Platinum Chemoresistance Research Articles

Macrophage Checkpoint Nanoimmunotherapy Has the Potential to Reduce Malignant Progression in Bioengineered In Vitro Models of Ovarian Cancer.

Most ovarian carcinoma (OvCa) patients present with advanced disease at the time of diagnosis. Malignant, metastatic OvCa is invasive and has poor prognosis, exposing the need for improved therapeutic targeting. High CD47 (OvCa) and SIRPα (macrophage) expression has been linked to decreased survival, making this interaction a significant target for therapeutic discovery. Even so, previous attempts have fallen short, limited by CD47 antibody specificity and efficacy. Macrophages are an important component of the OvCa tumor microenvironment and are manipulated to aid in cancer progression via CD47-SIRPα signaling. Thus, we have leveraged lipid-based nanoparticles (LNPs) to design a therapy uniquely situated to home to phagocytic macrophages expressing the SIRPα protein in metastatic OvCa. CD47-SIRPα presence was evaluated in patient histological sections using immunohistochemistry. 3D tumor spheroids generated on a hanging drop array with OVCAR3 high-grade serous OvCa and THP-1-derived macrophages created a representative model of cellular interactions involved in metastatic OvCa. Microfluidic techniques were employed to generate LNPs encapsulating SIRPα siRNA (siSIRPα) to affect the CD47-SIRPα signaling between the OvCa and macrophages. siSIRPα LNPs were characterized for optimal size, charge, and encapsulation efficiency. Uptake of the siSIRPα LNPs by macrophages was assessed by Incucyte. Following 48 h of 25 nM siSIRPα treatment, OvCa/macrophage heterospheroids were evaluated for SIRPα knockdown, platinum chemoresistance, and invasiveness. OvCa patient tumors and in vitro heterospheroids expressed CD47 and SIRPα. Macrophages in OvCa spheroids increased carboplatin resistance and invasion, indicating a more malignant phenotype. We observed successful LNP uptake by macrophages causing significant reduction in SIRPα gene and protein expressions and subsequent reversal of pro-tumoral alternative activation. Disrupting CD47-SIRPα interactions resulted in sensitizing OvCa/macrophage heterospheroids to platinum chemotherapy and reversal of cellular invasion outside of heterospheroids. Ultimately, our results strongly indicate the potential of using LNP-based nanoimmunotherapy to reduce malignant progression of ovarian cancer.

Abstract 575: Identification of SORL1 as a key regulator of chemoresistance in ovarian cancer

Abstract This study aimed to understand the molecular mechanism of platinum chemoresistance in recurrent ovarian tumors with a focus on the SORL1-regulated pathways. SORL1 is an intracellular sorting receptor that binds to HER2 in breast cancer; this promotes HER2 recycling to the cell surface, which ultimately impacts the progression and treatment of breast cancer. The role of SORL1 in ovarian cancer has not been extensively studied. Method: RNA was extracted from 15 pairs of matched primary and recurrent high grade serous ovarian tumors collected from patients. Human transcriptomic microarray was used to identify the differentially expressed genes. The identified genes were validated by RT-QPCR and western blot in five ovarian cancer cell lines that were treated or untreated with carboplatin. To study the effects of identified target gene SORL1, overexpression and knockdown in ovarian cancer cell lines were established using SORL1 plasmid and shRNA. Proliferation and response to carboplatin were analyzed using celltiter Glo and caspase-3/7 activity assays respectively. EGFR and FGFR4 were identified as the interacting proteins of SORL1 in ovarian cancer cells through FGF1 and EGF1 treatment and co-immunoprecipitation. A proximity ligation assay was performed to validate their interactions. Finally, a xenograft mouse model was used to compare the tumor forming abilities and the response to carboplatin of SORL1-knockdown ovarian cancer cells and the control cancer cells in vivo. Protein expression of SORL1, EGFR, and FGFR4 in the collected tumors was determined via western blot. Result: Transcriptomic analysis of matched human primary and recurrent ovarian tumors identified SORL1 as an upregulated gene in recurrent tumors compared to primary samples. SORL1 expression was also upregulated in ovarian cancer cells surviving carboplatin treatment. Proliferation assays demonstrated that SORL1-overexpressing and knockdown cell lines had increased and inhibited proliferative capabilities respectively. Caspase-3 activity assays showed that SORL1-overexpressing and knockdown cell lines had reduced and increased sensitivity to carboplatin treatment respectively. SORL1 was found to bind to and promote the expression of EGFR and FGFR4. When injected subcutaneously into nude mice, SORL1-knockdown cells had reduced tumor forming capabilities and increased sensitivity to carboplatin. SORL1-knockdown caused the tumors to express lower levels of SORL1, EGFR, and FGFR4 proteins in comparison with the control tumors. Conclusion: This study demonstrates that SORL1 regulates the EGFR and FGF4 pathways to promote the growth and carboplatin resistance of ovarian cancer cells. Targeting this pathway has potential to aid in the treatment of platinum-resistant ovarian cancer. Citation Format: Miranda Mansolf, Ziyan Jian, Fangfang Bi, Tobias M. Hartwich, Yang Yang-Hartwich. Identification of SORL1 as a key regulator of chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 575.

