Trastuzumab-resistant Cell Lines Research Articles

Efficacy and delivery strategies of the dual Rac/Cdc42 inhibitor MBQ-167 in HER2 overexpressing breast cancer

Trastuzumab and trastuzumab-based treatments are the standard of care for breast cancer patients who overexpress the human epidermal growth factor receptor 2 (HER2). However, patients often develop resistance to trastuzumab via signaling from alternative growth factor receptors that converge to activate guanine nucleotide exchange factors (GEFs) that in turn activate the Rho GTPases Rac and Cdc42. Since Rac and Cdc42 have been implicated in high tumor grade and therapy resistance, inhibiting the activity of Rac and Cdc42 is a rational strategy to overcome HER2-targeted therapy resistance. Therefore, our group developed MBQ-167, a dual Rac/Cdc42 inhibitor with IC50s of 103 nM and 78 nM for Rac and Cdc42, respectively, which is highly effective in reducing cell and tumor growth and metastasis in breast cancer cell and mouse models. Herein, we created a trastuzumab resistant variant of the SKBR3 HER2 positive breast cancer cell line and show that Rac activation is a central mechanism in trastuzumab resistance. Next, we tested the potential of targeting MBQ-167 to HER2 overexpressing trastuzumab-resistant cell lines in vitro, and show that MBQ-167, but not trastuzumab, reduces cell viability and induces apoptosis. When MBQ-167 was targeted to mammary fatpad tumors established from HER2 overexpressing cells via immunoliposomes functionalized with trastuzumab, MBQ-167 and MBQ-167-loaded liposomes show equal efficacy in reducing the viability of trastuzumab-resistant cells, inhibiting tumor growth in mouse xenografts, and reducing metastasis to lungs and liver. This study demonstrates the efficacy of MBQ-167 as an alternative therapeutic in HER2 overexpressing cancers, delivered either in free form or in liposomes.

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.

Novel HER2-targeted therapy to overcome trastuzumab resistance in HER2-amplified gastric cancer

Trastuzumab is used to treat HER2-amplified metastatic gastric cancer; however, most patients become trastuzumab-resistant within a year. Knowledge of the mechanisms underlying trastuzumab resistance is required to overcome this limitation. Here, we aimed to elucidate this resistance mechanism using four trastuzumab-resistant (TR) cell lines and investigate the efficacy of HER2-targeted therapies to overcome treatment resistance. Each TR cell line had different phenotypic characteristics. Interestingly, HER2 expression remained as high as the parental cell lines in TR cell lines, suggesting that HER2-targeted agents were still useful. As expected, three tyrosine kinase inhibitors (lapatinib, neratinib, and tucatinib) and one antibody–drug conjugate (trastuzumab deruxtecan: T-DXd) exhibited good antitumor effects against TR cell lines. We further investigated the potential biological mechanism of T-DXd. When treated with trastuzumab or T-DXd, HER2 or its downstream signals were disrupted in parental cell lines, but not in TR cell lines. Moreover, T-DXd induced the expression of pH2A.X and cPARP and caused cell cycle arrest in the S or G2-M phase in TR cell lines. T-DXd showed promising antitumor activity in both parental and TR cell lines, suggesting that it is a potential candidate for overcoming trastuzumab resistance.

Identification of a novel potent CDK inhibitor degrading cyclinK with a superb activity to reverse trastuzumab-resistance in HER2-positive breast cancer in vivo

