Trastuzumab-resistant Cell Lines Research Articles

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<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.

Alpha-crystallin B chains in trastuzumab-resistant breast cancer cells promote endothelial cell tube formation through activating mTOR

The specific human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibody trastuzumab shows considerable clinical efficacy in patients with HER2-overexpressing breast cancer. However, about 20% of patients who receive trastuzumab in the adjuvant setting relapse, and approximately half of patients with metastatic HER2-positive breast cancer develop resistance to trastuzumab within 1 year. Although the mechanism of trastuzumab resistance has been explored broadly, whether and how angiogenesis participates in trastuzumab resistance is unclear. Here, we examined the association between angiogenesis and trastuzumab resistance by using a trastuzumab-resistant cell line (SKBR3-TR). Compared with that from the parental trastuzumab-sensitive SKBR3 cells, the culture supernatant from SKBR3-TR cells significantly increased the sprouting of endothelial cells. To identify intercellular features that contribute to the induction of endothelial tube formation, proteomics revealed that α-crystallin B chain (αB-crystallin) was upregulated in SKBR3-TR cells. Moreover, silencing of αB-crystallin significantly repressed SKBR3-TR-induced tube formation, and knockdown of αB-crystallin in SKBR3-TR cells suppressed the activation of mechanistic target of rapamycin (mTOR) in endothelial cells. In addition, treatment with rapamycin, an inhibitor of mTOR, reversed the SKBR3-TR-induced promotion of tube formation. In summary, αB-crystallin enhanced the ability of SKBR3-TR cells to activate mTOR in endothelial cells and thus promote angiogenesis.

YES1 as a Therapeutic Target for HER2-Positive Breast Cancer after Trastuzumab and Trastuzumab-Emtansine (T-DM1) Resistance Development.

Trastuzumab-emtansine (T-DM1) is a therapeutic agent molecularly targeting human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC), and it is especially effective for MBC with resistance to trastuzumab. Although several reports have described T-DM1 resistance, few have examined the mechanism underlying T-DM1 resistance after the development of acquired resistance to trastuzumab. We previously reported that YES1, a member of the Src family, plays an important role in acquired resistance to trastuzumab in HER2-amplified breast cancer cells. We newly established a trastuzumab/T-DM1-dual-resistant cell line and analyzed the resistance mechanisms in this cell line. At first, the T-DM1 effectively inhibited the YES1-amplified trastuzumab-resistant cell line, but resistance to T-DM1 gradually developed. YES1 amplification was further enhanced after acquired resistance to T-DM1 became apparent, and the knockdown of the YES1 or the administration of the Src inhibitor dasatinib restored sensitivity to T-DM1. Our results indicate that YES1 is also strongly associated with T-DM1 resistance after the development of acquired resistance to trastuzumab, and the continuous inhibition of YES1 is important for overcoming resistance to T-DM1.

Abstract 1090: Overcoming trastuzumab resistance using trastuzumab deruxtecan (T-DXd), a HER2 targeting antibody drug conjugate, in HER2 amplified gastric cancer

