Trastuzumab-resistant JIMT-1 Cells Research Articles

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

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

The HSP90 inhibitor HVH-2930 exhibits potent efficacy against trastuzumab-resistant HER2-positive breast cancer.

Rationale: Resistance to targeted therapies like trastuzumab remains a critical challenge for HER2-positive breast cancer patients. Despite the progress of several N-terminal HSP90 inhibitors in clinical trials, none have achieved approval for clinical use, primarily due to issues such as induction of the heat shock response (HSR), off-target effects, and unfavorable toxicity profiles. We sought to examine the effects of HVH-2930, a novel C-terminal HSP90 inhibitor, in overcoming trastuzumab resistance. Methods: The effect of HVH-2930 on trastuzumab-sensitive and -resistant cell lines in vitro was evaluated in terms of cell viability, expression of HSP90 client proteins, and impact on cancer stem cells. An in vivo model with trastuzumab-resistant JIMT-1 cells was used to examine the efficacy and toxicity of HVH-2930. Results: HVH-2930 was rationally designed to fit into the ATP-binding pocket interface cavity of the hHSP90 homodimer in the C-terminal domain of HSP90, stabilizing its open conformation and hindering ATP binding. HVH-2930 induces apoptosis without inducing the HSR but by specifically suppressing the HER2 signaling pathway. This occurs with the downregulation of HER2/p95HER2 and disruption of HER2 family member heterodimerization. Attenuation of cancer stem cell (CSC)-like properties was associated with the downregulation of stemness factors such as ALDH1, CD44, Nanog and Oct4. Furthermore, HVH-2930 administration inhibited angiogenesis and tumor growth in trastuzumab-resistant xenograft mice. A synergistic effect was observed when combining HVH-2930 and paclitaxel in JIMT-1 xenografts. Conclusion: Our findings highlight the potent efficacy of HVH-2930 in overcoming trastuzumab resistance in HER2-positive breast cancer. Further investigation is warranted to fully establish its therapeutic potential.

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.

PNSA, a Novel C-Terminal Inhibitor of HSP90, Reverses Epithelial-Mesenchymal Transition and Suppresses Metastasis of Breast Cancer Cells In Vitro.

Metastasis accounts for the vast majority of deaths in breast cancer, and novel and effective treatments to inhibit cancer metastasis remain urgently developed. The expression level of heat shock protein 90 (HSP90) in invasive breast cancer tissue is higher than in adjacent non-cancerous tissue. In the present study, we investigated the inhibitory effect of penisuloxazin A (PNSA), a novel C- terminal inhibitor of HSP90, on metastasis of breast cancer cells and related mechanism in vitro. We found that PNSA obviously affected adhesion, migration, and invasion of triple-negative breast cancer (TNBC) MDA-MB-231 cells and Trastuzumab-resistant JIMT-1 cells. Furthermore, PNSA was capable of reversing epithelial–mesenchymal transformation (EMT) of MDA-MB-231 cells with change of cell morphology. PNSA increases E-cadherin expression followed by decreasing amounts of N-cadherin, vimentin, and matrix metalloproteinases9 (MMP9) and proteolytic activity of matrix metalloproteinases2 (MMP2) and MMP9. Comparatively, the N-terminal inhibitor of HSP90 17-allyl-17-demethoxygeldanamycin (17-AAG) had no effect on EMT of MDA-MB-231 cells. PNSA was uncovered to reduce the stability of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) proteins and thereby inhibiting their downstream signaling transductions by inhibition of HSP90. In addition, PNSA reduced the expression of programmed cell death-ligand 1 (PD-L1) to promote natural killer (NK) cells to kill breast cancer cells with a dose far less than that of cytotoxicity to NK cell itself, implying the potential of PNSA to enhance immune surveillance against metastasis in vivo. All these results indicate that PNSA is a promising anti-metastasis agent worthy of being studied in the future.

Abstract 4935: T-DM1-resistant cells gain high invasive activity via EGFR and integrin cooperated pathways

Abstract Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate developed to treat trastuzumab-resistant cancer. Despite initial favorable outcomes, most patients eventually cease to respond by developing acquired resistance to T-DM1. Currently, there is no targeted therapy to treat T-DM1-resistant cancer. To explore novel therapeutic targets to overcome T-DM1 resistance, we generated T-DM1-resistant cells using trastuzumab-resistant JIMT1 cells. We found that the loss of human epidermal growth factor receptor 2 (HER2) confers T-DM1 resistance, which in turn activates a compensatory mechanism that increases epidermal growth factor receptor (EGFR) expression. Upregulation of EGFR increases the protein levels of α5β1 integrin, resulting in enhanced motility and invasion of T-DM1-resistant cells. However, silencing β1 integrin expression by siRNA or β1 integrin blockage induced by an inhibitory antibody, MAB 13, significantly increases invasion of T-DM1-resistant cells. The discovery of functional cooperation between EGFR and integrin in regulating cell growth and invasion provides an opportunity to develop novel therapeutic strategy by dual-targeting EGFR and specific integrin to overcome T-DM1 resistance. We now try to identify downstream molecules involved in the increased invasion using next generation sequencing (NGS) technology in β1 integrin-deficient T-DM1 resistant cells. Citation Format: Yukinori Endo, Zachary Olson, Wen Jin Wu. T-DM1-resistant cells gain high invasive activity via EGFR and integrin cooperated pathways [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 4935.

Abstract 115: Flubendazole targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer with trastuzumab resistance

Abstract Background and Propose: HER2-positive breast cancers are associated with an aggressive phenotype and trastuzumab resistance leading to poor clinical outcomes. Flubendazole (FLU) is a potent anthelmintic agent that inhibits microtubule polymerization, and also exhibits anticancer activity in several cancer types. The objective of the present study was to investigate the effect of FLU on apoptosis, HER2/Akt signaling pathway, cancer stem cell (CSC)-like properties and trastuzumab-resistance in HER2-positive breast cancer cells. Methods: FLU-induced antitumor effects were characterized by MTS assay, Sub-G1 and G2/M-phase arrest population, Annexin-V analysis, ALDH1 activity assay, Western blotting and immunofluorescence. For in vivo study, trastuzumab-resistant JIMT1 cells were injected into the mammary fat pads of BALB/c nude mice. We then determined the tumor growth and CSC-like properties. Results and conclusion: FLU significantly reduced cell viability and induced apoptosis with accompanied by activation of caspase-3, -7, and -8, as well as PARP cleavage in HER2-positive cell lines (MDA-MB-453, SKBR3 and BT474). FLU-induced apoptosis is associated with significant decreases in expression levels of HER2, p-HER2 (Tyr1221/1222), HER3, and p-HER3 (Tyr1289). FLU treatment also resulted in G2/M-phase arrest of the cell cycle, as evidenced by a marked downregulation of phospho-Histone H3 expression. These results were closely related to the decline of CSC-like properties, coinciding with the suppression of ALDH1 activity. These effects were also observed in trastuzumab-resistant JIMT-1 cells, and significant reductions in tumor growth and ALDH1A1 downregulation were observed in xenograft models together with downregulation of HER2 expression in vivo. Our findings suggest that FLU may be potentially effective for the treatment of HER2-positive breast cancer with trastuzumab resistance. Note: This abstract was not presented at the meeting. Citation Format: Daeil Sung, Youngkwan Cho, Eunhye Oh, Tae-Min Cho, Yoon-Jae Kim, Ji Young Kim, Jae hong Seo. Flubendazole targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer with trastuzumab resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 115. doi:10.1158/1538-7445.AM2017-115

Abstract B069: The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC

Abstract Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab')2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies.Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be.The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition. Citation Format: Gábor Tóth, Árpád Szöőr, László Simon, Yosef Yarden, János Szöllősi, György Vereb. The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B069.

Three-dimensional microtissues essentially contribute to preclinical validations of therapeutic targets in breast cancer.