CDK12 is overexpressed in HER2-positive breast cancers and promotes tumorigenesis and trastuzumab resistance. Thus CDK12 is a good therapeutic target for the HER2-positive breast tumors resistant to trastuzumab. We previously reported a novel purine-based CDK inhibitor with an ability to degrade cyclinK. Herein, we further explored and synthesized new derivatives, and identified a new potent pan-CDK inhibitor degrading cyclinK (32e). Compound 32e potently inhibited CDK12/cyclinK with IC50 = 3 nM, and suppressed the growth of the both trastuzumab-sensitive and trastuzumab-resistant HER2-positive breast cancer cell lines (GI50's = 9–21 nM), which is superior to a potent, clinical pan-CDK inhibitor dinaciclib. Moreover, 32e (10, 20 mg/kg, ip, twice a week) showed a dose-dependent inhibition of tumor growth and a more dramatic anti-cancer effect than dinaciclib in mouse in vivo orthotopic breast cancer model of trastuzumab-resistant HCC1954 cells. Kinome-wide inhibition profiling revealed that 32e at 1 μM exhibits a decent selectivity toward CDK-family kinases including CDK12 over other wildtype protein kinases. Quantitative global proteomic analysis of 32e-treated HCC1954 cells demonstrated that 32e also showed a decent selectivity in degrading cyclinK over other cyclins. Compound 32e could be developed as a drug for intractable trastuzumab-resistant HER2-positive breast cancers. Our current study would provide a useful insight in designing potent cyclinK degraders.

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.

Targeting CXCR4 abrogates resistance to trastuzumab by blocking cell cycle progression and synergizes with docetaxel in breast cancer treatment

BackgroundAlthough trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms.MethodsImmunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms.ResultsUsing a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel.ConclusionsOur findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.

MiR-146a-5p Promotes Angiogenesis and Confers Trastuzumab Resistance in HER2+ Breast Cancer

Trastuzumab treatment has significantly improved the prognosis of HER2-positive breast cancer patients. Despite this, resistance to therapy still remains the main clinical challenge. In order to evaluate the implication of microRNAs in the trastuzumab response, we performed a microRNA array in parental and acquired trastuzumab-resistant HER2-positive breast cancer cell lines. Our results identified miR-146a-5p as the main dysregulated microRNA. Interestingly, high miR-146a-5p expression in primary tumor tissue significantly correlated with shorter disease-free survival in HER2-positive breast cancer patients. The gain- and loss-of-function of miR-146a-5p modulated the response to trastuzumab. Furthermore, the overexpression of miR-146a-5p increased migration and angiogenesis, and promoted cell cycle progression by reducing CDKN1A expression. Exosomes from trastuzumab-resistant cells showed a high level of miR-146a-5p expression compared with the parental cells. In addition, the co-culture with resistant cells' exosomes was able to decrease in sensitivity and increase the migration capacities in trastuzumab-sensitive cells, as well as angiogenesis in HUVEC-2 cells. Collectively, these data support the role of miR-146a-5p in resistance to trastuzumab, and demonstrate that it can be transferred by exosomes conferring resistance properties to other cells.

Abstract 3698: Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive breast cancer

Abstract Background: Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several potential reasons that cause trastuzumab resistance, we still have poor knowledge about the changes in cell itself as well as its interaction with tumor microenvironment (TME) components during resistance formation. Here we suggested that altered histone modification reprograms polyunsaturated fatty acids (PUFAs) metabolism, which reduces cellular PUFAs and stimulates prostaglandin E2 (PGE2) production. Excessive PGE2 may further affect both cancer cells as well as immune cells in TME. Materials and Methods: Secondary trastuzumab-resistant cell line SKBR3_HR was generated from sensitive SKBR3 cells by raising trastuzumab concentration gently from 1ug/ml to 20ug/ml in 30 weeks. Cell viabilities were measured by CCK8 test. CUT&amp;Tag with anti-H3K4me3 and anti-H3K27me3 antibodies were applied to make sequencing libraries. Total RNA and protein were collected for transcriptome and proteome analysis. Activity scores of metabolism processes were defined and calculated as the relative gene expression value averaged over all genes in this pathway in certain cell types. Oxidized PUFAs and PGE2 were assessed by flow cytometry with BODIPY-C11 and ELISA, respectively. Results: SKBR3_HR cells showed similar viability as primary resistant JIMT1 cells and were significantly higher than original SKBR3 cells. Pathway analysis indicated reduced PUFAs synthesis and activated arachidonic acid metabolism. As a consequence, oxidized PUFAs decreased while PGE2 accumulated heavily in SKBR3_HR cells, which may promote tumor and vascular endothelial cell proliferation but interrupt immune cell activity in TME. Transcriptional activity of two critical PUFAs synthesis-related genes, FASN and SCD, were reduced in resistant cells, while two key genes in arachidonic acid metabolism, PTGS1 and PTGES, were activated. H3K4me3 and H3K27me3 play opposite but critical rules in transcriptional regulation. During resistance formation, changes happened on 3256 H3K27me3 peaks as well as 316 H3K4me3 peaks. In detail, although little H3K27me3 changes were found at the promoter regions of four genes (FASN, SCD, PTGS1 and PTGES), reduced H3K4me3 levels may inhibit FASN and SCD expression while raised levels could stimulate PTGS1 and PTGES transcription. Conclusions: Combining the transcriptome and histone modification data, we have a deeper understanding of the cellular adaptions to trastuzumab. Altered trimethylation of H3K4 but not H3K27 remodels the expressions of FASN, SCD, PTGS1 and PTGES, drives the reprogramming of PUFAs-associated metabolism, and finally reduces cellular PUFAs while stimulates PGE2 production. High PGE2 level may affect the activities of tumor and TME-related cells. Citation Format: Yongmei Yin, Ningjun Duan, Yijia Hua, Shuang Hu. Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive 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 3698.