Abstract HER2 is amplified in 7% to 20% of gastric cancer (GC) and is associated with poor clinical outcome. Trastuzumab, HER2-targeting antibody, has already been successfully employed for the treatment of HER2 amplified metastatic GC patients. However, most GC patients who achieved an initial response to the trastuzumab-based regimen developed resistance within 1 year after treatment. In this study, we aimed to investigate the efficacy and biological mechanism of trastuzumab deruxtecan (T-DXd) for overcoming trastuzumab resistance in HER2 amplified GC cell lines. We established 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. Next, we compared the anti-tumor efficacy of trastuzumab and T-DXd (Daiichi Sankyo, Japan), a HER2-targeting antibody-drug conjugate (ADC), in parental and trastuzumab resistant cell lines. Drug sensitivities were determined by the inhibition rates (IR) at 100μg/ml using cell viability assay. Both in baseline and in post-treatment drug responses, expression levels of proteins such as HER2, Erk, Akt, H2A.X and cPARP were detected by western blotting. The parental cell lines were sensitive to trastuzumab, while four trastuzumab resistant (TR) cell lines demonstrated significant resistance to trastuzumab (p<0.05). Compared with the parental cell lines, pHER2 expression was increased in trastuzumab resistant cell lines, and HER2 expression was decreased but still remained at a high level, suggesting that T-DXd can bind to HER2. As expected, T-DXd was sensitive in both parental and trastuzumab resistant cell lines. In detail, sensitivity to T-DXd in parental and resistant cells were as follows: YCC-33 vs YCC-33TR (79.6% vs 90.7%), YCC-38 vs YCC-38TR (94.4% vs 76.5%), NCI-N87 vs NCI-N87TR (83.8% vs 82.6%), and SNU-216 vs SNU-216TR (58.3% vs 42.6%). When treated with T-DXd to trastuzumab resistant cell lines, the expression levels of HER2 and HER2-related molecules showed various patterns for each cell line, and the expression levels of pH2A.X or cPARP were increased compared to trastuzumab treatment. It seems that T-DXd acts to cause DNA damage and induce apoptosis in parental and trastuzumab resistant cell lines. In summary, the established trastuzumab resistant cell lines retained high HER2 expression, and both HER2 amplified parental and trastuzumab resistant cell lines were sensitive to T-DXd. Our data indicated that T-DXd may have a potential therapeutic effect to overcome trastuzumab resistance in GC. Citation Format: Juin Park, Inhye Jeong, Sun Kyoung Kang, Seo Young Yu, Woo Sun Kwon, Tae Soo Kim, Jihyun Hwang, Hyun Cheol Chung, Sun Young Rha. Overcoming trastuzumab resistance using trastuzumab deruxtecan (T-DXd), a HER2 targeting antibody drug conjugate, in HER2 amplified gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1090.

Abstract 1376: Dosing frequency/PD/efficacy relationship of MEN1611 in HER2 amplified, PIK3CA mutated, and refractory to Trastuzumab xenograft model of breast cancer

Abstract The PI3K-AKT-mTOR pathway is one of the most dysregulated pathways in human cancers, with PIK3CA, the gene encoding the p110α catalytic isoform of PI3K, representing the most frequently mutated gene of this network. In breast tumors, PIK3CA mutations occur in about 20% to 40% of HER2-positive and HER2-negative respectively. The lack of driver oncogenic function, hypothesized for PIK3CA mutations, paralleled by the poor clinical benefit showed by monotherapy with PI3K inhibitors in solid tumors, suggests the use of rational combinations with optimized dosing to improve PI3K inhibitor efficacy. Here we investigate the activity of the PI3K α/β selective and δ-sparing inhibitor MEN1611 in once-daily (QD) at 3.2 mg/kg versus twice-daily (BID) dosing at 1.6 mg/kg, both as a single agent and in combination with trastuzumab, to examine the correlation between drug regimen, target effect, and tumor response. The in vivo studies were investigated with the HER2 amplified, PIK3CA mutated (p.C420R), and trastuzumab-resistant JIMT-1 breast cancer cell line. To compare the effect of QD versus BID MEN1611 treatment, we evaluated the magnitude of PI3K signaling at the tumor site, by measuring the levels of pAKT and pS6 at different time points, at the beginning and the end of treatment. Anti-tumor activity was evaluated in terms of tumor volume inhibition (TVI) and by the modified response evaluation criteria in solid tumors (mRECIST). In monotherapy, although QD dosing produced a slightly longer pathway inhibition (10 vs 8 hours) than the BID, the maximum magnitude achieved was comparable. The analysis of anti-tumor efficacy, showed non-significant differences between the two schedules in terms of tumor volume inhibition (52% versus 45% for the QD and the BID respectively), confirming the PD data. In combo, BID administration was able to keep the pathway efficiently downregulated, counteracting a rebound effect observed at the 24 hour time point in the QD group of animals. Moreover, efficacy evaluation showed significant tumor-regression activity in the BID group of animals compared to the QD (79% versus 43% respectively). Although further studies are needed to evaluate time-dependent drug exposure in tumor and plasma samples, these preliminary findings support the BID clinical schedule of MEN1611 in the B-PRECISE-01 clinical trial (NCT03767335). Citation Format: Alessio Fiascarelli, Giuseppe Merlino, Stefania Capano, Alessandro Bressan, Mario Bigioni, Andrea Pellacani, Monica Binaschi, Massimiliano Salerno. Dosing frequency/PD/efficacy relationship of MEN1611 in HER2 amplified, PIK3CA mutated, and refractory to Trastuzumab xenograft model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1376.

