Trastuzumab-resistant Breast Cancer Research Articles

Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Abstract PO3-24-05: Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer

Abstract Background Trastuzumab has shown great effectiveness in HER2 positive breast cancer treatment, but about 50% patients would undergo resistance during or after treatment. Although previous research has suggested several potential reasons for trastuzumab resistance, the metabolic reprogramming during resistance formation remains largely unclear. Here we identified aberrant ferroptosis associated cysteine metabolism in trastuzumab resistant HER2 positive breast cancer, which might become a novel target for overcoming resistance. Methods Trastuzumab sensitive HER2 positive breast cancer cell SKBR3 and resistant JITM1 were obtained for transcriptomics, proteomics, metabolomics and epigenomics analysis. Gene silencing was mediated by siRNAs. CUT&Tag was applied to compare H3K4me3 and H3K27me3 binding regions. DNA methylation levels and different methylated regions were evaluated by WGBS-seq. CRISPRi with dCas9-DNMT3A was applied to regulate specific DNA methylation in CpG islands. Lipid ROS was measured by flow cytometry with BODIPY-C11. Results Joint analyses of transcriptomics and proteomics according to ferroptosis pathways revealed downregulated glutathione metabolism, glutamate transmembrane and homocysteine metabolism processes, as well as upregulated fatty acid metabolism and iron metabolism pathways in JIMT1. Metabolomics verified that JIMT1 increased cysteine metabolism and decreased glutathione metabolism. SLC7A11 expression and GSH/GSSG ratio were increased in JIMT1, while no difference was observed in free cysteine. JIMT1 featured significant higher UGC codon usage bias and increased cysteinyl-tRNA synthetase. The abundance of H3K4me3 other than H3K27me3 in SLC7A11 promoter region was found increased in JIMT1, and the 5-mC level of CpG islands in SLC7A11 promoter region was shown decreased. Using dCas9-DNMT3A, the methylation of SLC7A11 promoter was enhanced and SLC7A11 expression was reduced in JIMT1. Inhibition of SLC7A11 by siRNAs, CRISPRi or Erastin all indicated a higher ferroptosis sensitivity in JIMT1. Conclusion Trastuzumab resistant HER2 positive breast cancer features aberrant cysteine metabolism resulting from altered H3K4me3 modification and DNA methylation in SLC7A11 promoter region. This might provide novel targets for further anti-HER2 treatment. Citation Format: Yongmei Yin, Yijia Hua, Ningjun Duan, Chunxiao Sun, Fan Yang. Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-05.

MiR-770-5p-induced cellular switch to sensitize trastuzumab resistant breast cancer cells targeting HER2/EGFR/IGF1R bidirectional crosstalk

miR-770-5p-induced cellular switch to sensitize trastuzumab resistant breast cancer cells targeting HER2/EGFR/IGF1R bidirectional crosstalk

An Imbalance in Histone Modifiers Induces tRNA-Cys-GCA Overexpression and tRF-27 Accumulation by Attenuating Promoter H3K27me3 in Primary Trastuzumab-Resistant Breast Cancer.

tRNA-derived fragments (tRFs) play crucial roles in cancer progression. Among them, tRF-27 has been identified as a key factor in promoting naïve trastuzumab resistance in HER2-positive breast cancer. However, the origin of tRF-27 remains uncertain. In this study, we propose that the upregulated expression of specific cysteine tRNAs may lead to the increased accumulation of tRF-27 in trastuzumab-resistant JIMT1 cells. Mechanistically, the reduced inhibitory H3K27me3 modification at the promoter regions of tRF-27-related tRNA genes in JIMT1 cells, potentially resulting from decreased EZH2 and increased KDM6A activity, may be a critical factor stimulating the transcriptional activity of these tRNA genes. Our research offers fresh insights into the mechanisms underlying elevated tRF-27 levels in trastuzumab-resistant breast cancer cells and suggests potential strategies to mitigate trastuzumab resistance in clinical treatments.

