Sensitivity Of Breast Cancer Cells Research Articles

Glycogen synthase 1 targeting reveals a metabolic vulnerability in triple-negative breast cancer

BackgroundHypoxia-induced glycogen turnover is implicated in cancer proliferation and therapy resistance. Triple-negative breast cancers (TNBCs), characterized by a hypoxic tumor microenvironment, respond poorly to therapy. We studied the expression of glycogen synthase 1 (GYS1), the key regulator of glycogenesis, and other glycogen-related enzymes in primary tumors of patients with breast cancer and evaluated the impact of GYS1 downregulation in preclinical models.MethodsmRNA expression of GYS1 and other glycogen-related enzymes in primary breast tumors and the correlation with patient survival were studied in the METABRIC dataset (n = 1904). Immunohistochemical staining of GYS1 and glycogen was performed on a tissue microarray of primary breast cancers (n = 337). In four breast cancer cell lines and a mouse xenograft model of triple-negative breast cancer, GYS1 was downregulated using small-interfering or stably expressed short-hairpin RNAs to study the effect of downregulation on breast cancer cell proliferation, glycogen content and sensitivity to various metabolically targeted drugs.ResultsHigh GYS1 mRNA expression was associated with poor patient overall survival (HR 1.20, P = 0.009), especially in the TNBC subgroup (HR 1.52, P = 0.014). Immunohistochemical GYS1 expression in primary breast tumors was highest in TNBCs (median H-score 80, IQR 53–121) and other Ki67-high tumors (median H-score 85, IQR 57–124) (P < 0.0001). Knockdown of GYS1 impaired proliferation of breast cancer cells, depleted glycogen stores and delayed growth of MDA-MB-231 xenografts. Knockdown of GYS1 made breast cancer cells more vulnerable to inhibition of mitochondrial proteostasis.ConclusionsOur findings highlight GYS1 as potential therapeutic target in breast cancer, especially in TNBC and other highly proliferative subsets.

METTL3-mediated m6A modification of lnc KCNQ1OT1 promotes doxorubicin resistance in breast cancer by regulating miR-103a-3p/MDR1 axis

ABSTRACT Doxorubicin (DOX) resistance in breast cancer (BC) poses a huge challenge for the therapeutic effect on BC. Lnc KCNQ1OT1 play crucial roles in chemotherapy resistance. However, the role and mechanism of lnc KCNQ1OT1 in DOX resistance BC have not been investigated, which merits further exploration. Based on MCF‐7 and MDA-MB-231 cells, MCF‐7/DOX and MDA-MB-231/DOX cells were established using gradient concentrations of DOX. IC50 values and cell viability were determined using MTT. Cell proliferation was investigated by colony formation. Flow cytometry was performed to examine cell apoptosis and cell cycle. Gene expression was examined using qRT-PCR and western blot. The interactions among METTL3, lnc KCNQ1OT1, miR-103a-3p, and MDR1 were validated with MeRIP-qPCR, RIP, and dual-luciferase reporter gene assays. The results showed that Lnc KCNQ1OT1 was highly expressed in DOX-resistant BC cells, and lnc KCNQ1OT1 depletion could enhance DOX sensitivity in BC cells and DOX-resistant BC cells. Besides, lnc KCNQ1OT1 was modulated by MELLT3 in the manner of m6A modification. MiR-103a-3p could interact with lnc KCNQ1OT1 and MDR1. Overexpression of MDR1 abolished the impacts of lnc KCNQ1OT1 depletion on DOX resistance in BC. In conclusion, our results unveiled that in BC cells and DOX-resistant BC cells, lnc KCNQ1OT1 could be mediated by METTL3 through m6A modification to elevate and stabilize its expression, further inhibitingmiR-103a-3p/MDR1 axis to promote DOX resistance, which might provide novel thought to overcome DOX resistance in BC.

Targeting and Sensitization of Breast Cancer Cells to Killing with a Novel Interleukin-13 Receptor α2-Specific Hybrid Cytolytic Peptide.

Highly metastatic breast cancers, such as triple-negative subtypes (TNBC), require the most effective treatments. Since interleukin-13 receptor (IL-13R)α2 is reportedly over-expressed in some cancers, we investigated here its expression and the feasibility of therapeutically targeting this receptor in breast cancer using a novel hybrid cytolytic peptide (Pep-1-Phor21) consisting of IL-13Rα2-binding (Pep-1) and cytolytic (Phor21) domains. This study demonstrates that particularly TNBC tissues and cells display the prominent expression of IL-13Rα2. Furthermore, Pep-1-Phor21 induced the rapid necrosis of tumor cells expressing cell-surface IL-13Rα2. Notably, IL-13Rα2 expression was found to be epigenetically regulated in breast cancer cells in that the inhibition of histone deacetylase (HDAC) or DNA methyltransferase (DNMT) upregulated IL-13Rα2 expression, thereby sensitizing them to Pep-1-Phor21. IL-13Rα2-negative non-malignant cells were refractory to these epigenetic effects. Consistent with its cytolytic activity, Pep-1-Phor21 readily destroyed IL-13Rα2-expressing breast cancer spheroids with HDAC or DNMT inhibition, further enhancing cytolytic activity. Therefore, the Pep-1-Phor21-mediated targeting of IL-13Rα2 is a potentially novel therapeutic strategy for TNBC. Given that tumor cells can be selectively sensitized to Pep-1-Phor21 via the epigenetic up-regulation of IL-13Rα2, a combined adjuvant approach involving Pep-1-Phor21 and epigenetic inhibitors may be an effective strategy.

