Development Of Chemotherapy Resistance Research Articles

ADAR1 promotes cisplatin resistance in intrahepatic cholangiocarcinoma by regulating BRCA2 expression through A-to-I editing manner.

Aberrant A-to-I RNA editing, mediated by ADAR1 has been found to be associated with increased tumourigenesis and the development of chemotherapy resistance in various types of cancer. Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive malignancy with a poor prognosis, and overcoming chemotherapy resistance poses a significant clinical challenge. This study aimed to clarify the roles of ADAR1 in tumour resistance to cisplatin in iCCA. We discovered that ADAR1 expression is elevated in iCCA patients, particularly in those resistant to cisplatin, and associated with poor clinical outcomes. Downregulation of ADAR1 can increase the sensitivity of iCCA cells to cisplatin treatment, whereas its overexpression has the inverse effect. By integrating RNA sequencing and Sanger sequencing, we identified BRCA2, a critical DNA damage repair gene, as a downstream target of ADAR1 in iCCA. ADAR1 mediates the A-to-I editing in BRCA2 3'UTR, inhibiting miR-3157-5p binding, consequently increasing BRCA2 mRNA and protein levels. Furthermore, ADAR1 enhances cellular DNA damage repair ability and facilitates cisplatin resistance in iCCA cells. Combining ADAR1 targeting with cisplatin treatment markedly enhances the anticancer efficacy of cisplatin. In conclusion, ADAR1 promotes tumour progression and cisplatin resistance of iCCA. ADAR1 targeting could inform the development of innovative combination therapies for iCCA.

Abstract 4232: Exploring chemotherapy resistance in pancreatic cancer: Insights from a 3D organoid model

Abstract Pancreatic cancer is marked by a dismal prognosis, with a 5-year survival rate at approximately 10%. A key contributor to its lethality is the development of chemotherapy resistance, followed by early metastasis. However, studies elucidating the mechanisms behind chemotherapy resistance in pancreatic cancer are limited. Previous research has relied on 2D cell line models, which present limitations, such as uniform cell types that fail to capture tumor heterogeneity and an inaccurate representation of clinical realities, wherein resistance often develops gradually rather than through continuous exposure to high drug concentrations. In this study, we developed a more sophisticated organoid model of chemotherapy resistance. Six organoids from three patients were matched to study chemotherapy resistance in a personalized context. Comprehensive whole-exome sequencing and transcriptomic analyses were performed, along with an examination of chemotherapy resistance protein expression and EMT-related markers. Notably, differential expression levels of CK19 and Vimentin were observed between samples. Our analysis also focused on the expression of the ATP-Binding Cassette Subfamily G Member 2 (ABCG2) gene in chemoresistant organoids. Western blot analysis indicated a significant increase in ABCG2 expression in these organoids compared to controls. According to the Moffitt RNA signature, the primary and corresponding chemoresistant organoids retained their molecular subtype classifications both before and after developing chemoresistance. Gene set enrichment analysis uncovered significant upregulation of pathways related to DNA repair and cell cycle regulation, particularly the G2M checkpoint, in chemoresistant organoids. Furthermore, gene ontology functional analysis suggested a notable overrepresentation of activities related to amino acid transmembrane transport and extracellular matrix constituents, underscoring their potential role in the chemoresistance phenotype. Citation Format: Hee Seung Lee. Exploring chemotherapy resistance in pancreatic cancer: Insights from a 3D organoid model [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 4232.

Breast cancer development coordinated by changes in microenvironment setting

Breast cancer is the most common malignancy in women worldwide, and one of the leading causes of cancer death. This disease shows a significant heterogeneity due to its genomic and histological diversity. Breast cancer is classified by pathologic features (i.e. histological subtype, tumor grade) and gene expression profiles (i.e. molecular subtypes). There are complex mechanisms implicated in its progression and the development of chemotherapy resistance. In recent times, tumor stroma is increasingly being recognized as an important factor which influences tumor pathogenesis and progression. Tumor-stromal cells interactions are involved in many phases of tumor growth, by modulating different cellular processes. Tumor-infiltrating lymphocytes are proven to be clinically significant as they correlate with good prognosis, especially in triple-negative and HER2-positive breast cancer patients. However, tumor-infiltrating lymphocytes are just one of the many components of the tumor microenvironment, which includes fibroblasts, macrophages, adipocytes, vascular cells etc., but also non-cellular components. One of the main cellular components of the tumor microenvironment are the fibroblasts which are activated and differentiated into breast cancer associated fibroblasts. They secrete many growth factors, cytokines, and chemokines which influence tumor growth and dissemination. Tumor microenvironment could be a source of new biomarkers with a potential predictive and prognostic significance. This review highlights the tumor microenvironment as an important contributor to the process of cancer development with an overview of the main components and the potential impact on the prognosis of breast cancer. It’s important to expand our understanding and knowledge of tumor-stromal signalling processes which may lead to the development of more successful and individualized therapeutic strategies.