Development and evaluation of a human CD47/HER2 bispecific antibody for Trastuzumab-resistant breast cancer immunotherapy

The treatment for trastuzumab-resistant breast cancer (BC) remains a challenge in clinical settings. It was known that CD47 is preferentially upregulated in HER2+ BC cells, which is correlated with drug resistance to trastuzumab. Here, we developed a novel anti-CD47/HER2 bispecific antibody (BsAb) against trastuzumab-resistant BC, named IMM2902. IMM2902 demonstrated high binding affinity, blocking activity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and internalization degradation effects against both trastuzumab-sensitive and trastuzumab-resistant BC cells in vitro. The in vivo experimental data indicated that IMM2902 was more effective than their respective controls in inhibiting tumor growth in a trastuzumab-sensitive BT474 mouse model, a trastuzumab-resistant HCC1954 mouse model, two trastuzumab-resistant patient-derived xenograft (PDX) mouse models and a cord blood (CB)-humanized HCC1954 mouse model. Through spatial transcriptome assays, multiplex immunofluorescence (mIFC) and in vitro assays, our findings provided evidence that IMM2902 effectively stimulates macrophages to generate C-X-C motif chemokine ligand (CXCL) 9 and CXCL10, thereby facilitating the recruitment of T cells and NK cells to the tumor site. Moreover, IMM2902 demonstrated a high safety profile regarding anemia and non-specific cytokines release. Collectively, our results highlighted a novel therapeutic approach for the treatment of HER2+ BCs and this approach exhibits significant anti-tumor efficacy without causing off-target toxicity in trastuzumab-resistant BC cells.

Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.

Exosomal Linc00969 induces trastuzumab resistance in breast cancer by increasing HER-2 protein expression and mRNA stability by binding to HUR

BackgroundBreast cancer (BC) is the most common malignant disease in female patients worldwide. In HER-2+ BC patients, trastuzumab therapy is associated with a better prognosis. However, many HER-2+ BC patients experience recurrence or metastasis because of trastuzumab resistance. The mechanisms underlying trastuzumab resistance remain unclear. Recently, substantial evidence has suggested that exosomes are associated with drug resistance, and lncRNAs have attracted increasing attention due to their potential role in the regulation of trastuzumab resistance.MethodsWe collected the exosomes from the plasma of BC patients with and without trastuzumab resistance, sequenced the whole transcriptomes, identified differentially expressed lncRNAs, and identified lncRNA Linc00969, which was overexpressed in trastuzumab-resistant patients. Then, we established trastuzumab-resistant BC cell lines and explored the role of exosomal Linc00969 in trastuzumab resistance in vitro and in vivo by silencing or overexpressing Linc00969 and performing a series of functional analyses. Furthermore, to explore the mechanism by which exosomal Linc00969 contributes to trastuzumab resistance, we measured changes in HER-2, HUR and autophagy-related protein expression levels after regulating Linc00969 expression. In addition, we investigated the interaction between Linc00969 and HUR via pull-down and RIP assays and the effect of HUR on HER-2 expression and trastuzumab resistance after blocking HUR.ResultsWe first found that exosomal lncRNA Linc00969 was overexpressed in trastuzumab-resistant BC patients and that exosome-mediated Linc00969 transfer could disseminate trastuzumab resistance in BC. Then, we found that silencing Linc00969 could reduce trastuzumab resistance and that overexpressing Linc00969 could enhance trastuzumab resistance. Furthermore, our results showed that Linc00969 could upregulate HER-2 expression at the protein level and maintain the stability of HER-2 mRNA by binding to HUR. Additionally, we found that exosomal Linc00969 could regulate trastuzumab resistance by inducing autophagy.ConclusionsIn this study, we first identified that exosomal lncRNA Linc00969 could induce trastuzumab resistance by increasing HER-2 protein expression and mRNA stability by binding to HUR, and Linc00969 might also be involved in trastuzumab resistance by inducing autophagy. Our results elucidate a novel mechanism underlying trastuzumab resistance, and Linc00969 might be a new target for improving the treatment of HER-2+ BC patients.

Trastuzumab-resistant breast cancer cells-derived tumor xenograft models exhibit distinct sensitivity to lapatinib treatment in vivo