Ovarian cancer ascites confers platinum chemoresistance to ovarian cancer cells

Ovarian cancer (OC), the second most common form of gynecologic malignancy, has a poor prognosis and is often discovered in the late stages. Platinum-based chemotherapy is the first line of therapy. Nevertheless, treatment OC has proven challenging due to toxicity and the development of acquired resistance to therapy. Tumor microenvironment (TME) has been associated with platinum chemoresistance.Malignant ascites has been used as OC tumor microenvironment and its ability to induce platinum chemoresistance has been investigated.Our results suggest that exposure to OC ascites induces platinum chemoresistance in 11 of 13 cases (85 %) on OC cells. In contrast, 75 % of cirrhotic ascites (3 of 4) failed to confer platinum chemoresistance to OC cells. Cytokine array analysis revealed that IL -6 and to a lesser extent HGF were enriched in OC ascites, whereas IL -22 was enriched in cirrhotic ascites. Pharmaceutical inhibitors targeting the IL -6/ JAK pathway were mildly effective in overcoming platinum chemoresistance induced by malignant ascites. In contrast, crizotinib, an HGF/c- MET inhibitor, and 2-hydroxyestradiol (2HE2) were effective in restoring platinum chemosensitivity to OC.Our results demonstrate the importance of OC ascites in supporting platinum chemoresistance and the potential of combination therapy to restore chemosensitivity of OC cells.

Tumor-Stroma Proportion to Predict Chemoresistance in Patients With Ovarian Cancer

Platinum-based chemotherapy is the backbone of standard-of-care treatment for patients with advanced-stage, high-grade serous carcinoma (HGSC), the most common form of ovarian cancer; however, one-third of patients have or acquire chemoresistance toward platinum-based therapies. To demonstrate the utility of tumor-stroma proportion (TSP) as a predictive biomarker of chemoresistance of HGSC, progression-free survival (PFS), and overall survival (OS). This prognostic study leveraged tumors from patients with HGSC in The Cancer Genome Atlas (TCGA) cohort (1993-2013) and an independent cohort of resected clinical specimens from patients with HGSC (2004-2014) available in diagnostic and tissue microarray formats from the University of Tübingen in Germany. Data analysis was conducted from January 2021 to January 2024. Diagnosis of HGSC. Principal outcome measures were the ability of TSP to predict platinum chemoresistance, PFS, and OS. Using hematoxylin and eosin-stained slides from the Tübingen cohort (used for routine diagnostic assessment from surgical specimens) as well as tissue microarrays, representative sections of tumors for scoring of TSP were identified using previously evaluated cutoffs of 50% stroma or greater (high TSP) and less than 50% stroma (low TSP). Digitized slides from the TCGA Cohort were analyzed and scored in a similar fashion. Kaplan-Meier time-to-event functions were fit to estimate PFS and OS. The study included 103 patients (mean [SD] age, 61.6 [11.1] years) from the TCGA cohort and 192 patients (mean [SD] age at diagnosis, 63.7 [11.1] years) from the Tübingen cohort. In the TCGA cohort, there was no significant association of TSP levels with chemoresistance, PFS, or OS. However, in the Tübingen cohort, high TSP was associated with significantly shorter PFS (HR, 1.586; 95% CI, 1.093-2.302; P = .02) and OS (hazard ratio [HR], 1.867; 1.249-2.789; P = .002). Patients with chemoresistant tumors were twice as likely to have high TSP as compared to patients with chemosensitive tumors (HR, 2.861; 95% CI, 1.256-6.515; P = .01). In tissue microarrays from 185 patients from the Tübingen cohort, high TSP was again associated with significantly shorter PFS (HR, 1.675; 95% CI, 1.012-2.772 P = .04) and OS (HR, 2.491; 95% CI, 1.585-3.912; P < .001). In this prognostic study, TSP was a consistent and reproducible marker of clinical outcome measures of HGSC, including PFS, OS, and platinum chemoresistance. Accurate and cost-effective predictive biomarkers of platinum chemotherapy resistance are needed to identify patients most likely to benefit from standard treatments, and TSP can easily be implemented and integrated into prospective clinical trial design and adapted to identify patients who are least likely to benefit long-term from conventional platinum-based cytotoxic chemotherapy treatment at the time of initial diagnosis.

Secreted Soluble Factors from Tumor-Activated Mesenchymal Stromal Cells Confer Platinum Chemoresistance to Ovarian Cancer Cells.

