Trastuzumab-resistant Breast Cancer Cell Lines Research Articles

LncRNA ZNF649-AS1 promotes trastuzumab resistance and TAM-dependent PD-L1 expression in breast cancer by regulating EXOC7 alternative splicing

BackgroundTrastuzumab resistance is a serious clinical problem in the treatment of HER2-positive breast cancer (BC). The lncRNA ZNF649-AS1 was previously found to promote HER2-positive BC trastuzumab resistance. The study aims to explore the molecular mechanism of ZNF649-AS1 in HER2-positive BC trastuzumab resistance. MethodsTumor tissue and peripheral blood samples were collected from 20 HER2-positive BC patients with trastuzumab-resistant and non-resistant, respectively. Trastuzumab-resistant BC cell lines SKBR-3-TR and BT474-TR were established. RIP was employed to confirm the binding of ZNF649-AS1, PRPF8 and exocyst complex component 7 (EXOC7). RNA expression of EXOC7-L (Full length of EXOC7) and EXOC7-S (Spliceosome of EXOC7) were detected using agarose gel electrophoresis. Expressions of macrophage markers CD68+ CD206+ were measured by flow cytometry. ResultsZNF649-AS1 expression was upregulated in HER2-positive BC trastuzumab resistance. ZNF649-AS1 downregulation inhibited trastuzumab resistance in HER2-positive BC. ZNF649-AS1 regulated EXOC7 alternative splicing by binding with PRPF8. EXOC7-S knockdown suppressed trastuzumab resistance and TAM-dependent PD-L1 expression in HER2-positive BC. EXOC7-S overexpression abolished the effects of ZNF649-AS1 knockdown on trastuzumab resistance and TAM-dependent PD-L1 expression in HER2-positive BC. ConclusionZNF649-AS1 promoted trastuzumab resistance and TAM-dependent PD-L1 expression in HER2-positive BC via promoting alternative splicing of EXOC7 by PRPF8.

Accumulation of liposomes in metastatic tumor sites is not necessary for anti-cancer drug efficacy

BackgroundThe tumor microenvironment is profoundly heterogeneous particularly when comparing sites of metastases. Establishing the extent of this heterogeneity may provide guidance on how best to design lipid-based drug delivery systems to treat metastatic disease. Building on our previous research, the current study employs a murine model of metastatic cancer to explore the distribution of ~ 100 nm liposomes. MethodsFemale NCr nude mice were inoculated with a fluorescently labeled, Her2/neu-positive, trastuzumab-resistant breast cancer cell line, JIMT-1mkate, either in the mammary fat pad to create an orthotopic tumor (OT), or via intracardiac injection (IC) to establish tumors throughout the body. Animals were dosed with fluorescent and radio-labeled liposomes. In vivo and ex vivo fluorescent imaging was used to track liposome distribution over a period of 48 h. Liposome distribution in orthotopic tumors was compared to sites of tumor growth that arose following IC injection. ResultsA significant amount of inter-vessel heterogeneity for DiR distribution was observed, with most tumor blood vessels showing little to no presence of the DiR-labelled liposomes. Further, there was limited extravascular distribution of DiR liposomes in the perivascular regions around DiR-positive vessels. While all OT tumors contained at least some DiR-positive vessels, many metastases had very little or none. Despite the apparent limited distribution of liposomes within metastases, two liposomal drug formulations, Irinophore C and Doxil, showed similar efficacy for both the OT and IC JIMT-1mkate models. ConclusionThese findings suggest that liposomal formulations achieve therapeutic benefits through mechanisms that extend beyond the enhanced permeability and retention effect.

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.

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.