Stellera chamaejasme extract against multidrug resistance of breast cancer cell line MCF-7

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.

Nebivolol Sensitizes BT-474 Breast Cancer Cells to FGFR Inhibitors.

The fibroblast growth factor receptor (FGFR) signaling pathway is abnormally activated in human cancers, including breast cancer. Therefore, targeting the FGFR signaling pathway is a potent strategy to treat breast cancer. The purpose of this study was to find drugs that could increase sensitivity to FGFR inhibitor effects in BT-474 breast cancer cells, and to investigate the combined effects and underlying mechanisms of these combinations for BT-474 breast cancer cell survival. Cell viability was measured by MTT assay. Protein expression was determined by western blot analysis. mRNA expression was detected by Real-time PCR. Drug synergy effect was determined by isobologram analysis. Nebivolol, a third generation β1-blocker, synergistically increased the sensitivity of BT-474 breast cancer cells to the potent and selective FGFR inhibitors erdafitinib (JNJ-42756493) and AZD4547. A combination of nebivolol and erdafitinib markedly reduced AKT activation. Suppression of AKT activation using specific siRNA and a selective inhibitor further enhanced cell sensitivity to combined treatment with nebivolol and erdafitinib, whereas SC79, a potent activator of AKT, reduced cell sensitivity to nebivolol and erdafitinib. Enhanced sensitivity of BT-474 breast cancer cells to nebivolol and erdafitinib was probably associated with down-regulation of AKT activation. Combined treatment with nebivolol and erdafitinib is a promising strategy for breast cancer treatment.

Prolactin-induced protein (PIP) increases the sensitivity of breast cancer cells to drug-induced apoptosis

We have previously shown that high expression of prolactin-induced protein (PIP) correlates with the response of breast cancer (BC) patients to standard adjuvant chemotherapy (doxorubicin and cyclophosphamide), which suggests that the absence of this glycoprotein is associated with resistance of tumor cells to chemotherapy. Therefore, in the present study, we analyzed the impact of PIP expression on resistance of BC cells to anti-cancer drugs and its biological role in BC progression. Expression of PIP and apoptotic genes in BC cell lines was analyzed using real-time PCR and Western blotting. PIP was detected in BC tissue specimens using immunohistochemistry. The tumorigenicity of cancer cells was analyzed by the in vivo tumor growth assay. Apoptotic cells were detected based on caspase-3 activation, Annexin V binding and TUNEL assay. The interaction of PIP with BC cells was analyzed using flow cytometry. Using two cellular models of BC (i.e. T47D cells with the knockdown of the PIP gene and MDA-MB-231 cells overexpressing PIP), we found that high expression of PIP resulted in (1) increased sensitivity of BC cells to apoptosis induced by doxorubicin (DOX), 4-hydroperoxycyclophosphamide (4-HC), and paclitaxel (PAX), and (2) improved efficacy of anti-cancer therapy with DOX in the xenograft mice model. Accordingly, a clinical study revealed that BC patients with higher PIP expression were characterized by longer 5-year overall survival and disease-free survival. Subsequent studies showed that PIP up-regulated the expression of the following pro-apoptotic genes: CRADD, DAPK1, FASLG, CD40 and BNIP2. This pro-apoptotic activity is mediated by secreted PIP and most probably involves the specific surface receptor. This study demonstrates that a high expression level of PIP sensitizes BC cells to anti-cancer drugs. Increased sensitivity to chemotherapy is the result of pro-apoptotic activity of PIP, which is evidenced by up-regulation of specific pro-apoptotic genes. As high expression of PIP significantly correlated with a better response of patients to anti-cancer drugs, this glycoprotein can be a marker for the prognostic evaluation of adjuvant chemotherapy.