Ovarian cancer (OC) ranks as the second most common type of gynecological malignancy, has poor survival rates, and is frequently diagnosed at an advanced stage. Platinum-based chemotherapy, such as carboplatin, represents the standard-of-care for OC. However, toxicity and acquired resistance to therapy have proven challenging for the treatment of patients. Chemoresistance, a principal obstacle to durable response in OC patients, is attributed to alterations within the cancer cells, and it can also be mediated by the tumor microenvironment (TME). In this study, we report that conditioned medium (CM) derived from murine and human stromal cells, MS-5 and HS-5, respectively, and tumor-activated HS-5, was active in conferring platinum chemoresistance to OC cells. Moreover, CM derived from differentiated murine pre-adipocyte (3T3-L1), but not undifferentiated pre-adipocyte cells, confers platinum chemoresistance to OC cells. Interestingly, CM derived from tumor-activated HS-5 was more effective in conferring chemoresistance than was CM derived from HS-5 cells. Various OC cells exhibit variable sensitivity to CM activity. Exploring CM content revealed the enrichment of a number of soluble factors in the tumor-activated HS-5, such as soluble uPAR (SuPAR), IL-6, and hepatocyte growth factor (HGF). FDA-approved JAK inhibitors were mildly effective in restoring platinum sensitivity in two of the three OC cell lines in the presence of CM. Moreover, Crizotinib, an ALK and c-MET inhibitor, in combination with platinum, blocked HGF's ability to promote platinum resistance and to restore platinum sensitivity to OC cells. Finally, exposure to 2-hydroxyestardiol (2HE2) was effective in restoring platinum sensitivity to OC cells exposed to CM. Our results showed the significance of soluble factors found in TME in promoting platinum chemoresistance and the potential of combination therapy to restore chemosensitivity to OC cells.

DNA Methylation Biomarkers for Prediction of Response to Platinum-Based Chemotherapy: Where Do We Stand?

Simple SummaryPlatinum-based agents are one of the most widely used chemotherapy drugs for various types of cancer. However, one of the main challenges in the application of platinum drugs is resistance, which is currently being widely investigated. Epigenetic DNA methylation-based biomarkers are promising to aid in the selection of patients, helping to foresee their platinum therapy response in advance. These biomarkers enable minimally invasive patient sample collection, short analysis, and good sensitivity. Hence, improved methodologies for the detection and quantification of DNA methylation biomarkers will facilitate their use in the choice of an optimal treatment strategy.Platinum-based chemotherapy is routinely used for the treatment of several cancers. Despite all the advances made in cancer research regarding this therapy and its mechanisms of action, tumor resistance remains a major concern, limiting its effectiveness. DNA methylation-based biomarkers may assist in the selection of patients that may benefit (or not) from this type of treatment and provide new targets to circumvent platinum chemoresistance, namely, through demethylating agents. We performed a systematic search of studies on biomarkers that might be predictive of platinum-based chemotherapy resistance, including in vitro and in vivo pre-clinical models and clinical studies using patient samples. DNA methylation biomarkers predictive of response to platinum remain mostly unexplored but seem promising in assisting clinicians in the generation of more personalized follow-up and treatment strategies. Improved methodologies for their detection and quantification, including non-invasively in liquid biopsies, are additional attractive features that can bring these biomarkers into clinical practice, fostering precision medicine.

TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells.

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

Glutathione peroxidase 4-dependent glutathione high-consumption drives acquired platinum chemoresistance in lung cancer-derived brain metastasis.

BackgroundPlatinum‐based chemotherapy is effective in inducing shrinkage of primary lung cancer lesions; however, it shows finite therapeutic efficacy in patients suffering from brain metastasis (BM). The intrinsic changes of BM cells, which contribute to the poor results remain unknown.MethodsPlatinum drug‐sensitivity was assessed by utilizing a preclinical BM model of PC9 lung adenocarcinoma cells in vitro and in vivo. High consumption of glutathione (GSH) and two associated upregulated proteins (GPX4 and GSTM1) in BM were identified by integrated metabolomics and proteomics in cell lines and verified by clinical serum sample. Gain‐of‐function and rescue experiments were implemented to reveal the impact and mechanism of GPX4 and GSTM1 on the chemosensitivity in BM. The interaction between GPX4 and GSTM1 was examined by immunoblotting and immunoprecipitation. The mechanism of upregulation of GPX4 was further uncovered by luciferase reporter assay, immunoprecipitation, and electrophoretic mobility shift assay.ResultsThe derivative brain metastatic subpopulations (PC9‐BrMs) of parental cells PC9 developed obvious resistance to platinum. Radically altered profiles of BM metabolism and protein expression compared with primary lung cancer cells were described and GPX4 and GSTM1 were identified as being responsible for the high consumption of GSH, leading to decreased chemosensitivity by negatively regulating ferroptosis. Besides, GSTM1 was found regulated by GPX4, which was transcriptionally activated by the Wnt/NR2F2 signaling axis in BM.ConclusionsCollectively, our findings demonstrated that Wnt/NR2F2/GPX4 promoted acquired chemoresistance by suppressing ferroptosis with high consumption of GSH. GPX4 inhibitor was found to augment the anticancer effect of platinum drugs in lung cancer BM, providing novel strategies for lung cancer patients with BM.

