inhibited cell proliferation, caused cell cycle arrest, induced cellular stress, and apoptosis, increased DNA damage, and suppressed cell invasion in ARK1 and SPEC2 cells. Treatment of both cells with 1 mM N-acetylcysteine reversed ascorbate-induced apoptosis and inhibition of cell proliferation. The combination of ascorbate and carboplatin produced significant synergistic effects in inhibiting cell proliferation and invasion, inducing cellular stress, causing DNA damage, and enhancing cleaved caspase 3 levels compared to each compound alone in both cells. ConclusionsAscorbate has potent antitumor activity and acts synergistically with carboplatin through its pro-oxidant effects. Clinical trials of ascorbate combined with carboplatin as adjuvant treatment of uterine serous carcinoma are worth exploring.

In Vivo and In Vitro Efficacy of Trastuzumab Deruxtecan in Uterine Serous Carcinoma.

Uterine serous carcinoma (USC) is a rare, biologically aggressive variant of endometrial cancer with a high recurrence rate and poor prognosis. HER2 overexpression (3+ positivity) by IHC and/or FISH ERBB2 gene amplification is detected in approximately one-third of patients with USC. Clinical trials incorporating trastuzumab with standard chemotherapy have recently demonstrated improved progression-free and overall survival in advanced-stage or recurrent USC that overexpresses HER2. However, a large number of patients with USC eventually developed resistance to trastuzumab. Trastuzumab deruxtecan (T-DXd) is a novel HER2-directed antibody-drug conjugate with a topoisomerase I inhibitor payload recently approved by the Food and Drug Administration (FDA) for multiple tumor indications. Here, we investigated the in vitro and in vivo efficacy of T-DXd in primary USC cell lines and xenografts with different HER2 expression. T-DXd-induced cell growth suppression in HER2-overexpressing cell lines in vitro, increased early and late apoptosis as assessed by annexin and propidium iodide staining, and, similarly to trastuzumab, T-DXd-induced significant antibody-dependent cellular cytotoxicity in the presence of peripheral blood lymphocytes. While negligible activity was detected against USC cell lines with low HER2 expression, T-DXd demonstrated significant bystander killing against USC tumors with low/negligible HER2 when such cells were admixed with HER2 3+ tumor cells in vitro. T-DXd showed tumor growth suppression in in vivo USC PDX models that overexpress HER2 at 3+ levels, prolonging survival when compared with controls, with minimal toxicity. Future clinical trials are warranted in patients with USC failing trastuzumab treatment.

Ipatasertib, an oral AKT inhibitor, in combination with carboplatin exhibits anti-proliferative effects in uterine serous carcinoma

Purpose Uterine serous carcinoma (USC) exhibits worse survival rates compared to the endometrioid subtype, and there is currently no effective treatment options for recurrence of this disease after platinum-based chemotherapy. Activation of PIK3CA/AKT/mTOR signaling pathway is a common biological feature in USC. Materials and Methods Ipatasertib (IPAT) is an investigational, orally administered, ATP-competitive, highly selective inhibitor of pan AKT that has demonstrated anti-proliferative activity in a variety of tumor cells and tumor models. In this study, we used IPAT, carboplatin and their combination to investigate the anti-tumor activity in SPEC-2 and ARK-1 cells. Results Our results indicate that IPAT combined with carboplatin at low doses was more effective at reducing proliferation, inducing apoptosis and causing cellular stress than IPAT or carboplatin alone. In particular, inhibition of the PIK3CA/AKT/mTOR pathway and induction of DNA damage were involved in the synergistic inhibition by combination treatment of cell viability in USC cells treated with the combination. Furthermore, IPAT in combination with carboplatin significantly reduced cell adhesion and inhibited cell invasion. Conclusions These findings suggest that the combination of IPAT and carboplatin has potential clinical implications for developing new USC treatment strategies.

Issue Information

Molecular OncologyVolume 17, Issue 1 p. 1-2 Issue InformationOpen Access Issue Information First published: 04 January 2023 https://doi.org/10.1002/1878-0261.13231AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Confocal microscopy image showing p-FAK-Y397 staining in ARK-1, a uterine serous carcinoma cell line. DAPI (blue), p-FAK-Y397 (green) and phalloidin (red). Read the research article by Isabel C. Lopez-Mejia, Jordi Pijuan et al. in pp. 98–118. Volume17, Issue1January 2023Pages 1-2 RelatedInformation

Oxidative stress-induced FAK activation contributes to uterine serous carcinoma aggressiveness.