Abstract 1079: A synthetic lethal strategy using PARP and ATM inhibition for overcoming trastuzumab-resistance in HER2-positive cancers

Abstract Background: DNA damage response (DDR) pathway is essential for maintaining genomic stability, cell survival, and tumorigenesis. However, the roles of DDR pathway in HER2-positive tumors remain unclear. This study aims to discover the function of DDR in trastuzumab-resistant (TR) cells to better understand HER2-biology and to suggest novel treatment strategy using DDR-targeting agents in HER2-positive TR tumors. Methods: Four HER2-positive TR cell lines (NCI-N87TR, SNU-216TR, SNU-2670TR, and SNU-2773TR) and their corresponding parental cells (NCI-N87, SNU-216, SNU-2670, and SNU-2773) were subject to immunoblotting to identify expression profiles of DDR proteins. Proteasome inhibitor MG-132 and cycloheximide chase assay were used for understanding PARP1 dynamics in TR cells. MTT assay and colony-forming assay were performed to evaluate antiproliferative effect of PARP inhibitor (PARPi, olaparib), ATM inhibitor (ATMi, AZD0156) and ATR inhibitor (ATRi, Ceralasertib). DNA damage and homologous recombination repair (HRR) efficiency were measured by comet assay and immunofluorescence respectively. Cell cycle analysis using flow cytometry was performed to assess drug-induced apoptosis. Results: Increase of PARP1 expression was observed in all four TR cells. Decreased proteasomal degradation of PARP1 was found in TR cells compared to parental cells. Olaparib showed better anti-proliferative effect in TR cells compared to parental cells. After treating with olaparib, there was a defect in formation of Rad51 foci, instead, there was induction of phosphorylation of ATM and ATR in TR cells, which might attenuate the effects of olaparib in TR cells. Combination of olaparib with AZD0156 showed stronger anti-tumor effect than olaparib monotherapy in TR cells. Moreover, synthetic lethal interaction was observed by olaparib/AZD0156 combination in TR cells. Interestingly, this synthetic lethal interaction was exclusively occurred in TR cells not in parental cells. Even though olaparib and ceralasertib combination showed better anti-tumor effect than each single drug in some TR cells, synthetic lethal interaction was not observed by olaparib/ceralasertib combination in TR cells. Conclusion: Dual inhibition of PARP1 and ATM could be a good strategy to overcome the resistance to trastuzumab in HER2-positive cancers using its synthetic lethal interaction. Citation Format: Kyoung-Seok Oh, Ah-Rong Nam, Ju-Hee Bang, Hye-Rim Seo, Jae-Min Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. A synthetic lethal strategy using PARP and ATM inhibition for overcoming trastuzumab-resistance in HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1079.

WEE1 inhibition reverses trastuzumab resistance in HER2-positive cancers.

To date, many efforts have been made to understand the resistance mechanism of trastuzumab in human epidermal growth factor receptor 2 (HER2)-positive breast and gastric cancer. However, there is still a huge unmet medical need for patients with trastuzumab resistance. In our study, we generated four trastuzumab-resistant (HR) cancer cell lines from ERBB2-amplified gastric and biliary tract cancer cell lines (SNU-216, NCI-N87, SNU-2670, and SNU-2773). Here, we found higher PD-L1 expression in trastuzumab-resistant (HR) HER2-positive cancer cells than in parental cells, and blocking PD-L1 reversed the resistance to trastuzumab in HR cells. Trastuzumab upregulated PD-L1 expression via NF-κB activation in both parental and HR cells, however, led to DNA damage only in parental cells. The WEE1 inhibitor adavosertib, which downregulates PD-L1 expression, enhanced trastuzumab efficacy by blocking BRCA1-CMTM6-PD-L1 signals and the HER2-CDCP-1-SRC axis. Additionally, the levels of galectin-9, CD163, FoxP3, and CTLA-4 were diminished by blocking WEE1 in the presence of human PBMCs in vitro. Taken together, the strategy of co-targeting HER2 and WEE1 could overcome resistance to trastuzumab in HER2-positive cancers, supporting further clinical development in HER2-positive cancer patients.