Jumonji domain containing 5 is a potential prognostic indicator in non-small cell lung cancer patients who received platinum-based chemotherapy

Background: Platinum-based chemotherapy is the cornerstone of non-small cell lung cancer (NSCLC) therapy. However, the molecular mechanisms and predictive markers of platinum chemoresistance have not been fully understood. Our recent study revealed that Jumonji domain containing 5 (JMJD5) expression in cells was elevated under DNA damage by alkylating agent or UV radiation, which suggests a potential role of JMJD5 in DNA damage related chemoresistance. However, the role of JMJD5 in NSCLC chemotherapy has not been reported. In this study, we demonstrated JMJD5 as a potential prognostic indicator in NSCLC patients who received platinum-based chemotherapy. Methods: JMJD5 protein expression level in tumor and adjacent normal tissues were detected by immunohistochemistry. Samples were from primary NSCLC patients who received platinum-based chemotherapy after surgical resection. Survival curves were presented by the Kaplan-Meier method and p value was acquired by log-rank test. Multivariate analysis was tested by Cox proportional-hazards regression method. Results: Elevated JMJD5 expression was found in 27.2% cases of tumor tissues (22/81), and high JMJD5 expression were significantly associated with poor overall survival time (OS) [HR =2.881 (1.774–9.121), P=0.001] and progression-free survival time (PFS) [HR =2.255 (1.417–5.886), P=0.004] in NSCLC patients who received platinum-based chemotherapy. In multivariate analyses, JMJD5 was proved to be an independent prognostic indictor for shorter OS [HR =2.339 (1.158–4.724), P=0.018] and PFS [HR =2.031 (1.095–3.767), P=0.025). Conclusions: High JMJD5 expression indicated a worse prognosis in NSCLC patients who received platinum-based chemotherapy, and JMJD5 may serve as a novel predictive marker in NSCLC chemotherapy.

RNAi and CRISPR/Cas9-based screens identify mismatch repair genes involved in platinum chemoresistance

Abstract Background Among cancer therapies relying on the defects in DNA repair mechanisms, the platinum agents (e.g. cisplatin or carboplatin) are most widely used. Despite being effective in majority of patients, the tumor often relapses, and these originally sensitive cancers acquire resistance towards platinum-based drugs. The mechanism of resistance development is not well understood and there is no clinical approach to prevent or diagnose it. Therefore, there is an urgent need for better understanding of DNA repair pathway interactions affecting cell response and resistance to these drugs. Methods The main aim of this study was to identify DNA repair pathways involved in drug sensitivity and resistance by RNAi knockdown and CRISPR/Cas9 knockout screenings. We tested a library of genes involved in different DNA repair pathways in the hTERT RPE-1 cell line. Silencing by RNAi and CRISPR-based knockout of the target genes were followed with drug treatments to identify repair pathway crosstalks. Results We found that silencing of ERCC1 and FANCD2 genes drastically increase cell sensitivity to cisplatin as well as towards PARP inhibitor Olaparib. By performing CRISPR screen, we have found that MSH2 (Mismatch Repair (MMR) gene) inactivation in ERCC1KO/KO cells alleviate cisplatin sensitivity of these cells but not the sensitivity towards Olaparib. These results suggest that in clinical settings, inactivation of MMR might provide a mechanism of the resistance development towards cisplatin but not towards PARP inhibitors. Therefore, knowledge about the activity of DNA repair pathways could be used to select appropriate treatments for patients. Conclusions We suggest that the comprehensive analysis of DNA repair pathway efficiencies, preferably achieved by cancer whole-genome sequencing could provide a diagnostic tool for treatment selection and could be applied in day-to-day cancer diagnostics. Legal entity responsible for the study Adam Mickiewicz University, Poznan, Poland. Funding Foundation for Polish Science. Disclosure All authors have declared no conflicts of interest.

Jumonji domain containing 5 is a potential prognostic indicator in non-small cell lung cancer patients who received platinum-based chemotherapy.

