HER2-positive Breast Cancer Cell Lines Research Articles

IRX4204 Induces Senescence and Cell Death in HER2-positive Breast Cancer and Synergizes with Anti-HER2 Therapy.

Rexinoids, agonists of nuclear retinoid X receptor (RXR), have been used for the treatment of cancers and are well-tolerated in both animals and humans. However, the usefulness of rexinoids in treatment of breast cancer remains unknown. This study examines the efficacy of IRX4204, a highly specific rexinoid, in breast cancer cell lines and preclinical models to identify a biomarker for response and potential mechanism of action. IRX4204 effects on breast cancer cell growth and viability were determined using cell lines, syngeneic mouse models and primary PDX tumors. In vitro assays of cell cycle, apoptosis, senescence, and lipid metabolism were used to uncover a potential mechanism of action. Standard anti-HER2 therapies were screened in combination with IRX4204 on a panel of breast cancer cell lines to determine drug synergy. IRX4204 significantly inhibits the growth of HER2-positive breast cancer cell lines, including trastuzumab and lapatinib resistant JIMT-1 and HCC1954. Treatment with IRX4204 reduced tumor growth rate in the MMTV-ErbB2 mouse and HER2-positive PDX model by 49% and 44% respectively. Mechanistic studies revealed IRX4204 modulates lipid metabolism and induces senescence of HER2-positive cells. In addition, IRX4204 demonstrates additivity and synergy with HER2 targeted monoclonal antibodies, tyrosine kinase inhibitors, and antibody drug conjugates. These findings identify HER2 as a biomarker for IRX4204 treatment response and demonstrate a novel use of RXR agonists to synergize with current anti-HER2 therapies. Furthermore, our results suggest that RXR agonists can be useful for the treatment of anti-HER2 resistant and metastatic HER2-positive breast cancer.

Abstract 536: Breaking through ADCs resistance: Constructing preclinical enhertu-induced resistant model

Abstract Antibody-drug conjugates (ADCs) are compounds that combine the specificity of monoclonal antibodies (mAbs) with the cytotoxic payload of toxic substances. In recent years, ADCs have rapidly developed in the field of cancer therapy, particularly in the treatment of HER2-positive breast cancer. One representative example is Trastuzumab deruxtecan (Enhertu), which has become a new benchmark for anti-HER2 ADCs due to its excellent performance in clinical trials. Despite the impressive clinical outcomes of ADCs, some tumors in patients may still develop resistance to these drugs. Therefore, it is crucial to understand the mechanisms of resistance and develop strategies to overcome ADCs resistance. To achieve these goals, it is essential to establish and utilize preclinical models to study these resistance mechanisms. Preclinical models of resistance to ADCs have mainly been generated using human cultured cell lines. JIMT-1 is a HER2-positive breast cancer cell line that exhibits primary resistance to the anti-HER2 antibody trastuzumab and also shows resistance to trastuzumab-derived ADC T-DM1. However, we found that Enhertu significantly inhibited tumor growth in JIMT-1 xenograft models. We subjected JIMT-1 tumor tissues to continuous passage in mice and administered long-term drug induction. The tumor growth rate gradually increased, and the drug sensitivity decreased, resulting in the acquisition of an Enhertu-resistant tumor model. The obtained resistant tumor tissues not only include tumor cells themselves but also comprise fibroblasts, extracellular matrix, and other cellular components, enabling a more comprehensive investigation into the interactions between different cells within the tumor microenvironment (TME). We believe that the establishment of preclinical Enhertu-resistant models helps predict resistance that has not yet emerged clinically and provides references for future drug development. Citation Format: Yuan Fang, Huiyi Wang, Jun Xing, Lu Yang, Jing Zhao, Xiang Gao, Cunxiang Ju. Breaking through ADCs resistance: Constructing preclinical enhertu-induced resistant model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 536.

Protecting the Brain: Novel Strategies for Preventing Breast Cancer Brain Metastases through Selective Estrogen Receptor β Agonists and In Vitro Blood-Brain Barrier Models.