BackgroundResistance to HER2-targeted therapies, including the monoclonal antibody trastuzumab and tyrosine kinase inhibitor lapatinib, frequently occurs and currently represents a significant clinical challenge in the management of HER2-positive breast cancer. We previously showed that the trastuzumab-resistant SKBR3-pool2 and BT474-HR20 sublines were refractory to lapatinib in vitro as compared to the parental SKBR3 and BT474 cells, respectively. The in vivo efficacy of lapatinib against trastuzumab-resistant breast cancer remained unclear.ResultsIn tumor xenograft models, both SKBR3-pool2- and BT474-HR20-derived tumors retained their resistance phenotype to trastuzumab; however, those tumors responded differently to the treatment with lapatinib. While lapatinib markedly suppressed growth of SKBR3-pool2-derived tumors, it slightly attenuated BT474-HR20 tumor growth. Immunohistochemistry analyses revealed that lapatinib neither affected the expression of HER3, nor altered the levels of phosphorylated HER3 and FOXO3a in vivo. Interestingly, lapatinib treatment significantly increased the levels of phosphorylated Akt and upregulated the expression of insulin receptor substrate-1 (IRS1) in the tumors-derived from BT474-HR20, but not SKBR3-pool2 cells. ConclusionsOur data indicated that SKBR3-pool2-derived tumors were highly sensitive to lapatinib treatment, whereas BT474-HR20 tumors exhibited resistance to lapatinib. It seemed that the inefficacy of lapatinib against BT474-HR20 tumors in vivo was attributed to lapatinib-induced upregulation of IRS1 and activation of Akt. Thus, the tumor xenograft models-derived from SKBR3-pool2 and BT474-HR20 cells serve as an excellent in vivo system to test the efficacy of other HER2-targeted therapies and novel agents to overcome trastuzumab resistance against HER2-positive breast cancer.

Abstract 3900: In vivo inhibition of metastatic HER2 positive trastuzumab resistant breast cancer using engineered destabilized 3UTR ARE of HER2 improves survival outcomes

Abstract Breast cancer is the most common form of cancer in women with mortality of about 58% in developing countries. HER2+ breast cancers are treated with trastuzumab in various forms in combination with chemotherapy and has been successful in targeting HER2 overexpression in HER2+ breast cancer and improving survival as a standard first line therapy for more than a decade. However, about 25% of early and 75% of late stage HER2 driven breast cancers are resistant to trastuzumab. For these patients, the clinical outcomes are grim. We report the destabilization of HER2 3‘UTR ARE which degraded oncogene HER2 transcript, protein expression, HER2 dependent kinases and interactome. In vitro, we transfected the engineered destabilizing HER2 constructs into BT474 clone 5 trastuzumab resistant breast cancer and within days, we inhibited cancer cell growth and degraded HER2 transcript and protein. The degradation of HER2 leads to loss of many kinases especially YES1 and WNK1 that are known causes of chemoresistance in HER2 positive trastuzumab resistant breast cancer. In vivo, we administered the destabilizing constructs as naked constructs or as complexed with nanocages to mice bearing tumors of BT474 clone 5 trastuzumab resistant cells. We found that our constructs, both the naked and nanocage-delivered, conferred significant survival (p values: 0.0178 and 0.00492) to about 60% of the exceptionally surviving mice compared to the controls. Taken together, we have developed a new therapy to target trastuzumab resistant HER2+ metastatic breast cancers with increased survival outcomes. Citation Format: Chidiebere U. Awah, Joo Sun Mun, Baris Boylu, Alooka Paragodaarachchi, Chika Ochu, Hiroshi Matsui, Olorunseun O. Ogunwobi. In vivo inhibition of metastatic HER2 positive trastuzumab resistant breast cancer using engineered destabilized 3UTR ARE of HER2 improves survival outcomes. [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 3900.

Antitumor effect of a novel humanized MUC1 antibody-drug conjugate on triple-negative breast cancer

Breast cancer is the most common malignant cancer in women. Triple-negative breast cancer (TNBC) has a poorer prognosis than other subtypes and is challenging to treat. MUC1 is a therapeutic target in breast and pancreatic cancer. We developed a novel humanized antibody that specifically binds MUC1 expressed in breast cancer cells and conjugated a humanized MUC1 (HzMUC1) antibody to monomethyl auristatin (MMAE). HzMUC1-MMAE showed an anti-proliferative effect on HER2 positive trastuzumab-resistant breast cancer. Immunoprecipitation indicated that HzMUC1 recognized native MUC1 in TNBC cells. Confocal microscopy showed that HzMUC1 bound MUC1 on the surface of TNBC cells, and the conjugates exhibited the same binding ability to HCC70 as unconjugated HzMUC1 by cell-based ELISA. Treatment of TNBC cells with HzMUC1-MMAE reduced growth of MUC1-positive cells and induced G2/M cell cycle arrest and apoptosis. In a mouse model of breast cancer, HzMUC1-MMAE significantly reduced the growth of tumors established by subcutaneous injection of HCC70 TNBC cells. Therefore, HzMUC1-ADC has therapeutic potential for TNBC.