Down-regulation of long non-coding RNA HOTAIR sensitizes breast cancer to trastuzumab

This study aimed to investigate the roles and possible molecular mechanisms of long non-coding RNA HOTAIR in regulating resistance to trastuzumab in breast cancer. Trastuzumab-resistant breast cancer cell line SK-BR-3-TR was assayed for the expression of HOX antisense intergenic RNA (HOTAIR), epithelial-mesenchymal transition (EMT)-related proteins or genes. Methylation levels of TGF- β, PTEN and cyclin-dependent kinase inhibitor 1B (or P27) were determined. In trastuzumab-resistant cell line, the mRNA level of HOTAIR was significantly up-regulated; in addition, the expression of TGF-β, Snail and Vimentin was also up-regulated, E-cadherin was down-regulated while the expression of HER2, PI3K, AKT, mTOR and MAPK in the HER2 receptor pathway and phosphorylation level of HER2 receptor remained unchanged, the methylation levels of the PTEN gene and TGF-β were increased and decreased, respectively. RNA interference downregulated the HOTAIR level and sensitized the cells to trastuzumab. It also resulted in down-regulation of TGF-β, Snail, Vimentin, p-AKT, p-APK and CyclinD1 and up-regulation of E-cadherin, PTEN and P27. Besides, the methylation levels of the PTEN gene and TGF-β were reduced and increased, respectively. Mouse models grafted with SK-BR-3-TR grew faster than with SK-BR-3-TS and siHOTAIR-SK-BR-3-TR.

ANKRD44 Gene Silencing: A Putative Role in Trastuzumab Resistance in Her2-Like Breast Cancer.

Trastuzumab is an effective therapeutic treatment for Her2-like breast cancer; despite this most of these tumors develop resistance to therapy due to specific gene mutations or alterations in gene expression. Understanding the mechanisms of resistance to Trastuzumab could be a useful tool in order to identify combinations of drugs that elude resistance and allow a better response for the treated patients. Twelve primary biopsies of Her2+/hormone receptor negative (ER-/PgR-) breast cancer patients were selected based on the specific response to neoadjuvant therapy with Trastuzumab and their whole exome was sequenced leading to the identification of 18 informative gene mutations that discriminate patients selectively based on response to treatment. Among these genes, we focused on the study of the ANKRD44 gene to understand its role in the mechanism of resistance to Trastuzumab. The ANKRD44 gene was silenced in Her2-like breast cancer cell line (BT474), obtaining a partially Trastuzumab-resistant breast cancer cell line that constitutively activates the NF-kb protein via the TAK1/AKT pathway. Following this activation an increase in the level of glycolysis in resistant cells is promoted, also confirmed by the up-regulation of the LDHB protein and by an increased TROP2 protein expression, found generally associated with aggressive tumors. These results allow us to consider the ANKRD44 gene as a potential gene involved in Trastuzumab resistance.

Abstract P5-03-15: Mechanism of activation of PPP1R1B drug-resistance gene in trastuzumab-resistant Her2+ breast cancer

Abstract Trastuzumab (Herceptin) resistance is a persistent challenge in the treatment of metastatic Her2+ breast cancer, with an average relapse time of 1 year after treatment. Human breast cancer cell-lines (SKBr3 and BT474) selected for trastuzumab resistance have a PPP1R1B gene (chr17:39,626,924-39,636,626) that is transcriptionally activated compared to their drug-sensitive counterparts. PPP1R1B codes for two related protein products, Darpp-32 (long) and t-Darpp (short), that are overexpressed in 50% of clinical breast cancer samples in comparison to normal breast tissue; reintroduction of t-Darpp into drug-sensitive cells confers drug resistance. The mechanism controlling overexpression of these transcripts is unknown. To gain insight into this mechanism we used ATAC-seq to map open chromatin areas in trastuzumab-resistant breast cancer cell lines and compared their open chromatin areas to those in the parental trastuzumab-sensitive counterparts. The PPP1R1B gene consists of 7 canonical exons. In each trastuzumab-resistant line there is two significantly increased open chromatin peaks, one within exon 2 and one within exon 3. We are performing deep sequencing (>200 million reads) to identify transcription factor footprints in these peaks. Furthermore, we performed RNA-seq analysis and identified genes that are similarly regulated in the resistant lines in comparison to their drug-sensitive counterparts. In summary, these results shed light on an area of high clinical importance and point to potential drug targets to be developed to overcome trastuzumab resistance in Her2+ breast cancer. Citation Format: Momand J, Murad R, Ma X, Geng S, Mortazavi A. Mechanism of activation of PPP1R1B drug-resistance gene in trastuzumab-resistant Her2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-15.