A 3D microtissues using T47D and JIMT‐1 cells were generated to analyze tissue‐like response of breast cancer cells after combined human epidermal growth factor receptor 2 (HER2)‐targeted treatment and radiation. Following lentiviral knockdown of HER2, we compared growth rate alterations using 2D monolayers, 3D microtissues, and mouse xenografts. Additionally, to model combined therapeutic strategies, we treated HER2‐depleted T47D cells and 3D microtissues using trastuzumab (anti‐HER2 antibody) in combination with irradiation. Comparison of HER2 knockdown with corresponding controls revealed growth impairment due to HER2 knockdown in T47D 2D monolayers, 3D microtissues, and xenografts (after 2, 12, and ≥40 days, respectively). In contrast, HER2 knockdown was less effective in inhibiting growth of trastuzumab‐resistant JIMT‐1 cells in vitro and in vivo. Combined administration of trastuzumab and radiation treatment was also analyzed using T47D 3D microtissues. Administration of both, radiation (5 Gy) and trastuzumab, significantly enhanced the growth inhibiting effect in 3D microtissues. To improve the predictive power of potential drugs—as single agents or in combination—here, we show that regarding tumor growth analyses, 3D microtissues are highly comparable to outcomes derived from xenografts. Considering increased limitations for animal experiments on the one hand and strong need of novel drugs on the other hand, it is indispensable to include highly reproducible 3D microtissue platform in preclinical analyses to validate more accurately the capacity of future drug‐combined radiotherapy.

Abstract 4244: Inhibition of HER2 and IGF2 triggers cell death in trastuzumab-resistant HER2 positive JIMT1 cells

Abstract Breast cancer is the second leading cause of death in women. Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) is detected in 20-30% of breast cancers. Increased levels of HER2 are linked to poor prognosis, more aggressive phenotype, and resistance to chemotherapy and hormonal treatment. HER2 is part of a four member family of cell surface receptors that are implicated in transmission of signals controlling cell growth and differentiation. HER2 has an intracellular domain with tyrosine kinase catalytic activity and dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor(IGF1R) in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the IGF1R, insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). Previous findings in our lab demonstrate an increased expression of IGF2 in trastuzumab-resistance cell line JIMT1 when compared to trastuzumab-sensitive cell lines BT474, SKBR3, and ZR75-30. We hypothesis that to overcome trastuzumab-resistance in JIMT1 cell lines, dual inhibition of HER2 and IGF2 is necessary to induce cell death. When JIMT1 cells were treated with resveratrol, both HER2 and IGF2 mRNA and protein levels were reduced and a decrease in cell viability was observed. We also analyzed the phosphorylation levels of HER2 and IGF1R. Our studies suggest that blocking both HER2 and IGF2 is necessary to decrease cell viability in trastuzumab-resistant JIMT1 cells. Citation Format: Xousaen M. Helu, Daisy De Leon. Inhibition of HER2 and IGF2 triggers cell death in trastuzumab-resistant HER2 positive JIMT1 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4244. doi:10.1158/1538-7445.AM2014-4244

Archazolid-B Provides Alternative Therapy for Trastuzumab-Resistant ErbB2 Positive Breast Cancer

Trastuzumab is the most commonly used humanized monoclonal antibody for specific therapy of breast cancer. Even though its clinical introduction was a breakthrough, a large fraction of the treated tumors are or become resistant. It is well known that receptor internalization and recycling are crucial for ErbB2 mediated signal transduction. Accordingly, inhibition of its molecular machinery could provide alternatives for tumor treatment. Vacuolar H+-ATPase (V-ATPase) is involved in the regulation of endocytotic/recycling pathways. Archazolid-B was described as a potent V-ATPase inhibitor, which induces apoptosis and impairs migration of tumor cells. Based on these observations we investigated the effect of Archazolid on trastuzumab-resistant Jimt-1 cells in vitro and in vivo. 10nM of Archazolid caused decreased membrane ErbB2 expression, which was accompanied by the reduced relative phosphorylation on Y1248 and intracellular accumulation of the receptors. As an in vivo model, SCID mice were transplanted with Jimt-1 xenografts and treated with Archazolid. Following administration of the V-ATPase inhibitor there was a significant decrease in tumor growth compared to control tumors. Confocal microscopic images of tumor sections showed heterogeneous distribution of the proliferation marker Ki67 positivity. Tumor areas with low Ki67 nuclear expression showed intracellular accumulation of ErbB2 molecules similarly to the in vitro experiments, compared to high Ki67 expressing areas with prominent membrane ErbB2 expression. This heterogeneity might be due to diverse tumor-vascularization. Our results demonstrate that Archazolid can be used for in vitro and in vivo tumor growth inhibition based on its interference with ErbB2 recycling.