Abstract P1-13-20: Reprogramming of oncogenic signal pathways and metabolism during trastuzumab resistance formation of HER2 positive breast cancer

Abstract Background The formation of resistance against trastuzumab deeply affects the treatment of HER2 positive breast cancer. Although studies have demonstrated several possible reasons that cause the trastuzumab resistance, the precise changes of cell process as well as the interaction between cancer cells and tumor microenvironment during the formation of resistance are still poorly understood. Here we figured out several crucial changes including oncogenic signal pathways as well as metabolism processes especially amino acids and polyunsaturated fatty acids (PUFAs) during the establishment of trastuzumab resistance. We further suggested that the inducers of ferroptosis may be promising reagents for trastuzumab resistant HER2 positive breast cancer. Methods The trastuzumab resistant cell was generated from a sensitive cell line SKBR3. To simulate the adaptation process of HER2 positive tumor to anti-tumor drug, the concentration of trastuzumab was changed gently to prevent cell death. After 8 weeks treatment, trastuzumab concentration in medium was raised from 1μg/ml to 6μg/ml, and a “persister” cell line SKBR3_HP was obtained. The original cell line SKBR3 was cultured for 8 weeks as well to exclude any changes that caused by culture condition. And each cell line includes 3 biological repeats. The viability of two cell lines is measured by CCK8 cell proliferation test with different concentration of trastuzumab. Total RNA of both SKBR3 and SKBR3_HP cells were prepared for sequencing. Activity scores of oncogenic signal pathways as well as metabolism processes were defined and calculated as the relative gene expression value averaged over all genes in this pathway in certain cell type. Flow cytometry was applied for reactive oxygen species measurement with BODIPY-C11. Results After 8 weeks treatment of trastuzumab, the persister cell line SKBR3_HP showed a significant higher viability than original sensitive cell line SKBR3. Oncogenic signal pathway analysis revealed that RTK-Ras, MYC and HIPPO pathways turn into a more active state in SKBR3_HP cells, as well as the upregulation of several cell cycle genes, which are all the response to the blockage of HER2 signal cascade and together maintains cell proliferation. Beside signal pathway, the reprogramming of metabolism is also the consequence of cell adaptation to trastuzumab. Minor changes in energy metabolism, such as glycolysis, citrate cycle and oxidative phosphorylation were observed in SKBR3_HP cells. However, amino acid metabolism, including the synthesis of phenylalanine, histidine, arginine, proline, cysteine and methionine, was largely enhanced in SKBR3_HP cells. Fatty acid, specifically the metabolism of PUFAs, such as linoleic acid, alpha-linolenic acid and arachidonic acid, was activated during the formation of trastuzumab resistance, which was confirmed by the lipid metabolomics data that most n-3/n-6 PUFAs decreased in SKBR3_HP cells. As cysteine and PUFAs metabolism might closely associate with cellular redox balances, erastin and RSL3 were applied to interrupt the intake of cysteine and potentiate the lipid peroxidation process, respectively. A situation of higher ferroptosis sensitivity accompanies raising peroxidated lipids could be detected in SKBR3_HP cells. Conclusion By analyzing the transcriptome and metabolomics data of trastuzumab sensitive and persister cell lines, we pointed out that the changes of oncogenic signal pathway, together with metabolism variation, particularly amino acids and PUFAs, are all the cellular adaptations to high trastuzumab environment. And the higher ferroptosis sensitivity of persister cells could be a valuable treatment target. Citation Format: Ningjun Duan, Yijia Hua, Shuang Hu, Yongmei Yin. Reprogramming of oncogenic signal pathways and metabolism during trastuzumab resistance formation of 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 P1-13-20.

PDPN positive CAFs contribute to HER2 positive breast cancer resistance to trastuzumab by inhibiting antibody-dependent NK cell-mediated cytotoxicity