Abstract P6-12-09: Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines

Abstract Introduction: NER is a pan-HER tyrosine kinase inhibitor (TKI) approved for the treatment of HER2+ BC in the adjuvant setting following trastuzumab and in combination with capecitabine for advanced disease. Resistance to small molecule TKIs like NER can develop in the clinic. Pre-clinical studies have highlighted that retinoic acid can inhibit BC growth and modulate HER2 signalling pathways. The RAR family of nuclear transcription factors consists of RARα, RARβ and RARγ. The synthetic retinoic acid fenretinide (FN) acts as a pan-RAR agonist, while AGN194310 (AG) acts as a pan-RAR antagonist. In order to investigate the anti-proliferative potential of co-targeting RAR and HER2 pathways in sensitive and resistant BC cell line models, we examined the effect of FN and AG in combination with NER in two HER2+, estrogen receptor-negative, trastuzumab-resistant cell lines HCC1569 and HCC1954, and their NER-resistant (NR) sub-lines HCC1569-NR and HCC1954-NR. Methods: HCC1569 and HCC1954 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. NR cell lines were generated by continuous exposure to 150nM NER for 6 months. 10 mM stock solutions of FN (H7779-Sigma), AG (SML2665-Sigma) and NER (supplied by Puma Biotechnology, Inc) were made in DMSO. Proliferation was measured as percentage growth versus DMSO control using an acid phosphatase based assay after 5 days drug exposure. The half-maximal inhibitory concentration (IC50) was calculated for each drug using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50) using CalcuSyn. Values &amp;lt; 1 represent a synergistic effect, a value of 1 is additive and values &amp;gt; 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research found that the NR cell lines were &amp;gt;10-fold resistant to NER (HCC1569-NR IC50 0.44 ± 0.1 μM, HCC1954-NR IC50 0.198 ± 0.019 μM) compared to the parental HCC1569 (IC50 0.018 ± 0.015 μM) and HCC1954 (IC50 0.017 ± 0.001 μM) cell lines. Pan-RAR agonism by FN had a potent anti-proliferative effect in the HCC1569 (FN IC50 0.22 ± 0.02 μM) and the HCC1569-NR cell lines (FN IC50 0.28 ± 0.13 μM), with the HCC1954 and HCC1954-NR cell lines proving less sensitive (IC50 6.47 ± 1.3 μM and 1.9 ± 0.2 μM, respectively). When combined with NER, FN produced a strong antagonistic effect in the HCC1569 cell line (CI value: 15.63 ± 9.5) and a strong synergistic effect in the HCC1954 cell line (CI value: 0.42 ± 0.06). For the NR cell line models, the NER/FN combination proved synergistic (HCC1569-NR, CI value: 0.84 ± 0.46) or additive (HCC1954-NR, CI value: 0.97 ± 0.15). Next, we wanted to assess the impact of antagonising rather than activating RAR activity in our cell line models. All four cell lines were less sensitive to antagonist AG (IC50 &amp;gt;8μM for all cell lines) compared to FN. The addition of AG to NER resulted in responses diametrically opposed to the FN/NER combination. The AG/NER combination produced a strong synergistic effect in the HCC1569 cell line (CI value: 0.52 ± 0.17), an antagonistic effect in the HCC1954 cell line (CI value: 2.1 ± 0.4) and an antagonistic effect in both NR cell lines (HCC1954-NR, CI value: 2.69 ± 0.6 and HCC1569-NR, CI value: 1.58 ± 0.12). Conclusions: This pre-clinical study suggests involvement of the RAR signalling pathway in response to NER and the development of NR. Results also suggest pan-RAR agonism, rather than pan-RAR antagonism, as a potential therapeutic strategy to overcome NR. Further investigation is warranted to determine how targeting the RAR signalling pathway may assist in the treatment of HER2+ BC. Citation Format: Debbie O’Reilly, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines [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 P6-12-09.

