Mechanisms Of Resistance In Cancer Research Articles

Poly (ADP-ribose) polymerase inhibitor therapy and mechanisms of resistance in epithelial ovarian cancer.

Advanced epithelial ovarian cancer is the commonest cause of gynaecological cancer deaths. First-line treatment for advanced disease includes a combination of platinum-taxane chemotherapy (post-operatively or peri-operatively) and maximal debulking surgery whenever feasible. Initial response rate to chemotherapy is high (up to 80%) but most patients will develop recurrence (approximately 70-90%) and succumb to the disease. Recently, poly-ADP-ribose polymerase (PARP) inhibition (by drugs such as Olaparib, Niraparib or Rucaparib) directed synthetic lethality approach in BRCA germline mutant or platinum sensitive disease has generated real hope for patients. PARP inhibitor (PARPi) maintenance therapy can prolong survival but therapeutic response is not sustained due to intrinsic or acquired secondary resistance to PARPi therapy. Reversion of BRCA1/2 mutation can lead to clinical PARPi resistance in BRCA-germline mutated ovarian cancer. However, in the more common platinum sensitive sporadic HGSOC, the clinical mechanisms of development of PARPi resistance remains to be defined. Here we provide a comprehensive review of the current status of PARPi and the mechanisms of resistance to therapy.

Current applications of tumor local ablation (TLA) combined with immune checkpoint inhibitors in breast cancer treatment.

Breast cancer is one of the most common cancers in women globally, posing significant challenges to treatment because of the diverse and complex pathological and molecular subtypes. The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the treatment of breast cancer, particularly for triple-negative breast cancer (TNBC), significantly improving patient outcomes. However, the overall tumor response rate remains suboptimal due to drug resistance to ICIs. This resistance is primarily due to the immune-suppressive tumor microenvironment (TME), tumor cells' ability to evade immune surveillance, and other complex immune regulatory mechanisms. To address these challenges, clinical researchers are actively exploring combinatorial therapeutic strategies with ICIs. Tumor local ablation (TLA) technology is anticipated to overcome resistance to ICIs and enhance therapeutic efficacy by ablating tumor tissue, releasing tumor antigens, remodeling the TME, and stimulating local and systemic immune responses. Combination therapy with TLA and ICIs has demonstrated promising results in preclinical breast cancer studies, underscoring the feasibility and importance of addressing drug resistance mechanisms in breast cancer. This provides novel strategies for breast cancer treatment and is expected to drive further advancements in the field.

Analysis of miRNAs: Biomarkers for HER2-Targeted Breast Cancer Therapy

Breast cancer remains a leading lethal cancer in women globally, with the HER2+ positive subtype associated with a higher chance of therapeutic resistance. Treatments tailored specifically to this subtype, such as targeted therapies, have significantly developed over the past decade. However, the issue of drug resistance to targeted drugs underlines the necessity of better treatment approaches. Recent studies have focused on microRNAs (miRNAs), single-stranded non-coding RNAs, as critical regulators in drug resistance mechanisms. These miRNAs, capable of influencing various cellular processes, have emerged as critical modulators in numerous diseases, including different cancer types, particularly breast cancer. This article reviews current methodologies in the study of miRNAs within the context of HER2-positive breast cancer, from the selection of study models and sample extraction to comprehensive analysis methods. Our objective is to highlight the potential that miRNAs hold as biomarkers capable of regulating drug resistance in this specific cancer subtype. Moreover, we also discuss the importance of integrating advanced study models alongside the latest bioinformatics tools to enrich this research domain. Furthermore, this article evaluates the use of clinical samples and cell line models for studying miRNAs in this field, outlining the advantages and limitations of each method and proposing a refined approach to research design. The main contribution of this work is the establishment of a detailed taxonomy of research strategies that address current challenges while also outlining promising future directions, particularly focused on elucidating the regulatory mechanisms of miRNAs in therapeutic resistance in breast cancer. In addition, by underscoring the necessity of employing a diverse array of study models and capitalizing on bioinformatics advancements, this article seeks to uncover the complex interactions between miRNAs and drug resistance mechanisms in breast cancer. Ultimately, our goal is to pave the way toward overcoming therapeutic resistance, thereby improving the prognosis for patients afflicted with HER2-positive breast cancer.