Abstract P2-03-08: Preclinical evaluation of the PI3Kα/δ inhibitor, copanlisib in HER2+ breast cancer: A proof of concept study

Abstract Purpose: The PI3K-AKT-mTORC1/C2 pathway is frequently activated in HER2+ breast cancer and upregulation of this pathway is a key mechanism of trastuzumab resistance. However, attempts to indirectly target this pathway by using the allosteric mTOR inhibitor everolimus have had limited clinical success. Here, we present the results of a preclinical study of the PI3K α/δ (dominant) inhibitor copanlisib alone and in combination with T-DM1, in HER2 amplified cell lines including a model with acquired resistance to trastuzumab. Method: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of copanlisib alone and in combination with T-DM1 were evaluated in a panel of HER2 amplified (ER+ or ER-), and HER2 amplified/PIK3CA mutated cell lines, as well as trastuzumab-resistant breast cancer cell lines. Results: 1) Copanlisib inhibited PI3K, mTOR and their downstream signaling molecules in HER2 amplified/PIK3CA WT or PIK3CA mutated as well as in trastuzumab-resistant cell lines, 2) interestingly, copanlisib also inhibited RAS-MAPK signaling in the earlier time points, 3) copanlisib caused a strong differential growth inhibition in HER2 amplified BC cell lines by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition was greater when copanlisib was combined with T-DM1, 4) administration of copanlisib induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner, 5) unlike everolimus copanlisib blocked HIF1α accumulation in hypoxic condition and this blocking effect was reversed by prior treatment with the proteasome inhibitor, carfilzomib and 6) copanlisib also attenuated HER2 amplified cell migration, an important phenotypic feature for metastasis. Conclusions: Copanlisib is highly effective (blocks proliferation, induces apoptosis, and inhibits PI3K and its downstream signaling targets) in HER2 amplified breast cancer cell lines including trastuzumab-resistant and PIK3CA mutated cell lines. The addition of copanlisib to T-DM1 might represent an improved treatment strategy for patients with refractory metastatic HER2+ breast cancer. Citation Format: De P, Carlson JH, Dey N, Leyland-Jones B. Preclinical evaluation of the PI3Kα/δ inhibitor, copanlisib in HER2+ breast cancer: A proof of concept study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-08.

DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer.

The majority of patients develop resistance against suppression of HER2-signaling mediated by trastuzumab in HER2 positive breast cancer (BC). HER2 overexpression activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. MAPK phosphatases (MKPs) are essential regulators of MAPKs and participate in many facets of cellular regulation, including proliferation and apoptosis. We aimed to identify whether differential MKPs are associated with resistance to targeted therapy in patients previously treated with trastuzumab. Using gene chip data of 88 HER2-positive, trastuzumab treated BC patients, candidate MKPs were identified by Receiver Operator Characteristics analysis performed in R. Genes were ranked using their achieved area under the curve (AUC) values and were further restricted to markers significantly associated with worse survival. Functional significance of the two strongest predictive markers was evaluated in vitro by gene silencing in HER2 overexpressing, trastuzumab resistant BC cell lines SKTR and JIMT-1. The strongest predictive MKPs were DUSP4/MKP-2 (AUC=0.75, p=0.0096) and DUSP6/MKP-3 (AUC=0.77, p=5.29E-05). Higher expression for these correlated to worse survival (DUSP4: HR=2.05, p=0.009 and DUSP6: HR=2, p=0.0015). Silencing of DUSP4 had significant sensitization effects – viability of DUSP4 siRNA transfected, trastuzumab treated cells decreased significantly compared to scramble-siRNA transfected controls (SKTR: p=0.016; JIMT-1: p=0.016). In contrast, simultaneous treatment with DUSP6 siRNA and trastuzumab did not alter cell proliferation. Our findings suggest that DUSP4 may represent a new potential target to overcome trastuzumab resistance.