Determining the Factors Predicting the Response to Anti-HER2 Therapy in HER2-Positive Breast Cancer Patients.

We aimed to identify the differently expressed genes or related pathways associated with good responses to anti-HER2 therapy and to suggest a model for predicting drug response in neoadjuvant systemic therapy with trastuzumab in HER2-positive breast cancer patients. This study was retrospectively analyzed from consecutively collected patient data. We recruited 64 women with breast cancer and categorized them into 3 groups: complete response (CR), partial response (PR), and drug resistance (DR). The final number of patients in the study was 20. RNA from 20 core needle biopsy paraffin-embedded tissues and 4 cultured cell lines (SKBR3 and BT474 breast cancer parent cells and cultured resistant cells) was extracted, reverse transcribed, and subjected to GeneChip array analysis. The obtained data were analyzed using Gene Ontology, Kyoto Gene and Genome Encyclopedia, Database for Annotation, Visualization and Integrated Discovery. In total, 6,656 genes differentially expressed between trastuzumab-susceptible and trastuzumab-resistant cell lines were identified. Among these, 3,224 were upregulated and 3,432 were downregulated. Expression changes in 34 genes in several pathways were found to be related to the response to trastuzumab-containing treatment in HER2-type breast cancer, interfering with adhesion to other cells or tissues (focal adhesion) and regulating extracellular matrix interactions and phagosome action. Thus, decreased tumor invasiveness and enhanced drug effects might be the mechanisms explaining the better drug response in the CR group. This multigene assay-based study provides insights into breast cancer signaling and possible predictions of therapeutic response to targeted therapies such as trastuzumab.

PKC-mediated phosphorylation and activation of the MEK/ERK pathway as a mechanism of acquired trastuzumab resistance in HER2-positive breast cancer.

Protein expression, activation and stability are regulated through inter-connected signal transduction pathways resulting in specific cellular states. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins (phospho-protein profile) in key signal transduction pathways, in breast cancer cellular models of trastuzumab resistance. To this effect, we utilized a multiplex, bead-based protein assay, DigiWest®, to measure around 100 proteins and protein modifications using specific antibodies. The main advantage of this methodology is the quantification of multiple analytes in one sample, utilising input volumes of a normal western blot. The intrinsically trastuzumab-resistant cell line JIMT-1 showed the largest number of concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β catenin stabilization by inhibitory phosphorylation of GSK3β, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance cellular models. The overexpression of upstream RAS/RAF, however, was confined to JIMT 1; meanwhile, in a cellular model of acquired trastuzumab resistance generated in this study (T15), entry into the ERK pathway seemed to be mostly mediated by PKCα activation. This is a novel observation and merits further investigation that can lead to new therapeutic combinations in HER2-positive breast cancer with acquired therapeutic resistance.