Exosome-delivered circRNA circSYT15 contributes to cisplatin resistance in cervical cancer cells through the miR-503-5p/RSF1 axis

ABSTRACT The development of chemotherapy resistance is a major obstacle for cervical cancer (CC) patients. Exosome-mediated transfer of circular RNAs (circRNAs) was found to have relevance to the CC. This study is designed to explore the role and mechanism of exosomal circRNA synaptotagmin 15 (circSYT15) on cisplatin (DDP) resistance in CC. Cell proliferation ability and apoptosis rate were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), colony formation, and flow cytometry assays. CircSYT15, microRNA-503-5p (miR-503-5p), Remodeling spacing factor 1 (RSF1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Exosomes were analyzed by a transmission electron microscope and nanoparticle tracking analysis. CD63, CD81, TSC101, Bcl-2, Bax, C-caspase 3, and RSF1 protein levels were examined by western blot assay. The binding between miR-503-5p and circSYT15 or RSF1 was predicted by circBank or Starbase and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP). The biological role of exosomal circSYT15 in DDP resistance of CC in vivo. CircSYT15 was upregulated in the DDP-resistant CC cells and exosomes isolated from DDP-resistant CC cells. CircSYT15 knockdown repressed the proliferation and drug resistance of CC and induced apoptosis in CC cells. Exosomes shuttled circSYT15 act as a sponge to affect RSF1 expression, thereby promoting proliferation and drug resistance and repressing apoptosis of sensitive CC cells. Exosomal circSYT15 boost DDP resistance of cervical cancer in vivo. Exosome-mediated transfer of circSYT15 enhanced DDP resistance in CC partly by targeting the miR-503-5p/RSF1 axis, providing a foundation for future clinical applications of CC drug resistance.

Inhibition of Ribonucleotide Reductase Induces Endoplasmic Reticulum Stress and Apoptosis, Leading to the Death of Docetaxel-resistant Prostate Cancer Cells.

The development of chemotherapy resistance in prostate cancer (PCa) patients poses a significant obstacle to disease progression. Ribonucleotide reductase is a crucial enzyme for cell division and tumor growth. Triapine, an inhibitor of ribonucleotide reductase, has shown strong anti-tumor activity in various types of cancers. However, the effect of triapine on docetaxel-resistant (DR) human PCa cells has not been explored previously. This study aimed to examine the potential anti-proliferative effects of triapine in PC3-DR (docetaxel-resistant) cells. Cell viability was determined by the MTT test, and apoptosis and cell cycle progression were analyzed by image-based cytometer. mRNA and protein expression were assessed by RT-qPCR and western blot, respectively. Triapine administration significantly reduced PC3 and PC3-DR cells' survival, while the cytotoxic effect was higher in PC3-DR cells. Cell death resulting from inhibition of ribonucleotide reductase was mediated by endoplasmic reticulum stress, induction of apoptosis, and cell cycle arrest. The findings were supported by the upregulation of caspases, Bax, Bak, P21, P27, P53, TNF-α, FAS, and FASL, and downregulation of Bcl2, Bcl-XL, cyclin-dependent kinase 2 (CDK2), CDK4, cyclins, and heat shock proteins expression. According to the data, the reduction of ABC transporter proteins and NF-ĸB expression may play a role in triapine-mediated cytotoxicity in docetaxel-resistant cells. Based on our findings, triapine emerges as a promising chemotherapeutic approach for combating docetaxel- resistant prostate cancer.