Abstract 550: p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors

Abstract The p53 tumor suppressor mutations resulting in loss of function control tumor proliferation in 50% of human cancers. TP53 missense and nonsense mutations are present in about 50% and 7-12% of the p53 driven cancer, respectively. To date there is no effective treatment for rescuing p53 function in cancers harboring p53 nonsense mutations. We have developed a new potent strategy combining mRNA with a tumor selective nanocarrier, to rescue of p53 tumor suppressor functions as potential therapeutic approach in cancers. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides that form stable neutral nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring different types of p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (weekly, 0.5 &amp; 1.0mg/kg) was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and lung H358 (p53 null/KRASG12C) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment was evaluated on PARPi resistant SUM-149PT and OVCAR-8 and PARPi sensitive MDA-MB436 cells. ADGN-531/AMG-510 synergy was evaluated on H358 and M-PACA cells and H358 mouse xenograft. ADGN-531 markedly delay the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. ADGN-531 mediated p53 function rescue is directly correlated to the type of p53 mutation in the cancer cells. ADGN-531 induces 80% cell proliferation inhibition in p53 null/nonsense cells and 20 to 50% in p53 missense cells. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition. ADGN-531 (1.0mg/kg) induces 40-60% tumor regression SaOs2, SW403 and H358 xenografted mouse model. We demonstrated a synergistic combination between ADGN-531/AMG-510 a KRASG12C inhibitor in human lung carcinoma. ADGN-531 (0.5mg/kg, weekly)/AMG-510 (10mg/kg, daily) induces about 80% tumor regression, higher than with either single agents. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to Veliparib. ADGN-531 treatments are well tolerated, no sign of clinical toxicity or inflammatory response was detected after single or repeated administrations. ADGN-531 is effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy or in combination together with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Khadidja Boularache, Melanie Guidetti, Veronique Josserand, Neil Desai. p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors [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 550.

Ribosome Biogenesis Regulator 1 Homolog (RRS1) Promotes Cisplatin Resistance by Regulating AEG-1 Abundance in Breast Cancer Cells

Many ribosomal proteins are highly expressed in tumors and are closely related to their diagnosis, prognosis and pathological characteristics. However, few studies are available on the correlation between ribosomal proteins and chemoresistance. RRS1 (human regulator of ribosome synthesis 1), a critical nuclear protein involved in ribosome biogenesis, also plays a key role in the genesis and development of breast cancer by protecting cancer cells from apoptosis. Given that apoptosis resistance is one of the causes of the cisplatin resistance of tumor cells, our aim was to determine the relationship between RRS1 and cisplatin resistance in breast cancer cells. Here, we report that RRS1 is associated with cisplatin resistance in breast cancer cells. RRS1 silencing increased the sensitivity of MCF-7/DDP cells to cisplatin and inhibited cancer cell proliferation by blocking cell cycle distribution and enhancing apoptosis. AEG-1 (astrocyte elevated gene-1) promotes drug resistance by interfering with the ubiquitination and proteasomal degradation of MDR1 (multidrug resistance gene 1), thereby enhancing drug efflux. We found that RRS1 binds to and stabilizes AEG-1 by inhibiting ubiquitination and subsequent proteasomal degradation, which then promotes drug efflux by upregulating MDR1. Furthermore, RRS1 also induces apoptosis resistance in breast cancer cells through the ERK/Bcl-2/BAX signaling pathway. Our study is the first to show that RRS1 sensitizes breast cancer cells to cisplatin by binding to AEG-1, and it provides a theoretical basis to improve the efficacy of cisplatin-based chemotherapy.

TIPE2 sensitizes breast cancer cells to paclitaxel by suppressing drug-induced autophagy and cancer stem cell properties.

Drug resistance is a great obstacle to the clinical application of paclitaxel (PTX) in breast cancer treatment. Chemoresistance can be either primary or acquired. Multifarious factors are related to drug resistance. Among these factors, drug-induced autophagy has been shown to contribute to acquired chemoresistance in cancer cells. Additionally, cancer stem cells (CSCs) drive primary chemoresistance. Recent advances regarding TIPE2 demonstrate that TIPE2 enhances osteosarcoma and non-small cell lung cancer cell sensitivity to cisplatin. However, the role of TIPE2 in PTX resistance in breast cancer cells has not been elucidated. Here, the in vitro and in vivo study demonstrated that TIPE2 sensitized breast cancer cells to PTX by suppressing drug-induced autophagy and CSC properties. Mechanistically, we found that TIPE2 activated the AKT/mTOR signalling pathway and inhibited the TAK1/MAPK signalling pathway to suppress drug-induced autophagy. Moreover, TIPE2 inhibited TAK1/NF-κB activation to reduce breast CSC properties. Collectively, our results first elucidated the inhibitory role of TIPE2 in breast cancer chemoresistance. Thus, TIPE2 may be a new target for breast cancer chemotherapy.

PARP Inhibition Sensitizes Breast Cancer Cells to Eribulin.