The anti-ErbB2 antibody H2-18 and the pan-PI3K inhibitor GDC-0941 effectively inhibit trastuzumab-resistant ErbB2-overexpressing breast cancer.

Trastuzumab, an anti-ErbB2 humanized antibody, brings benefit to patients with ErbB2-amplified metastatic breast cancers. However, the resistance to trastuzumab is common. Our previously reported H2-18, an anti-ErbB2 antibody, potently induced programmed cell death in trastuzumab-resistant breast cancer cells. Here, we aim to investigate the antitumor efficacy of H2-18 in combination with the pan-PI3K inhibitor GDC-0941 in trastuzumab-resistant breast cancer cell lines. The results showed that H2-18 and GDC-0941 synergistically inhibited the in vitro proliferation of BT-474, SKBR-3, HCC-1954 and HCC-1419 breast cancer cells. H2-18 plus GDC-0941 showed significantly enhanced programmed cell death-inducing activity compared with each drug used alone. The combination of H2-18 and GDC-0941 did not increase the effect of single agent on ROS production, cell cycle and ErbB2 signaling. Importantly, the in vivo antitumor efficacy of H2-18 plus GDC-0941 was superior to that of single agent. Thus, the enhanced in vivo antitumor efficacy of H2-18 plus GDC-0941 may mainly be attributable to its increased programmed cell death-inducing activity. Collectively, H2-18 plus GDC-0941 could effectively inhibit tumor growth, suggesting the potential to be translated into clinic as an efficient strategy for ErbB2-overexpressing breast cancers.

Yes1 signaling mediates the resistance to Trastuzumab/Lap atinib in breast cancer

BackgroundOverexpression of human epidermal growth factor receptor 2 (HER2) is observed in approximately 15–23% of breast cancers and these cancers are classified as HER2-positive breast cancer. Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer and has improved patient overall survival. However, acquired resistance to trastuzumab is still a critical issue in breast cancer treatment. We previously established a trastuzumab-resistant breast cancer cell line (named as BT-474-R) from a trastuzumab-sensitive HER2-amplified cell line BT-474. Lapatinib is also a molecular-targeted drug for HER2-positive breast cancer, which acquired the resistance to trastuzumab. Acquired resistance to lapatinib is also an issue to be conquered.MethodsWe established trastuzumab/lapatinib-dual resistant cell line (named as BT-474-RL2) by additionally treating BT-474-R with lapatinib. We analyzed the mechanisms of resistance to trastuzumab and lapatinib. Besides, we analyzed the effect of the detected resistance mechanism in HER2-positive breast cancer patients.ResultsProto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R and BT-474-RL2. Silencing of Yes1 by siRNA induced both BT-474-R and BT-474-RL2 to restore the sensitivity to trastuzumab and lapatinib. Pharmaceutical inhibition of Yes1 by the Src inhibitor dasatinib was also effective to restore the sensitivity to trastuzumab and lapatinib in the two resistant cell lines. Combination treatment with dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, the combination treatments induced G1-phase cell-cycle arrest and apoptosis. Consistent with cell line data, high expression of Yes1 mRNA was correlated with worse prognosis in patients with HER2-positive breast cancer.ConclusionYes1 plays an important role in acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancer. Our data suggest that pharmacological inhibition of Yes1 may be an effective strategy to overcome resistance to trastuzumab and lapatinib.