Breast cancer brain metastasis (BCBM) is a challenging condition with limited treatment options and poor prognosis. Understanding the interactions between tumor cells and the blood-brain barrier (BBB) is critical for developing novel therapeutic strategies. One promising target is estrogen receptor β (ERβ), which promotes the expression of key tight junction proteins, sealing the BBB and reducing its permeability. In this study, we investigated the effects of 17β-estradiol (E2) and the selective ERβ agonist diarylpropionitrile (DPN) on endothelial and cancer cells. Western blot analysis revealed the expression patterns of ERs in these cell lines, and estrogen treatment upregulated claudin-5 expression in brain endothelial cells. Using in vitro models of the BBB, we found that DPN treatment significantly increased BBB tightness about suppressed BBB transmigration activity of representative Her2-positive (BT-474) and triple-negative (MDA-MB-231) breast cancer cell lines. However, the efficacy of DPN treatment decreased when cancer cells were pre-differentiated in the presence of E2. Our results support ERβ as a potential target for the prevention and treatment of BCBM and suggest that targeted vector-based approaches may be effective for future preventive and therapeutic implications.

Targeting HER2-positive breast cancer cells by a combination of dasatinib and BMS-202: Insight into the molecular pathways

BackgroundRecent investigations have reported the benefits of using a tyrosine kinase inhibitor, dasatinib (DA), as well as programmed death-ligand 1 (PD-L1) inhibitors in the management of several solid tumors, including breast cancer. Nevertheless, the outcome of the combination of these inhibitors on HER2-positive breast cancer is not explored yet.MethodsHerein, we investigated the impact of DA and PD-L1 inhibitor (BMS-202) combination on HER2-positive breast cancer cell lines, SKBR3 and ZR75.ResultsOur data reveal that the combination significantly inhibits cell viability of both cancer cell lines as compared to monotreatment. Moreover, the combination inhibits epithelial-mesenchymal transition (EMT) progression and reduces cancer cell invasion by restoring E-cadherin and β-catenin expressions and loss of vimentin, major biomarkers of EMT. Additionally, the combination reduces the colony formation of both cell lines in comparison with their matched control. Also, the combination considerably inhibits the angiogenesis of the chorioallantoic membrane model compared with monotreatment. Molecular pathway analysis of treated cells shows that this combination blocks HER2, AKT, β-catenin, and JNK1/2/3 activities.ConclusionOur findings implicate that a combination of DA and BMS-202 could have a significant impact on the management of HER2-positive breast cancer.

Comparison of trastuzumab emtansine, trastuzumab deruxtecan, and disitamab vedotin in a multiresistant HER2-positive breast cancer lung metastasis model

Human epidermal growth factor 2 (HER2)-positive breast cancer with lung metastases resistant to targeted agents is a common therapeutic challenge. Absence of preclinical lung metastasis models that are resistant to multiple anti-HER2 targeted drugs hampers the development of novel therapies. We established a novel HER2-positive breast cancer cell line (L-JIMT-1) with a high propensity to form lung metastases from the parenteral JIMT-1 cell line by injecting JIMT-1 cells into immunodeficient SCID mice. Lung metastases developed in all mice injected with L-JIMT-1 cells, and more rapidly and in greater numbers compared with the parental JIMT-1 cells. L-JIMT-1 cells expressed more epidermal growth factor receptor and HER2 than JIMT-1 cells. L-JIMT-1 cells were resistant to all five tyrosine kinase inhibitors tested in vitro (afatinib, erlotinib, lapatinib, sapitinib, and tucatinib). When we compared JIMT-1 and L-JIMT-1 sensitivity to three HER2-targeting antibody-drug conjugates (ADCs) trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (T-DXd), and disitamab vedotin (DV) in vitro, JIMT-1 cells were resistant T-DXd, partially sensitive to T-DM1, and sensitive to DV, while L-JIMT-1 cells were resistant to both T-DM1 and T-DXd, but moderately sensitive to DV. In a mouse model, all three ADCs inhibited the growth of L-JIMT-1 lung metastases compared to a vehicle, but DV and T-DXd more strongly than T-DM1, and DV treatment led to the smallest tumor burden. The L-JIMT breast cancer lung metastasis model developed may be useful in the evaluation of anti-cancer agents for multiresistant HER2-positive advanced breast cancer.