PLOD3 contributes to HER-2 therapy resistance in gastric cancer through FoxO3/Survivin pathway

Human epidermal growth factor receptor 2 (HER-2), a famous therapeutic target for breast cancer, is also associated with an increased risk of recurrence and poor outcomes of other malignancies, including gastric cancer. Yet the mechanism of HER-2 therapy resistance remains controversial due to the heterogeneity of gastric adenocarcinoma. We know, Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 3 (PLOD3), a key gene coding enzymes that catalyze the lysyl hydroxylation of extracellular matrix collagen, plays an important contributor to HER-2 targeting agent Trastuzumab resistance in gastric cancer. Herein, we analyzed clinical samples of gastric cancer patients and gastric cancer cell lines and identified PLOD3, unveiled that depletion of PLOD3 leads to decreased cell proliferation, tumor growth and Trastuzumab sensitivity in these Trastuzumab resistant GC cell lines. Clinically, increased PLOD3 expression correlates with decreased Trastuzumab therapy responsiveness in GC patients. Mechanistically, we show that PLOD3 represses tumor suppressor FoxO3 expression, therefore upregulating Survivin protein expression that contributes to Trastuzumab resistance in GC. Therefore, our study identifies a new signaling axis PLOD3-FoxO3- Survivin pathway that may be therapeutically targeted in HER-2 positive gastric cancer.

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.

Abstract 5290: BSI-001, a novel anti-HER2 antibody exhibiting potent synergistic efficacy with trastuzumab

Abstract Background: Trastuzumab and pertuzumab are anti-HER2 antibodies that bind to two different epitopes of HER2. Although pertuzumab itself did not exhibit substantial clinical benefit as monotherapy, it was approved to be used in combination with trastuzumab because of its ability to enhance the clinical efficacy of trastuzumab. Currently, the combination of trastuzumab and pertuzumab is being used as first-line treatment for HER2-positive metastatic breast cancer. However, while the combination of trastuzumab and pertuzumab has been effective, there is still opportunity for greater effectiveness by identifying anti-HER2 mAbs that will be better synergistic partners of trastuzumab, allowing for better efficacy and/or broader indications. Methods: A SynAb࣪ Platform was used to identify BSI-001, an anti-HER2 mAb, through trastuzumab-based synergistic functional screening. A competitive ELISA was used to perform epitope binning of the anti-HER2 antibodies. BSI-001 was further characterized in combination with trastuzumab for its bioactivity in vitro, including inhibition of cell proliferation and antigen-mediated internalization of HER2 positive cell lines. The synergistic effect of BSI-001 with trastuzumab was also evaluated in trastuzumab-resistant cell lines. Multiple animal models were then employed to investigate the synergistic antitumor activity of the combination in vivo. Results: BSI-001 is the best functional partner of trastuzumab identified through the SynAb࣪ platform. BSI-001 binds to a unique epitope, distinct from the binding epitopes of trastuzumab and pertuzumab. It exhibits superior bioactivity and efficacy in vitro and in vivo when combined with trastuzumab. The combination of BSI-001 and trastuzumab inhibits HER2-positive cancer cell growth more potently than that of trastuzumab and pertuzumab. The combination of BSI-001 with trastuzumab also shows inhibition of cell growth of the trastuzumab-resistant cell line BT-474 Clone 5, whereas the combination of pertuzumab and trastuzumab did not. In addition, BSI-001 is able to synergize with trastuzumab to promote antigen-antibody internalization. In animal models, BSI-001 demonstrates superior synergistic antitumor activity when combined with trastuzumab as compared to pertuzumab. Summary: The combination of BSI-001 and trastuzumab showed significantly better in vitro and in vivo activity than the combination of trastuzumab and pertuzumab. In addition, because of the activity of the BSI-001/trastuzumab combination in a trastuzumab-resistant cancer cell line, there is the potential for this combination to be used to treat patients with trastuzumab-resistant cancer or patients who experienced relapse following trastuzumab and pertuzumab treatment. Citation Format: Shukai Xia, Zeyu Peng, Xiaoyu Ding, Wanjian Gu, Yujie Zhong, Jinyu Liu, Hongyan Li, Xiaoyao Hao, Xiaodong F. Liu, Mark Z. Ma, Lan Luo, Chunni Zhang, Mingjiu Chen. BSI-001, a novel anti-HER2 antibody exhibiting potent synergistic efficacy with trastuzumab [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 5290.