Combined SRC inhibitor saracatinib and anti-ErbB2 antibody H2-18 produces a synergistic antitumor effect on trastuzumab-resistant breast cancer

Despite of the effectiveness of the anti-ErbB2 humanized antibody trastuzumab, trastuzumab resistance emerges as a major and common clinical problem. Thus, circumventing trastuzumab resistance has become an urgent need. Recently, Src inhibitor saracatinib has drawn great attention for its key role in trastuzumab response. As shown in our previous study, H2-18, an anti-ErbB2 antibody, could potently induce programmed cell death (PCD) in trastuzumab-resistant breast cancer cells. Here we combined H2-18 and a Src inhibitor, saracatinib, and studied the antitumor activity of this drug combination in trastuzumab-resistant breast cancer cell lines. The results showed that H2-18 and saracatinib could synergistically inhibit cell proliferation of BT-474, SKBR-3, HCC-1954 and HCC-1419 breast cancer cell lines in vitro. H2-18 plus saracatinib could also inhibit the HCC-1954 tumor growth more effectively in vivo than each drug alone. H2-18 plus saracatinib showed a significantly more potent PCD-inducing activity compared with either H2-18 or saracatinib alone. We conclude that enhanced PCD may contribute to the superior antitumor efficacy of this combination therapy. The combination of H2-18 and SRC inhibitor has the potential to be translated into clinic.

Abstract 4392: Yes1 is the key molecule for the resistance to trastuzumab in breast cancer, and dasatinib overcomes the resistance

Abstract Background: Overexpression of human epidermal growth factor receptor 2 (HER2) was observed in approximately 15-23% of breast cancers and they are classified as HER2-positive breast cancer. Trastuzumab is a therapeutic drug for the first choice for HER2-positive breast cancer, showing good response. However, acquired resistance to trastuzumab is one of the critical clinical issues in breast cancer treatment, especially in the patients with recurrent breast cancer. Therefore, it is necessary to develop the effective therapy to overcome the resistance. In this study, we established a trastuzumab-resistant breast cancer cell line from a trastuzumab-sensitive cell line with HER2 amplification (BT-474). We analyzed the mechanisms of resistance to trastuzumab and demonstrated the anti-tumor effect of dasatinib. Methods: Trastuzumab-resistant breast cancer cell line (BT-474-R) was established by treating BT-474 cells for long-term exposure with increasing doses of trastuzumab (from 0.1 μg/mL up to 40 μg/mL). Expression and activation of HER2 and its related molecules were investigated using western blotting and real-time PCR. Cell viability was evaluated using MTS assay. Cell cycle was analyzed using flow cytometry. Results: Proto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R. HER2 and Akt were also activated. Silencing of Yes1 by siRNA induced BT-474-R to recover sensitivity to trastuzumab. Pharmaceutical inhibition of Yes1 by Src inhibitor dasatinib was also effective to recovery sensitivity to trastuzumab in BT-474-R. Combination treatment of dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, these combination treatments induced G1-phase cell-cycle arrest and apoptosis. Conclusion: Yes1 plays an important role in acquired resistance to trastuzumab in HER2-positive breast cancer. Our data also suggest that pharmacological inhibition of Yes1 may become the new strategy to overcome resistance to trastuzumab. Citation Format: Hiromasa Yamamoto, Tatsuaki Takeda, Hirotaka Kanzaki, Ken Suzawa, Kei Namba, Hiroki Sato, Hidejiro Torigoe, Mototsugu Watanabe, Yuho Maki, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Yoshihisa Kitamura, Toshiaki Sendo, Shinichi Toyooka. Yes1 is the key molecule for the resistance to trastuzumab in breast cancer, and dasatinib overcomes the resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4392.