Uterine serous carcinoma (USC) is an aggressive form of endometrial cancer (EC), characterized by its high propensity for metastases. In fact, while endometrioid endometrial carcinoma (EEC), which accounts for 85% of EC, presents a good prognosis, USC is the most frequently fatal. Herein, we used for the first time a peptide-based tyrosine-kinase-activity profiling approach to quantify the changes in tyrosine kinase activation between USC and EEC. Among the tyrosine kinases highly activated in USC, we identified focal adhesion kinase (FAK). We conducted mechanistic studies using cellular models. In a USC cell line, targeting FAK either by inhibitors PF-573228 and defactinib (VS-6063) or by gene silencing limits 3D cell growth and reduces cell migration. Moreover, results from our studies suggest that oxidative stress is increased in USC tumors compared to EEC ones. Reactive oxygen species (ROS) induce tyrosine phosphorylation of FAK and a concomitant tyrosine phosphorylation of paxillin, a mediator of FAK signal transduction. Mechanistically, by tracking hundreds of individual cells per condition, we show that ROS increased cell distance and migration velocity, highlighting the role of ROS-FAK-PAX signaling in cell migration. Both defactinib and ROS scavenger N-acetylcysteine (NAC) revert this effect, pointing toward ROS as potential culprits for the increase in USC cell motility. A proof of concept of the role of FAK in controlling cell growth was obtained in in vivo experiments using cancer-tissue-originated spheroids (CTOS) and a patient-derived orthotopic xenograft model (orthoxenograft/PDOX). Defactinib reduces cell proliferation and protein oxidation, supporting a pro-tumoral antioxidant role of FAK, whereas antioxidant NAC reverts FAK inhibitor effects. Overall, our data points to ROS-mediated FAK activation in USC as being responsible for the poor prognosis of this tumor type and emphasize the potential of FAK inhibition for USC treatment.

In Vivo and In Vitro Efficacy of Enhertu in Uterine Serous Carcinoma (047)

Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Graphical Abstract View Large Image Figure Viewer Download Hi-res image Graphical Abstract View Large Image Figure Viewer Download Hi-res image Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted. Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted.

Impact of the glutathione synthesis pathway on sulfasalazine-treated endometrial cancer.

Glutathione is an antioxidant that has an important role in chemotherapeutic drug resistance in cancer. Cysteine is synthesized from cystine and is transported into the cell via the xCT antiporter. Another pathway for synthesizing cysteine involves intracellular methionine. We determined whether targeting the xCT represents a promising strategy for the treatment of endometrial cancer and identified factors that predict efficacy of this treatment strategy. In uterine serous carcinoma (USC) cell lines, the combination of cisplatin and the xCT inhibitor, sulfasalazine, significantly inhibited cell growth compared with single-agent cisplatin or sulfasalazine. Sulfasalazine treatment significantly decreased intracellular glutathione levels and induced apoptosis when combined with cisplatin in USC cell lines. On the one hand, the effectiveness of combined cisplatin and sulfasalazine was not evident in endometrioid carcinoma. USC cell lines exhibited increased expression of xCT and decreased expression of cystathionine gamma lyase (CGL), which is an enzyme involved in the synthesis of cysteine from methionine. On the other hand, endometrioid carcinoma cell lines exhibited increased CGL expression or decreased xCT expression. These findings suggest that using a glutathione synthesis pathway-based approach for selecting subjects for sulfasalazine treatment may be an effective strategy for circumventing glutathione-related chemotherapeutic drug resistance in endometrial carcinoma.

In vitro and in vivo activity of DHES0815A, an antibody-drug conjugate targeting HER2/neu in uterine serous carcinoma

<h3>Objectives:</h3> Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2-amplified USC tumors at a distinct epitope from that bound by trastuzumab, pertuzumab, and T-DM1 (trastuzumab emtansine) after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A as a single agent against primary USC cell lines and xenografts. <h3>Methods:</h3> Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for <i>C-erbB2</i> gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability in USC primary cell lines after exposure to DHES0815A, the non-targeted ADC, the unconjugated antibody MHES0488A, and T-DM1 were evaluated using flow cytometry-based-assays. <i>in vivo</i> activity of these respective agents was tested against HER2/neu overexpressing USC (ARK2) xenografts. <h3>Results:</h3> High HER2/neu protein expression by IHC and <i>C-erbB2</i> gene amplification by FISH was seen in 25% (3/12) of the primary USC cell lines. Primary tumor cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (<i>p<0.05</i>). DHES0815A caused growth-inhibition and increased survival in HER2/neu overexpressing xenografts when compared to controls (<i>p<0.05</i>). The animals also tolerated DHES0815A treatment with no significant change in their body weight [Figure 1]. <h3>Conclusions:</h3> DHES0815A as a single agent is both highly selective and toxic to USC tumors overexpressing HER2/neu both <i>in vitro</i> and <i>in vivo.</i> HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel and less toxic treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy.

