Radioresistant Breast Cancer Research Articles

Ginkgetin enhances breast cancer radiotherapy sensitization by suppressing NRF2-HO-1 axis activity.

Breast cancer (BC) is a critical threat to women's lives. Radiotherapy (RT) is a pivotal treatment modality for BC, but the failure of RT due to radioresistance is still not well facilitated. Ginkgetin (GK) has a potent anti-tumor activity intimately associated with ferroptosis. This study applied in vitro and in vivo experimental models to ascertain the GK mechanism of action on BC radioresistance. The outcomes reported that GK could inhibit BC cell growth and increase apoptosis. In addition, when BC cells generated radioresistance, GK promoted ferroptosis of radioresistant BC cells by mitigating NRF2 expression, suppressing HO-1 and NQO1 expression, increasing the intracellular content of reactive oxygen species (ROS) and ferrous ions, accelerating the glutathione (GSH) depletion, and decreasing GPX4 expression. Notably, GK can damage intracellular mitochondria and cause a substantial increase in ferrous ions in BC cells. Therefore, GK shows immense potential for enhancing breast cancer radiotherapy sensitivity, which may provide pivotal evidence for subsequent RT sensitization.

Non-coding RNAs modulation in breast cancer radioresponse: mechanisms and therapeutic implications.

Breast cancer is the most frequent type of cancer in women, with significant incidence and fatality rates. Radiation therapy is an important therapeutic option for breast cancer patients. However, tumor cells' resistance to radiation can limit therapy efficacy, resulting in recurrence and death. Non-coding RNAs (ncRNAs) are aclass of small RNA molecules that do not translate into proteins but can affect the translation of target mRNA. Several investigations on breast cancer have demonstrated abnormal expression of ncRNAs in response to radiation. Non-coding RNAs are essential in controlling numerous processes such as DNA damage response, cancer stem cell pathways, cell cycle regulation, cell death, and inflammation. Dysregulation of ncRNAs after irradiation influences radiosensitivity or radioresistance of breast cancer cells. Understanding the molecular mechanisms underlying Radiation response can lead to innovative treatment ways to reduce breast cancer radioresistance and increase radiotherapy's efficacy. This review summarizes current research on ncRNA dysregulation following irradiation and analyzes ncRNAs' function and mechanism in modifying breast cancer cell radiosensitivity and radioresistance.

Dual Roles of microRNA-122 in Hepatocellular Carcinoma and Breast Cancer Progression and Metastasis: A Comprehensive Review.

microRNA-122 (miR-122) plays crucial yet contrasting roles in hepatocellular carcinoma (HCC) and breast cancer (BC), two prevalent and aggressive malignancies. This review synthesizes current research on miR-122's functions in these cancers, focusing on its potential as a diagnostic, prognostic, and therapeutic target. A comprehensive literature search was conducted using PubMed, Web of Science, and Scopus databases. In HCC, miR-122 is downregulated in most cases, suppressing oncogenic pathways and reducing tumor growth and metastasis. Restoring miR-122 levels has shown promising therapeutic potential, increasing sensitivity to treatments like sorafenib. In contrast, in BC, miR-122 plays a pro-metastatic role, especially in triple-negative breast cancer (TNBC) and metastatic lesions. miR-122's ability to influence key pathways, such as the Wnt/β-catenin and NF-κB pathways in HCC, and its role in enhancing the Warburg effect in BC underline its significance in cancer biology. miR-122, a key factor in breast cancer radioresistance, suppresses tumors in radiosensitive cells. Inhibiting miR-122 could reverse resistance and potentially overcome radiotherapy resistance. Given its context-dependent functions, miR-122 could serve as a potential therapeutic target, where restoring or inhibiting its expression may help in treating HCC and BC, respectively. The dual roles of miR-122 underscore its significance in cancer biology and its potential in precision medicine.