Abstract 724: Anti-tumor effect of Dasatinib in HER2 positive breast cancer with Trastuzumab resistance

Abstract [Background] Overexpression of human epidermal growth factor receptor 2 (HER2) was observed in approximately 20%-25% of breast cancers and they are classified in HER2-positive breast cancer. Trastuzumab is a therapeutic drug for the first choice for HER2-positive breast cancer, showing good response. However, acquired resistance to trastuzumab is one of the critical clinical issues in breast cancer treatment, especially in the patients with recurrent breast cancer. Therefore it is necessary to develop the effective therapy to overcome resistance. In this study, we established a trastuzumab-resistant breast cancer cell line from a trastumab-sensitive cell line with HER2 amplification (BT-474), and demonstrated anti-tumor effect of dasatinib in the breast cancer cell with trastuzumab resistance. [Methods] Trastuzumab-resistant breast cancer cell line (BT-474-R) was established by treating BT-474 cells for long-term exposing with trastuzumab. We performed cell viability assay to analyze drug sensitivity. Then, we performed quantitative PCR and western blotting to investigate expression and activation of HER2 and related molecules. Cell viability assay and signal analysis were performed with or without trastuzumab and/or dasatinib treatment. [Results] Proto-oncogene tyrosine-protein kinase Src was up-regulated and activated in BT-474-R. Signaling molecules, such as Akt and MAPK, were also activated. Dasatinib, which is known as a Src inhibitor, suppressed the activation of signal molecules and cell survival of BT-474-R. Moreover, dasatinib treatment recovered sensitivity to trastumab in the BT-474-R with down-regulation of Src phosphorylation. [Conclusion] Src plays an important role in acquired resistance to trastumab in breast cancer. Our data also suggest that inhibition of Src may become the new strategy to overcome trastuzumab resistance. Citation Format: Tatsuaki Takeda, Hirotaka Kanzaki, Shinichi Toyooka, Mototsugu Watanabe, Tomoaki Ohtsuka, Ken Suzawa, Shinsuke Hashida, Yuho Maki, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Yoshihisa Kitamura, Toshiaki Sendo. Anti-tumor effect of Dasatinib in HER2 positive breast cancer with Trastuzumab resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 724. doi:10.1158/1538-7445.AM2015-724

Abstract 4661: The regulatory mechanism of fibronectin in EGFR and/or HER2 positive breast cancer

Abstract Fibronectin (FN) is a glycoprotein of the extracellular matrix and plays a major role in cell adhesion, migration and oncogenic transformation. Aberrant FN expression is associated with poor prognosis in a variety of cancers including breast cancer. However, the regulatory mechanism between FN and HER2 is not fully elucidated in breast cancer. Our clinical data showed that both disease free survival (DFS) and overall survival (OS) was lower in EGFR (+) and HER2 (+) breast cancer patients than in EGFR (+) breast cancer patients. To verify the causing of poor prognosis, we analyzed secreted proteins in EGFR and/or HER2 overexpressed MDA-MB231 cells. Our results showed that the level of FN expression was significantly increased in HER2 overexpressed MDA-MB231 cells. On the other hand, the induction of FN by HER2 overexpression was decreased by trastuzumab (HER2 monoclonal antibody) and AG825 (HER2 inhibitor), respectively. In addition, we observed that the expression of FN was dose-dependently increased by EGFR ligands in SKBR3 cells which are expressed endogenous EGFR and HER2. EGF ligands-induced FN expression also decreased through the suppression of HER2 in SKBR3 cell. However, these effects did not detect in trastuzumab-resistant breast cancer cell line, JIMT-1. Taken together, we demonstrated that the induction of FN by HER2 correlates poor clinical outcome in breast cancer patients. Citation Format: Myeongjin Jeon. The regulatory mechanism of fibronectin in EGFR and/or HER2 positive breast cancer. [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 4661. doi:10.1158/1538-7445.AM2014-4661

Bispecific Antibody to ErbB2 Overcomes Trastuzumab Resistance through Comprehensive Blockade of ErbB2 Heterodimerization

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. However, resistance to trastuzumab is common. Heterodimerization between ErbB2 and other ErbBs may redundantly trigger cell proliferation signals and confer trastuzumab resistance. Here, we developed a bispecific anti-ErbB2 antibody using trastuzumab and pertuzumab, another ErbB2-specific humanized antibody that binds to a distinct epitope from trastuzumab. This bispecific antibody, denoted as TPL, retained the full binding activities of both parental antibodies and exhibited pharmacokinetic properties similar to those of a conventional immunoglobulin G molecule. Unexpectedly, TPL showed superior ErbB2 heterodimerization-blocking activity over the combination of both parental monoclonal antibodies, possibly through steric hindrance and/or inducing ErbB2 conformational change. Further data indicated that TPL potently abrogated ErbB2 signaling in trastuzumab-resistant breast cancer cell lines. In addition, we showed that TPL was far more effective than trastuzumab plus pertuzumab in inhibiting the growth of trastuzumab-resistant breast cancer cell lines, both in vitro and in vivo. Importantly, TPL treatment eradicated established trastuzumab-resistant tumors in tumor-bearing nude mice. Our results suggest that trastuzumab-resistant breast tumors remain dependent on ErbB2 signaling and that comprehensive blockade of ErbB2 heterodimerization may be an effective therapeutic avenue. The unique potential of TPL to overcome trastuzumab resistance warrants its consideration as a promising treatment in the clinic.

Trastuzumab-Resistant Cells Rely on a HER2-PI3K-FoxO-Survivin Axis and Are Sensitive to PI3K Inhibitors

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.

Abstract 337: In vitro evaluation of dual pan-PI3-kinase/mTOR inhibitor in HER2 overexpressing breast cancer cells

Abstract The HER/EGFR is a target of significant interest in human cancer due to the high frequency and broad spectrum aberrations in the pathway observed in human tumors, including breast tumors. Following ligand induced homodimeraziation/ heterodimeraziation HER2 transmits its signal mainly via PI3Kinase-AKT-mTOR and RAS-RAF-ERK1/2 pathways that promote proliferation, survival and angiogenesis. PI3Kinase-AKT-mTOR is the most frequently activated oncopathway in human cancer (De P and Leyland -Jones B, JCO Editorial in Press; Holbro T 2003 PNAS 100: 8933). Tumor cells with hyperactive PI3Kinase-AKT-mTOR pathway are not only resistant to external stresses that can induce cell death and also to chemotherapy. Literature suggests that hyyperactivation of PI3Kinase-AKT-mTOR pathway is one the major causes for the development of de novo or acquired resistance to trastuzumab (Berns K et al 2007 Cancer Cell 12: 395-402; Junttila TT et al 2009 Cancer Cell 15: 429-440). The importance of PI3kinase pathway in Her2 amplified and/or trastuzumab resistance breast cancer implies that interruption of this pathway is important in order for HER2-directed therapies to exert an anticancer effect. The present study was carried out to investigate the preclinical efficacy of a dual pan-PI3K plus mTOR inhibitor, BEZ235 alone or in combination with trastuzumab in HER2 overexpressing trastuzumab-sensitive and trastuzumab-resistant breast cancer cell lines. Here we utilized HER2 overexpressing SKBR3, BT474 and trastuzumab -resistant BT474 cells as preclinical models. Our results show 1) BEZ235 time dependently and dose dependently suppressed the pathway downstream of PI-3Kinase including p-AKT, p-mTOR and p-P70S6Kinase in both trastuzumab-sentsitive and trastuzumab-resistance breast cancer cell lines at 50nM, 100nM and 200nM concentrations and 100% inhibition was observed at higher concentration 2) inhibition of AKT and its downstream effectors were more pronounced when trastuzumab was treated along with BEZ235 even at lower concentrations, 3) the well described AKT activation due to mTOR inhibition was prevented by higher doses of BEZ235 (at 200 nm concentration) treatment, and 4) unlike other pan-PI3Kinase inhibitor (e.g. LY294002, SF1126), BEZ235 has no effect on hypoxia-mediated stabilization of hypoxia-inducible factor −1α (HIF1α) however, heregulin-induced HIF1α synthesis which is an important angiogenic modulator in breast cancer cells, was significantly decreased (> 70%) following the treatment of BEZ235. Our in vitro data provide evidence that clinically pertinent dual pan-PI3K plus mTOR inhibitor can reverse trastuzumab resistance in breast cancer cells, and further support to carry out in vivo efficacy studies (tumor growth and tumor-induced angiogenesis) with BEZ 235 alone or in combination with trastuzumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 337.