Abstract LB281: HIRA suppression is a mechanism of chemotherapy resistance in ovarian cancer

Abstract Ovarian cancer accounts for the fifth most common cause of cancer death among women, despite being relatively rare. This is in large because majority of cases are diagnosed at advanced stage and frequently recur after primary treatment. Despite numerous efforts to develop effective targeted therapies and immunotherapies for ovarian cancer the standard of care remains chemotherapies. We postulate that part of the problem is the paucity of preclinical research and clinical trials focused on dormant persister ovarian cancer cells that remain after chemotherapy treatments. Emerging evidence supports the idea that persister cancer cells undergo a period of dormancy following chemotherapy which enables their survival and drive late recurrence. Thus, there is dire need to understand the pathways that empower the establishment of dormancy following chemotherapies in ovarian cancer. Epigenetic reprogramming is notoriously known to enable cell fate transitions and the establishment of drug resistance. An important layer of epigenetic regulation frequently overlooked in cancer is the regulation of nucleosome composition via histone variants. Importantly, analysis of data from the human protein atlas revealed that histone H3.3 chaperone, Histone Regulator Protein A (HIRA) expression inversely correlated with poor prognosis, suggesting that HIRA suppression might play a role in the development of chemotherapy resistance. Here, we demonstrate that HIRA is a major regulator of cell fate transitions in ovarian cancer cells in response to chemotherapies. Our data indicates that HIRA levels are suppressed in response chemotherapies in cancer cells. Genetic suppression of HIRA in ovarian cancer cells triggered a non-poliferative state characterized by the induction of canonical dormancy markers (p27, an increase in p38 phosphorylation) and cell cycle arrest. Accordingly, HIRA suppression in these cancer cells renders them less sensitive to the standard of care chemotherapy agent paclitaxel. Together, our data demonstrates that HIRA levels play an important role in controlling cell fate decisions in ovarian cancer and regulate chemotherapy sensitivity. Citation Format: Maya Yasukawa, Stanislav Drapela, Didem Ilter, Mian M. Shahzad, Ana P. Gomes. HIRA suppression is a mechanism of chemotherapy resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB281.

Abstract 3789: Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance

Abstract Up to 90% of failures in chemotherapy treatment are associated with metastatic-related drug resistance. Extracellular vesicles (EVs) are believed to play a role in the spread of chemotherapy resistance through the horizontal transfer of oncoproteins and noncoding RNA species, but the underlying mechanisms for this are not fully understood. Here, we study EV biogenesis, release, and uptake in the context of breast cancer chemoresistance. Specifically, we characterized the bioactive cargoes in EVs secreted by chemoresistant triple negative breast cancer cells and chemoresistant estrogen receptor-positive breast cancer cell lines. Preliminary data show that these EVs contain unusually high levels of circular RNAs, small RNA species formed covalently through backsplicing and are typically noncoding. When chemosensitive cells absorb these circRNA-enriched EVs, they experience a significant increase in resistance phenotypes. Moreover, knockdown of the circRNAs via antisense oligonucleotides results in decreased chemoresistance, suggesting a role of circRNAs in the development of chemotherapy resistance. Previous studies have shown that tumor-derived exosomal circRNAs may confer resistance by sponging microRNAs. We are exploring what the circRNAs do mechanistically to promote tumor survival, and clarifying these mechanisms as a whole will serve to potentially provide therapeutic targets against chemotherapy resistance and cancer recurrence. Citation Format: Harrison Ngue, Jitendra Shrestha, Hyejin Kim, Shobha Vasudevan. Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance. [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 3789.

Temozolomide, Simvastatin and Acetylshikonin Combination Induces Mitochondrial-Dependent Apoptosis in GBM Cells, Which Is Regulated by Autophagy

Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have had more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol-lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple-combination therapy of TMZ, Simvastatin (Simva), and acetylshikonin, and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species, mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that a TMZ/Simva/ASH combination therapy induced significantly more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to the partial inhibition of autophagy flux (accumulation of LC3β-II and a decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux, as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for a more effective treatment of GBM, but further investigations are required for a better identification of the mechanisms.