BackgroundPlatinum-based chemotherapy is the cornerstone of non-small cell lung cancer (NSCLC) therapy. However, the molecular mechanisms and predictive markers of platinum chemoresistance have not been fully understood. Our recent study revealed that Jumonji domain containing 5 (JMJD5) expression in cells was elevated under DNA damage by alkylating agent or UV radiation, which suggests a potential role of JMJD5 in DNA damage related chemoresistance. However, the role of JMJD5 in NSCLC chemotherapy has not been reported. In this study, we demonstrated JMJD5 as a potential prognostic indicator in NSCLC patients who received platinum-based chemotherapy.MethodsJMJD5 protein expression level in tumor and adjacent normal tissues were detected by immunohistochemistry. Samples were from primary NSCLC patients who received platinum-based chemotherapy after surgical resection. Survival curves were presented by the Kaplan-Meier method and p value was acquired by log-rank test. Multivariate analysis was tested by Cox proportional-hazards regression method.ResultsElevated JMJD5 expression was found in 27.2% cases of tumor tissues (22/81), and high JMJD5 expression were significantly associated with poor overall survival time (OS) [HR =2.881 (1.774–9.121), P=0.001] and progression-free survival time (PFS) [HR =2.255 (1.417–5.886), P=0.004] in NSCLC patients who received platinum-based chemotherapy. In multivariate analyses, JMJD5 was proved to be an independent prognostic indictor for shorter OS [HR =2.339 (1.158–4.724), P=0.018] and PFS [HR =2.031 (1.095–3.767), P=0.025).ConclusionsHigh JMJD5 expression indicated a worse prognosis in NSCLC patients who received platinum-based chemotherapy, and JMJD5 may serve as a novel predictive marker in NSCLC chemotherapy.

Analysis of Differentially Expressed MicroRNAs and Circulating Tumor Cells as Predictive Biomarkers of Platinum Chemoresistance in Primary Ovarian Carcinomas: A Prospective Study

Lesson Learned.Circulating tumor cells, microRNA markers, or other biomarkers merit examination as part of correlative scientific analyses in prospective clinical trials.Background.Platinum chemotherapy resistance occurs in approximately 25% of patients with ovarian carcinoma; however, no biomarkers of ovarian carcinoma chemoresistance have been validated. We performed a prospective trial designed to identify tumor‐based predictive biomarkers of platinum resistance.Methods.Tumor specimens were collected from 29 women with newly diagnosed histopathologically proven primary ovarian carcinoma. Of these, 23 women had specimens accessible for assessment and outcome data available regarding chemosensitive versus chemoresistance status via review of the medical record. Tumor slices were stained with antibodies against two microRNAs (miRNAs 29b and 199a) differentially expressed in chemoresistant ovarian cancer cell lines. Additionally, blood samples obtained at the time of diagnosis were analyzed for the presence of circulating tumor cells (CTCs).Results.The average age of the patients was 64 years, and 82.6% had high‐grade epithelial carcinomas. The baseline median CA‐125 was 464 (range 32–2,782). No statistically significant differences were observed in miR29b or 199a expression in platinum‐resistant/refractory versus platinum‐sensitive tumors. Furthermore, the presence of CTCs was not found to be statistically significantly predictive of eventual platinum resistance.Conclusion.Our analysis showed no differences in miR29b and 199a expression, and differences in baseline CTCs in women with newly diagnosed ovarian tumors were not statistically significant.

Abstract 3029: Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer

Abstract [Introduction] Ovarian cancer (OvCa) is one of the main causes of gynecologic cancer death in many countries and the number of patients is increasing. The standard treatment for advanced OvCa is cytoreductive surgery and adjuvant chemotherapy. The key drug for OvCa chemotherapy is platinum. However more than 70% of advanced stage OvCa relapse within 5 years, and the major issue to treat them is platinum resistance. We previously reported that Annexin (Anx) A4 protein was associating with chemoresistance to platinum-based cancer drugs. Nucleic acid therapeutics is promising therapy to target molecule existing intra cell membrane. The aim of this study is to evaluate the usefulness of antisense oligonucleotides (ASOs) and toovercome the platinum chemoresistance with ASOs by suppressing the expression of Anx A4 in cancer cells. [Methods] Cell line: To assess AnxA4 ASOs, two human ovarian cancer cell lines, RMG-1 and OVISE, which have platinum resistance and strongly express Anx A4, were used in each experiment. Anx A4 ASOs: We selected targeting Annexin A4 mRNA sequence in silico and made 16 ASOs. To enhance the stability, 2’, 4’-bridged nucleic acid were inserted in both 3’and 5’ends. And we analyzed suppression of Annexin A4 in ovarian clear cell cancer cell lines in vitro using real time PCR and western blotting. Platinum resistance verification: In vitro, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50values) were calculated. In vivo, we used ICRnu/nu mice xenografted subcutaneously with RMG-I and OVISE cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice. [Result] Of the 16 types of antisense oligonucleotides, we focused on 2 type of ASO, #7, #9 which had stronger knockdown effect for Anx A4 expression. In RMG-I cell, IC50value of Anx A4 ASO transfected cells were 6.42 µM in compared with 11.12 µM in control ASO transfected cells. (p=0.012) In OVISE cells, IC50value was also significantly higher in transfected Anx A4 ASO cells. (10.30 µM, 4.66 µM, p&amp;lt;0.0001) Significantly more amount of platinum had accumulated in Anx A4 ASO transfected cells in compared with control ASO transfected cells and no treatment cells.In vivo, tumor growth of mice treated with cisplatin+Anx A4 ASOs were significantly inhibited compared to cisplatin + control ASOs in both the RMG-1 and OVISE cell lines xenograft mice. [Conclusion] By transfection of ANXA4 ASO, platinum resistance has been improved both in vitro and in vivo. Targeting ANX A4 with nucleic therapeutics could be an option for platinum resistant ovarian cancer. Citation Format: Satoshi Nakagawa, Reisa Kakubari, Shinya Matsuzaki, Yutaka Ueda, Kosuke Hiramatsu, Satoshi Serada, Minoru Fujimoto, Tadashi Kimura, Tetsuji Naka. Targeting Annexin A4 with antisense oligonucleotides improves platinum resistance of ovarian cancer [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 3029.