Abstract B030: ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model

Abstract Background: ABC101-1 is an antibody-drug conjugate (ADC) targeting receptor tyrosine-protein kinase erbB-2, also known as HER2. ABC101-1 consists of a humanized IgG1 monoclonal antibody called trastuzumab conjugated to a microtubulin inhibitor, DM1, through interchain cysteines with a drug-to-antibody ratio (DAR) of 8. Our novel non-cleavable linker platform using maleimide as a connector enabled the construction of homogeneous ABC101-1 with DAR of 8, via a straightforward process resulting in high conjugation yield. Employing non-cleavable linkers may ensure that the cytotoxic drug is delivered stably to tumor cells, enhancing the therapeutic efficacy of ADC. Herein, we describe anti-breast cancer activity through in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and binding affinity of ABC101-1. Methods: In vitro cytotoxicity of ADC and linker-payload was evaluated by measuring cell viability using Cell Count Kit-8 (CCK8) analysis. In vivo anti-tumor activity was investigated using SKBR3 cell line-derived xenograft (CDX) model. A pharmacokinetic (PK) profile was assessed by performing indirect ELISA to determine concentrations of total mAb in the serum. The binding affinity of ABC101-1 to HER2 protein was assessed using the indirect ELISA measuring the concentrations of total mAb. Results: In this study, ABC101-1 exhibited potent in vitro cytotoxicity with picomolar activity on HER2-positive breast cancer cell line. In the CDX breast cancer model, ABC101-1 led to stronger tumor growth inhibition in both early and late phases than ado-trastuzumab emtansine (DAR 3.5) at the same dose. Also, the favorable pharmacokinetic profile of ABC101-1 was observed, along with the preserved antigen-binding ability upon the conjugation to linker-payload. Conclusions: ABC101-1 displayed preclinical efficacy in in vitro and in vivo studies as well as a favorable PK, thereby suggesting its therapeutic potential as an anti-tumor drug in breast cancer therapy. This study would reveal that our novel linker platform is a promising and efficient strategy to develop effective and homogeneous ADCs with DAR 8. Our finding may support the further pre-clinical evaluation of ABC101-1. Citation Format: Seo Woo Kim, Hana Yu, Geon Ho Lee, Tae Gyu Lim, Minji Jeong, Hyeonjoo Hwang, Jee Eun Moon, Yong Beom Lee, Sung-Ha Jin, Jin Hyun Jeong. ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B030.

Endosomal recycling inhibitors downregulate estrogen receptor-alpha and synergise with endocrine therapies

PurposeBreast cancer (BC) accounts for roughly 30% of new cancers diagnosed in women each year; thus, this cancer type represents a substantial burden for people and health care systems. Despite the existence of effective therapies to treat BC, drug resistance remains a problem and is a major cause of treatment failure. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. Recent research indicates that inhibition of the endosomal recycling pathway, an intracellular membrane trafficking pathway that returns endocytosed proteins back to the plasma membrane, may be a promising strategy to downregulate clinically relevant cell surface proteins such as HER2 and HER3, and to overcome drug resistance.MethodsTo investigate the molecular mechanism of action of an endosomal recycling inhibitor (ERI) called primaquine, we performed a reverse-phase protein array (RPPA) assay using a HER2-positive breast cancer cell line. The RPPA findings were confirmed by Western blot and RT-qPCR in several BC cell lines. Novel drug combinations were tested by MTT cell viability and clonogenic assays.ResultsAmong the signalling molecules downregulated by ERIs were estrogen receptor-alpha (ER-α) and androgen receptor. We confirmed this finding in other breast cancer cell lines and show that downregulation occurs at the transcriptional level. We also found that ERIs synergise with tamoxifen, a standard-of-care therapy for breast cancer.DiscussionOur data suggest that combining ERIs with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

DNA Methylation-Regulated HOXC8's Role in HER2-Positive Breast Cancer Function and its Contribution to Herceptin Resistance.

The epidermal growth factor receptor 2 (HER2) is overexpressed in 30% of breast cancers, and this overexpression is strongly correlated with a poor prognosis. Herceptin is a common treatment for HER2-positive breast cancer; however, cancer cells tend to adapt gradually to the drug, rendering it ineffective. The study revealed an association between the methylation status of the Homeobox C8 (HOXC8) gene and tumor development. Therefore, it is of paramount importance to delve into the interaction between HOXC8 and HER2-positive breast cancer, along with its molecular mechanisms. This exploration holds significant implications for a deeper understanding of the pathophysiological processes underlying HER2-positive breast cancer. Tumor tissue and pathological data from patients with HER2-positive breast cancer were systematically collected. Additionally, the human HER2-positive breast cancer cell line, SKBR3, was cultured in vitro to assess both the expression level of HOXC8 and the degree of DNA methylation. The study aimed to explore the relationship between the relative expression of HOXC8 and the clinical characteristics of breast cancer patients. The expression level of HOXC8 and the promoter methylation of HOXC8 were verified by methylation treatment of SKBR3 breast cancer cells. The regulation of HOXC8 was meticulously carried out, leading to the division of the cells into distinct groups. The study further analyzed the expression levels and biological capabilities within each group. Finally, the in vitro and in vivo sensitivity of the cells to Herceptin, a common treatment for HER2-positive breast cancer, was measured to assess the efficacy of the drug. In HER2-positive breast cancer cases characterized by poor methylation, there was an up-regulation of HOXC8. Its expression was found to be correlated with key clinical factors such as tumor size, lymph node status, clinical tumor, node, metastasis (cTNM) staging, and Herceptin resistance (p < 0.05). Upon methylation of breast cancer cells, there was a significant decrease in HOXC8 expression (p < 0.05). The study revealed that overexpression of HOXC8 resulted in increased proliferation, cloning, and metastasis of HER2-positive breast cancer cells, along with a reduced apoptosis rate (p < 0.05). Conversely, interference with HOXC8 expression reversed this scenario (p < 0.05). A Herceptin-resistant substrain, POOL2, was established using SKBR3 cells. Animal studies demonstrated that overexpressing HOXC8 accelerated tumor development and enhanced POOL2 cells' resistance to Herceptin (p < 0.05). However, following interference with HOXC8, POOL2 cells exhibited increased responsiveness to Herceptin, leading to a gradual reduction in tumor size (p < 0.05). In HER2-positive breast cancer, the expression of HOXC8 is elevated in a manner dependent on DNA methylation, and this elevated expression is closely linked to the pathology of the patient. Interfering with HOXC8 expression demonstrates the potential to partially inhibit the development and spread of breast cancer, as well as to alleviate resistance to Herceptin.