Toward understanding the roles of matrix metallopeptidase 1 in ovarian cancer

Ovarian Cancer (OC) is the leading cause of gynecologic cancer-related deaths worldwide. The leading risk factors for OC-related death are OC recurrence and the development of chemotherapy resistance. Investigation into molecular differences that distinguish primary from recurrent disease and the role of the tumor microenvironment (TME) in OC progression may help identify therapeutic targets. Gene expression microarray data comparisons between 21 primary and 21 recurrent OC specimens (16 matched pairs) showed significantly increased expression of Matrix Metallopeptidase 1 (MMP1) in the recurrent specimens (p = 0.03). We, therefore, investigated MMP1 expression regulation and how endogenous and exogenous MMP1 expression influences OC cell proliferation, migration/invasion and chemosensitivity. Both endogenous MMP1 knockdown and low levels of exogenous MMP1 increased cell proliferation of the OC cell line, CAOV2 (p < 0.01 and p < 0.001, respectively). Furthermore, CAOV2 cells cultured with low exogenous MMP1 exhibited increased invasion (p = 0.04 and p = 0.002, respectively, for two shRNA-conditioned mediums, shMMP1-1 or shMMP1-2) and faster migration by wound healing assay relative to controls without MMP1 knockdown. CAOV2 MMP1 knockdown cells were also more resistant than controls to carboplatin (p = 0.04) and paclitaxel (p = 0.017). To explore the functions of cancer environmental MMP1 in different cancer cells, 3 OC cell lines (CAOV2, HEYA8 and SKOV3) were tested for their proliferation when cultured under a low MMP1 conditioned medium. Interestingly, while the proliferation was increased in CAOV2 and HEYA8 cells, it was reduced when SKOV3 OC cells were cultured with low exogenous MMP1 (CAOV2: * p = 0.01, HEYA8: **** p = 0.0004, SKOV3: ** p = 0.002). These results likely reflect inherent MMP1 expression variability in OC tissues and cell lines that is at least partly dependent on other endogenous parameters of the TME, including pH, metabolic state, and oxygenation, all of which were found to alter levels of endogenous MMP1. Given the ability of MMP1 to promote oncogenic or tumor-suppressive behaviors, further study will be necessary to better understand how MMP1 contributes to promoting or restraining tumor progression in an individualized manner.

SETD2 detection may reveal response to induction therapy and survival profile in acute myeloid leukemia patients

ABSTRACTObjective:SET domain containing protein 2 (SETD2) involves in the progression and development of chemotherapy resistance in acute myeloid leukemia (AML). Hence, this study aimed to investigate the relationship of SETD2 expression with disease risk, features, treatment response, and survival profile in AML.Methods:One-hundred and sixty primary AML patients were retrospectively analyzed. Their bone marrow (BM) samples before and after induction therapy were retrieved for SETD2 detection by RT-qPCR. Moreover, SETD2 expression in BM samples of 20 disease controls (DCs) were also determined.Results:SETD2 expression was downregulated in AML patients compared to DCs (P < 0.001). Higher SETD2 expression related to white blood cells ≤10 × 109/L despite not reaching statistical significance (P = 0.062). One-hundred and nineteen (74.4%) AML patients achieved complete response (CR), while the remaining 41 (25.6%) did not achieve that. Furthermore, increased SETD2 expression was associated with CR achievement (P = 0.015). Survival analyses displayed that SETD2 high (vs. low) was related to prolonged event-free survival (EFS) (P = 0.001) and overall survival (OS) (P = 0.021). Moreover, increased SETD2 quartile was correlated with favorable EFS (P = 0.004) and OS (P = 0.042). After adjustment using multivariate Cox’s regression analysis, higher SETD2 quartile was independently related to prolonged EFS [hazard ratio (HR): 0.766, P = 0.013] and OS (HR: 0.669, P = 0.013). It was also noticed that SETD2 expression was elevated during the induction therapy (P < 0.001).Conclusion:Detection of SETD2 may assist in estimating treatment response and survival profile in AML patients.

Promising Phyto-Antioxidant Methanolic Extract from Canarium Odontophyllum Miq. (Dabai) Leaves against UVB Induced B164A5 Melanoma Cells as a Potential Skin Chemoprevention Agent

The high incidence and mortality of skin cancer along with the development of chemotherapy resistance have urged researchers to look into alternative strategies to combat cancer. Chemoprevention is one of the well-researched strategies using natural products. Canarium odontophyllum Miq. (dabai) is a local seasonal fruit that is mainly found in Sarawak, Malaysia. The leaves of the fruit harbor many medicinal useful phytochemicals that provide a new insight for chemoprevention. The present research has been planned to study the chemopreventive activity of methanolic extract of C. odontophyllum Miq. leaves on UVB induced B164A5 melanoma cells through its antioxidant profiles. The extract displayed statistically significant (p&lt;0.05) antioxidant activity by enhancing superoxide dismutase, SOD (1023.02 ± 106.74 U/mg protein) and catalase (0.12 ± 0.003 U/mg protein) activities compared to negative control. Low oxidative damage was also observed whereby the protein carbonyl and malondialdehyde levels were significantly reduced (p&lt;0.05), 1.69 ± 0.296 nmol/mg protein and 1.181 ± 0.03 nmol/mg protein respectively. In conclusion, the extract exhibited a promising skin cancer chemoprevention activity through its significant antioxidant activities. These findings evidently pave the path for further investigations in chemoprevention strategy.

Quantification of spatial pharmacogene expression heterogeneity in breast tumors.