Trastuzumab is a humanized monoclonal antibody, and has been clinical employed to treat human epidermal growth factor receptor 2 (HER2) positive breast cancer. However, drug resistance to trastuzumab remains a challenge due to the generally uncharacterized interactive immune responses within the tumor tissue. In this study, by means of single-cell sequencing, we identified a novel podoplanin-positive (PDPN+) cancer-associated fibroblasts (CAFs) subset, which was enriched in trastuzumab resistant tumor tissues. Furthermore, we found that PDPN+ CAFs promote resistance to trastuzumab in HER2+ breast cancer by secreting immunosuppressive factors indoleamine 2,3-dioxygenase 1 (IDO1) as well as tryptophan 2,3-dioxygenase 2 (TDO2), thereby suppressing antibody-dependent cell-mediated cytotoxicity (ADCC), which was mediated by functional NK cells. A dual inhibitor IDO/TDO-IN-3 simultaneously targeting IDO1 and TDO2 showed a promising effect on reversing PDPN+ CAFs-induced suppression of NK cells mediated ADCC. Collectively, a novel subset of PDPN+ CAFs was identified in this study, which induced trastuzumab resistance in breast cancer of HER2+ status via inhibiting ADCC immune response mediated by NK cells, hinting that PDPN+ CAFs could be a novel target of treatment to increase the sensitivity of HER2+ breast cancer to trastuzumab.

Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our invitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.

Therapeutic Response Monitoring with 89Zr-DFO-Pertuzumab in HER2-Positive and Trastuzumab-Resistant Breast Cancer Models.

Immuno-positron emission tomography (PET) has great potential to evaluate the target expression level and therapeutic response for targeted cancer therapy. Immuno-PET imaging with pertuzumab, due to specific recognition in different binding sites of HER2, could be useful for the determination of the therapeutic efficacy of HER2-targeted therapy, trastuzumab, and heat shock protein 90 (HSP90) inhibitor, in HER2-expressing breast cancer. The aim of this study is to evaluate the feasibility of monitoring therapeutic response with 89Zr-DFO-pertuzumab for the treatment of HER2-targeted therapeutics, trastuzumab, or the HSP90 inhibitor 17-DMAG, in trastuzumab-resistant JIMT-1 breast cancer models. We prepared an immuno-PET imaging agent using desferoxamine (DFO)-pertuzumab labeled with 89Zr and performed the biodistribution and PET imaging in breast cancer xenograft models for monitoring therapeutic response to HER2-targeted therapy. 89Zr-DFO-pertuzumab was successfully prepared and showed specific binding to HER2 in vitro and clearly visualized HER2 expressing JIMT-1 tumors. 89Zr-DFO-pertuzumab had prominent tumor uptake in HER2 expressing JIMT-1 tumors. JIMT-1 tumors showed trastuzumab-resistant and HSP90 inhibitor sensitive characterization. In immuno-PET imaging, isotype antibody-treated JIMT-1 tumors had similar uptake in trastuzumab-treated JIMT-1 tumors, but 17-DMAG-treated JIMT-1 tumors showed greatly reduced uptake compared to vehicle-treated tumors. Additionally, HER2 downregulation evaluated by immuno-PET imaging was verified by western blot analysis and immunofluorescence staining which resulted in a significant reduction in the tumor’s HER2 level in 17-DMAG-treated JIMT-1 tumors. 89Zr-DFO-pertuzumab immuno-PET may be clinically translated to select pertinent patients for HER2-targeted therapy and to monitor the therapeutic response in HER2-positive cancer patients under various HER2-targeted therapeutics treatments.

Abstract 896: An antifungal, Itraconazole, induces cell death by targeting HER2 signaling and stem-like properties in trastuzumab-resistant HER2-positive breast cancer