Trastuzumab-Resistant Breast Cancer Cells Remain Sensitive to the Auger Electron–Emitting Radiotherapeutic Agent 111In-NLS-Trastuzumab and Are Radiosensitized by Methotrexate

Our goals in this study were to determine whether (111)In-trastuzumab coupled to peptides harboring nuclear localizing sequences (NLSs) could kill trastuzumab-resistant breast cancer cell lines through the emission of Auger electrons and whether the combination of radiosensitization with methotrexate (MTX) would augment the cytotoxicity of this radiopharmaceutical. Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate for reaction with NLS peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with (111)In. HER2 expression was determined by Western blot and by radioligand binding assay using (111)In-trastuzumab in a panel of breast cancer cell lines, including SK-BR-3, MDA-MB-231 and its HER2-transfected subclone (231-H2N), and 2 trastuzumab-resistant variants (TrR1 and TrR2). Nuclear importation of (111)In-NLS-trastuzumab and (111)In-trastuzumab in breast cancer cells was measured by subcellular fractionation, and the clonogenic survival of these cells was determined after incubation with (111)In-NLS-trastuzumab, (111)In-trastuzumab, or trastuzumab (combined with or without MTX). Survival curves were analyzed according to the dose-response model, and the radiation-enhancement ratio was calculated from the survival curve parameters. The expression of HER2 was highest in SK-BR-3 cells (12.6 x 10(5) receptors/cell), compared with 231-H2N and TrR1 cells (6.1 x 10(5) and 5.1 x 10(5) receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 x 10(5) and 0.6 x 10(5) receptors/cell, respectively). NLS peptides increased the nuclear uptake of (111)In-trastuzumab in MDA-MB-231, 231-H2N, TrR1, and TrR2 cells from 0.1%+/-0.01%, 2.5%+/-0.2%, 2.8%+/-0.7%, and 0.5%+/-0.1% to 0.5%+/-0.1%, 4.6%+/-0.1%, 5.2%+/-0.6%, and 1.5%+/-0.2%, respectively. The cytotoxicity of (111)In-NLS-trastuzumab on breast cancer cells was directly correlated with the HER2 expression densities of the cells. On a molar concentration basis, the effective concentration required to kill 50% of 231-H2N and TrR1 cells for (111)In-NLS-trastuzumab was 9- to 12-fold lower than for (111)In-trastuzumab and 16- to 77-fold lower than for trastuzumab. MDA-MB-231 and TrR2 cells were less sensitive to (111)In-NLS-trastuzumab or (111)In-trastuzumab, and both cell lines were completely insensitive to trastuzumab. The radiation-enhancement ratio induced by MTX for 231-H2N and TrR1 cells after exposure to (111)In-NLS-trastuzumab was 1.42 and 1.68, respectively. Targeted Auger electron radioimmunotherapy with (111)In-NLS-trastuzumab can overcome resistance to trastuzumab, and MTX can potently enhance the sensitivity of HER2-overexpressing breast cancer cells to the lethal Auger electrons emitted by this radiopharmaceutical.

Genomic and gene expression characterization of a novel trastuzumab-resistant breast cancer cell line

Genomic and gene expression characterization of a novel trastuzumab-resistant breast cancer cell line