Abstract 5079: Comparative proteome analysis for predicting the prognosis of trastuzumab-based therapy

Abstract Trastuzumab, a HER2-targeting antibody, has markedly altered the clinical course of the disease in HER2 + gastric cancer, which is 10-15% of metastatic GC. However, the majority of patients develop resistance within one year of treatment. Therefore, understanding the mechanisms of trastuzumab resistance is urgently needed to overcome trastuzumab resistance. This study explored the characteristics of trastuzumab resistant cell lines through comparative proteome analysis. We previously reported establishment of four trastuzumab resistant cell lines from HER2 amplified GC cell lines by trastuzumab dose-escalation treatment; YCC-33TR, YCC-38TR, NCI-N87TR, and SNU-216TR. Formalin-fixed paraffin-embedded tissue samples were obtained 10 GC patients prior to trastuzumab therapy. The patients were first stratified into poor and good responders based on whether their tumors had or had not progressed. For analysis of differentially expressed proteins (DEPs), label-free quantitative proteomic profiling was performed using high-resolution LC-MS system. Gene ontology (GO) and KEGG pathway enrichment analyses were conducted using DAVID database. Consistent with the previous results, we found that HER2 expression in TR cell lines remained as high as parental cell lines, also in proteomic profiling. As a result of proteomic analysis, total 5285 proteins were identified in 4 parental and 4 TR cell lines; 4854 common proteins, while 55 proteins unique to parental cell lines and 58 proteins unique to TR cell lines. Then, we did the hierarchical clustering with these DEPs to identify protein expression patterns. We observed that 129 and 62 proteins were upregulated in parental and TR cell lines, respectively (p&amp;lt;0.05). We identified C5orf51 and CENP-E, poor prognostic factors, were upregulated 200-fold in TR cell lines. Interestingly, we found that HLA-B, ITM2C, and PPP1R3D were highly expressed in TR cell lines and trastuzumab poor responders. In conclusion, we performed comparative proteome analysis in parental and TR cell lines and investigated the corresponding expression change. It may gain further novel insights into the mechanisms of trastuzuamb resistance. Citation Format: Juin Park, Woo Sun Kwon, Sun Kyoung Kang, Tae Soo Kim, Jihyun Hwang, Sang Woo Cho, Kyunggon Kim, Hyun Cheol Chung, Sun Young Rha. Comparative proteome analysis for predicting the prognosis of trastuzumab-based therapy [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 5079.

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 5334: Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers

Abstract Background: Human epithelial growth factor receptor 2 (HER2) is a well-established therapeutic target in HER2-positive breast and gastric cancer. Recently, novel HER2-targeting agents are being developed after trastuzumab, and HER2-targeted therapies have been attempted in across solid tumor types, including biliary tract cancer. In these aspects, it is essential to understand and elucidate the resistant mechanism of the HER2-targeting strategies. Yes-associated protein (YAP), a transcription factor of the Hippo pathway, function as proto-oncoproteins by inducing target genes involved in cancer cell survival and proliferation (Zhao B, et al. Genes Develop 2008). Also, YAP is emerging as a resistance mechanism of cytotoxic and targeted drugs. In this study, we explore the role of YAP in overcoming resistance to trastuzumab in HER2-positive cancer cells. Methods: Four trastuzumab-resistant (HR) cell lines were established using HER2-postive gastric and biliary tract cancer cell lines (N87, SNU216, SNU2670, and SNU2773) (Jin MH, et al. Mol Cancer Ther. 2017). YAP siRNA and Verteporfin (YAP-TEAD inhibitor) were used to downregulate YAP. MTT assay, cell cycle analysis, western blot and migration assay were performed to analyze the antitumor effects through YAP downregulation. In order to elucidate tumor cell immune-modulation by YAP, human PBMC co-culture was used and immune markers, such as programmed death-ligand 1 (PD-L1), were analyzed in HR cell lines. Results: Increase of receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) expression was observed in the HR cells. Relatively high expression of pYAP, YAP, and transcriptional co-activator with PDZ-binding motif (TAZ) were observed in the HR cells (SNU216HR, SNU2670HR and SNU2773HR) compared to parental cells. Reducing overexpressed YAP in HR cells resulted in tumor cell growth inhibition. In cell cycle analysis, the increase of subG1 phase through YAP downregulation was observed, and cyclinD, B, A and BCL-XL were effectively decreased. Migration was also inhibited by the decrease in YAP expression. By verteporfin treatment, changes in immune markers including increase of CD80 and decrease of PD-L1 was found. In addition, p-STAT3 and IL6, Which regulate PD-L1, were also decreased through the reduction of YAP. In HR cells treated with siYAP, there was a tendency to increase CD8+ T cells upon PBMC co-culture. Conclusion: YAP is upregulated in trastuzumab-resistant (HR) cells and upregulated YAP induce PD-L1 expression. Inhibition of YAP shows anti-tumor effects and modulates immune status. Collectively, our results suggest that inhibition of YAP is one of promising strategies to overcome trastuzumab resistance in HER2-positive cancers. Citation Format: Ah Rong Nam, Jeesun Yoon, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Tae-Yong Kim, Do-Youn Oh. Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers [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 5334.

Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer.

Cyclin-dependent kinases (CDKs) are a broad family of proteins involved in the cell cycle and transcriptional regulation. In this article, we explore the antitumoral activity of a novel proteolysis-targeting chimera (PROTAC) compound against CDK9. Breast cancer cell lines from different subtypes were used. Transcriptomic mapping of CDKs in breast cancer demonstrated that the expression of CDK9 predicted a detrimental outcome in basal-like tumors (HR = 1.51, CI = 1.08–2.11, p = 0.015) and, particularly, in the luminal B subtype with HER2+ expression (HR = 1.82, CI = 1.17–2.82, p = 0.0069). The novel CDK9 PROTAC, THAL-SNS-032, displayed a profound inhibitory activity in MCF7, T47D, and BT474 cells, with less effect in SKBR3, HCC1569, HCC1954, MDA-MB-231, HS578T, and BT549 cells. The three cell lines with HER2 overexpression and no presence of ER, SKBR3, HCC1569, and HCC1954 displayed an EC50 three times higher compared to ER-positive and dual ER/HER2-positive cell lines. BT474-derived trastuzumab-resistant cell lines displayed a particular sensitivity to THAL-SNS-032. Western blot analyses showed that THAL-SNS-032 caused a decrease in CDK9 levels in BT474, BT474-RH, and BT474-TDM1R cells, and a significant increase in apoptosis. Experiments in animals demonstrated an inverse therapeutic index of THAL-SNS-032, with doses in the nontherapeutic and toxic range. The identified toxicity was mainly due to an on-target off-tumor effect of the compound in the gastrointestinal epithelium. In summary, the potent and efficient antitumoral properties of the CDK9 PROTAC THAL-SNS-032 opens the possibility of using this type of compound in breast cancer only if specifically delivered to cancer cells, particularly in ER/HER2-positive and HER2-resistant tumors.

MTOR Inhibition and T-DM1 in HER2-Positive Breast Cancer.

Inhibition of mTOR increases the antitumor activity of T-DM1, supporting that the combination of mTOR inhibitors and antibody-drug conjugates warrants clinical evaluation in patients with HER2-positive breast cancer.

FTY720 in resistant human epidermal growth factor receptor 2-positive breast cancer

The prognosis of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer has considerably improved. However, no reliable treatment besides anti-HER2 strategies has been available. FTY720, a small-molecule compound used for treating refractory multiple sclerosis, has been reported to have beneficial effects against cancers. We therefore evaluated the efficacy of FTY720 in trastuzumab-resistant breast cancer cells and investigated the possible mechanism involved. This study evaluated morphological changes after FTY720 treatment. Antiproliferative WST-1 assays and LDH Cytotoxicity Assay Kits were used to determine the treatment effects of drugs, whereas Western blot analysis was used to evaluate protein expression. Apoptotic events were investigated through annexin V staining and TUNEL assays using flow cytometry. FTY720 was effective in trastuzumab-resistant breast cancer cell lines despite the presence of PIK3CA mutation. Studied on a xenograft mouse model, FTY720-treated groups had statistically significantly poorer HCC1954 xenograft growth in vivo compared with the control group. Our findings suggest that FTY720 can overcome resistance to trastuzumab therapy in patients with HER2-positive breast cancer, with FTY720 plus trastuzumab might offer even better efficacy in vitro and in vivo.