Abstract Trastuzumab resistance in HER2-positive breast cancer leads to a poorer prognosis and shorter overall survival. Identifying existing drugs for another purpose of use may be considered as a part of new approaches to overcome resistance in cancer biology. Itraconazole is a FDA-approved drug, which is one of anti-fungal drugs. This drug has been reported to exploit as a cancer therapeutic agent in several cancer types. We sought to investigate the anti-cancer effects of itraconazole on cell proliferation, apoptosis, autophagy and breast cancer stem cell-like properties in terms of challenging trastuzumab resistance in HER2-positive breast cancer. The effect of itraconazole on trastuzumab-resistant cell line, JIMT-1, in vitro was examined in aspects of cell viability, apoptosis, autophagy, and impact on cancer stem cells. As in vivo experimental model, trastuzumab-resistant JIMT-1 cells were implanted to generate xenografts to study anti-tumor efficacy of itraconazole. Treatment of itraconazole significantly suppressed the growth of JIMT-1 cells with marked induction of apoptosis. Itraconazole downregulated p185HER2 and truncated-p95HER2, and their phosphorylation levels in JIMT-1 cells. In addition, the outcome of the decrease in the levels of Beclin-1 and p62 and the increase of LC3 I/II after the exposure to itraconazole supported that itraconazole induced autophagy as well. Importantly, itraconazole not only killed proliferating tumor cells but also effectively eradicated cancer stem-like populations. To elucidate eradication of cancer stem-like population by itraconazole, ALDH1 activity assay and FACS analysis of CD44+/CD24- stem-like phenotype in JIMT-1 cells were carried out. As a result, stem-cell like populations were impaired as evidenced by a significant decrease in ALDH1 activity and CD44+/CD24- stem-like populations. Through in vivo mouse model, the physiological relevance of in vitro observations was confirmed. Evident inhibition of tumor burden and growth in trastuzumab-resistant xenografts was shown in consequence of itraconazole administration, accompanying by substantial downregulation of HER2, ALDH1 and microvessel density as well as dramatic decrease of p62 in vivo. No injury to liver or kidneys by itraconazol was found based on no statistically significant difference in serum levels of ALT, AST and BUN between vehicle- and itraconazole-administrated groups. We have demonstrated that itraconazole, which is FDA-approved drug as an anti-fungal drug, exerts anti-tumor activity in trastuzumab-resistant HER2-positive breast cancer by targeting cancer stem-like properties, suppression of the HER2 signaling as well as induction of autophagy. These findings support the notion that itraconazole might be a new strategic approach as a treatment for trastuzumab-resistant HER2-positive breast cancers. Citation Format: Jung Min Park, Soeun Park, Minsu Park, Seongjae Kim, Juyeon Seo, Dongmi Ko, Kee Dal Nam, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. An antifungal, Itraconazole, induces cell death by targeting HER2 signaling and stem-like properties in trastuzumab-resistant HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 896.

Abstract 1788: Engineered destabilized AU rich elements on the 3UTR of HER2 degrades HER2, inhibits proliferation, and induces apoptosis in HER2 positive trastuzumab resistant breast cancer cells

Abstract Resistance to molecular targeted therapy is a major challenge facing breast cancer patients and unfavorably impacts clinical outcomes, leading to hundreds of thousands of deaths yearly. Resistance to cancer therapies arises from many factors. One of the key factors driving resistance is oncogenes which initiate pro-growth, pro-survival and metastatic programs that enable cancers escape various therapies. In HER2+ breast cancer, the oncogene HER2 is the master driver of this tumor. Trastuzumab effectively targets HER2. While this is generally successful, some patients who have HER2+ breast cancer develop resistance to trastuzumab and there are limited treatment options for them. Moreover, for low HER2+ expressing breast cancer, there are limited treatment options for them. And in African American women who have HER2+ breast cancer, there is disparity in their treatment outcomes. We have discovered that 3’UTR of HER2 is enriched with poly U stabilizing AU rich elements (ARE). We have developed a novel technology wherein we engineered the stabilizing HER2 3’UTR ARE motifs to destabilizing motif in HER2+ and HER2+ trastuzumab resistant breast cancer cells to control HER2 transcript. We achieved complete degradation of HER2 within 4-8 days in HER2+ wildtype breast cancer cells and 9-11 days in HER2+ trastuzumab resistant breast cancer cells. We had loss of cancer cell viability by more than 90%. We also show that control of HER2 transcript downregulated HER2-dependent kinases, transcription factors, and HER2 interactome. Mechanistically, the control of HER2 transcript was achieved by destabilized ARE sequence specificity which triggered the proteins PARN, XRN1 and CNOT1 to degrade HER2 transcript and this led to induction of active caspase 3/7, reduction of cell size, and severe distortion and disruption of the cancer cell membrane leading to cell death. Taken together, we have a developed a novel approach to control HER2 transcript expression both on spatial and temporal scale. This novel approach offers applications for targeting HER2+ trastuzumab resistant breast cancer as well as other cancers resistant to HER2 targeted therapy and driven by pervasive oncogenic signals. Citation Format: Chidiebere U. Awah, Fu Dong, Fayola Levine, Leonard Ash, Yana Glemaud, Olorunseun Ogunwobi. Engineered destabilized AU rich elements on the 3UTR of HER2 degrades HER2, inhibits proliferation, and induces apoptosis in HER2 positive trastuzumab resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1788.

Abstract 2606: ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance

Abstract Inhibition of the DNA damage response (DDR) pathway is an attractive approach to target cancers cells. Unlike normal cells which relay on two cell cycle checkpoints to repair DNA damage, most tumor cells only depend on the G2 checkpoint because their G1 checkpoint is defective. Wee1 regulates the G2/M checkpoint and abrogating the G2 checkpoint by inhibiting Wee-1 in cancer cells with DNA damage can preferentially sensitize cancer cells to undergo mitotic catastrophe. We have developed and characterized ZN-c3, a potent and selective Wee-1 inhibitor with a differentiated kinase selectivity and pharmacokinetics profile. ZN-c3 was evaluated in ER-positive, triple negative, triple negative PARP inhibitor resistant, and Her-2 positive trastuzumab resistant breast cancers. In the ER-positive MCF-7 efficacy model, ZN-c3 demonstrated anti-tumor activity as a single agent as well as in combinations with a CDK4/6 inhibitor (Palbociclib) or ZN-c5, a selective estrogen receptor degrader (SERD). In the MDA-MB-436, a triple negative breast cancer tumor xenograft model, ZN-c3 demonstrated efficacy as a single agent and in combination with the PARP inhibitors Olaparib or Niraparib. We extended these combination studies by evaluating the efficacy of Niraparib in combination with ZN-c3 in patient-derived xenograft (PDX) triple negative breast tumors. ZN-c3 was efficacious as a single agent and the efficacy was enhanced in the combination with Niraparib. PARP inhibitors are effective and have been approved for treatment of some cancer types. However, most patients develop resistance to these agents. We sought to investigate whether ZN-c3 can overcome the acquired resistance to PARP inhibitors (Olaparib and Niraparib). We generated PARP inhibitor resistant breast tumor xenograft models. A significant tumor growth inhibition was observed in these resistant tumor xenograft models when treated with ZN-c3. These data suggest that ZN-c3 as a single agent can overcome the acquired PARP inhibitor resistance. Finally, we also evaluated ZN-c3 activity in a HER-2 positive trastuzumab resistant breast tumor xenograft model (JIMT-1) and ZN-c3 showed anti-tumor activity alone and in combination with trastuzumab. In summary, these results demonstrate that ZN-c3 is effective as a single agent and in combination with other targeted therapies in breast cancer models. Moreover, ZN-c3 can overcome the acquired resistance to PARP inhibitors and trastuzumab. ZN-c3 is currently in clinical trials for several indications and has demonstrated promising clinical activity and good tolerability. Citation Format: Jiali Li, Jianhui Ma, Petrus R. de Jong, Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Brant Boren, Sayee Hegde Hegde, Fernando Doñate, Ahmed A. Samatar. ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2606.

Abstract 5340: GATA3-VMP1 axis induces trastuzumab resistance via promoting autophagy in HER2-positive breast cancer cells

Abstract Trastuzumab is the most important agent for HER-2 positive breast cancer both in neoadjuvant/adjuvant and metastatic settings. However, primary or acquired resistance to trastuzumab often compromises the efficacy and impairs patients’ survival. Vacuole Membrane Protein-1(VMP1), a transmembrane protein located in the endoplasmic reticulum membrane, has been reported to induce chemotherapy resistance by promoting autophagy. Our preliminary study found that the expression of GATA Binding Protein 3(GATA3) and VMP1 are positively correlated in tissue samples from HER-2 positive breast cancer patients. GATA3 and VMP1 were significantly increased in both protein and mRNA levels in trastuzumab-resistant breast cancer cell lines as compared to the sensitive counterparts. Furthermore, the expression autophagy signature protein P62 significantly decreased in trastuzumab-resistant cell lines, while the expression of autophagy activation protein ATG5 increased, indicating a close relationship between autophagy and trastuzumab resistance. In tissue samples from patients treated with neoadjuvant regiments containing trastuzumab, the expression of VMP1 significantly increased in the non-pCR (non-pathological complete response) group than the pCR group. Overexpression of VMP1 in wild-type breast cancer cells decreased their sensitivity to trastuzumab. On the contrary, when VMP1 was knocked-down in trastuzumab-resistance cells, the sensitivity of cell lines to trastuzumab was increased. Moreover, overexpressed GATA3 in wild-type HER-2 positive cell lines, the expression of VMP1 increased as well as the expression of ATG5, the signature protein of autophagy, suggesting that the transcription factor GATA3 may play a role in the transcriptional activation of VMP1. The promoter sequence was predicted, and it was found that GATA3 regulatory core sequence "TGATA" existed in front of the transcription start site from -327bp to -322bp, indicating that GATA3 may transcriptionally activate the expression of VMP1 and promote the occurrence of autophagy. TCGA (The Cancer Genome Atlas) correlation analysis showed that, both VMP1 and GATA3 are positively correlated with classical autophagy related factors ATG2B, ATG7 and BCL1, indicating VMP1 and GATA3 may be involved in the positive regulation of autophagy in HER-2 positive breast cancer. Collectively, our results suggest that GATA3 transcriptionally activates VMP1 to promote autophagy, resulting in the induction of trastuzumab resistance in HER-2 positive breast cancer. Further mechanistic study on autophagy in contribution to trastuzumab resistance is warranted. Citation Format: hao-yu Lin, Yu-cui Gu, Zhen-hao Wang, Kai-yuan Huang, He-xing Sun, De Zeng, Yuan-ke Liang. GATA3-VMP1 axis induces trastuzumab resistance via promoting autophagy in HER2-positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5340.