New Challenges in Tumor Mutation Heterogeneity in Advanced Ovarian Cancer by a Targeted Next-Generation Sequencing (NGS) Approach.

Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III–IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD < 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor.

Tumor stroma proportion to predict platinum chemoresistance in primary ovarian carcinomas: A prospective study.

5581 Background: Platinum chemotherapy resistance occurs in approximately 25% of patients with ovarian carcinoma and represents a major barrier to effective care of this patient population. To date there are no effective nor validate predictive biomarkers of chemoresistance of ovarian carcinomas. We performed a prospective trial designed to enroll patients with ovarian masses suspicious for ovarian cancer, with the goal of identifying tumor-based predictive biomarkers of platinum resistance. Methods: 60 women were enrolled on the study. Tumor specimens were collected from 49 of these women with newly diagnosed pelvic masses, of which 29 were found to have histopathologically proven primary ovarian carcinoma. Of these primary malignant cases, 24 had specimens accessible for assessment of tumor-stroma proportion and data available regarding chemosensitive vs chemoresistance status via review of the medical record using a UMN IRB-approved protocol. Tumor slices were stained with H&amp;E and also for antibodies against two microRNAs (29b and 199a) differentially expressed in ovarian cancer cell lines. Tumor-stroma proportions were assessed by two experienced pathologists blinded to chemoresistance status, with &lt;50% stroma scored as low proportion, &gt;50% scored as high proportion. Results: The average age of assessed patients with malignant tumors was 62. 87.5% had high-grade epithelial carcinomas. Baseline median CA-125 was 416 (range 32-2782). 80% of ovarian cancer patients with chemoresistance had tumor stroma proportions &gt;50%; 73.7% of cancer patients with chemosensitive tumors had proportions &lt;50% (p-value: 0.047). Expression of miR29b or 199a did not significantly correlate with chemoresistance. Conclusions: Tumor-stroma proportion is a useful predictive biomarker of platinum chemoresistance. If validated in larger datasets, it would be a relatively inexpensive and helpful tool for tailoring treatment strategies and clinical decision-making in women with ovarian cancer.

Abstract 508: DNA repair status in a patient derived ovarian cancer xenobank

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy with a 5-year relative survival rate of 45%. The high mortality rate is in part due to the development of platinum chemoresistance occurring in more than 70% of patients after the first-line therapy. DNA repair capacity has been reported to be a key determinant for the cellular response to platinum agents. Since half of the high grade serous EOCs lacks Homologous Recombination repair, we aimed to profile the DNA repair status in a panel of well characterized 42 ovarian patient derived xenografts (PDXs) recently established in our laboratory and to correlate it with the in vivo response to a platinum based therapy. We evaluated by real time PCR (ABI-7900, Applied Biosystems) the mRNA levels of genes with a key role in Base Excision Repair (OGG1, POLB and PARP1), Homologous Recombination (BRCA1, PALB2, TP53BP1 and RAD51), Nucleotide Excision Repair (ERCC1, XPA, XPF, XPD and XPG), Fanconi Anemia pathway (FANCA, FANCC, FANCD2 and FANCF), Translesion Repair (POLEta), Mismatch Repair (MLH1), Microhomology End Joining (POLQ), Non Homologous End Joining (XRCC4, XRCC5, XRCC6 and XRCC7) and CDK12, a kinase regulating the transcription of some DNA repair genes. The methylation status of BRCA1, ERCC1, MLH1, XPA, XPG and FANCF was investigated by standard techniques. Our results show that the DNA repair genes considered were variably expressed in all the 42 PDXs analyzed, with no specific histotype-specific cluster of expression. The expression of PALB2, FANCC, FANCD2, OGG1, POLQ and RAD51 was found to correlate with the expression of at least six other genes. In high grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. BRCA1 was found to be hypermethylated in 51% of the xenografts. TP53 mutated PDXs showed statistically significant higher levels of POLQ, FANCD2, RAD51, and POLB genes. The expression of CDK12 [p=0.017], PALB2 [p=0.019] and XPF [p= 0.016] was negatively associated with the in vivo response to DDP, with resistant PDXs showing higher mRNA levels than responsive ones. We looked for association with overall survival in the TCGA data set and we found that high levels of CDK12 were associated with a worse overall survival in patients with a residual tumor after surgery minus than 2cm. These data suggest that some DNA repair genes can have a role in EOC patients’ response to DDP therapy. Particularly, CDK12 was significantly able to predict worse survival in patients undergoing optimal debulking surgery. Our xenobank will be a valid instrument to set up functional DNA repair assays, as suggested by preliminary data on primary cultures. Citation Format: Federica Guffanti, Maddalena Fratelli, Monica Ganzinelli, Francesca Ricci, Roberta Affatato, Maria Rosa Cappelletti, Daniele Generali, Francesca Bizzaro, Massimo Broggini, Raffaella Giavazzi, Giovanna Damia. DNA repair status in a patient derived ovarian cancer xenobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2017-508