Abstract 911: In vitro and in vivo activity of DHES0815A, an antibody-drug conjugate targeting HER2/neu in uterine serous carcinoma

Abstract Objective: Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2 overexpressing tumors at a distinct epitope from that bound by trastuzumab and pertuzumab after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A against primary USC cell lines and xenografts. Methods: Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for C-erbB2 gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability and bystander killing in USC cell lines after exposure to DHES0815A, the non-targeted ADC, and the unconjugated antibody (i.e. MHES0488A) were evaluated using flow cytometry-based-assays. In vivo activity of DHES0815A was tested against HER2/neu overexpressing USC xenografts. Results: High HER2/neu protein expression was seen in 25% (3/12) of the primary USC cell lines. USC cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (p&amp;lt;0.001). DHES0815A did not induce significant bystander killing of HER2/neu negative tumors when admixed with HER2/neu positive tumors. DHES0815A caused growth-inhibition and increased survival in USC HER2/neu overexpressing xenografts when compared to controls (p&amp;lt;0.01). Conclusions: DHES0815A is both highly selective and toxic to USC tumors overexpressing HER2/neu both in vitro and in vivo. HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy. Citation Format: Joan Tymon R. Tymon-Rosario, Elena Bonazzoli, Stefania Bellone, Arancha Manzano, Paola Manara, Luca Zammataro, Silvia Pelligra, Adele Guglielmi, Barbara Gnutti, Burak Zeybek, Justin Harold, Dennis Mauricio, Elena Ratner, Peter Schwartz, Alessandro D. Santin. In vitro and in vivo activity of DHES0815A, an antibody-drug conjugate targeting HER2/neu in uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 911.

XCT inhibitor sulfasalazine depletes paclitaxel-resistant tumor cells through ferroptosis in uterine serous carcinoma.

Glutathione (GSH) is a primary antioxidant that protects cells against reactive oxygen species (ROS), and high levels of GSH promote cancer cell survival and resistance to chemotherapy. The glutamine transporter xCT is essential for the intracellular synthesis of GSH, whereby xCT determines the intracellular redox balance. However, whether xCT inhibition can overcome GSH-mediated resistance to chemotherapeutic agents in uterine serous carcinoma (USC) remains unclear. Thus, the present study investigated the effect of the xCT inhibitor, sulfasalazine (SAS) on cytotoxicity in paclitaxel-sensitive and -resistant USC cell lines. The molecular mechanism by which SAS induces ferroptotic cell death in paclitaxel-resistant cells was assessed. The results of the cytotoxicity assay demonstrated that SAS was more cytotoxic in paclitaxel-resistant cells compared with in -sensitive cells; however, paclitaxel cytotoxicity was not enhanced in either of the USC cell lines. Immunoblotting analysis and the cell death assays performed using ferroptosis inhibitors indicated that SAS-mediated cell death was induced through ferroptosis, and not apoptosis, in paclitaxel-resistant cells. Furthermore, ROS production was increased in paclitaxel-resistant but not in -sensitive cells, even at low SAS concentration, and JNK was activated, which is a downstream target in the Ras signaling pathway. Knockdown of JNK reversed the inhibitory effect of SAS on cell proliferation and cell death. The synthetic lethal interaction between ROS accumulation and Ras effector JNK activation may be critical for enhancing the sensitivity to ferroptotic cell death mediated by xCT inhibitor, SAS. Taken together, the results of the present study suggest that xCT inhibition may be an effective treatment for patients with recurrent paclitaxel-resistant USC.

Abstract 4819: Sacituzumab Govitecan (IMMU-132) in uterine serous carcinoma

Abstract Objective: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with poor prognosis. Sacituzumab govitecan (IMMU-132) is a novel antibody-drug conjugate (ADC) targeting trophoblast antigen 2 (Trop-2), a cell surface glycoprotein highly expressed in many epithelial tumors including USC, to deliver SN-38, the active metabolite of irinotecan. The objective of this study was to preclinically evaluate the efficacy of IMMU-132 against primary USC cell lines and xenografts. Methods: Trop-2 expression in primary tumor cell lines and USC cell viability after exposure to IMMU-132 ADC (hRS7-CL2A-SN-38), non-targeting control ADC (h679-CL2A-SN-38), and naked antibody hRS7 IgG were evaluated using RT-PCR and flow-cytometry-based-assays. Antibody-dependent-cell-cytotoxicity (ADCC) against Trop-2+ and Trop-2- USC cell lines was evaluated in vitro using 4 hr Chromium release assays. Finally, in vivo activity of Sacituzumab govitecan was tested against Trop-2+ USC xenografts by 3 twice-a-week retro-orbital injection of 500 μg of IMMU-132, control-ADC, and hRS7 naked-IgG. Results: Overexpression of Trop-2 was detected in 67% (8 out of 12) of primary USC cell lines. Primary tumors overexpressing Trop-2 were significantly more sensitive (ie, lower IC50) to IMMU-132 (hRS7-CL2A-SN-38) when compared to control ADC (h679-CL2A-SN-38). Both sacituzumab govitecan (IMMU-132) and the naked antibody hRS7 induced high level of ADCC against Trop2+ USC cell lines while no cytotoxicity was detected against Trop-2 negative tumors. In vivo experiments comparing IMMU-132 activity to control ADC and hRS7 showed a dramatically improved tumor suppression and increased survival in IMMU-132 treated mice when compared to controls (P = 0.0001 and P = 0.0002, respectively). Conclusion: IMMU-132 demonstrated remarkable antitumor activity against biologically aggressive USC overexpressing Trop-2. Our preclinical results combined with the dramatic clinical response recently reported in an USC patient treated with IMMU-132 (https://doi.org/10.1016/j.gore.2018.05.009) strongly supports further clinical development of sacituzumab govitecan in USC patients with advanced/recurrent disease. (ie, clinical trial IND 140394). Furthermore, due to a cleavable linker that can cause bystander effect, sacituzumab govitecan could be also effective in tumors with heterogenous TROP-2 expression. Citation Format: Salvatore Lopez, Chanhee Han, Burak Zeybek, Elena Bonazzoli, Anna Bianchi, Paola Manara, Stefania Bellone, Aranzazu Manzano, Emanuele Perrone, Luca Zammataro, Gary Altwerger, Alessandro D. Santin. Sacituzumab Govitecan (IMMU-132) in uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4819.

Phosphorylation of STAT1 serine 727 enhances platinum resistance in uterine serous carcinoma.

Uterine serous carcinoma (USC) is a highly aggressive histological subtype of endometrial cancers harboring highly metastatic and chemoresistant features. Our previous study showed that STAT1 is highly expressed in USC and acts as a key molecule that is positively correlated with tumor progression, but it remains unclear whether STAT1 is relevant to the malicious chemorefractory nature of USC. In the present study, we investigated the regulatory role of STAT1 toward platinum-cytotoxicity in USC. STAT1 suppression sensitized USC cells to increase cisplatin-mediated apoptosis (p < 0.001). Furthermore, phosphorylation of STAT1 was prominently observed on serine-727 (pSTAT1-Ser727), but not on tyrosine-701, in the nucleus of USC cells treated with cisplatin. Mechanistically, the inhibition of pSTAT1-Ser727 by dominant-negative plasmid elevated cisplatin-mediated apoptosis by increasing intracellular accumulation of cisplatin through upregulation of CTR1 expression. TBB has an inhibitory effect on casein kinase 2 (CK2), which phosphorylate STAT1 at serine residues. Sequential treatment with TBB and cisplatin on USC cells greatly reduced nuclear pSTAT1-Ser727, enhanced intracellular accumulation of cisplatin, and subsequently increased apoptosis. Tumor load was significantly reduced by combination therapy of TBB and cisplatin in in vivo xenograft models (p < 0.001). Our results collectively suggest that pSTAT1-Ser727 may play a key role in platinum resistance as well as tumor progression in USC. Thus, targeting the STAT1 pathway via CK2 inhibitor can be a novel method for attenuating the chemorefractory nature of USC.

Inhibition of BET Bromodomain Proteins with GS-5829 and GS-626510 in Uterine Serous Carcinoma, a Biologically Aggressive Variant of Endometrial Cancer.