Chemotherapeutic drug concentrations vary across different regions of tumors and this is thought to be involved in development of chemotherapy resistance. Insufficient drug delivery to some regions of the tumor may be due to spatial differences in expression of genes involved in the disposition, transport, and detoxification of drugs (pharmacogenes). Therefore, in this study, we analyzed the spatial expression of 286 pharmacogenes in six breast cancer tissues using the recently developed Visium spatial transcriptomics platform to (1) determine if these pharmacogenes are expressed heterogeneously across tumor tissue and (2) to determine which pharmacogenes have the most spatial expression heterogeneity. The spatial transcriptomics technology sequences the transcriptome of 55 um diameter barcoded sections (spots) across a tissue sample. We analyzed spatial gene expression profiles of four biobank-sourced breast tumor samples in addition to two breast tumor sample datasets from 10× Genomics. We define heterogeneity as the interquartile range of read counts. Collectively, we identified 8887 spots in tumor regions, 3814 in stroma, 44 in lymphocytes, and 116 in normal regions based on pathologist annotation of the tissues. We showed statistically significant differences in expression of pharmacogenes in tumor regions compared to surrounding non-tumor regions. We also observed that the most heterogeneously expressed genes within tumor regions were involved in reactive oxygen species (ROS)handling and detoxification mechanisms. GPX4, GSTP1, MGST3, SOD1, CYP4Z1, CYB5R3, GSTK1, and NAT1 showed the most heterogeneous expression within tumor regions. The heterogeneous expression of these pharmacogenes may have important implications for cancer therapy due to their ability to impact drug distribution and efficacy throughout the tumor. Our results suggest that chemoresistance caused by expression of GPX4, GSTP1, MGST3, and SOD1 may be intrinsic, not acquired, since the heterogeneity is not specific to chemotherapy-treated samples or cell type. Additionally, we identified candidate chemoresistance pharmacogenes that can be further tested through focused follow-up studies.

Wnt/beta-catenin signaling confers ferroptosis resistance by targeting GPX4 in gastric cancer

The development of chemotherapy resistance is the most vital obstacle to clinical efficacy in gastric cancer (GC). The dysregulation of the Wnt/beta-catenin signaling pathway is critically associated with GC development and chemotherapy resistance. Ferroptosis is a form of regulated cell death, induced by an iron-dependent accumulation of lipid peroxides during chemotherapy. However, whether the Wnt/beta-catenin signaling directly controls resistance to cell death, remains unclear. Here, we show that the activation of the Wnt/beta-catenin signaling attenuates cellular lipid ROS production and subsequently inhibits ferroptosis in GC cells. The beta-catenin/TCF4 transcription complex directly binds to the promoter region of GPX4 and induces its expression, resulting in the suppression of ferroptotic cell death. Concordantly, TCF4 deficiency promotes cisplatin-induced ferroptosis in vitro and in vivo. Thus, we demonstrate that the aberrant activation of the Wnt/beta-catenin signaling confers ferroptosis resistance and suggests a potential therapeutic strategy to enhance chemo-sensitivity for advanced GC patients.

Comparison of Colorectal Cancer Stem Cells and Oxaliplatin-Resistant Cells Unveils Functional Similarities.

Colorectal cancer is the second most common cancer in women, the third in men, and an important cause of cancer-related mortality. Recurrence and the development of chemotherapy resistance are major hindrances for patients’ treatment. The presence of cancer stem cells with chemotherapy resistance able to generate proliferating tumor cells contributes to tumor recurrence and resistance. In addition, tumor cells can develop chemoresistance through adaptation mechanisms. In this article, cancer stem cells were isolated from HT29 and SW620 colorectal cancer cell lines. Oxaliplatin resistance was induced by a single drug treatment simulating the usual guidelines of patient treatment. A comparison of these two populations showed similarities since cancer stem cells presented increased oxaliplatin resistance, and resistant cells contained an increased number of cancer stem cells. Cancer stem cells isolated from resistant cells showed increased oxaliplatin resistance. Cell invasion capacity and epithelial-mesenchymal transition were increased both in cancer stem cells and oxaliplatin-resistant cells. mRNA expression analysis showed that both cell types shared a significant proportion of commonly regulated genes. In summary, the data presented indicate that colorectal cancer stem cells and oxaliplatin-resistant cells are highly related cell populations that might have interesting implications in the development of tumor recurrence and resistance to chemotherapy.

Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells.