Research On Befortinib: Characteristics, Role, And Development of Fourth-Generation EGFR-Tkis

Lung cancer is one of the most common cancers in terms of morbidity and mortality. Among them, non-small cell lung cancer (NSCLC) accounts for 90% of lung cancer patients. Epidermal Growth Factor Receptor (EGFR) mutations are also the most common type of driver gene mutation in advanced NSCLC. In Chinese NSCLC patients, the proportion of EGFR mutations was 28.2%, and this proportion increased to 50.2% in lung adenocarcinoma. All three generations of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors (EGFR-TKI) are effective therapeutics for lung cancer patients carrying EGFR-sensitive mutations or T790M mutations, each of which has its own advantages and characteristics, as well as its own limitations and adverse reactions. Befortinib, which is new to the market in 2023, has better Median progression-free survival (mPFS). However, three generations of EGFR-TKI cannot completely overcome the resistance mechanism of lung cancer. In addition to the T790M mutation, there are other mutations or mechanisms that cause lung cancer to become resistant to third-generation EGFR-TKI, such as C797S mutation, MET amplification, HER2 amplification, etc. At present, the fourth-generation of EGFR-TKI is still under continuous research and development.

Integration of phosphoinositide 3-kinase (PI3K) and transforming growth factor β1 (TGF-β1) signaling cascades: role in the therapeutic inefficiency of tamoxifen

Growth factors signaling cascades and their interaction with the central regulatory targets of tumor cells and estrogens are considered as the main mechanisms of hormonal resistance in breast cancer. The integration of the transforming growth factor β1 (TGF-β1) and PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling pathway may result in the activation of proliferation and, as a result, the development of an in-effective response to therapy and disease progression. The review summarizes a systematic analysis of the literature data on the role of TGF-β1 signaling in the mechanisms of tamoxifen resistance to in the aspect of interaction with the PI3K/Akt/mTOR. The interaction between the estrogen receptors α signaling and tamoxifen, the mechanisms of regulatory activation of TGF-β1 and PI3K/Akt/mTOR, as well as their contribution to the tamoxifen response are considered. The direct involvement of TGF-β1/PI3K in the mechanisms of tamoxifen resistance to determines the prospects for studying the effector of these cascades as molecular targets. The knowledge accumulated to date allows considering the TGF-β1/PI3K signaling pathway as a potential molecular tool for the search for effective strategies for blocking the resistance of tumor cells to tamoxifen.

Mechanisms of Resistance in Non-Small Cell Lung Cancer

Objectives: Treatment of patients with non-small cell lung cancer continues to evolve and the discovery and approval of tyrosine kinase inhibitors in patients with NSCLC and targetable alterations brought an important contribution in treating these patients. Material and Methods: This review aims to describe the main alterations that can affect the signaling pathways involved in NSCLC, present the therapy that can target these alterations and describe the mechanisms that can determine resistance to targeted therapy. Literature research was performed on PubMed and Google Scholar and 60 relevant articles were selected for the purpose of our review. Discussion: The data of the present research were structured in nine paragraphs, each one presenting the main alterations that can affect the signaling pathways in patients diagnosed with NSCLC, targeted therapy used in managing these cases and mechanisms of resistance to targeted therapy. Conclusions: Testing for molecular biomarkers should be mandatory for every newly diagnosed NSCLC patient in order to offer the most appropriate treatment.

Abstract C046: Rapid autopsy provides unique research opportunity to evaluate KRASG12C inhibitor resistance mechanisms in non-small cell lung cancer