Abstract 1397: Antisense oligo nucleotide of Annexin A4 improved platinum resistance in ovarian clear cell cancer

Abstract Introduction: Ovarian cancer in Japan are classified as clear cell carcinoma (CCC) more than 20 %, this percentage is higher than in Europe and United States. Besides, it is well known that CCC of ovary is highly resistant to cancer chemotherapy including carboplatin and paclitaxel treatment. We reported that Annexin A4 protein was overexpressed in ovarian CCC tissues by immunohistochemical analysis. Elevated Annexin A4 level has been detected in various epithelial cancer cell lines and have reported associating with chemoresistance to platinum-based cancer drugs. To overcome the platinum chemoresistance, we thought antisense oligonucleotides (ASOs) to be a good therapeutic option in a way of highly specific therapy for improving chemoresistance by suppressing the expression of Annexin A4 in cancer cells. Methods: We generated ASO targeting Annexin A4 with 2’, 4’-bridged nucleic acid. And we analyzed suppression of Annexin A4 in ASO-transfected RMG-I cell line (CCC) in vitro using real time PCR and western blotting. In 16 types of ASOs targeting Annexin A4, 2 ASOs were eligible. Cells were seeded in 96-well plates (2,000 cells per well). Next day, cells were transfected with ASOs using lipofectamine 2000 and were exposed to various concentrations of cisplatin (0 - 100 μM) for 72 hr. Then, drug concentrations resulting in a 50% inhibition of cell growth (IC50 values) were calculated. Intracellular platinum accumulation in Annexin A4 overexpressing cells was analyzed. To assess the improvement of platinum resistance in vivo, we used ICR nu/nu mice xenografted subcutaneously with RMG-I cells. Intraperitoneal injection of cisplatin 3mg/kg after intratumoral administration of ASO 1mg/kg each twice a week were given to xenograft mice. Results: By realtime PCR analysis, among strong 16 types of ASOs targeting Annexin A4, 2 ASOs showed strong knockdown efficiency (about 80% knockdown) compared to negative control ASOs. Western blotting analysis showing knockdown of Annexin A4 expression was observed in Annexin A4 ASO transfected cells compared to no treatment or control ASOs in vitro. ASO-transfected RMG-I cells was less resistant to cisplatin (IC50 = 3.3μM) compared with control cells (IC50 = 5.2μM) Same result were obtained with carboplatin. Platinum resistance was significantly improved in treated with Annexin A4 ASO and cisplatin compared to control ASO and cisplatin treated group in vivo. Conclusion: By transfection of ASOs targeting Annexin A4, platinum resistance have improved in vivo and in vitro, Annexin A4 have associated with efflux of platinum anti-tumor drug. In conclusion, antisense oligonucleotides for Annexin A4 will be a therapeutic option for ovarian clear cell carcinoma with chemoresistance to platinum antitumor drug. Citation Format: Reisa Kakubari, Satoshi Nakagawa, Tadashi Iwamiya, Eiji Kobayashi, Shinnya Matsuzaki, Yutaka Ueda, Kiyoshi Yoshino, Yuya Kasahara, Satoshi Obika, Tadashi Kimura, Satoshi Serada, Tetsuji Naka, Minoru Fujimoto. Antisense oligo nucleotide of Annexin A4 improved platinum resistance in ovarian clear cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2017-1397

NOTCH1 Signaling Regulates Self-Renewal and Platinum Chemoresistance of Cancer Stem-like Cells in Human Non-Small Cell Lung Cancer.

Cancer stem-like cells (CSC) are thought to drive tumor initiation, metastasis, relapse, and therapeutic resistance, but their specific pathogenic characters in many cancers, including non-small cell lung cancer (NSCLC), have yet to be well defined. Here, we develop findings that the growth factor HGF promotes CSC sphere formation in NSCLC cell populations. In patient-derived sphere-forming assays (PD-SFA) with HGF, CD49f and CD104 were defined as novel markers of lung CSC (LCSC). In particular, we isolated a subpopulation of CD166+CD49fhiCD104-Lin- LCSC present in all human specimens of NSCLC examined, regardless of their histologic subtypes or genetic driver mutations. This specific cell population was tumorigenic and capable of self-renewal, giving rise to tumor spheres in vitro and orthotopic lung tumors in immune-compromised mice. Mechanistic investigations established that NOTCH1 was preferentially expressed in this cell subpopulation and required for self-renewal via the transcription factor HES1. Through a distinct HES1-independent pathway, NOTCH1 also protected LCSCs from cisplatin-induced cell death. Notably, treatment with a γ-secretase inhibitor that blunts NOTCH1 function ablated self-renewing LCSC activity and restored platinum sensitivity in vitro and in vivo Overall, our results define the pathogenic characters of a cancer stem-like subpopulation in lung cancer, the targeting of which may relieve platinum resistance in this disease. Cancer Res; 77(11); 3082-91. ©2017 AACR.

MiR-205 and miR-218 expression is associated with carboplatin chemoresistance and regulation of apoptosis via Mcl-1 and Survivin in lung cancer cells

Lung cancer chemoresistance is the most frequent barrier in lung cancer therapy. Recent studies have indicated that microRNAs play a significant role in this mechanism and can function as either tumor suppressor or tumor promoters. However the effect of miRNA in lung cancer chemoresistance is poorly understood. Therefore, in the present study we investigated the role of two distinct miR members, the miR-205 and the tumor suppressor miR-218 in the proliferation, invasion and induction of apoptosis in lung cancer cells after carboplatin treatment. The results showed that miR-205 overexpression in A549 and H1975 lung cancer cells is concurrent with the down regulation of miR-218 and in linked with carboplatin sensitivity and chemoresistance. Interestingly, ectopic miR-218 overexpression reduced cell proliferation, invasion and migration of lung cancer cells, whereas miR-205 rescued the suppressive effect of miR-218 by altering the expression levels of the pro-apoptotic proteins PARP, Caspase 3, Bax and upregulating the anti-apoptotic markers Mcl-1 and Survivin. Taken together our findings imply that the miRNAs miR-205 and miR-218 play a key role in the development of lung cancer acquired chemoresistance and the tumor suppressor role of miR-218 in inhibiting lung cancer cell tumorigenesis and overcoming platinum chemoresistance is significant for future cancer therapeutic approaches.

Sensitization of lung cancer cells to cisplatin by β-elemene is mediated through blockade of cell cycle progression: antitumor efficacies of β-elemene and its synthetic analogs

The development of effective agents for overcoming platinum chemoresistance in lung carcinoma continues to have high priority. We have demonstrated recently that β-elemene, a novel antitumor compound, enhances cisplatin activity by triggering lung cancer cell death via apoptosis. Here, we investigated whether β-elemene acts synergistically with cisplatin to inhibit non-small cell lung cancer (NSCLC) cell proliferation by blocking cell cycle progression. β-Elemene substantially increased the suppressive effect of cisplatin on cell growth and proliferation in the NSCLC cell lines H460 and A549. Furthermore, β-elemene augmented cisplatin in the cell cycle arrest of NSCLC cells at G(2)/M. This was associated with upregulated checkpoint kinase (CHK2) expression and reduced CDC2 activity (i.e., increased phosphorylation of CDC2 on Tyr-15 and decreased phosphorylation of CDC2 on Thr-161). Moreover, β-elemene and cisplatin in combination clearly decreased the protein levels of cyclin B1 and CDC25C and increased the levels of p21(Cip1/Waf1), p27(Kip1), and GADD45 in these cells, compared with the effects of either agent alone at the same concentration. These results suggest that the β-elemene-enhanced inhibitory effect of cisplatin on lung carcinoma cell proliferation is regulated by a CHK2-mediated CDC25C/CDC2/cyclin B1 signaling pathway and leads to the blockade of cell cycle progression at G(2)/M. A comparison of the cytotoxic efficacies of β-elemene and three synthetic analogs (β-elemenol, β-elemenal, and β-elemene fluoride) in the two lung cancer cell lines revealed that β-elemenol and β-elemene fluoride had the same antitumor efficacy as β-elemene, whereas β-elemenal was appreciably more potent than β-elemene. Thus, although all three synthetic analogs of β-elemene considerably suppressed NSCLC cell growth and proliferation, β-elemenal may have greater potential as an anticancer alternative to β-elemene in treating lung cancer and other tumors.