Effects of Simultaneous Knockdown of HER2 and PTK6 on Malignancy and Tumor Progression in Human Breast Cancer Cells

Breast cancer is the most common malignancy in women of the Western world. One prominent feature of breast cancer is the co- and overexpression of HER2 and protein tyrosine kinase 6 (PTK6). According to the current clinical cancer therapy guidelines, HER2-overexpressing tumors are routinely treated with trastuzumab, a humanized monoclonal antibody targeting HER2. Approximately, 30% of HER2-overexpressing breast tumors at least initially respond to the anti-HER2 therapy, but a subgroup of these tumors develops resistance shortly after the administration of trastuzumab. A PTK6-targeted therapy does not yet exist. Here, we show for the first time that the simultaneous knockdown in vitro, compared with the single knockdown of HER2 and PTK6, in particular in the trastuzumab-resistant JIMT-1 cells, leads to a significantly decreased phosphorylation of crucial signaling proteins: mitogen-activated protein kinase 1/3 (MAPK 1/3, ERK 1/2) and p38 MAPK, and (phosphatase and tensin homologue deleted on chromosome ten) PTEN that are involved in tumorigenesis. In addition, dual knockdown strongly reduced the migration and invasion of the JIMT-1 cells. Moreover, the downregulation of HER2 and PTK6 led to an induction of p27, and the dual knockdown significantly diminished cell proliferation in JIMT-1 and T47D cells. In vivo experiments showed significantly reduced levels of tumor growth following HER2 or PTK6 knockdown. Our results indicate a novel strategy also for the treatment of trastuzumab resistance in tumors. Thus, the inhibition of these two signaling proteins may lead to a more effective control of breast cancer.

Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo

IntroductionTrastuzumab is widely used for the treatment of HER2-positive breast cancer. Despite encouraging clinical results, a significant fraction of patients are, or become, refractory to the drug. To overcome this, trastuzumab-DM1 (T-DM1), a newer, more potent drug has been introduced. We tested the efficacy and mechanisms of action of T-DM1 in nine HER2-positive breast cancer cell lines in vitro and in vivo. The nine cell lines studied included UACC-893, MDA-453 and JIMT-1, which are resistant to both trastuzumab and lapatinib.MethodsAlamarBlue cell-proliferation assay was used to determine the growth response of breast cancer cell lines to trastuzumab and T-DM1 in vitro. Trastuzumab- and T-DM1-mediated antibody-dependent cellular cytotoxicity (ADCC) was analysed by measuring the lactate dehydrogenase released from the cancer cells as a result of ADCC activity of peripheral blood mononuclear cells. Severe Combined Immunodeficient (SCID) mice were inoculated with trastuzumab-resistant JIMT-1 cells to investigate the tumour inhibitory effect of T-DM1 in vivo. The xenograft samples were investigated using histology and immunohistochemistry.ResultsT-DM1 was strongly growth inhibitory on all investigated HER2-positive breast cancer cell lines in vitro. T-DM1 also evoked antibody-dependent cellular cytotoxicity (ADCC) similar to that of trastuzumab. Outgrowth of JIMT-1 xenograft tumours in SCID mice was significantly inhibited by T-DM1. Histologically, the cellular response to T-DM1 consisted of apoptosis and mitotic catastrophe, the latter evidenced by an increased number of cells with aberrant mitotic figures and giant multinucleated cells.ConclusionsOur results suggest mitotic catastrophe as a previously undescribed mechanism of action of T-DM1. T-DM1 was found effective even on breast cancer cell lines with moderate HER2 expression levels and cross-resistance to trastuzumab and lapatinib (MDA-453 and JIMT-1).