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.

Recombinant anti-human epidermal growth factor receptor type 2 single-chain variable fragment-alpha-luffin fusion protein as a putative immunotoxin against human epidermal growth factor receptor type 2-positive breast cancer cells: an experimental research.

Breast cancer is one of the most frequent causes of cancer death in women. The application of immunotoxins to target overexpressed biomarkers on the surface of cancer cells and delivery of the toxin molecules into these cells has attracted too much attention during the last decade. This study was conducted to investigate the possible in-vitro cytotoxic and apoptotic activity of previously designed recombinant immunotoxin compromising anti-HER2 single-chain variable fragment (scFv) and alpha-luffin protein in human epidermal growth factor receptor type 2 (HER2)-positive and HER2-negative breast cancer cell lines. The previously designed recombinant immunotoxin and alpha-luffin protein were expressed in E. coli host cells and purified using Ni-affinity chromatography. The cytotoxicity of the proteins was tested through MTT and apoptosis studies on HER2-positive and HER2-negative breast cancer cell lines. Treatment of SKBR3 and MDA-MB-468 cells with the immunotoxin caused differential cytotoxicity and apoptotic events. Flow cytometry analysis indicated that the immunotoxin could arrest SKBR3 cells at the G0/G1 phase and induce apoptosis and cell death which were not observed in HER2-negative MDA-MB-468 cells. Annexin V/PI staining revealed late apoptotic events in SKBR3 cells treated with the immunotoxin which was different from the early apoptosis induced by the alpha-luffin protein alone. This immunotoxin could be a promising tool in developing new targeted therapeutic agents against HER2-positive cancer cells. Animal experiments are needed before making firmed conclusions.

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 6121: UCP2 inhibitor eradicates cancer stem-like population in trastuzumab-resistant HER2-positive breast cancer