NDUFA4L2 promotes trastuzumab resistance in HER2-positive breast cancer

Background:Trastuzumab (Herceptin) is the key systemic therapy for HER2-positive breast cancer. However, the initial response rate is limited to approximately 50% in patients. Moreover, most patients, especially at an advanced stage, eventually develop acquired resistance. Understanding the mechanisms of trastuzumab resistance is crucial for achieving better treatment outcome in this group of patients.Methods:A trastuzumab-resistant (TR) cell line was developed using the BT474 HER2-positive breast cancer cell line. Whole-transcriptome expression array was performed and the TR-related gene NDUFA4L2 was identified by differential expression analysis between BT474 and BT474-TR. Mitochondrial localization of NDUFA4L2 was confirmed by immunofluorescence and western blotting using mitochondrial fractionation. Mitochondrial function and energy metabolism were evaluated using Seahorse, ATP production, and lactate production assays, and cellular reactive oxygen species (ROS) levels were determined using DCFDA. NDUFA4L2 expression in patients was evaluated by immunohistochemistry, and relapse-free survival was analyzed using the Kaplan–Meier method.Results:NDUFA4L2 was highly expressed in the TR HER2-positive breast cancer cell line. High expression level of NDUFA4L2 was associated with shorter relapse-free intervals in trastuzumab-treated HER2-positive breast cancer patients. Overexpression of NDUFA4L2 enhanced Warburg effects, enhanced aerobic glycolysis, reduced oxygen consumption, and lowered ROS production. Mechanistically, overexpression of NDUFA4L2 facilitated mitochondrial relocalization of HER2 and suppressed ROS production, thus rendering cancer cells more resistant to trastuzumab treatment.Conclusions:We identified NDUFA4L2 as a new biomarker and potential therapeutic target for TR HER2-positive breast cancer.

Cyclin E overexpression confers resistance to trastuzumab through noncanonical phosphorylation of SMAD3 in HER2+ breast cancer

ABSTRACT The efficacy of trastuzumab, a treatment for HER2+ breast cancer, can be limited by the development of resistance. Cyclin E (CCNE) overexpression has been implicated in trastuzumab resistance. We sought to uncover a potential mechanism for this trastuzumab resistance and focused on a model of CCNE overexpressing HER2+ breast cancer and noncanonical phosphorylation of the TGF-β signaling protein, SMAD3. Network analysis of transcriptional activity in a HER2+, CCNE overexpressing, trastuzumab-resistant cell line (BT474R2) identified decreased SMAD3 activity was associated with treatment resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p < .01), and noncanonical phosphorylation of SMAD3 was increased in these CCNE-overexpressing cells. Also, in response to CDK2 inhibition, expression patterns linked to restored canonical SMAD3 signaling, including decreased cMyc and increased cyclin-dependent inhibitor, p15, were identified. The BT474R2 cell line was modified through overexpression of SMAD3 (BT474R2-SMAD3), a mutant construct resistant to CCNE-mediated noncanonical phosphorylation of SMAD3 (BT474R2-5M), and a control (BT474R2-Blank). In vitro studies examining the response to trastuzumab showed increased sensitivity to treatment for BT474R2-5M cells. These findings were then validated in NSG mice inoculated with BT474R2-5M cells or BT474R2 control cells. After treatment with trastuzumab, the NSG mice inoculated with BT474R2-5M cells developed significantly lower tumor volumes (p < .001), when compared to mice inoculated with BT474R2 cells. Taken together, these results indicate that for patients with HER2+ breast cancer, a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, could be treated with CDK2 inhibition to help enhance the efficacy of trastuzumab therapy.

The Hippo Transducer YAP/TAZ as a Biomarker of Therapeutic Response and Prognosis in Trastuzumab-Based Neoadjuvant Therapy Treated HER2-Positive Breast Cancer Patients.