Abstract TThe discovery of small molecule inhibitors that target mutant KRASG12C has revolutionized the treatment paradigm for KRASG12C-mutant non-small cell lung cancer (NSCLC). While the initial response rates to KRASG12C inhibitors (KRASG12Ci) are approximately 40%, the development of heterogenous resistance mechanisms remains a challenge. Understanding the heterogeneity of resistance mechanisms is critical in overcoming this obstacle. Autopsy samples provide valuable insights into therapy resistance and genomic evolution. Moffitt’s Rapid Tissue Donation (RTD) program offers patients with lung malignancies the opportunity to donate primary and metastatic tumor samples, as well as non-tumoral tissue samples and body fluids, for research purposes after their death. 16 patients with KRASG12C-mutant NSCLC enrolled into the RTD, and 15 were treated with KRASG12Ci. Postmortem samples from seven patients were collected to date, including 38 tumor samples from lung, liver, lymph nodes, soft tissue, adrenal, spleen, pancreas, brain and epicardium samples. Radiological measurements were used to evaluate therapy response in each lesion. 26/38 tumor samples were non-responding to treatment whereas the remaining 12 were stable/responding lesions. Among the seven patients, five had both non-responding and stable/responding lesions and the remaining two had only non-responding lesions. Whole exome sequencing (WES) and transcriptomic analyses (RNA-Seq) were performed in 7 tumoral samples and 1 non-tumoral liver sample from one single patient. KRASG12C mutation and PTENR55M mutations were found in all tumor lesions, irrespective of therapy response. A PIK3CAE542K mutation was present in all tumor lesions, except for one subcutaneous tumor that did not respond to treatment. In the non-responding lung tumor, as well as the stable metastatic lymph node, a MAP2K3A203S mutation was identified. Notably, the non-responding liver lesions exhibited an ERBB3A17G mutation, whereas the responding liver lesion did not. Gene expression analyses comparing non-responding and responding liver lesions revealed an upregulation of genes associated with hypoxia, epithelial mesenchymal transition, and inflammation in the non-responding lesions, while a subgroup of genes regulated by MYC was upregulated in the responding liver lesion. Additional genomic analyses of other autopsy samples are underway. Our findings reveal that therapy resistance may be driven by pathway alterations instead of genomic alterations. The collection of rapid autopsy specimens from primary and metastatic tissue sites from patients with KRASG12C-mutant NSCLC is essential to understand the heterogeneous KRASG12Ci mechanisms of resistance and explore biomarkers that may guide clinical decision-making in these patients. Citation Format: Hilal Ozakinci, Gina Nazario, Yaakov Stern, Hitendra S Solanki, Dung-Tsa Chen, Rose Trevor, Matthew Schabath, Paul Stewart, Jamie K Teer, Amer Beg, Theresa A Boyle, Eric Haura, Bruna Pellini. Rapid autopsy provides unique research opportunity to evaluate KRASG12C inhibitor resistance mechanisms in non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C046.

Abstract IA009: Antibody-lectin chimeras for glyco-immune checkpoint blockade

Abstract Despite the curative potential of checkpoint blockade immunotherapy, a majority of patients remain unresponsive to existing treatments. Glyco-immune checkpoints – interactions of cell-surface glycans with lectin, or glycan binding, immunoreceptors – have emerged as prominent mechanisms of immune evasion and therapeutic resistance in cancer. Here, we describe antibody-lectin chimeras (AbLecs), a modular platform for glyco-immune checkpoint blockade. AbLecs are bispecific antibody-like molecules comprising a tumor-targeting arm as well as a lectin “decoy receptor” domain that directly binds tumor glycans and blocks their ability to engage lectin receptors on immune cells. AbLecs enhanced cancer cell killing in vitro via macrophage phagocytosis and NK cell and granulocyte cytotoxicity compared to their parent monoclonal antibodies. AbLec treatment reduced tumor burden in vivo compared to treatment with the parent antibody in a humanized model of metastatic HER2+ cancer. Furthermore, AbLecs synergized with blockade of the “don’t eat me” signal CD47 for enhanced tumor killing. AbLecs can be readily designed to target numerous tumor-associated antigens and glyco-immune checkpoint ligands, and therefore represent a new modality of cancer immunotherapy. Citation Format: Jessica Stark. Antibody-lectin chimeras for glyco-immune checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA009.

DRMref: comprehensive reference map of drug resistance mechanisms in human cancer.