Abstract Purpose: Uncoupling protein 2 (UCP2) is a member of the mitochondrial anion carrier protein family that plays an important role in stabilizing the inner mitochondrial membrane potential (MMP, ΔΨm) and controlling reactive oxygen species (ROS) production. A selective UCP2 inhibitor, genipin is known to elicit cytotoxicity in several cancers, however, its effects on cancer stem cells (CSCs)-like properties and trastuzumab resistance in HER2-positive breast cancer cells have not been fully elucidated. In the present study, we sought to investigate the mechanism of action of genipin responsible for the induction of apoptosis and its effects on CSC-like features, expression of HER family member and trastuzumab resistance in HER2-positive breast cancer cells in vitro and in vivo. Experimental Designs: The effects of genipin on trastuzuamb-sensitive [BT474 and SKBR3] and trastuzumab-resistant [JIMT-1 and MDA-MB-453] HER2-positive breast cancer cell lines in vitro were evaluated for cell viability, Sub-G1, ROS, MMP, ALDH1 activity, CD44+/CD24- subpopulation and mammosphere formation. To confirm the physiological relevance of our in vitro observations, we explored the impact of genipin on tumor growth and angiogenesis and expression of p95HER2 and ALDH1A1 in trastuzumab-resistant xenograft model in vivo. Results: HER2-positive breast cancer cells harbored a higher level of UCP2, when compared to their counterparts. Genipin significantly downregulated UCP2 and mitochondrial dysfunction coinciding with increased ROS generation and disruption of MMP. These phenomena were accompanied with upregulation of the Bax/Bcl-2 ratio and activation of caspase-3 and caspase-7. Genipin treatment led to significant reduction in levels of truncated p95HER2, p-HER2, p-HER3 and p-Akt levels in both trastuzumab-sensitive and -resistant lines. Marked decline of CD44 and ALDH1A1 expression by genipin treatment was associated with attenuation of mammosphere-forming ability. UCP2 level is predominantly upregulated in CSC-enriched populations, while its knockdown significantly suppressed CSC-like characteristics concomitant with decreased ALDH1A1 and CD44 expression as well as impairment of ALDH1 activity. Genipin administration significantly retarded tumor growth and angiogenesis in trastuzumab-resistant JIMT-1 xenograft tumors. The antitumor effect occurred concomitantly with a decrease in Ki-67 proliferating index and enhancement of apoptosis. Furthermore, individuals receiving genipin exhibited markedly lower levels of p95HER2, full-length p185HER2, CD44 and ALDH1A1 expression compared to their control counterparts. Conclusion: To our knowledge, our findings are the first reported instance of genipin-induced suppression of CSC-like properties and HER2/HER3/Akt axis, implying that genipin treatment may have application in addressing trastuzumab resistance. Citation Format: Minsu Park, Soeun Park, Juyeon Seo, Dongmi Ko, Seongjae Kim, Yong Koo Kang, Kee Dal Nam, So Ra Seuk, Tae-Min Cho, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. UCP2 inhibitor eradicates cancer stem-like population in trastuzumab-resistant 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 6121.

Abstract 2489: Study the efficacy of hypomethylating agent in treating regular and brain metastasized HER2-positive breast cancer

Abstract Breast cancer is the second leading cause of cancer with the highest mortality rate in females in the United States. Late stage breast cancer can metastasize to different locations of the body, and patients with brain metastasis have a poor prognosis and short survival time. HER2-positive breast cancer, composed of ~15-20% of total cases, is aggressive with unfavorable clinical outcomes. Although HER2-targeted therapies are available, they are mostly ineffective against brain metastasis. To study tumor metastasis, we had established the breast cancer brain metastasis preclinical cell and mouse models. Our previous study has shown that the hypomethylating agent azacitidine (AZA) is effective in treating brain metastasis triple-negative breast cancer using in vitro cell and in vivo mouse models. Thus, we hypothesized that AZA will be effective to treat HER2-positive breast cancer. To test our hypothesis, we treated regular (JIMT-1) and brain metastasized (JIMT-1 Br) HER2-positive breast cancer cell lines with various concentrations of AZA, and the IC50 values, cell proliferation, and apoptosis were measured. Our results showed that train metastasized HER2-positive breast cancer JIMT-1 Br cells have similar growth rate compared to their parental regular breast cancer JIMT-1 cells. Regular breast cancer cells are more sensitive to AZA treatment, supported by (1) The IC50 value of AZA in JIMT-1 cells is significantly lower than that in JIMT-1 Br cells; (2) 72 hours of AZA treatment significantly inhibited cell grow in both cell lines, and the inhibition of JIMT-1 growth is greater than that of JIMT-1 Br cells; (3) AZA triggered apoptosis in both cell lines in a dose-dependent manner; (4) Higher concentrations of AZA induced a higher percentage of cell apoptosis in JIMT-1 compared to JIMT-1 Br cells. AZA treatment does not significantly change the expression of metastasis protein markers in either cell line. Epithelial-mesenchymal transition promotes cancer cell invasion and metastasis. Epithelial markers include cytokeratins and E-cadherin, and mesenchymal markers include N-cadherin and vimentin. We measured the expression of those markers in both cell lines after AZA treatment by Western blotting, and found that the expression of pan-cytokeratins and keratin 18 were equivalent between both cell lines upon AZA treatment. However, the expression of keratin 19, E-cadherin, N-cadherin, and vimentin were not detected in either cell line. In conclusion, in this study, we tested the in vitro effects of the hypomethylating agent AZA in both regular and brain metastasized HER2-positive breast cancer cells. We found that AZA inhibits cell growth and promotes apoptosis in both cell types, but the regular breast cancer cells are more sensitive to AZA treatment compared to brain metastasized cells, shown by the differences in IC50 values and extents of cell growth inhibition and apoptosis induction. Citation Format: Tuoen Liu, Christopher Butler, Kelsey Donoughe, Lauren Forchette, William Sebastian, Gabor Szalai, Paul Lockman. Study the efficacy of hypomethylating agent in treating regular and brain metastasized 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 2489.