Poly(ADP-ribose) polymerases 1 and 2 (PARP1, 2), and 3 mediate protein modifications that facilitate the recruitment of DNA repair factors to single and double strand breaks. PARP3 is unique in that it is also required for efficient mitotic progression and stabilization of the mitotic spindle. Eribulin, an anti-microtubule agent used clinically to treat breast cancer, exerts its cytotoxicity by altering microtubule dynamics resulting in cell cycle arrest and apoptosis. Herein, we hypothesize that the pan PARP inhibitor olaparib has the potential to enhance the cytotoxicity of eribulin by halting mitosis through inhibition of PARP3. The effect of olaparib on eribulin cytotoxicity was assessed using the Sulforhodamine (SRB) assay, with two triple negative breast cancer cell lines and an estrogen receptor positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer cell line. Alteration by the treatments on PARP3 activity and microtubule dynamics were assessed utilizing a chemiluminescent enzymatic assay and immunofluorescence, respectively. The effect of the treatments on cell cycle progression and apoptosis induction were assessed by flow cytometry using propidium iodide and Annexin V staining, respectively. Our results demonstrate that non-cytotoxic concentrations of olaparib sensitize breast cancer cells regardless of ER status. Mechanistically, our results indicate that olaparib potentiates eribulin-induced cell cycle arrest at the G2/M boundary, PARP3 inhibition and microtubule destabilizing resulting in mitotic catastrophe and apoptosis. In breast cancer (regardless of ER status) settings, treatment outcomes could be improved by the incorporation of olaparib in eribulin treatment regimens.

An Angiogenesis-Related lncRNA Signature Is Associated with Prognosis and Tumor Immune Microenvironment in Breast Cancer.

Angiogenesis is crucial in the development and progression of tumors. This study examined the relationship between angiogenesis-related lncRNAs (AR-lncRNAs) and breast cancer (BC) immunity and prognosis. We used univariate Cox regression analysis to obtain AR-lncRNAs closely related to BC prognosis. Cluster analysis of BC patients was performed using non-negative matrix factorization (NMF) analysis according to the expression of AR-lncRNAs that were prognostically relevant. An AR-lncRNA risk model (AR-lncM) was created using LASSO regression analysis to predict the prognosis and survival of BC patients. Subsequently, the effect of LINC01614 on cell migration and invasion was verified by Transwell and Western blot assays, and the CCK-8 assay detected its impact on cell sensitivity to tamoxifen. Finally, we obtained 17 AR-lncRNAs from the TCGA database that were closely associated with the prognosis of BC patients. Based on the expression of these AR-lncRNAs, BC patients were divided into five clusters using NMF analysis. Cluster 1 was found to have a better prognosis, higher expression of immune checkpoints, and higher levels of immune cell infiltration. Furthermore, an AR-LncM model was created using ten prognostic-related AR-lncRNAs. The model's risk predictive performance was validated using survival analysis, timeROC curves, and univariate and multivariate Cox analysis. The most interesting gene in the model, LINC01614, was found to regulate epithelial-mesenchymal transition (EMT) and tamoxifen sensitivity in BC cells, implying that LINC01614 could be a potential therapeutic target for BC patients.

Genomic landscape of metastatic breast cancer (MBC) patients with methylthioadenosine phosphorylase (MTAP) loss

Homozygous deletion of MTAP upregulates de novo synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer cells to DNSP inhibitors such as methotrexate, L-alanosine and pemetrexed. 7,301 cases of MBC underwent hybrid-capture based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Tumor cell PD-L1 expression was determined by IHC (Dako 22C3). 208 (2.84%) of MBC featured MTAP loss. MTAP loss patients were younger (p = 0.002) and were more frequently ER- (30% vs. 50%; p < 0.0001), triple negative (TNBC) (47% vs. 27%; p < 0.0001) and less frequently HER2+ (2% vs. 8%; p = 0.0001) than MTAP intact MBC. Lobular histology and CDH1 mutations were more frequent in MTAP intact (14%) than MTAP loss MBC (p < 0.0001). CDKN2A (100%) and CDKN2B (97%) loss (9p21 co-deletion) were significantly associated with MTAP loss (p < 0.0001). Likely associated with the increased TNBC cases, BRCA1 mutation was also more frequent in MTAP loss MBC (10% vs. 4%; p < 0.0001). As for immune checkpoint inhibitors biomarkers, higher TMB >20 mut/Mb levels in the MTAP intact MBC (p < 0.0001) and higher PD-L1 low expression (1-49% TPS) in the MTAP loss MTAP (p = 0.002) were observed. MTAP loss in MBC has distinct clinical features with genomic alterations (GA) affecting both targeted and immunotherapies. Further efforts are necessary to identify alternative means of targeting PRMT5 and MTA2 in MTAP-ve cancers to benefit from the high-MTA environment of MTAP-deficient cancers.

Mir-153-3p Modulates the Breast Cancer Cells' Chemosensitivity to Doxorubicin by Targeting KIF20A.

Breast cancer is considered the solid tumor most sensitive to chemotherapy. However, it can become resistant to various chemotherapeutic drugs, including doxorubicin, which triggers cell death by intercalation between DNA bases, free radical formation, and topoisomerase II inhibition. When drug resistance develops, several miRNAs are dysregulated, suggesting that miRNAs may play a significant role in resistance formation. In the current study, we investigated how doxorubicin sensitivity of breast cancer cells is affected by miR-153-3p and its target gene. The MTT method was used to determine the chemo-sensitizing effect of miR-153-3p on doxorubicin in MCF-7 and MDA-MB-231 cell lines. Results of Western blot and dual luciferase confirmed that miR-153-3p targets KIF20A and decreases its expression. Transwell and flow cytometry experiments showed that miR-153-3p and doxorubicin together had higher effects on MCF-7 and MDA-MB-231 cell proliferation, migration, and invasion, as well as increasing apoptosis and arresting cells in the G1 phase. Proteins related to apoptosis and the cell cycle exhibited the same tendency. Intracellular vesicle formation was inhibited and RAB26 was also downregulated by treatment with miR-153-3p alone or in combination with doxorubicin. Doxorubicin's ability to suppress tumors may be enhanced by miR-153-3p, according to in vivo studies. According to our findings, miR-153-3p has a direct effect on KIF20A and may regulate the formation of intracellular vesicles, which in turn makes breast cancer cells more susceptible to doxorubicin.

Abstract P2-16-04: MNX1 promotes tumor sensitivity to HER2-targeted therapy in HER2-positive breast cancer

Abstract Background: Female breast cancer has been the world’s leading cancer incidence among women. HER2-positive breast cancer accounts for about 15%~20% of all breast cancer, with a high degree of malignancy and easy metastasis and recurrence. With the development of medical technology and the continuous innovation of HER2 targeting drugs, patients with HER2-positive breast cancer have more treatment options and their prognosis had been greatly improved. However, there is still a lack of biomarkers for HER2-positive early breast cancer in clinical practice. Methods: This study prospectively collected single-center (Fudan University Shanghai Cancer Center, FUSCC) preoperative core needle biopsy samples of breast cancer patients from July 2017 to July 2018 who received neoadjuvant paclitaxel, carboplatin plus with trastuzumab (PCH). The patients were divided into pCR and non-PCR groups, and 81 patients were enrolled. The differential expression of genes was screened and validated by RNA-seq. The gene expression data for GSE181574 (this database has a total of 105 cases of breast cancer tissue samples were collected by core needle biopsy before neoadjuvant treatment. 52 cases underwent Ado-trastuzumab emtansine plus pertuzumab, 9 cases of paclitaxel plus trastuzumab, and 44 cases of paclitaxel plus trastuzumab and pertuzumab), GSE52707 (SK-BR-3 Lapatinib resistance cell group vs control group), and GSE15043 (BT474 Herceptin-resistant cell group vs control group), were downloaded from the Gene Expression Omnibus (GEO) database. Cell cloning formation, proliferation assay, and drug sensitivity experiments were conducted in MNX1 ectopic and knockdown cell lines. Co-IP assay, RNA-Seq, and ChIP-Seq analysis were used to explore the downstream pathways that MNX1 might be involved. Results: High-throughput sequencing results of core needle biopsy samples from 81 HER2-positive breast cancer patients were divided into pCR and non-pCR groups. Using DEseq2 packet analysis to screen differentially expressed genes, P-value &amp;lt; 0.05, | log2FoldChange | &amp;gt; 1 as the filter, there were 620 up-regulated genes and 715 down-regulated genes in the pCR group. The same method was used for the analysis of the GSE181574 data set. Combined the two datasets used P values and log2FoldChange (P-value &amp;lt; 0.05, | log2FoldChange | &amp;gt; 1.2) as selection criteria, we found the MNX1 and PNMT expressions were significantly higher in the pCR group. Subsequently, differential analysis of GSE52707 and GSE15043 datasets showed that MNX1 and PNMT expression levels were reduced in drug-resistant cell lines. Considering the P-value and | log2FoldChange |, we ultimately selected motor neuron and pancreatic homeobox 1(MNX1) gene as the target factors for follow-up studies. In the cloning formation and proliferation assay, overexpression of MNX1 inhibits proliferation and clonal formation of HER2-positive breast cancer cells. In the drug sensitivity experiments overexpression of MNX1 enhances the sensitivity of HER2-positive breast cancer cells to tyrosine kinase inhibitors (TKI) such as lapatinib and pyrotinib. MNX1 knockdown reduces the sensitivity of HER2-positive breast cancer cells to TKI. Through RNA-seq and ChIP-seq, we found that CD-M6PR might be a downstream target gene regulated by MNX1. Verification by Dual-Luciferase Reporter Assay MNX1 was found to positively regulate CD-M6PR transcription. By Co-IP assay, it was found that MNX1 interacted with EEF1D, and EEF1D stabilized MNX1. MNX1 and CD-M6PR affect the proliferation and drug sensitivity of breast cancer cells by down-regulating the PI3K-AKT-mTOR pathway. Conclusion: Overexpression of MNX1 increases the sensitivity of HER-2 positive cells to TKI. MNX1 binds to EEF1D and is stabilized by EEF1D. MNX1 positively regulates M6PR and affects the proliferation of breast cancer cells and the sensitivity of breast cancer cells to TKI through the PI3K-AKT-mTOR pathway. Citation Format: Wei-Ru Chi, Bingqiu Xiu, Min Xiong, Pei Li, Qi Zhang, Lun Li, Jianjing Hou, Xujie Zhou, Yuting Sang, Ming Chen, Liyi Zhang, Jingyan Xue, Yayun Chi, Jiong Wu. MNX1 promotes tumor sensitivity to HER2-targeted therapy in HER2-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-04.

Abstract P3-11-08: Inhibition of Sirtuin 3 Sensitizes Breast Cancer Cells to Fulvestrant

Abstract Endocrine therapy can lead to nutrient deprivation by reducing glucose and glutamine uptake and total cellular ATP production. The ability of cells to bypass this metabolic stress is fundamental to how they regulate BC growth and acquire endocrine resistance. Sirtuins (SIRTs) are NAD+-dependent deacylases and regulate a widerange of intracellular processes including metabolism. SIRT3, activated by calorie restriction, modulates mitochondrial adaptation to low energy input. It has a key role in mitochondrial integrity and function, regulating cell survival, death and metabolic pathways, regulating the shift to amino-acid and fatty-acid catabolism. SIRT3 can maintain ROS levels at the appropriate levels for sustaining a proliferative phenotype, preventing apoptosis and promoting carcinogenesis. A role of SIRT3 in epigenetic regulation has also been reported. We used Differential Dependency Network analysis to compare the wiring of the SIRTs and key metabolism-related genes in matched antiestrogen- sensitive vs. resistant breast cancer (BC) cells, which identified several novel signaling hubs including CDKN2A (p16), linking SIRT3 to cell proliferation. The ability of SIRTs to sense and respond to changes in energy, coupled with their deacetylase/deacylase functions, could provide a mechanism for the cell to rewire signaling and maintain a newly acquired drug-resistant phenotype. Using endocrine sensitive (LCC1) and resistant (LCC9) BC cells, we confirmed the higher expression of SIRT3 in LCC9, compared to LCC1 cells. Upregulation of SIRT3 expression within 24 hrs of Fulvestrant (ICI 1 µM) treatment (p=0.0176) in LCC1 was shown, but no effect was observed in LCC9 cells. Conversely, treatment with 17β-estradiol (E2- 10 nM) did not significantly affect SIRT3 expression in either LCC1 or LCC9 cells. Inhibition of SIRT3 acitivity, using the LC0296 inhibitor (15 µM), reduced growth rate in LCC1 and SIRT3-silenced LCC9 cells under treatment with ICI (1 nM and 1 µM, respectively) (p&amp;lt; 0.0001, p&amp;lt; 0.001 respectively). Treatment with SIRTUIN 3 inhibitor and ICI induced apoptosis in LCC1 and SIRT3-silenced LCC9 cells compared to the untreated control and ICI only treated cells (p&amp;lt; 0.001). The combination SIRT3 inhibitor (15 µM) plus ICI (0.5 µM) increased ROS production in LCC9-SIRT3 silenced cells in 24 hours of treatment compared to the untreated control and ICI only treated cells (p&amp;lt; 0.001). SIRT3 inhibitor (15 µM) decreased LCC1 cell spheroid formation (p&amp;lt; 0.05), however it didn’t show additive effect to the effect of ICI (0.5 µM) in the same conditions. The combination of SIRT3 inhibitor (15 µM) and ICI (1 µM) reduced the growth of LCC9 SIRT3-silenced cells in detached condition compared to untreated control and ICI only treated cells (p&amp;lt; 0.001). The results show that SIRT3 may have a role in Fulvestrant response in BC cells; the mechanism is under investigation in our laboratory. Citation Format: Karla Andrade de Oliveira, Surojeet Sengupta, Lu Jin, Fabia de Oliveira Andrade, Melike Ozgu-Onal, Anil Yadav, Robert Clarke. Inhibition of Sirtuin 3 Sensitizes Breast Cancer Cells to Fulvestrant [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-08.

Abstract P1-13-19: ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in HER2 positive breast cancer

Abstract Background: Trastuzumab combined with pertuzumab is the main therapy for HER2 positive breast cancer, but poor patient response due to drug resistance remains a clinical challenge. Organoids derived from tumor patients can highly maintain the heterogeneity of the original tumor, and have been used in drug sensitivity testing and new drug development. To screen for genes that associated with trastuzumab or pertuzumab drug resistance, this study used HER2-positive breast cancer organoids to establish a model of trastuzumab combined with pertuzumab and conducted high-throughput sequencing to screen the differential genes. We carried out further research to explore the effect of ESAM on drug sensitivity of HER2-targeted therapy and clarify the specific regulatory mechanisms and potential therapeutic targets of ESAM. Method: 8 HER2 positive breast cancer organoids were cultured. Immunohistochemistry, hematoxylin-eosin staining and Immunofluorescence were performed to identify the consistency between organoids and primitive tumors. According to drug sensitivity results of organoids to trastuzumab combined with pertuzumab, organoids were divided into a relatively sensitive group and a relatively insensitive group. Trastuzumab combined with pertuzumab was regularly used to stimulate the two groups. After 3 cycles, high-throughput transcriptome sequencing was used to evaluate their gene expression to find a gene responsible for trastuzumab and pertuzumab resistance. METABRIC and our center database were used to determine the prognostic value of ESAM. ESAM overexpression and knockdown stably transfected cell lines were constructed in SK-BR-3 and BT474 breast cancer cell lines by lentivirus. Colony formation assay, CCK-8 assay, organoid model and mouse xenograft model were conducted to examine the influence of ESAM on proliferation and trastuzumab or pertuzumab sensitization in vitro and in vivo. RNA-seq and GSEA analysis were performed to investigate the downstream pathways of ESAM. Western blot was used to confirm the relationship between ESAM and mTOR pathway. Small interfering RNA and mTOR/PI3K pathway inhibitors were used to confirm the function of mTOR in ESAM-induced drug resistance of trastuzumab. Results: HER2 positive breast cancer organoids can maintain the pathological characteristics of primitive tumors. The results of high-throughput transcriptome show that ESAM is significantly up-regulated in the relatively insensitive group compared to the relatively sensitive group. METABRIC and our center database suggest that high ESAM mRNA expression was associated with poor OS, RFS, DFS and PFS of HER2-positive breast cancer patients. CCK-8 and cell colony formation assay confirm that ESAM can promote the proliferation of HER2-positive breast cancer cells and inhibit the drug sensitivity of HER2-positive breast cancer cells to trastuzumab and pertuzumab. GSEA analysis shows high ESAM can activate mTORC1 signaling pathway. Western blotting analysis proves the expression of ESAM is positively correlated with mTOR pathway. Inhibition of mTOR pathway in ESAM overexpression cells can suppress ESAM-mediated proliferation and reverse the drug sensitivity of cells to trastuzumab and pertuzumab. In vivo, overexpression of ESAM can promote the proliferation of mouse mammary tumors and reduce the sensitivity of mouse mammary tumors to trastuzumab, combined use of PI3K inhibitor could reverse drug sensitivity of mouse mammary tumors to trastuzumab. Conclusion: High ESAM expression is associated with poor prognosis in patients with HER2 positive breast cancer. ESAM can activate mTOR pathway, promote cell proliferation and reduce the sensitivity of HER2 positive breast cancer cells to trastuzumab and pertuzumab, which can be inhibited by the application of PI3K/mTOR inhibitors. Keywords: ESAM; HER2 positive breast cancer; HER2 targeted therapy; mTOR; drug sensitivity. Citation Format: Jiong Wu, Xujie Zhou, Yayun Chi, Jingyan Xue. ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in HER2 positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-19.

Abstract P6-10-01: The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4

Abstract Immunotherapy has dramatically impacted cancer therapy, but it has been challenging to apply immunotherapy to estrogen receptor (ER) positive breast cancer and many other solid tumors that do not display neoantigens. One way to target these tumors is to induce necrosis, which robustly activates immune cells, inducing immunogenic cell death. However, anticancer therapy-induced necrosis was primarily characterized by morphological changes, and the molecular drivers of necrosis were largely obscure. To probe necrosis, we used our necrosis inducing anticancer agents, the small molecules BHPI and second-generation ErSO, which kill cancer cells by hyperactivating the anticipatory unfolded protein response (a-UPR). In orthotopic mouse xenografts, ErSO induces complete regression without recurrence of large, therapy-resistant primary ER positive breast tumors, of most lung, bone, and liver metastases, near complete regression of challenging breast cancer brain metastases and robust responses in PDX and patient derived organoids (PDOs) models. ErSO also induces complete or near complete regression in mouse xenograft models of ER positive ovarian and endometrial cancer. Using genome wide CRISPR-Cas9 screens with negative selection against our necrosis-inducing a-UPR hyperactivators, BHPI and ErSO, we identified the calcium-activated, ATP-inhibited, plasma membrane sodium channel, Transient Receptor Potential Melastatin Member 4 (TRPM4) as critical for anticancer therapy induced necrosis. TRPM4 knockout in multiple models abolished ErSO-induced ATP depletion, sustained UPR activation, cell swelling, necrotic cell death and increased migration of immune cells. Notably, knockout of TRPM4 completely abolished the ability of ErSO to induce regression of ER positive breast tumors in mice. Supporting a broad role for the TRPM4 pathway in anticancer therapy induced necrosis, rapid cancer cell death induced by four necrosis-inducing cancer therapies unrelated to ErSO, that range from FDA-approved to preclinical, is strongly reversed by TRPM4 knockout. ErSO treatment induces migration of macrophage into regressing tumors. Medium from cancer cells killed by necrosis-inducing ErSO, but not by an apoptosis inducer, dramatically increases macrophage migration and activation, as shown by induction of pro-inflammatory cytokines. This work identifies a protein that plays a pivotal role in the action of diverse anticancer therapies inducing immunogenic necrosis. Since increasing levels of TRPM4 increase sensitivity of breast cancer cells to killing by ErSO, TRPM4 is a novel biomarker whose levels can be used to identify patients most likely to benefit from ErSO and other necrosis-inducing cancer therapies. Citation Format: Santanu Ghosh, Rachel Yang, Darjan Duraki, Ji Eun Kim, Junyao Zhu, Mara Livezey, Matthew Boudreau, Ben H. Park, TImothy Fan, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-01.

Abstract P2-16-01: The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells

Abstract Background: Breast cancer is the most commonly diagnosed non-cutaneous malignancy in American women and one of the leading causes of cancer deaths. Breast cancer can be divided into several subtypes, the most aggressive of which is Triple-Negative Breast Cancer (TNBC), a disease with few targeted therapies. TP53 mutations are found in 80% or more of TNBCs. However, direct targeting of mutant TP53 has been difficult. To identify drugs that can specifically induce the death of TP53-mutant breast cancers, we conducted a drug screen in TP53-mutant and TP53-wild type breast cancer cells. Through this combined in silico and in vitro drug screen, we discovered that TP53 mutant TNBC cells have an increased sensitivity to KIF11 inhibition as compared to TP53 wild-type cells. Hypothesis: We hypothesize that TP53 mutational status confers sensitivity of triple-negative breast cancer cells to KIF11 inhibition. Methods: We obtained data on TP53 mutational status, KIF11 mRNA expression levels, and clinical characteristics from publicly-available TCGA, METABRIC, and CCLE datasets. To demonstrate the effect of KIF11 inhibition on cell growth, we treated TP53 mutant and wild-type breast cancer cell lines with the small molecule KIF11 inhibitor SB-743921 and KIF11 siRNAs. Cell counts were obtained through staining with DAPI or Hoechst nuclear stains and imaging on the MetaXPress PICO instrument. To determine protein expression of p53 and Kif11 across various cell lines, western blotting was performed. We then investigated the biological mechanisms of growth inhibition by DRAQ7 staining and flow cytometry analysis following Annexin V-PI staining. Results: Using cell growth assays we demonstrated that TP53 mutant cells are more sensitive to KIF11 inhibition. We next utilized cell lines in which TP53 mutations had been introduced into TP53 wild-type cells to show that overexpression of a TP53 mutant gene can sensitize cells to KIF11 inhibition. Using assays of proliferation and apoptosis, we showed that TP53 mutant breast cancer cells treated with a KIF11 inhibitor undergo cell death. Using publicly-available datasets of breast cancers, we showed that KIF11 is upregulated in TNBCs and TP53 mutant cancers, and that high expression of KIF11 in breast cancer is correlated with poorer clinical prognosis. Conclusions: Our results show that the Kif11 protein is essential for the survival of TP53 mutant TNBC cells. Inhibitors of Kif11 induce death of TP53 mutant TNBCs and thus, this kinesin-like protein involved in cell spindle mechanics is a potential target for the treatment of these aggressive cancers. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers TL1TR003169 and UL1TR003167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the John Charles Cain Endowment. Citation Format: Amanda Lanier, Abhijit Mazumdar, William Tahaney, Powel Brown. The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-01.

OCT4B1 Dysregulation Is a Key Factor in Different Sensitivity of Normal Cells and Breast Cancer Cells to the Curcumin

OCT4B1 Dysregulation Is a Key Factor in Different Sensitivity of Normal Cells and Breast Cancer Cells to the Curcumin

MED27 plays a tumor-promoting role in breast cancer progression by targeting KLF4.

The mediator complex usually cooperates with transcription factors to be involved in RNA polymerase II-mediated gene transcription. As one component of this complex, MED27 has been reported in our previous studies to promote thyroid cancer and melanoma progression. However, the precise function of MED27 in breast cancer development remains poorly understood. Here, we found that MED27 was more highly expressed in breast cancer samples than in normal tissues, especially in triple-negative breast cancer, and its expression level was elevated with the increase in pathological stage. MED27 knockdown in triple-negative breast cancer cells inhibited cancer cell metastasis and stemness maintenance, which was accompanied by downregulation of the expression of EMT- and stem traits-associated proteins, and vice versa in non-triple-negative breast cancer. Furthermore, MED27 knockdown sensitized breast cancer cells to epirubicin treatment by inducing cellular apoptosis and reducing tumorsphere-forming ability. Based on RNA-seq, we identified KLF4 as the possible downstream target of MED27. KLF4 overexpression reversed the MED27 silencing-mediated arrest of cellular metastasis and stemness maintenance capacity in breast cancer in vitro and in vivo. Mechanistically, MED27 transcriptionally regulated KLF4 by binding to its promoter region at positions -156 to +177. Collectively, our study not only demonstrated the tumor-promoting role of MED27 in breast cancer progression by transcriptionally targeting KLF4, but also suggested the possibility of developing the MED27/KLF4 signaling axis as a potential therapeutic target in breast cancer.