BackgroundWe explored the therapeutic and prognostic effect of YAP/TAZ intensityinHER2-positive breast cancer patients. We also investigated the relationship between YAP/TAZ expression and Trastuzumab-resistance.MethodsWe collected clinicopathological information from 397 cases. We evaluated therapeutic and prognostic effect of YAP/TAZ and other variables. We also cultivated Trastuzumab-resistance cell lines and explored relationship between YAP/TAZ and Trastuzumab-resistance.ResultsOver-expression of YAP/TAZ was remarkable in Trastuzumab-resistant cells, and so did HER3 and HER2/HER3 heterodimer. Inhibition of YAP/TAZ expression reversed Trastuzumab-resistance.YAP/TAZ deficiency contributed to favorable therapeutic response, and so did hormone receptor insufficiency and chemotherapy dosage inferiority. Deficient YAP/TAZ intensity and abundant hormone receptor intensity contributed to better survival. Over-expression of YAP/TAZ was obvious in recurrent cases in comparison with their matching primary lesions. Prognostic superiority of insufficient YAP/TAZ intensity was more outstanding in hormone receptor negative cases. Over-expression of YAP/TAZ and HER3 was generally synchronous. Absence of HER3 expression in residual lesions might correlate with better breast cancer-free survival.ConclusionsOver-expression of YAP/TAZ as well as HER-3 and HER2/HER3 heterodimer was synchronously remarkable in Trastuzumab-resistant cell lines. Inhibition of YAP/TAZ expression reversed Trastuzumab resistance. Deficient YAP/TAZ intensity as well as insufficient hormone receptor intensity and high chemotherapy dosage contributed to favorable therapeutic response. Deficient YAP/TAZ intensity and abundant hormone receptor intensity contributed to better survival, and so did absence of HER3expression in residual lesions. Prognostic superiority of YAP/TAZ expression depended on hormone receptor status. Cases with synchronous over-expression of YAP/TAZ and HER3 suffered poor survival, which revealed the potential effect of YAP/TAZ-HER2/HER3 crosstalk in prognosis of HER2-positive patients.

Abstract 1915: Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling

Abstract Introduction The widespread clinical use of monoclonal antibodies such as trastuzumab in personalized medicine, targeting upstream proteins such as receptors with cascading effects on downstream signaling, elicits the importance of studying the phosphoproteome in the investigation of resistance to targeted therapy. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins in key signal transduction pathways, in cellular models of resistance. Method DigiWest®, a Luminex® xMAP® bead-based high-throughput protein profiling method, was utilized to measure 194 proteins and phospho-proteins from 12µg of total protein, obtained from HER2-positive cell lines which were trastuzumab-sensitive (SKBR3), intrinsically resistant (JIMT-1, UACC-893) and with acquired resistance (T15, obtained by long-term culture of SKBR3 in 15µg/mL trastuzumab). The resulting median fluorescence intensities (MFI) were normalized to total protein content. Relative quantification was done against SKBR3 and a log2 fold-change of ±1 was considered to be significant. For each of the three trastuzumab-resistant cell lines, targets which were differentially expressed were organized into established signal transduction pathways, and specific phosphosite expression values were used to determine whether each protein was under- or over-activated. Results A total of 115 proteins were expressed in at least one of the HER2-positive cell lines at a normalized MFI above the set threshold. JIMT-1 showed the most concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β-catenin stabilization, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance – it was over-activated in all the three trastuzumab-resistant cell lines. The overexpression of upstream RAS, however, was confined to the intrinsically resistant cell lines JIMT-1 and UACC-893; meanwhile, in T15, entry into the ERK pathway was mostly mediated by pan-PKC overexpression. PKCs, especially PKCα, were also highly activated in UACC-893. β-catenin stabilization was another mechanism observed only in the intrinsically trastuzumab-resistant cell lines. Conclusion Both distinct and overlapping mechanisms are involved in intrinsic and acquired trastuzumab resistance. Multiple pathways can also co-exist to promote cell survival, as was mostly exemplified by JIMT-1, derived from an aggressive, basal-like tumor. Furthermore, different models of intrinsic resistance (JIMT-1 vs UACC-893) showed significant differences in differentially activated signal transduction pathways. DigiWest® provided a simple and robust multiplexing solution for this phospho-proteomic study. Citation Format: Jeanesse Scerri, Felix Ruoff, Markus Templin, Godfrey Grech, Christian Scerri. Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1915.

Preliminary safety, efficacy and pharmacokinetics (PK) results of KN026, a HER2 bispecific antibody in patients (pts) with HER2-positive metastatic breast cancer.

1041 Background: KN026 is a novel bispecific antibody that simultaneously binds to two distinct HER2 epitopes, the same domains as trastuzumab (ECD4) and pertuzumab (ECD2). It blocks ligand-dependent and independent tumor growth and enhances HER2 receptor internalization. In preclinical studies, KN026 showed activity in trastuzumab plus pertuzumab resistant tumor cell lines. This first-in-human study evaluated the safety/tolerability, pharmacokinetics (PK), and preliminary efficacy of KN026 monotherapy. Methods: This dose-escalation and expansion study enrolled Chinese patients (pts) with metastatic breast cancer who have failed prior anti-HER2 therapy. All pts intravenously received KN026 monotherapy at ascending dose of 5 mg/kg (QW), 10 mg/kg (QW), 20 mg/kg (Q2W) or 30 mg/kg (Q3W). Dose limiting toxicity (DLT) evaluation period was 28 days for QW and Q2W, and 21 days for Q3W. Efficacy evaluation was performed by RECIST 1.1 every 6 weeks and safety assessment according to CTCAE v 4.03. Results: As of the Jan, 22, 2020, 63 pts [median age: 54 years (31~69)] enrolled and 62 pts were included in the efficacy analysis. 41 pts remained on treatment and 22 pts discontinued treatment due to disease progression (n = 21) and adverse events (n = 1). The median treatment duration was 12 weeks (range: 4~62 weeks). Median prior lines of therapies are 3 (range: 1~15), and median prior lines of HER2 target therapies are 2 (range: 1~12). No DLTs were observed. Treatment-related AEs (TRAEs) occurred in 49 pts and 4 pts experienced 4 grade 3 TRAE (hypertension, infusion related reaction, transaminases increased and ventricular arrhythmia). The common (≥ 10%) TRAE were pyrexia (23.8%), diarrhea (19.0%), aspartate aminotransferase increased (15.9%), neutrophil count decreased (11.1%) and white blood cell count decreased (11.1%). The objective response rate at recommended Phase 2 dose levels (n = 56) was 32.1% (95% CI 20.3, 46.0) and disease control rate 76.8% (95% CI 63.6, 87.0). Pharmacokinetic analysis showed exposure (Cmax and AUC0-t) of KN026 increased by dose. The recommended Phase 2 dose (RP2D) were 20 mg/kg Q2W and 30 mg/kg Q3W. Conclusions: KN026 is well tolerated and has demonstrated encouraging anti-tumor activity in HER2-positive breast cancer patients who have failed standard anti-HER2 therapies. The recommended Phase 2 dose (RP2D) of KN026 were 20 mg/kg Q2W and 30 mg/kg Q3W. Phase II trials in various HER2-positive and HER2-low/intermediate solid tumors are currently ongoing. Clinical trial information: NCT03619681 .

MiR-126 reduces trastuzumab resistance by targeting PIK3R2 and regulating AKT/mTOR pathway in breast cancer cells.

MicroRNAs (miRNAs) have been found to play a key role in drug resistance. In the current study, we aimed to explore the potential role of miR‐126 in trastuzumab resistance in breast cancer cells. We found that the trastuzumab‐resistant cell lines SKBR3/TR and BT474/TR had low expression of miR‐126 and increased ability to migrate and invade. The resistance, invasion and mobilization abilities of the cells resistant to trastuzumab were reduced by ectopic expression of miR‐126 mimics. In comparison, inhibition of miR‐126 in SKBR3 parental cells had the opposite effect of an increased resistance to trastuzumab as well as invasion and migration. It was also found that miR‐126 directly targets PIK3R2 in breast cancer cells. PIK3R2‐knockdown cells showed decreased resistance to trastuzumab, while overexpression of PIK3R2 increased trastuzumab resistance. In addition, our finding showed that overexpression of miR‐126 reduced resistance to trastuzumab in the trastuzumab‐resistant cells and that inhibition of the PIK3R2/PI3K/AKT/mTOR signalling pathway was involved in this effect. SKBR3/TR cells also showed increased sensitivity to trastuzumab mediated by miR‐126 in vivo. In conclusion, the above findings demonstrated that overexpression of miR‐126 or down‐regulation of its target gene may be a potential approach to overcome trastuzumab resistance in breast cancer cells.