Anticancer effect of zanubrutinib in HER2-positive breast cancer cell lines

Small molecule Bruton’s tyrosine kinase (BTK) inhibitors have been developed for the treatment of various haemato-oncological diseases, and ibrutinib was approved as the first BTK inhibitor for anticancer therapy in 2013. Previous reports proved the receptor kinase human epidermal growth factor receptor 2 (HER2) to be a valid off-target kinase of ibrutinib and potentially other irreversible BTK inhibitors, as it possesses a druggable cysteine residue in the active site of the enzyme. These findings suggest ibrutinib as a candidate drug for repositioning in HER2-positive breast cancer (BCa). This subtype of breast cancer belongs to one of the most common classes of breast tumours, and its prognosis is characterized by a high rate of recurrence and tumour invasiveness. Based on their similar kinase selectivity profiles, we investigated the anticancer effect of zanubrutinib, evobrutinib, tirabrutinib and acalabrutinib in different BCa cell lines and sought to determine whether it is linked with targeting the epidermal growth factor receptor family (ERBB) pathway. We found that zanubrutinib is a potential inhibitor of the HER2 signalling pathway, displaying an antiproliferative effect in HER2-positive BCa cell lines. Zanubrutinib effectively inhibits the phosphorylation of proteins in the ERBB signalling cascade, including the downstream kinases Akt and ERK, which mediate key signals ensuring the survival and proliferation of cancer cells. We thus propose zanubrutinib as another suitable candidate for repurposing in HER2-amplified solid tumours.

Abstract P1-11-10: Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (HER2+) breast cancer (BC) cell lines

Abstract Background: Targeting the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors with small molecule TKIs lapatinib, neratinib and tucatinib, monoclonal antibodies like trastuzumab and more recently with ADCs like T-DM1 and T-DXd has resulted in improved survival rates for patients with HER2+ BC. TKIs are primarily utilised in trastuzumab-refractory disease. Neratinib, and the pan-HER TKI afatinib, bind irreversibly to all members of the HER family, while lapatinib binds reversibly to HER2 and EGFR, and tucatinib binds reversibly to HER2 alone. Previous work in our lab has shown that TKIs like lapatinib are capable of modulating tumour surface HER2 levels and increasing trastuzumab load on tumours. Thus, there is a rationale for the use of TKI/ADC combinations in HER2+ BC. Using innately trastuzumab-resistant HER2+ BC cell lines, this in vitro study aims to assess the anti-proliferative potential of HER2-targeting ADCs T-DM1 or T-DXd in combination with the TKIs afatinib, lapatinib, neratinib or tucatinib. Methods: HCC1569 and HCC1954 cells (HER2+, estrogen receptor (ER)-negative; innately trastuzumab-resistant) were grown in RPMI1640/10% FBS at 37°C and 5% CO2. The anti-proliferative effects of afatinib, lapatinib, neratinib and tucatinib; of T-DM1 and T-DXd; and of each TKI/ADC combination thereof were assessed in these cells via 5-day acid phosphatase. ADCs were obtained from Saint Vincent’s University Hospital, Dublin, and TKIs were purchased from commercial sources. Calcusyn software was used to generate IC50 values and combination index (CI) values at ED50. CI values &amp;gt; 1 represent an antagonistic combination, CI values = 1 are additive, and CI values &amp;lt; 1 are synergistic. All assays were carried out in triplicate. Results: The HCC1569 cell line was more sensitive to all four TKIs (IC50 values were 12.8 ± 0.3, 453.8 ± 47.1 nM, 4.7 ± 1.4 and 381.5 ± 37.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively) compared to the HCC1954 cell line (IC50 22.3 ± 2.8, 652.4 ± 36.0, 57.0 ± 6.3 and 2365.1 ± 185.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively). Despite their innate trastuzumab resistance, both cell lines displayed high sensitivity to T-DM1 (IC50 25.2 ± 8.7 ng/mL for HCC1569 and IC50 30.4 ± 3.2 ng/mL for HCC1954) and to T-DXd (IC50 16.0 ± 3.2 ng/mL for HCC1569 and IC50 36.8 ± 8.7 ng/mL for HCC1954). In both cell lines, co-treatment with ADCs and the irreversible TKIs afatinib and neratinib resulted in additive or synergistic responses, while the combination of the ADCs with the reversible TKIs lapatinib and tucatinib resulted in mild to moderate antagonism (Table 1). Conclusions: In this pre-clinical study, T-DM1 and T-DXd consistently exhibited antagonistic interactions with reversible HER2-targeting TKIs, and synergy/additivity in combination with the irreversible TKIs tested. Future work will explore the underlying mechanisms of this observed synergy and antagonism, including impacts of TKIs on HER2 expression and real-time ADC internalisation rates. Table 1. CI values +/- Std Dev for each TKI/ADC treatment combination in HCC1569 and HCC1954 cells Citation Format: Niall Ashfield, Amira F. Mahdi, Neil T. Conlon, John Crown, Denis M. Collins. Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (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 P1-11-10.