Aim: The development of chemotherapy resistance is the major obstacle in the treatment of advanced prostate cancer (PCa). Extracellular vesicles (EVs) secretion plays a significant role among different mechanisms contributing to chemoresistance. Hence, inhibition of EVs release may increase the efficacy of chemotherapeutic drugs against PCa. Methods: Paclitaxel (PTX) resistant PCa cells (PC3-R and DU145-R) were treated with GW4869, a known exosome biogenesis inhibitor. EVs were isolated from the conditioned media by ExoQuick-based precipitation method and characterized for concentration and size distribution by nanoparticle tracking analysis. The effect of GW4869 treatment on the survival and growth of PCa cells was assessed by MTT, and colony formation assays in vitro, and ectopic PC3-R xenografts in male athymic nude mice in vivo. The effect of other EV biogenesis inhibitors, imipramine and dimethyl amiloride (DMA), treatment was also analyzed on the survival of PC3-R cells. Results: GW4869 (10-20 µM) treatment of PTX resistant PCa cells significantly reduced the release of small EVs (50-100 nm size range) while increasing the release of larger EVs (> 150 nm in size), and inhibited their clonogenicity. Moreover, GW4869 (5-20 µM) treatment (24-72h) significantly inhibited the survival of PC3-R cells in a dose-dependent manner. We observed a similar growth inhibition with both imipramine (5-20 µg/mL) and DMA (5-20 µg/mL) treatment in PC3-R cells. Furthermore, GW4869 treatment (IP) in mice bearing PC3-R xenografts significantly reduced the tumor weight (65% reduction, P = 0.017) compared to the vehicle-treated control mice without causing any noticeable toxicity. Conclusion: Inhibiting the release of EVs could sensitize the resistant PCa cells to chemotherapy.

Long non-coding RNA CTSLP8 mediates ovarian cancer progression and chemotherapy resistance by modulating cellular glycolysis and regulating c-Myc expression through PKM2

PurposeLong non-coding RNAs (lncRNAs) play vital roles in tumor progression and resistance. Ovarian cancer (OC), a common gynecological cancer, is associated with poor prognosis as it can progress to peritoneal metastasis and develop resistance to chemotherapy. This study aimed to examine the role of lncRNAs in the development of chemotherapy resistance in OC.MethodsThe clinical samples were divided into chemotherapy-sensitive and chemotherapy-resistant groups based on the chemotherapy response at follow-up. The glycolysis levels in the two groups were analyzed using positron emission tomography/computed tomography (PET/CT) scanning and immunohistochemistry. GEO dataset analysis revealed the expression of CTSLP8 in chemotherapy-resistant patients with OC. Two pairs of normal and diamminodichloroplatinum (DDP)-resistant cells were transfected with CTSLP8 overexpression and knockdown constructs to examine the functions of CTSLP8 in the OC cells and elucidate the underlying mechanisms. The in vivo effect of CTSLP8 overexpression and knockdown on the chemotherapy response of tumors was examined using a mouse subcutaneous tumor model. The tissue chips were subjected to fluorescence in situ hybridization and immunohistochemical (IHC) staining to examine the correlation among CTSLP8 expression, DDP resistance, and prognosis in OC.ResultsThe dataset analysis demonstrated that CTSLP8 was upregulated in chemotherapy-resistant tumor tissues. CTSLP8 promoted the proliferation and development of DDP resistance in the OC cells. Moreover, CTSLP8 promoted c-Myc expression by facilitating the binding of PKM2 to the promoter region of c-Myc, thereby upregulating glycolysis. The analysis of tissue chips revealed that the upregulation of CTSLP8 was associated with the development of DDP resistance and poor prognosis in patients with OC.ConclusionsThese findings indicate that CTSLP8 forms a complex with PKM2 to regulate c-Myc, and this action results in the upregulation of cellular glycolysis, consequently promoting OC progression and development of chemotherapy resistance.Headlights1. CTSLP8 was upregulated in the chemotherapy-resistant tumor tissues.2. CTSLP8 promoted the proliferation and cisplatin resistance in the OC cells.3. CTSLP8 promoted glycolysis by facilitating the binding of PKM2 to the promoter region of c-Myc.4. Inhibition of CTSLP8 or the combination of c-Myc inhibitors with cisplatin were potential therapeutic strategies for chemotherapy-resistant of OC.

Abstract 1410: Chemotherapy resistance pathways identified in ovarian cancer patients treated with neoadjuvant chemotherapy using single cell RNA sequencing of matched tumor biopsies

Abstract Background: Women with ovarian cancer suffer from a high rate of mortality due to the development of chemotherapy resistance. To better understand the mechanisms of chemotherapy resistance we initiated a prospective study of women with ovarian cancer, which we refer to as the Ovarian Cancer Precision Medicine Initiative (OCPMI) where we collect tumor biopsies and perform comprehensive molecular analyses. Our goal is to use the molecular data from these specimens to understand the mechanisms of chemo-resistance and use that information to improve therapeutic approaches. Methods: In this study, we report our analysis of pairs of matched tumor specimens taken from seven ovarian cancer patients enrolled in OCPMI who were treated using neoadjuvant chemotherapy (NACT). The first tumor specimen was taken at time of initial diagnostic biopsy (pre-NACT), while the matched second tumor biopsy was taken after NACT administration (post-NACT), during interval debulking surgery. All fourteen tumor biopsies were subjected to single cell RNA sequencing to generate gene expression data on 54,827 pre-NACT cells and 30,661 post-NACT cells. Bioinformatic techniques were used to compare the post-NACT tumor cells that had experienced chemotherapy to the chemo-naïve pre-NACT tumor cells. Gene/pathway enrichment tests were performed to identify genes and pathways that correlated with the chemo resistance phenotype. Results: In all matched biopsies we found that the majority of cells in the chemo-experienced post-NACT samples could be mapped to a subset of the cells in the chemo-naïve, pre-NACT samples based on gene expression patterns. We hypothesize that this subset of pre-NACT cells are the precursor cells that are able to withstand chemotherapy and could be the cause of recurrence. Analysis of the genes and signaling pathways that were upregulated in the chemo-resistant subset of pre-NACT cells identified potential novel therapeutic targets including TNFa signaling, JAK-STAT signaling, interferon signaling, TGFb signaling, and STK33 kinase activation. Conclusions: Single cell RNA sequencing combined with sequential biopsies provides an unprecedented ability to understand mechanisms of response to chemotherapy. This type of analysis can be used to identify novel therapeutic approaches targeting the chemo-resistance phenotype. Citation Format: Timothy K. Starr, Shobhana Talukdar, Zenas Chang, Jason Cepela, Christopher Tastad, Mihir Shetty, Isaac Schneider, Jun Woo, Marissa Macchietto, Christine Henzler, Ying Zhang, Joshua Baller, Constantin Aiferis, Sarah Munro, Andrew C. Nelson, Jinhua Wang, Boris J. Winterhoff. Chemotherapy resistance pathways identified in ovarian cancer patients treated with neoadjuvant chemotherapy using single cell RNA sequencing of matched tumor biopsies [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 1410.

Induction of Divergent Cell Death Pathways by Urea and Carbohydrazide Derivatives.

The complexity of cancer biology and the development of chemotherapy resistance are two main obstacles to cancer treatment and necessitate novel anticancer molecules that target different cell death pathways. Modulation of Endoplasmic Reticulum (ER) stress and subsequent activation of the Unfolded Protein Response (UPR) has been proposed as a potential chemotherapeutic target, as prolonged ER stress can lead to cell death via apoptosis or necrosis. The present study aims to evaluate the molecular mechanism underlying the cytotoxic activity of selected urea and carbohydrazide derivatives. Cell proliferation assays were performed on HeLa, Capan-1, MCF-7, HCC-1937, and MRC-5 cell lines by WST-1 assay. The expression levels of selected ER stress, autophagy, and apoptosis marker proteins were compared by immunoblotting to characterize the underlying mechanism of cytotoxicity. Flow cytometry was used to detect apoptosis. Of the tested cytotoxic compounds, 3a, 4a, 5a, 6a, and 1b dramatically and 5b moderately increased ER stress-related CHOP protein levels. Interestingly, 5b but not 3a, 4a, 5a, 6a, or 1b increased the expression of proapoptotic proteins such as cleaved PARP-1 and cleaved caspase-3 and -7. The flow-cytometry analysis further confirmed that the cytotoxic activity of 5b but not the other compounds is mediated by apoptosis, demonstrated by a significant increase in the percentage of late apoptotic cells (7-AAD/annexin V double-positive cells). Our results suggest that changing a substituent from trifluoromethyl to nitro in urea and carbohydrazide core structure alters the cell death mechanism from apoptosis to an apoptosis-independent cell death pathway. This study shows an example of how such simple modifications of a core chemical structure could cause the induction of divergent cell death pathways.

Chemotherapy-elicited exosomal miR-378a-3p and miR-378d to promote breast cancer stemness and chemoresistance via the activation of EZH2/STAT3 signaling.

e12615 Background: Not all breast cancer (BC) patients who receive neoadjuvant chemotherapy achieve a pathologic complete response (pCR), but the reasons for this are unknown. Previous studies have shown that exosomes produced in the tumor microenvironment in response to chemotherapy promote a chemotherapy-resistant phenotype in tumors. However, the role of BC chemo-elicited exosomes in regulating chemoresistance is poorly understood. Methods: Using commercial kits, serum exosomes were extracted from patients before neoadjuvant chemotherapy, after one cycle of chemotherapy and after four cycles of chemotherapy consisting of doxorubicin (DOX) and paclitaxel (PTX). Their miRNAs were sequenced, and the correlation between the sequencing results and chemotherapy effects was further verified by RT-qPCR using patient serum exosomes. Cell Counting Kit-8 (CCK-8) was used to detect chemosensitivity. Stemness was assessed by CD44+/CD24- population analysis and mammosphere formation assays. Chromatin immunoprecipitation (ChIP) experiments were performed to verify the binding of signal transducer and activator of transcription 3 (STAT3) to the promoter of miRNAs. Results: Here, we provide clinical evidence that chemotherapy-elicited exosomal miR-378a-3p and miR-378d are closely related to the chemotherapy response and that exosomes produced by BC cells after stimulation with DOX or PTX deliver miR-378a-3p and miR-378d to neighboring cells to activate WNT and NOTCH stemness pathways and induce drug resistance by targeting Dickkopf 3 (DKK3) and NUMB. In addition, STAT3, which is enhanced by zeste homolog 2 (EZH2), bound to the promoter regions of miR-378a-3p and miR-378d, thereby increasing their expression in exosomes. More importantly, chemotherapeutic agents combined with the EZH2 inhibitor tazemetostat reversed chemotherapy-elicited exosome induced drug resistance in a nude mouse tumor xenograft model. Conclusions: This study revealed a novel mechanism of acquired chemoresistance whereby chemotherapy activates the EZH2/STAT3 axis in BC cells, which then secrete chemotherapy-elicited exosomes enriched in miR-378a-3p and miR-378d. These exosomes are absorbed by chemotherapy-surviving BC cells, leading to activation of WNT and NOTCH stem cell pathways via the targeting of DKK3 and NUMB and subsequently resulting in drug resistance. Therefore, blocking this adaptive mechanism during chemotherapy may reduce the development of chemotherapy resistance and maximize the therapeutic effect.

High expression of ABCG2 is associated with chemotherapy resistance of osteosarcoma

ObjectivesPrevious studies showed overexpression of ABCG2 in a variety of tumor tissues, which could potentially indicate the probability of chemotherapy resistance. This study aimed to reveal the role of ABCG2 in the development of chemotherapy resistance and the prognosis of osteosarcoma (OS).MethodsSixty-eight OS patients were included in this study. Tumor tissues were collected for each patient during surgery. DOX-resistant OS cell lines were induced by consecutive exposure of gradually increasing concentration of DOX to the parental cell lines. Lentivirus was used for the knockdown of ABCG2 in OS cells. Cells were treated with the gradient concentration of DOX, and the viability was assessed by CCK8 assay. Total RNA was isolated from the tumor tissues or tumor cells, and the expression of ABCG2 was analyzed by qPCR. The relationship between ABCG2 expression and clinicopathological characteristics of the patients was analyzed using Student’s t test or the Chi-square test. The overall survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. p < 0.05 was considered statistically significant.ResultsDOX-resistant OS cells were successfully established through continuous exposure to DOX. Forty-eight hours after DOX exposure, the IC 50 value of DOX-resistant HOS cells and DOX-resistant U2OS was 3.5 μM and 3.25 μM, respectively. By contrast, those of the untreated HOS and U2OS cells were 1.15 μM and 0.93 μM, respectively (p < 0.01). The mRNA expression level of ABCG2 was significantly increased in DOX-resistant cell lines. The CCK-8 assay showed that the DOX-resistant HOS cells and DOX-resistant U2OS cells transfected with ShABCG2 were more sensitive to the DOX treatment than those transfected with ShCtrl. Analysis of gene expression in OS tissues showed remarkably higher expression of ABCG2 as compared with adjacent normal tissues (p < 0.01). Patients with high expression level of ABCG2 had obviously decreased overall survival time than the patients with normal expression (p < 0.01).ConclusionsABCG2 expression level was significantly associated with the resistance to chemotherapy and the overall survival of OS patients. ABCG2 may be a promising therapeutic target for OS patients.