Drug resistance poses a significant challenge in cancer treatment. Despite the initial effectiveness of therapies such as chemotherapy, targeted therapy and immunotherapy, many patients eventually develop resistance. To gain deep insights into the underlying mechanisms, single-cell profiling has been performed to interrogate drug resistance at cell level. Herein, we have built the DRMref database (https://ccsm.uth.edu/DRMref/) to provide comprehensive characterization of drug resistance using single-cell data from drug treatment settings. The current version of DRMref includes 42 single-cell datasets from 30 studies, covering 382 samples, 13 major cancer types, 26 cancer subtypes, 35 treatment regimens and 42 drugs. All datasets in DRMref are browsable and searchable, with detailed annotations provided. Meanwhile, DRMref includes analyses of cellular composition, intratumoral heterogeneity, epithelial-mesenchymal transition, cell-cell interaction and differentially expressed genes in resistant cells. Notably, DRMref investigates the drug resistance mechanisms (e.g. Aberration of Drug's Therapeutic Target, Drug Inactivation by Structure Modification, etc.) in resistant cells. Additional enrichment analysis of hallmark/KEGG (Kyoto Encyclopedia of Genes and Genomes)/GO(Gene Ontology) pathways, as well as the identification of microRNA, motif and transcription factors involved in resistant cells, is provided in DRMref for user's exploration. Overall, DRMref serves as a unique single-cell-based resource for studying drug resistance,drug combination therapy and discovering novel drug targets.

The Role of Cyanidin-3-O-glucoside in Modulating Oxaliplatin Resistance by Reversing Mesenchymal Phenotype in Colorectal Cancer.

Epithelial-mesenchymal transition (EMT) plays an important role in the biological and biochemical processes of cells, and it is a critical process in the malignant transformation, and mobility of cancer. Additionally, EMT is one of the main mechanisms contributing to chemoresistance. Resistance to oxaliplatin (OXA) poses a momentous challenge in the chemotherapy of advanced colorectal cancer (CRC) patients, highlighting the need to reverse drug resistance and improve patient survival. In this study, we explored the response of cyanidin-3-O-glucoside (C3G), the most abundant anthocyanin in plants, on the mechanisms of drug resistance in cancer, with the purpose of overcoming acquired OXA resistance in CRC cell lines. We generated an acquired OXA-resistant cell line, named HCT-116-ROx, by gradually exposing parental HCT-116 cells to increasing concentrations of OXA. To characterize the resistance, we performed cytotoxicity assays and shape factor analyses. The apoptotic rate of both resistant and parental cells was determined using Hoechst 33342/Propidium Iodide (PI) fluorescence staining. Migration capacity was evaluated using a wound-healing assay. The mesenchymal phenotype was assessed through qRT-PCR and immunofluorescence staining, employing E-cadherin, N-cadherin, and Vimentin markers. Resistance characterization announced decreased OXA sensitivity in resistant cells compared to parental cells. Moreover, the resistant cells exhibited a spindle cell morphology, indicative of the mesenchymal phenotype. Combined treatment of C3G and OXA resulted in an augmented apoptotic rate in the resistant cells. The migration capacity of resistant cells was higher than parental cells, while treatment with C3G decreased the migration rate of HCT-116-ROx cells. Analysis of EMT markers showed that HCT-116-ROx cells exhibited loss of the epithelial phenotype (E-cadherin) and gain of the mesenchymal phenotype (N-cadherin and Vimentin) compared to HCT-116 cells. However, treatment of resistant cells with C3G reversed the mesenchymal phenotype. The morphological observations of cells acquiring oxaliplatin resistance indicated the loss of the epithelial phenotype and the acquisition of the mesenchymal phenotype. These findings suggest that EMT may contribute to acquired OXA resistance in CRC. Furthermore, C3G decreased the mobility of resistant cells, and reversed the EMT process, indicating its potential to overcome acquired OXA resistance.

Broad-spectrum kinome profiling identifies CDK6 upregulation as a driver of lenvatinib resistance in hepatocellular carcinoma

Increasing evidence has demonstrated that drug resistance can be acquired in cancer cells by kinase rewiring, which is an obstacle for efficient cancer therapy. However, it is technically challenging to measure the expression of protein kinases on large scale due to their dynamic range in human proteome. We employ a lysine-targeted sulfonyl fluoride probe, named XO44, which binds to 133 endogenous kinases in intact lenvatinib-resistant hepatocellular carcinoma (HCC) cells. This analysis reveals cyclin-dependent kinase 6 (CDK6) upregulation, which is mediated by ERK/YAP1 signaling cascade. Functional analyses show that CDK6 is crucial in regulation of acquired lenvatinib resistance in HCC via augmentation of liver cancer stem cells with clinical significance. We identify a noncanonical pathway of CDK6 in which it binds and regulates the activity of GSK3β, leading to activation of Wnt/β-catenin signaling. Consistently, CDK6 inhibition by palbociclib or degradation by proteolysis targeting chimeras (PROTACs) is highly synergistic with lenvatinib in vitro. Interestingly, palbociclib not only exerts maximal growth suppressive effect with lenvatinib in lenvatinib-resistant HCC models but also reshapes the tumor immune microenvironment. Together, we unveil CDK6 as a druggable target in lenvatinib-resistant HCC and highlight the use of a chemical biology approach to understand nongenetic resistance mechanisms in cancer.

Natural resistance to cancer: A window of hope.

As healthcare systems are improving and thereby the life expectancy of human populations is increasing, cancer is representing itself as the second leading cause of death. Although cancer biologists have put enormous effort on cancer research so far, we still have a long way to go before being able to treat cancers efficiently. One interesting approach in cancer biology is to learn from natural resistance and/or predisposition to cancer. Cancer-resistant species and tissues are thought-provoking models whose study shed light on the inherent cancer resistance mechanisms that arose during the course of evolution. On the other hand, there are some syndromes and factors that increase the risk of cancer development, and revealing their underlying mechanisms will increase our knowledge about the process of cancer formation. Here, we review natural resistance and predisposition to cancer and the known mechanisms at play. Further insights from these natural phenomena will help design future cancer research and could ultimately lead to the development of novel cancer therapeutic strategies.

MiRNA-214-3p stimulates carcinogen-induced mammary epithelial cell apoptosis in mammary cancer-resistant species

Mammary cancer incidence varies greatly across species and underlying mechanisms remain elusive. We previously showed that mammosphere-derived epithelial cells from species with low mammary cancer incidence, such as horses, respond to carcinogen 7, 12-Dimethylbenz(a)anthracene-induced DNA damage by undergoing apoptosis, a postulated anti-cancer mechanism. Additionally, we found that miR-214-3p expression in mammosphere-derived epithelial cells is lower in mammary cancer-resistant as compared to mammary cancer-susceptible species. Here we show that increasing miR-214 expression and decreasing expression of its target gene nuclear factor kappa B subunit 1 in mammosphere-derived epithelial cells from horses abolishes 7,12-Dimethylbenz(a)anthracene-induced apoptosis. A direct interaction of miR-214-3p with another target gene, unc-5 netrin receptor A, is also demonstrated. We propose that relatively low levels of miR-214 in mammosphere-derived epithelial cells from mammals with low mammary cancer incidence, allow for constitutive gene nuclear factor kappa B subunit 1 expression and apoptosis in response to 7, 12-Dimethylbenz(a)anthracene. Better understanding of the mechanisms regulating cellular responses to carcinogens improves our overall understanding of mammary cancer resistance mechanisms.

5O Deciphering resistance mechanisms in cancer: Insights from the final report of MATCH-R study with focus on molecular drivers

5O Deciphering resistance mechanisms in cancer: Insights from the final report of MATCH-R study with focus on molecular drivers

UP55 - The role of the unfolded protein response in mechanisms of treatment resistance in prostate cancer

UP55 - The role of the unfolded protein response in mechanisms of treatment resistance in prostate cancer

1331P Resistance mechanisms in EGFR-mutated advanced non-small cell lung cancer (aNSCLC) patients (pts) treated with first-line osimertinib (osi) and monitoring plasma mutational load: Preliminary results of REM study

1331P Resistance mechanisms in EGFR-mutated advanced non-small cell lung cancer (aNSCLC) patients (pts) treated with first-line osimertinib (osi) and monitoring plasma mutational load: Preliminary results of REM study

METTL3 promotes drug resistance to oxaliplatin in gastric cancer cells through DNA repair pathway.

Gastric cancer (GC) poses a significant threat to human health and remains a prevalent form of cancer. Despite clinical treatments, the prognosis for Gastric cancer patients is still unsatisfactory, largely due to the development of multidrug resistance. Oxaliplatin (OXA), a second-generation platinum drug, is commonly recommended for adjuvant and palliative chemotherapy in Gastric cancer; however, the underlying mechanisms of acquired resistance to Oxaliplatin in Gastric cancer patients are not yet fully understood. In this study, we aimed to explore the potential mechanisms of Oxaliplatin resistance in Gastric cancer by employing bioinformatics analysis and conducting in vitro experiments. Specifically, we focused on investigating the role of methyltransferase-like 3 (METTL3). Our findings revealed that the knockdown of METTL3 significantly impeded the proliferation and migration of Gastric cancer cells. METTL3 knockdown induced apoptosis in OXA-resistant Gastric cancer cells and enhanced their sensitivity to Oxaliplatin. Furthermore, we found that DNA repair pathways were significantly activated in OXA-resistant Gastric cancer cells, and METTL3 knockdown significantly inhibited DNA repair pathways. Another important finding is that METTL3 knockdown and OXA-induced Gastric cancer cell death are additive, and the targeted METTL3 can assist Oxaliplatin treatment. Collectively, our findings suggest that METTL3 knockdown can augment the sensitivity of Gastric cancer cells to Oxaliplatin by impeding DNA repair processes. Consequently, targeting METTL3 holds great promise as a viable adjuvant strategy in the treatment of Gastric cancer patients.

Drug resistance mechanism and reversal strategy in lung cancer immunotherapy.

Among all malignant tumors, lung cancer has the highest mortality and morbidity rates. The non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the most common histological subtypes. Although there are a number of internationally recognized lung cancer therapy regimens, their therapeutic effects remain inadequate. The outlook for individuals with lung carcinoma has ameliorated partly thanks to the intensive study of the tumor microenvironment and immune checkpoint inhibitors. Numerous cancers have been effectively treated with immunotherapy, which has had positive therapeutic results. Global clinical trials have validated that PD-1/PD-L1 inhibitors are effective and safe for treating lung cancer either independently or in combination, and they are gradually being recommended as systemic treatment medications by numerous guidelines. However, the immunotherapy resistance restricts the immunotherapy efficacy due to the formation of tumor immunosuppressive microenvironment and tumor mutations, and immunotherapy is only effective for a small percentage of lung cancer patients. To summarize, while tumor immunotherapy is benefiting an increasing number of lung cancer patients, most of them still develop natural or acquired resistance during immunotherapy. Consequently, a crucial and urgent topic is understanding and tackling drug resistance triggered by immunotherapy in lung cancer treatment. This review will outline the presently recognized mechanisms of immunotherapy resistance and reversal strategies in lung cancer.

Identifying mechanisms of therapeutic resistance in low-grade serous ovarian cancer (2282)

Identifying mechanisms of therapeutic resistance in low-grade serous ovarian cancer (2282)

Review of possible mechanisms of radiotherapy resistance in cervical cancer.

Radiotherapy is one of the main treatments for cervical cancer. Early cervical cancer is usually considered postoperative radiotherapy alone. Radiotherapy combined with cisplatin is the standard treatment for locally advanced cervical cancer (LACC), but sometimes the disease will relapse within a short time after the end of treatment. Tumor recurrence is usually related to the inherent radiation resistance of the tumor, mainly involving cell proliferation, apoptosis, DNA repair, tumor microenvironment, tumor metabolism, and stem cells. In the past few decades, the mechanism of radiotherapy resistance of cervical cancer has been extensively studied, but due to its complex process, the specific mechanism of radiotherapy resistance of cervical cancer is still not fully understood. In this review, we discuss the current status of radiotherapy resistance in cervical cancer and the possible mechanisms of radiotherapy resistance, and provide favorable therapeutic targets for improving radiotherapy sensitivity. In conclusion, this article describes the importance of understanding the pathway and target of radioresistance for cervical cancer to promote the development of effective radiotherapy sensitizers.