Purpose: Uterine serous carcinoma (USC) is a rare and aggressive variant of endometrial cancer. Whole-exome sequencing (WES) studies have recently reported c-Myc gene amplification in a large number of USCs, suggesting c-Myc as a potential therapeutic target. We investigated the activity of novel BET bromodomain inhibitors (GS-5829 and GS-626510, Gilead Sciences Inc.) and JQ1 against primary USC cultures and USC xenografts.Experimental Design: We evaluated c-Myc expression by qRT-PCR in a total of 45 USCs including fresh-frozen tumor tissues and primary USC cell lines. We also performed IHC and Western blot experiments in 8 USC tumors. USC cultures were evaluated for sensitivity to GS-5829, GS-626510, and JQ1 in vitro using proliferation, viability, and apoptosis assays. Finally, the in vivo activity of GS-5829, GS-626510, and JQ1 was studied in USC-ARK1 and USC-ARK2 mouse xenografts.Results: Fresh-frozen USC and primary USC cell lines overexpressed c-Myc when compared with normal tissues (P = 0.0009 and 0.0083, respectively). High c-Myc expression was found in 7 of 8 of primary USC cell lines tested by qRT-PCR and 5 of 8 tested by IHC. In vitro experiments demonstrated high sensitivity of USC cell lines to the exposure to GS-5829, GS-626510, and JQ1 with BET inhibitors causing a dose-dependent decrease in the phosphorylated levels of c-Myc and a dose-dependent increase in caspase activation (apoptosis). In comparative in vivo experiments, GS-5829 and/or GS-626510 were found more effective than JQ1 at the concentrations/doses used in decreasing tumor growth in both USC-ARK1 and USC-ARK2 mouse xenograft models.Conclusions: GS-5829 and GS-626510 may represent novel, highly effective therapeutics agents against recurrent/chemotherapy-resistant USC-overexpressing c-Myc. Clinical studies with GS-5829 in patients with USC harboring chemotherapy-resistant disease are warranted. Clin Cancer Res; 24(19); 4845-53. ©2018 AACR.

Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel.

IntroductionUterine serous carcinoma (USC) is more aggressive than other subtypes of endometrial carcinoma and is associated with a poor prognosis. We analyzed the metabolomic profile of USC with acquired resistance to paclitaxel.ResultsGlutathione (GSH) concentration in PTX-1 cells was higher than in USPC-1 cells. In addition, GSH concentration in the USPC-1 cells increased after treatment with paclitaxel but was unchanged in PTX-1 cells. Glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P) concentrations in PTX-1 cells were higher than those in USPC-1 cells. G6P concentration in the USPC-1 cells was unchanged after treatment with paclitaxel, while it decreased in PTX-1 cells.ConclusionOur results indicate that increased GSH and glucose metabolism may be related to acquiring resistance to paclitaxel in USC and thus may be targets for anti-USC therapy.Materials and MethodsWe compared metabolic profiles and reactions to paclitaxel in both a wild type USC cell line (USPC-1) and PTX-1, a cell line derived from USPC-1 which acquired paclitaxel resistance, using a capillary electrophoresis CE-MS/MS system.

Abstract 383: Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel

Abstract Introduction: Uterine serous carcinoma (USC) is more aggressive than other subtypes of endometrial carcinoma and is associated with a poor prognosis. We analyzed the metabolomic profile of USC with acquired resistance to paclitaxel. Method: We compared metabolic profiles and reactions to paclitaxel in both a wild-type USC cell line (USPC-1) and PTX-1, a cell line derived from USPC-1 that acquired paclitaxel resistance, using a capillary electrophoresis CE-MS/MS system. Results: Glutathione (GSH) concentration in PTX-1 cells was higher than in USPC-1 cells. In addition, GSH concentration in the USPC-1 cells increased after treatment with paclitaxel but was unchanged in PTX-1 cells. Glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P) concentrations in PTX-1 cells were higher than those in USPC-1 cells. G6P concentration in the USPC-1 cells was unchanged after treatment with paclitaxel, while it decreased in PTX-1 cells. Conclusion: Our results indicate that increased GSH and glucose metabolism may be related to acquiring resistance to paclitaxel in USC and thus may be targets for anti-USC therapy. Citation Format: Manabu Seino, Tsuyoshi Ohta, Hirotsugu Sakaki, Takeshi Sudo, Satoru Nagase. Metabolomic analysis of uterine serous carcinoma with acquired resistance to paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 383.

Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo.

Background:Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets.Methods:Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs.Results:We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs.Conclusions:Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.

PIK3CA oncogenic mutations represent a major mechanism of resistance to trastuzumab in HER2/neu overexpressing uterine serous carcinomas

We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment.

Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines In Vitro and In Vivo

HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC) and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib, and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA-mutated and PIK3CA wild-type HER2/neu-amplified USC cell lines. Cell viability and cell-cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC xenografts. We found both single-agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long-lasting growth inhibition in both USC xenografts when compared with single-agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0-G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single-agent-treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA and pan-ErbB inhibitor-resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild-type PIK3CA resistant to chemotherapy.