Molecular mechanisms of flavonoids and their modulatory effects against breast cancer: A scoping review

Background: Breast cancer is the most prevalent malignancy among women. It is a disease whose incidence and mortality rates are on the upsurge globally. Debilitating effects, cost and resistance to available chemotherapeutic interventions render them unideal. Dietary phytochemicals have been shown to have preventive and therapeutic effects. Research continues to affirm the role of flavonoids as potential chemotherapeutic agents in combating the disease. Understanding modulation of key cellular signalling pathways by flavonoids presents promising molecular targets that may be leveraged to develop better chemotherapeutic agents for breast cancer. Methods: To describe the in vitro and in vivo modulatory effects of flavonoids on molecular anti-cancer mechanisms we searched three databases. We included original articles describing modulation of cell signalling processes such as; cell cycle, apoptosis, autophagy, angiogenesis, invasion and migration which are involved in tumorigenesis. The search guidelines such as; year of publication, search strategy, study design and language informed article selection. Results: Thirty-six articles were reviewed. Modulatory effects of six subclasses of flavonoids on breast cancer tumorigenic pathways were reported. The effects included enhanced apoptosis, attenuation of; angiogenesis, cell cycle, invasion, migration and metastasis. For instance, pectolinarigenin inhibited signal transducer and activator of transcription 3 (stat3) signalling pathway in triple negative breast cancer. Whereas, sideritoflavone caused cell cycle arrest and inhibited migration in trastuzumab resistant breast cancer cells. Additionally, quercetin and ampelopsin inhibited stemness features in triple negative breast cancer cells. Conclusion: Evidently flavonoids showed significant modulatory effects on cellular signalling pathways crucial for breast cancer progression. The ability of flavonoids to act on a wide range of mechanisms as well as on aggressive breast cancer types presents an array of hope. We recommend that further studies be done to ascertain the applicability of these compounds in treatment of breast cancers.

A novel circular RNA confers trastuzumab resistance in human epidermal growth factor receptor 2-positive breast cancer through regulating ferroptosis.

HER2-positive breast cancer is an aggressive subtype of breast cancer, characterized by high malignancy and poor prognosis. Trastuzumab, the first HER2-targeted monoclonal antibody therapy, has a crucial role in a curative setting in HER2-positive breast cancer. However, frequent drug resistance inhibits its clinical efficacy. Herein, by performing circular RNA (circRNA) profiling, we identified a novel circRNA, circ-BGN, as a key contributor in trastuzumab resistance. Circ-BGN was evidently increased in trastuzumab-resistant breast cancer cells and tissues, linking to poor overall survival. Knockdown of circ-BGN inhibited breast cancer cell viability and notably restored its sensitivity to trastuzumab. Further, we found that circ-BGN could directly bind to OTUB1 and SLC7A11, enhancing OTUB1-mediated SLC7A11 deubiquitination and thereby inhibiting ferroptosis, a newly recognized form of cell death that is distinct from apoptosis, necrosis, and autophagy. Moreover, erastin, a small-molecule ferroptosis inducer, could effectively restore the anti-tumor effect of trastuzumab. Pre-clinically, the orthotopic tumor model showed that erastin significantly reduced tumor volume generated by trastuzumab-resistant breast cancer cells, which was more pronounced after combined circ-BGN knockdown. Collectively, our data reveal a novel circRNA controlling trastuzumab resistance via regulation of ferroptosis, providing a promising therapeutic strategy for trastuzumab-resistant breast cancer patients.