Abstract P5-02-21: RAB5 is a generic biomarker for ADC efficacy

Abstract Antibody-drug conjugates (ADCs) have demonstrated impressive activity in recent clinical trials in breast cancer. Such targeted therapeutics strongly depends on the presence of target molecules on the tumor cells, and the presence of such target molecules may determine the response of ADCs. However, ADCs are also dependent on cellular uptake, and factors regulating endocytosis as well as intracellular trafficking may strongly influence ADC activity. We have recently demonstrated that the activity of the HER2 targeted ADC trastuzumab emtansine (T-DM1) is dependent on the expression of RAB5A, a protein regulating endocytosis (1). A significant correlation between Rab5A expression and T-DM1 efficacy was found in a panel of HER2 expressing breast- and ovarian cancer cell lines. This result was verified in the I-SPY2 clinical trial where patients with high RAB5A expression were more likely to achieve a pathological complete response following T-DM1 as a neoadjuvant. The result was further validated in patients treated with T-DM1 in the Kamilla study where patients with a high RAB5A had a longer progression free survival. All ADCs should in principle be dependent on endocytosis to exert their activity. This triggered the investigation of proteins regulating endocytosis as predictive biomarkers for ADCs in general. METHODS: HER2-positive breast and ovarian cancer cell lines were evaluated with respect to the sensitivity and efficacy of treatment with T-DM1, trastuzumab deruxtecan, sacituzumab govitecan and the targeted toxin MH3B1/rGel.. The expression levels of proteins involved in endocytosis and endocytic trafficking including RAB4A, RAB5A and RAB11A were investigated in addition to the molecular drug targets (HER2 and TROP2). Cellular drug sensitivity was correlated to the expression levels of the investigated proteins using both RNA and protein as readout. RESULTS: The early endosome marker RAB5A, was found to correlate positively to the activity of trastuzumab deruxtecan, sacituzumab govitecan and MH3B1/rGel in the HER2 positive cell line panel, confirming the importance of RAB5A expression for the activity of these drugs. A significant correlation was found between RAB5A and drug efficacy using both protein and RNA as a readout. CONCLUSION: The present results indicate RAB5A as a generic predictive biomarker for both ADCs and targeted toxins which both depend on cellular uptake for cytotoxic efficacy. The results supports using both protein and RNA as a readout for RAB5A expression and point towards the development of a RAB5A stratification procedure for ADC and targeted toxin treatment. 1. Engebraaten O, Yau C, Berg K, Borgen E, Garred O, Berstad MEB, Fremstedal ASV, DeMichele A, Veer LV, Esserman L, Weyergang A. RAB5A expression is a predictive biomarker for trastuzumab emtansine in breast cancer. Nature communications 2021;12(1):6427 doi 10.1038/s41467-021-26018-z. Citation Format: Olav Engebraaten, Astrid Medhus, Ane Longva, Anette Weyergang, Kristian Berg. RAB5 is a generic biomarker for ADC efficacy [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 P5-02-21.

Syndecan-4 regulates the HER2-positive breast cancer cell proliferation cells via CK19/AKT signalling

Despite the use of the highly specific anti-HER2 receptor (trastuzumab) therapy, HER2-positive breast cancers account for 20–30% of all breast cancer carcinomas, with HER2 status a challenge to treatment interventions. The heparan sulfate proteoglycans (HSPGs) are prominently expressed in the extracellular matrix (ECM), mediate breast cancer proliferation, development, and metastasis with most studies to date conducted in animal models. This study examined HSPGs in HER2-positive human breast cancer cell lines and their contribution to cancer cell proliferation. The study examined the cells following enhancement (via the addition of heparin) and knockdown (KD; using short interfering RNA, siRNA) of HSPG core proteins. The interaction of HSPG core proteins and AKT signalling molecules was examined to identify any influence of this signalling pathway on cancer cell proliferation. Our findings illustrated the HSPG syndecan-4 (SDC4) core protein significantly regulates cell proliferation with increased BC cell proliferation following heparin addition to cultures and decreased cell number following SDC4 KD. In addition, along with SDC4, significant changes in CK19/AKT signalling were identified as mediators of BC HER2-positive BC cell proliferation. This study provides evidence for a cell growth regulatory axis involving HSPGs/CK19 and AKT that represents a potential molecular target to prevent proliferation of HER2-positive breast cancer cells.