Advantages of single high-dose radiation therapy compared with conventional fractionated radiation therapy in overcoming radioresistance

Background Radioresistance is a major clinical challenge in cancer treatment, as it reduces the effectiveness of radiation therapy (RT). While advances in radiation delivery have enabled the clinical use of high-dose hypofractionated RT, its impact on radioresistant tumors remains unclear. This study aimed to compare the effects of single high-dose RT with conventional fractionated RT on radioresistant breast cancer cells and explore the underlying mechanisms. Methods Radioresistant cell lines were previously established by exposing SK-BR-3 and MCF-7 cells to 48 Gy and 70 Gy of radiation, respectively, in multiple fractions. We compared the effects of 2 Gy × 5 and 7 Gy × 1 fractions on these cells using clonogenic survival assays and western blot analysis. In vivo antitumor effects were assessed in SR tumor-bearing BALB/c mice irradiated with either 2 Gy × 5 or 7 Gy × 1 fractions. Results 7 Gy x1 was more efficient at killing radioresistant breast cancer cells than 2 Gy x5. Furthermore, the 7 Gy x1 fraction produced higher levels of reactive oxygen species (ROS) and decreased the expression of radioresistance factors such as p-STAT3, ACSL4, FOXM1, RAD51, Bcl-xL, and survivin. Consistent with the in vitro studies, the 7 Gy × 1 fraction also showed superior antitumor effects in SR tumor-bearing BALB/c mice. Conclusions Single high-dose RT offers superior advantages over conventional fractionated RT in regard to overcoming radioresistance, supporting its potential as a promising treatment for recurrent tumors.

Significant Impact of let-7d MicroRNA on Breast Cancer Cell Lines Post-Radiation Treatment

Introduction: Radiotherapy is a common treatment for breast cancer treatment, that induces DNA damage. These DNA damages are addressed through various repair pathways, which regulate the DNA repair systems and confer radio-resistance. Non-coding RNAs are a big proportion of genome transcripts without the potential to encode proteins. Related studies demonstrated that radiation affects the expression of non-coding RNAs. Let-7d, a tumor suppressor in numerous cancers, has some target genes that play a role in the DNA repair system. Materials and Methods: Human breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in a Dulbecco's Modified Eagle Medium. The exponentially growing cells were treated with some doses of X-rays. After radiation treatment and cell harvesting, RNA was extracted, and cDNA synthesis was done. The let-7d miRNA expression changes were calculated with real-time quantitative reverse transcription polymerase chain reaction. Results: The results implied that radiation caused increased let-7d expression in breast cancer cell lines after radiation treatment. In addition, the results showed that 24 h after radiation, the expression of let-7d in the radioresistant cell line was higher than the radiosensitive one; 48 h after radiation, the expression of let-7d in the radiosensitive cell line was higher than the other one. Conclusions: The results demonstrated that radiation treatment increased let-7d miRNA expression in both radiosensitive and radio-resistant breast cancer cell lines. Therefore, let-7d might be involved in the radiosensitivity of breast cancer.

Gp78-regulated KAP1 phosphorylation induces radioresistance in breast cancer by facilitating PPP1CC/PPP2CA ubiquitination

Adjuvant radiation therapy is a common treatment for breast cancer, yet its effectiveness is often limited by radioresistance in patients. Identifying novel targets to combat this radioresistance is imperative. Recent investigations show that gp78 is upregulated in drug-resistant breast cancer cells. Our study reveals that gp78 markedly increased the phosphorylation of KAP1 and promoted DNA damage repair caused by ionizing radiation. Mechanistically, gp78 degrades phosphatases (PPP1CC/PPP2CA) in a ubiquitination-dependent manner. PPP1CC and PPP2CA are crucial regulators of KAP1 phosphorylation in response to DNA damage. Therefore, gp78 leads to a notable elevation in the phosphorylation of KAP1 by degrading phosphatases, thereby promoting the DNA damage repair process and increasing the radioresistance of tumor cells. The identification of gp78 as a pivotal regulator in radioresistance suggests a promising avenue for intervention. Combining blockade strategies targeting gp78 holds a signification potential for reversing radioresistance and improving the efficacy of breast cancer radiotherapy.

Quercetin and its derivatives from lotus (Nelumbo nucifera) seedpod extract combat radioresistance by suppressing ACSL4.

Radioresistance poses a significant obstacle in cancer treatment. Lotus seedpod extract (LSE) has demonstrated anticancer effects in various cancer cells. However, its potential against radioresistant tumors remains unclear. In this study, we aimed to investigate the effect of LSE on radioresistant breast cancer cells, explore the underlying mechanism, and identify the major constituents responsible for its cytotoxic effect. LSE, extracted using 70% ethanol, exhibited selective cytotoxic effects against radioresistant breast cancer cells compared with their parental cells. Chemical analysis identified quercetin and its derivatives, hyperoside and miquelianin, as the major constituents responsible for these selective effects. Notably, quercetin displayed the most potent cytotoxicity against radioresistant breast cancer cells compared with hyperoside and miquelianin. Further investigation revealed that these compounds inhibited the activation of DNA repair systems, leading to the accumulation of DNA damage and the induction of apoptosis. Importantly, they efficiently suppressed the expression of ACSL4, a factor previously associated with radioresistance. In an in vivo study, quercetin exhibited a significant suppression of tumor growth in radioresistant tumor-bearing mice. Taken together, our findings highlight the potential of LSE and its major constituents, quercetin and its derivatives, in overcoming radioresistance in breast cancer. This study provides compelling evidence to support the use of LSE as a medicinal source for the future adjunctive therapy to combat radioresistance in breast cancers.

Integrated Transcriptome Analysis of Radioresistant Cells Revealed Genes and Pathways Predictive Of Tumor Response to Radiotherapy and Chemotherapy in Breast Cancer

AbstractBreast cancer cells exposed to radiotherapy frequently develop radiation resistance through molecular and phenotypic changes. While there is evidences of pathways controlling radioresistance, the evolution of diverse cell phenotypes and transcriptional changes as mediators of radioresistance in breast cancer are restricted. Moreover, the effectiveness of the chemotherapy on radioresistant cells remains uncertain. In this work, an isogenic model of radioresistant breast cancer cells (RR cells) is used to study this phenotype. RR cells show high survival rates after radiation, moreover, RR cells of the triple negative breast cancer (TNBC) subtype show a significantly advanced invasiveness phenotype. Notably, RR cells are significantly sensitive to chemotherapy by inhibit cell survival and promote apoptosis. Transcriptomics and gene co‐expression network analysis identify differentially expressed genes (DEGs) and hub genes related to survival and apoptosis pathways in the RR cells of luminal subtype, while in TNBC subtype, cell migration, cell differentiation, and immune pathways are enriched. Hub genes predict the failure of radiotherapy in breast cancer patients, but they are also related to pathological complete response after chemotherapy. Transcriptome changes during acquired radioresistance uncover genes and pathways associated to radio and chemotherapy response. These results demonstrate that radioresistant pathways may converge to develop collateral chemo‐sensitivity.

Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) has recently emerged as a promising therapeutic target in cancer. In this study, we explored the biological function of MAP4K4 in radioresistant breast cancer cells using two MAP4K4 inhibitors, namely PF06260933 and GNE-495. Radioresistant SR and MR cells were established by exposing SK-BR-3 and MCF-7 breast cancer cells to 48–70 Gy of radiation delivered at 4–5 Gy twice a week over 10 months. Surprisingly, although radioresistant cells were derived from two different subtypes of breast cancer cell lines, MAP4K4 was significantly elevated regardless of subtype. Inhibition of MAP4K4 with PF06260933 or GNE-495 selectively targeted radioresistant cells and improved the response to irradiation. Furthermore, MAP4K4 inhibitors induced apoptosis through the accumulation of DNA damage by inhibiting DNA repair systems in radioresistant cells. Notably, Inhibition of MAP4K4 suppressed the expressions of ACSL4, suggesting that MAP4K4 functioned as an upstream effector of ACSL4. This study is the first to report that MAP4K4 plays a crucial role in mediating the radioresistance of breast cancer by acting upstream of ACSL4 to enhance DNA damage response and inhibit apoptosis. We hope that our findings provide a basis for the development of new drugs targeting MAP4K4 to overcome radioresistance.

Innovative therapeutic strategies to overcome radioresistance in breast cancer.

Despite a comparatively favorable prognosis relative to other malignancies, breast cancer continues to significantly impact women's health globally, partly due to its high incidence rate. A critical factor in treatment failure is radiation resistance - the capacity of tumor cells to withstand high doses of ionizing radiation. Advancements in understanding the cellular and molecular mechanisms underlying radioresistance, coupled with enhanced characterization of radioresistant cell clones, are paving the way for the development of novel treatment modalities that hold potential for future clinical application. In the context of combating radioresistance in breast cancer, potential targets of interest include long non-coding RNAs (lncRNAs), micro RNAs (miRNAs), and their associated signaling pathways, along with other signal transduction routes amenable to pharmacological intervention. Furthermore, technical, and methodological innovations, such as the integration of hyperthermia or nanoparticles with radiotherapy, have the potential to enhance treatment responses in patients with radioresistant breast cancer. This review endeavors to provide a comprehensive survey of the current scientific landscape, focusing on novel therapeutic advancements specifically addressing radioresistant breast cancer.

Nuclear isoform of RAPH1 interacts with FOXQ1 to promote aggressiveness and radioresistance in breast cancer

Radioresistance limits the efficacy of radiotherapy against breast cancer, especially the most lethal subtype of breast cancer, triple-negative breast cancer (TNBC). Epithelial-to-mesenchymal transition (EMT) is closely related to tumor radioresistance. In this work, we attempted to identify the key EMT-related transcription factor(s) that can induce radioresistance in breast cancer cells. A set of 44 EMT transcription factors were analyzed in parental and radioresistant TNBC cell lines. The function of FOXQ1, a differentially expressed transcription factor, was determined in TNBC radioresistance. FOXQ1-interacting proteins were identified by co-immunoprecipitation and mass spectrometry. Compared with parental cells, FOXQ1 was significantly upregulated in radioresistant TNBC cells. Silencing of FOXQ1 increased the radiosensitiviy of radioresistant TNBC cells both in vitro and in vivo. FOXQ1 associated with a nuclear isoform of RAPH1 (named RAPH1-i3) in radioresistant TNBC cells. Overexpression of RAPH1-i3 enhanced TNBC cell proliferation and migration, and most interestingly, induced radioresistance in parental TNBC cells when co-expressed with FOXQ1. Similar findings were observed in estrogen receptor-positive breast cancer cell lines that had co-expression of RAPH1-i3 and FOXQ1. Mechanistically, co-expression of RAPH1-i3 and FOXQ1 activated STAT3 signaling and increased the expression of CCND1, MCL1, Bcl-XL, and MMP2. Depletion of RAPH1-i3 impaired the radioresistance of radioresistant TNBC cells. Additionally, RAPH1-i3 upregulation was associated with advanced tumor stage and reduced disease-free survival in TNBC patients. These results collectively show that RAPH1-i3 interacts with FOXQ1 to promote breast cancer progression and radioresistance. RAPH1-i3 and FOXQ1 represent therapeutic targets for the treatment of breast cancer including TNBC.

Network-Derived Radioresistant Breast Cancer Target with Candidate Inhibitors from Brown Algae: A Sequential Assessment from Target Selection to Quantum Chemical Calculation.

Despite significant progress in early detection and treatment, a few aggressive breast cancers still exhibit resistance to therapy. This study aimed to identify a therapeutic target for radioresistant breast cancer (RRbc) through a protein network from breast cancer genes and to evaluate potent phytochemicals against the identified target. Our approach includes the integration of differential expression genes from expression datasets to create a protein network and to use survival analysis to identify the crucial RRbc protein in order to discover a therapeutic target. Next, the phytochemicals sourced from brown algae were screened through molecular docking, ADME (absorption, distribution, metabolism, and excretion), molecular dynamics (MD) simulation, MM-GBSA, and quantum mechanics against the identified target. As a result of our protein network investigation, the proto-oncogene c-KIT (KIT) protein was identified as a potent radioresistant breast cancer target. Further, phytochemical screening establishes that nahocol-A1 from brown algae has high binding characteristics (-8.56 kcal/mol) against the KIT protein. Then, quantum chemical analysis of nahocol-A1 provided insights into its electronic properties favorable for protein binding. Also, MD simulation comprehends the conformational stability of the KIT-nahocol-A1 complex. Overall, our findings suggest nahocol-A1 could serve as a promising therapeutic candidate for radioresistant breast cancer.

Comparative Proteomic Analysis of Irradiation-Induced Radioresistant Breast Cancer Cells Using Label-Free Quantitation.

Breast cancer poses severe threats to human health as radioresistance becomes increasingly prevalent. The mechanisms of radioresistance are hard to expound completely. This study aims to explore proteomic changes of radioresistance, which will help elucidate the potential mechanisms responsible for breast cancer radioresistance and explore potential therapeutic targets. A radioresistant breast cancer cell line was established by repeated irradiation. Liquid Chromatograph Mass Spectrometer (LC-MS) was used to quantify protein expression. Proteomic changes associated with radioresistance were evaluated by proteomic analysis. Further, cell radioresistance and several identified proteins were verified in in vitro experiments. In the study, more than 3000 proteins were detected, 243 of which were identified as up-regulated proteins and another 633 as down-regulated proteins. Gene Ontology (GO) enrichment analysis indicated that these proteins were mainly expressed in the lysosome and ribosome, associated with coenzyme binding and the structural constituent of the ribosome, involved in mitotic cytokinesis and ribonucleoprotein complex biogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that many biological processes were extensively altered, particularly spliceosome and thermogenesis. It is worth noting that the functions and pathways related to ribosomes were significantly enriched, therefore ribosomal proteins (RPL6 and RPS13) were identified through western blot and highly expressed in relatively radiosensitive cells. Additionally, several identified proteins, including S100A4, RanBP9, and ISG15, were also verified to be differentially expressed in different radiosensitive cells. Our results provide a framework for further studies into the mechanisms of radioresistance and serve as a basis to construct a predictive model of radioresistance in breast cancer. Ribosome may participate in the radioresistance of breast cancer, which provides new insights into the proteomic characteristics of the mechanisms of radioresistance.

Role of The circ-HIPK3, circ-PVT1, miR-25, and miR-149 in Response of Breast Cancer Cells to Ionizing Radiation.

Determining cellular radiosensitivity of breast cancer (BC) patients through molecular markers before radiation therapy (RT) allows accurate prediction of individual's response to radiation. The aim of this study was therefore to investigate the potential role of epigenetic biomarkers in breast cancer cellular radiosensitivity. In this experimental study, we treated two BC cell lines, MDA-MB 231 and MCF-7, with doses of 2, 4, and 8Gy of irradiation for 24 and 48 hours. Expression levels of circ-HIPK3, circ-PVT1, miR-25, and miR- 149 were quantified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Significance of the observations was statistically verified using one-way ANOVA with a significance level of P<0.05. Annexin V-FITC/PI binding assay was utilized to measure cellular apoptosis. The rate of cell apoptosis was significantly higher in MCF-7 cells compared to MDA-MB-231 cells at doses of 4Gy and 8Gy (P=0.013 and P=0.004, respectively). RNA expression analysis showed that circ-HIPK3 was increased in the MDA-MB-231 cell line compared to the MCF-7 cell line after exposure to 8Gy for 48 hours. Expression of circ-PVT1 was found to be higher in MDA-MB-231 cells compared to MCF-7 cells after exposure to 8Gy for 24 hours, likewise after exposure to 4Gy and 8Gy for 48 hours. After exposing 8Gy, expression of miR-25 was increased in MDA-MB-231 cells compared to MCF-7 cells at 24 and 48 hours. After exposing 8Gy dose, expression of miR-149 was increased in MCF-7 cells compared to MDA-MB-231 cells at 24 and 48 hours. circ-HIPK3, circ-PVT1, and miR-25 played crucial roles in the mechanisms of radioresistance in breast cancer. Additionally, miR-149 was involved in regulating cellular radiosensitivity. Therefore, these factors provided predictive information about a tumor's radiosensitivity or its response to treatment, which could be valuable in personalizing radiation dosage.

Coordination-driven FBXW7 DNAzyme-Fe nanoassembly enables a binary switch of breast cancer cell cycle checkpoint responses for enhanced ferroptosis-radiotherapy.

Radiotherapy is a mainstream modality for breast cancer treatment that employs ionizing radiation (IR) to damage tumor cell DNA and elevate ROS stress, which demonstrates multiple clinically-favorable advantages including localized treatment and low invasiveness. However, breast cancer cells may activate the p53-mediated cell cycle regulation in response to radiotherapy to repair IR-induced cellular damage and facilitate post-treatment survival. F-Box and WD Repeat Domain Containing 7 (FBXW7) is a promoter of p53 degradation and critical nexus of cell proliferation and survival events. Herein, we engineered a cooperative radio-ferroptosis-stimulatory nanomedicine through coordination-driven self-assembly between ferroptosis-inducing Fe2+ ions and FBXW7-inhibiting DNAzymes and further modification of tumor-targeting dopamine-modified hyaluronic acid (HA). The nanoassembly could be selectively internalized by breast cancer cells and disintegrated in lysosomes to release the therapeutic payload. DNAzyme readily abolishes FBXW7 expression and stabilizes phosphorylated p53 to cause irreversible G2 phase arrest for amplifying post-IR tumor cell apoptosis. Meanwhile, the p53 stabilization also inhibits the SLC7A11-cystine-GSH axis, which combines with the IR-upregulated ROS levels to amplify Fe2+-mediated ferroptotic damage. The DNAzyme-Fe-HA nanoassembly could thus systematically boost the tumor cell damaging effects of IR, presenting a simple and effective approach to augment the response of breast cancer to radiotherapy. STATEMENT OF SIGNIFICANCE: To overcome the intrinsic radioresistance in breast cancer, we prepared co-assembly of Fe2+ and FBXW7-targeted DNAzymes and modified surface with dopamine conjugated hyaluronic acid (HA), which enabled tumor-specific FBXW7-targeted gene therapy and ferroptosis therapy in IR-treated breast cancers. The nanoassembly could be activated in acidic condition to release the therapeutic contents. Specifically, the DNAzymes could selectively degrade FBXW7 mRNA in breast cancer cells to simultaneously induce accumulation of p53 and retardation of NHEJ repair, eventually inducing irreversible cell cycle arrest to promote apoptosis. The p53 stabilization would also inhibit the SLC7A11/GSH/GPX4 axis to enhance Fe2+ mediated ferroptosis. These merits could act in a cooperative manner to induce pronounced tumor inhibitory effect, offering new approaches for tumor radiosensitization in the clinics.

Mechanisms of Intrinsic Radioresistance in Breast Cancer Identify Potential Therapeutic Vulnerabilities

Clinical management of breast cancer (BC) includes radiation therapy (RT) for most women, though the molecular mechanisms that underly RT response and intrinsic radioresistance are poorly understood. Both in vitro and in vivo models aid in our understanding of radiobiology, and we hypothesized that transcriptional changes caused by radiation in vitro in BC cell lines would be recapitulated in an in vivo mouse xenograft model and uncover targetable mechanisms of radioresistance in BC. Radiosensitivity was measured with clonogenic survival assays in 16 cell lines. RNA-seq experiments in vitro and in vivo were performed in an RT resistant (SUM-159) and RT sensitive (ZR-75) cell line 24 hrs after 4 Gy or after 2 Gy x 6 fractions, respectively. Differentially expressed genes (DEGs) were identified from RNA-seq data with DeSeq2 followed by pathway analysis with iPathwayGuide. RT sensitivity was subtype independent in 16 BC cell lines, with SUM-159 radioresistant (SF 0.88) and ZR-75-1 radiosensitive (SF 0.29). There were 75 unique pathways that were significantly altered after RT in SUM-159 cells (53 pathways in vivo only, 36 pathways in vitro only, 14 both conditions; adjusted p-value < 0.05) and 85 unique pathways that were significantly altered after RT in ZR-75-1 cells (16 pathways in vivo only, 72 in vivo only, 3 both conditions; adjusted p-value < 0.05). Pathways that were significantly affected in both cell lines exclusively in the in vitro condition include canonical RT response pathways such as cell cycle, cellular senescence, and DNA replication, though the direction of DEGs were opposite in the two cell lines for each of these pathways. The IL-17 signaling pathway was significantly altered for both cell lines in vivo. Of the pathways that were significantly altered in both conditions for SUM-159 cells, inflammation, including chemokine signaling pathway and cytokine-cytokine receptor interaction, were among the most significant. Significantly more cytokines were upregulated following RT in vivo than in vitro. Cytokines were not upregulated in ZR-75-1 cells in vitro or in vivo. Taken together, the significant changes in the IL-17 pathway and the upregulation of cytokines only in vivo indicate a potential of the tumor microenvironment in the in vivo condition that the in vitro condition lacks. Increased heterogeneity in vivo relative to in vitro may also explain the absence of several canonical RT response pathways in the in vivo conditions for each cell line. Notably, the opposite direction of DEG changes in the canonical RT response pathways between the 2 cell lines with disparate radiosensitivity levels may point to important biologic vulnerabilities that may be targeted in the resistant SUM-159 cells. Future studies are underway using additional BC cell lines and single-cell analysis to better understand RT response heterogeneity.

E2F3 induces DNA damage repair, stem-like properties and therapy resistance in breast cancer

Therapy resistance is a major hurdle to the treatment of human malignant tumors. Both DNA damage repair and stem-like properties contribute to chemoresistance and radioresistance. E2F transcription factor 3 (E2F3) is overexpressed in breast cancer tissues, and promotes proliferation of breast cancer cells. Higher E2F3 level is associated with shorter survival of breast cancer patients. Functional studies further showed that E2F3 promotes S-phage entry, DNA replication, DNA damage repair and stem-like properties. Accordingly, E2F3 knockdown sensitizes breast cancer cells to DNA-damaging agents Adriamycin, Cisplatin, Olaparib and X-ray. Forkhead box M1 (FOXM1) is a downstream molecule of E2F3 signaling, mediating the effects of E2F3 on breast cancer cells. In an m6A methyltransferase METTL14-dependent manner, YTH RNA binding protein F2 (YTHDF2) increase E2F3 mRNA stability and expression, promotes DNA damage repair and induces therapy resistance. These data demonstrate that YTHDF2-E2F3 pathway is a novel target to overcome chemoresistance and radioresistance in breast cancer.

LINC00963-FOSB-mediated transcription activation of UBE3C enhances radioresistance of breast cancer cells by inducing ubiquitination-dependent protein degradation of TP73

BackgroundThe ubiquitin protein ligase E3C (UBE3C) has been reported to play an oncogenic role in breast cancer (BRCA). This work further investigates the effect of UBE3C on the radioresistance of BRCA cells.MethodsMolecules linking to radioresistance in BRCA were identified by analyzing two GEO datasets, GSE31863 and GSE101920. UBE3C overexpression or knockdown was induced in parental or radioresistant BRCA cells, followed by irradiation treatment. The malignant properties of cells in vitro, and the growth and metastatic activity of cells in nude mice, were analyzed. Downstream target proteins, as well as upstream transcriptional regulators of UBE3C, were predicted by bioinformatics tools. Molecular interactions were confirmed by immunoprecipitation and immunofluorescence assays. Furthermore, artificial alterations of TP73 and FOSB were induced in the BRCA cells for functional rescue assays.ResultsAccording to bioinformatics analyses, UBE3C expression was linked to radioresistance in BRCA. UBE3C knockdown in radioresistant BRCA cells reduced while its overexpression in parental BRCA cells increased the radioresistance of cells in vitro and in vivo. UBE3C, which induced ubiquitination-dependent protein degradation of TP73, was transcriptionally activated by FOSB. The radioresistance of cancer cells was blocked by TP73 overexpression or FOSB knockdown. Additionally, LINC00963 was found to be responsible for the recruitment of FOSB to the UBE3C promoter for transcription activation.ConclusionThis work demonstrates that LINC00963 induces nuclear translocation of FOSB and the consequent transcription activation of UBE3C, which enhances radioresistance of BRCA cells by inducing ubiquitination-dependent protein degradation of TP73.Graphical

Abstract 2401: Molecular mechanisms of intrinsic radioresistance in breast cancer

Abstract Background: Clinical management of BC includes radiation therapy (RT), with most women receiving RT as part of their treatment. Although effective, many women develop locoregional recurrence, including a disproportionate number of women with triple-negative or inflammatory BC. Unfortunately, the molecular mechanisms that underly RT response and intrinsic radioresistance are poorly understood. We hypothesized that transcriptomic and proteomic changes that occur after ionizing radiation in intrinsically radiosensitive and resistant BC models would offer mechanistic insight into mediators of this differential response. Methods: Intrinsic radiosensitivity across all 10 cell lines was measured with clonogenic survival assays as the surviving fraction (SF) after 2 Gy RT. Gene expression changes were assessed by RNA-Seq 24 hours after 4 Gy RT. For long-course RT, cell lines were treated with fractionated RT (2 Gy x 5 fractions). For in vivo mouse xenograft experiments mice received fractionated RT (2 Gy x 6 fractions). Differential gene expression analysis with DeSeq2 was performed on all samples, followed by pathway analysis with Advaita Bioinformatics’ iPathwayGuide. Protein was collected 1, 12, and 24 hours after RT for RPPA analysis evaluating expression changes in 100 proteins and phospho-proteins with SuperCurve. Results: Clonogenic survival identified a wide range of radiation sensitivity in human BC cell lines (SF 83% - 19%) with no significant correlation (r %lt 0.3) to intrinsic BC subtype. The most highly affected pathways in both resistant and sensitive cell lines 24 hours after RT include cell cycle, cellular senescence, and estrogen signaling pathways. For the long-course RT samples, several pathways were significantly altered in fractionated samples only, including MAPK and Hippo signaling and EGFR tyrosine kinase inhibitor resistance. From the in vivo experiments, pathways uniquely affected in the in vivo samples include IL-17 signaling and transcriptional misregulation in cancer. From the proteomic data, we found that proteins including p53, Bcl-2 family proteins, and cell cycle proteins exhibit expression changes after 1 hour. A significant number of pathways (N=69, p %lt 0.01, FDR 0.05) were affected in radioresistant BC models compared to radiosensitive cell lines and these pathways may underlie intrinsic radioresistance. Conclusions: Ionizing radiation induces transcriptomic and proteomic expression changes that differ between intrinsically sensitive and resistant BC models in both single fraction and fractionated studies. Pathways identified in these analyses offer potential insight into the mechanisms underlying intrinsic radioresistance and suggest biologic vulnerabilities that may be targeted to more effectively treat women at a high risk of local BC recurrence. Genome wide CRIPSR-Cas9 screens are currently underway in these breast cancer models to confirm these vulnerability targets. Citation Format: Breanna N. McBean, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Lynn M. Lerner, Connor Ward, Meilan Liu, Alan P. Boyle, Corey W. Speers. Molecular mechanisms of intrinsic radioresistance in breast cancer [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 2401.

Nodakenin Induces ROS-Dependent Apoptotic Cell Death and ER Stress in Radioresistant Breast Cancer

Angelica gigas exerts powerful anti-tumor and anti-cancer effects in various cancer cell types. However, there have been few studies regarding the anti-cancer effect of nodakenin, a bioactive compound of Angelica gigas, in vivo and in vitro on breast cancers. I found that nodakenin, in a concentration-dependent manner, inhibits breast cancer cell viability and decreases the tumor volume in mice. Additionally, nodakenin induces caspase-3-dependent apoptosis in breast cancer cells; however, the combination of Z-VAD-FMK and nodakenin suppresses the caspase-3-dependent apoptotic cell death. Furthermore, nodakenin mediates apoptotic cell death via the PERK-mediated signaling pathway and calcium (Ca2+) release, and nodakenin combined with thapsigargin induces synergistic cell death by inhibiting sarco/endoplasmic reticulum (ER) Ca2+-ATPase. However, knockdown of PERK or CHOP inhibits Ca2+ generation and caspase-dependent apoptosis in nodakenin-treated breast cancer cells. Nodakenin induces ROS and Ca2+ generation, ER stress, and apoptotic cell death; however, the knockdown of Nox4 inhibits ROS generation and ER stress- and caspase-dependent apoptotic cell death. In addition, nodakenin combined with radiation overcomes radioresistance in radioresistant breast cancer cells by suppressing epithelial-mesenchymal transition phenotypes, including the decrease in E-cadherin and the increase in N-cadherin and vimentin. Therefore, these findings indicate that nodakenin may be a novel therapeutic strategy for breast cancers.