HER2 mRNA Levels, Estrogen Receptor Activity and Susceptibility to Trastuzumab in Primary Breast Cancer.

While the results thus far demonstrate the clinical benefit of trastuzumab in breast cancer (BC), some patients do not respond to this drug. HER2 mRNA, alone or combined with other genes/biomarkers, has been proven to be a powerful predictive marker in several studies. Here, we provide evidence of the association between HER2 mRNA levels and the response to anti-HER2 treatment in HER2-positive BC patients treated with adjuvant trastuzumab and show that this association is independent of estrogen receptor (ER) tumor positivity. While HER2 mRNA expression was significantly correlated with HER2 protein levels in ER-negative tumors, no correlation was found in ER-positive tumors, and HER2 protein expression was not associated with relapse risk. Correlation analyses in the ER-positive subset identified ER activity as the pathway inversely associated with HER2 mRNA. Associations between HER2 levels and oncogene addiction, as well as between HER2 activation and trastuzumab sensitivity, were also observed in vitro in HER2-positive BC cell lines. In ER-positive but not ER-negative BC cells, HER2 transcription was increased by reducing ligand-dependent ER activity or inducing ER degradation. Accordingly, HER2 mRNA levels in patients were found to be inversely correlated with blood levels of estradiol, the natural ligand of ER that induces ER activation. Moreover, low estradiol levels were associated with a lower risk of relapse in HER2-positive BC patients treated with adjuvant trastuzumab. Overall, we found that HER2 mRNA levels, but not protein levels, indicate the HER2 dependency of tumor cells and low estrogen-dependent ER activity in HER2-positive tumors.

Trastuzumab-based near-infrared photoimmunotherapy in xenograft mouse of breast cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel form of cancer treatment using conjugates of antibody against overexpressed antigens in cancers and photoabsorber IRDye700DX. HER2 is overexpressed in various cancers, for which molecular targeted therapy such as trastuzumab has been developed. The present study investigated the efficacy potential of HER2-targeted NIR-PIT using trastuzumab-IRDye700DX conjugate (Tra-IR700) in HER2-positive breast cancer. We first examined the reactivity of Tra-IR700 and the cytotoxicity of NIR-PIT in vitro. HER2-positive BT-474 and SK-BR-3 cells and HER2-negative BT-20 cells were used. Tra-IR700 fluorescence was only observed in HER2-positive breast cancer cell lines, and the fluorescence was localized to the cell surface. Furthermore, HER2-positive breast cancer cell lines treated with NIR-PIT showed swelling and blebbing shortly after irradiation, and eventually increased PI-positive dead cells. Next, tumor accumulation of Tra-IR700 and tumor damage by NIR-PIT were examined in vivo. Tra-IR700 was administered intravenously to a xenograft model in which BT-474 cells were implanted subcutaneously in BALB/c nude mice. Tra-IR700 fluorescence was the highest in tumor tissue 1day after administration, and the fluorescence was localized to the cell membrane of tumor cells. At this time point, NIR-PIT resulted in diffuse necrosis of tumor tissues 1day after irradiation. These results suggest that NIR-PIT with Tra-IR700 induces a highly selective therapeutic effect in a HER2-positive breast cancer model. NIR-PIT using Tra-IR700 is expected to be a novel treatment for HER2-positive cancers, including breast cancer.

Disentangling ERBB Signaling in Breast Cancer Subtypes-A Model-Based Analysis.

Simple SummaryBreast cancer subtypes are characterized by the expression and activity of estrogen-, progesterone- and HER2-receptors and differ by the treatment as well as patient prognosis. Tumors of the HER2-subtype overexpress this receptor and are successfully targeted with anti-HER2 therapies. We wanted to know if the HER2-receptor and the downstream signaling network act similarly also in the other subtypes and if this network could potentially be a therapeutic target beyond the HER2-positive subtype. To this end, we quantitatively assessed the wiring of signaling events in the individual subtypes to unravel the characteristics of HER-signaling. Our data along with a model-based analysis suggest that major parts of the intracellular signal transduction network are unchanged between the different breast cancer subtypes and that the clinical differences mostly come from the different levels at which these receptors are present in tumor cells as well as from the particular mutations that are present in individual tumors.Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand–drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype.