Role In Trastuzumab Resistance Research Articles

LncRNA ZNF649-AS1 Induces Trastuzumab Resistance by Promoting ATG5 Expression and Autophagy

The regulatory mechanism of long non-coding RNAs (lncRNAs) in trastuzumab resistance is not well established to date. In this research, we identified differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer. HiSeq sequencing and quantitative real-time PCR were performed to identify the dysregulated lncRNAs. Mass spectrometry, RNA fluorescence in situ hybridization (RNA-FISH), and immunoprecipitation assays were performed to identify the direct interactions between ZNF649-AS1 and other associated targets, such as polypyrimidine tract binding protein 1 (PTBP1) and autophagy related 5 (ATG5). Our results showed that ZNF649-AS1 was more highly expressed in trastuzumab-resistant cells compared to sensitive cells. Increased expression of ZNF649-AS1 was associated with a poorer response and shorter survival time of breast cancer patients. ZNF649-AS1 was upregulated by H3K27ac modification at the presence of trastuzumab treatment, and knockdown of ZNF649-AS1 reversed trastuzumab resistance via modulating ATG5 expression and autophagy. Mechanically, ZNF649-AS1 was associated with PTBP1 protein, which further promoted the transcription activity of the ATG5 gene. In conclusion, we demonstrated that H3K27ac modification-induced upregulation of ZNF649-AS1 could cause autophagy and trastuzumab resistance through associating with PTBP1 and promoting ATG5 transcription.

RETRACTED ARTICLE: Exosome-mediated lncRNA AFAP1-AS1 promotes trastuzumab resistance through binding with AUF1 and activating ERBB2 translation

BackgroundAlthough trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Recent evidence suggests that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis and chemoresistance. However, the regulatory mechanism of lncRNAs in trastuzumab resistance is not well established to date. In this research, we identified the differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer.MethodsLncRNA microarray and qRT-PCR were performed to identify the dysregulated lncRNAs. Transmission electron microscopy, differential ultracentrifugation and qRT-PCR were used to verify the existence of exosomal AFAP1-AS1 (actin filament associated protein 1 antisense RNA 1). Bioinformatics prediction, RNA fluorescence in situ hybridization (RNA-FISH) and immunoprecipitation assays were performed to identify the direct interactions between AFAP1-AS1 and other associated targets, such as AU-binding factor 1 (AUF1) and ERBB2. Finally, a series gain- or loss-functional assays were done to prove the precise role of AFAP1-AS1 in trastuzumab resistance.ResultsAFAP1-AS1 was screened out due to its higher expression in trastuzumab-resistant cells compared to sensitive cells. Increased expression of AFAP1-AS1was associate with poorer response and shorter survival time of breast cancer patients. AFAP1-AS1 was upregulated by H3K27ac modification at promoter region, and knockdown of AFAP1-AS1 reversed trastuzumab resistance. Moreover, extracellular AFAP1-AS1 secreted from trastuzumab resistant cells was packaged into exosomes and then disseminated trastuzumab resistance of receipt cells. Mechanically, AFAP1-AS1 was associated with AUF1 protein, which further promoted the translation of ERBB2 without influencing the mRNA level.ConclusionExosomal AFAP1-AS1 could induce trastuzumab resistance through associating with AUF1 and promoting ERBB2 translation. Therefore, AFAP1-AS1 level may be useful for prediction of trastuzumab resistance and breast cancer treatment.

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.

MiR-200c inhibits TGF-β-induced-EMT to restore trastuzumab sensitivity by targeting ZEB1 and ZEB2 in gastric cancer.

Gastric cancer is the fifth most common malignancy in the world, with Eastern Asia as one of areas with the highest incidence rates. Trastuzumab, a HER2-targeting antibody, combined with chemotherapy has been successfully employed for the gastric cancer patients with HER2 overexpression/amplification. However, trastuzumab resistance is a major problem in clinical practice. Here we observed that the trastuzumab-resistant gastric cancer cell line NCI-N87/TR expressed high levels of epithelial-mesenchymal transition factors and demonstrated increased migration and invasion capability compared with NCI-N87 cells. Downregulated E-cadherin and increased N-cadherin, TGF-β, ZEB1, ZEB2, TWIST1, and Snail were detected in NCI-N87/TR cells. We also found that miR-200c was downregulated in NCI-N87/TR cells compared with parental cells NCI-87 by qRT-PCR. Treatment with TGF-β downregulated the expression of miR-200c and upregulated ZEB2, and significantly decreased the trastuzumab sensitivity of NCI-N87 cells. miR-200c restored trastuzumab sensitivity and inhibited migration and invasion through suppressing ZEB1 and ZEB2. In summary, TGF-β/ZEB2 axis plays an encouraging role in trastuzumab resistance of gastric cancer, while miR-200c overexpression downregulates ZEB1/ZEB2 and resensitizes drugs resistance. Our findings might provide a potential therapeutic strategy for trastuzumab resistance of gastric cancer.

The erbB3- and IGF-1 receptor-initiated signaling pathways exhibit distinct effects on lapatinib sensitivity against trastuzumab-resistant breast cancer cells.

Both erbB3 and IGF-1 receptor (IGF-1R) have been shown to play an important role in trastuzumab resistance. However, it remains unclear whether erbB3- and IGF-1R-initiated signaling pathways possess distinct effects on the sensitivity of lapatinib, a dual tyrosine kinase inhibitor against both EGFR and erbB2, in trastuzumab-resistant breast cancer. Here, we show that the trastuzumab-resistant SKBR3-pool2 and BT474-HR20 breast cancer sublines, as compared the parental SKBR3 and BT474 cells, respectively, exhibit refractoriness to lapatinib. Knockdown of erbB3 inhibited Akt in SKBR3-pool2 and BT474-HR20 cells, significantly increased lapatinib efficacy, and dramatically re-sensitized the cells to lapatinib-induced apoptosis. In contrast, specific knockdown of IGF-1R did not alter the cells' responsiveness to lapatinib. While the levels of phosphorylated Src (P-Src) were reduced upon IGF-1R downregulation, the P-Akt levels remained unchanged. Furthermore, a specific inhibitor of Akt, but not Src, significantly enhanced lapatinib-mediated anti-proliferative/anti-survival effects on SKBR3-pool2 and BT474-HR20 cells. These data indicate that erbB3 signaling is critical for both trastuzumab and lapatinib resistances mainly through the PI-3K/Akt pathway, whereas IGF-1R-initiated Src activation results in trastuzumab resistance without affecting lapatinib sensitivity. Our findings may facilitate the development of precision therapeutic regimens for erbB2-positive breast cancer patients who become resistant to erbB2-targeted therapy.

Alteration of gene expression in MDA-MB-453 breast cancer cell line in response to continuous exposure to Trastuzumab

Development of resistance against cancer therapeutic agents is a common problem in cancer management. Trastuzumab resistance is one of the challenges in management of HER-2-positive breast cancer patients resulting in breast cancer progression, metastasis, and patient poor outcome. The aim of this study is to determine the alteration in gene expression in response to Trastuzumab resistance after long-term exposure to Trastuzumab. The Trastuzumab-resistant MDA-MB-453 (MDA-MB-453/TR) cell line was developed by exposing cells to 10μM Trastuzumab continuously for 6months. Sensitivity toward Trastuzumab was tested using cell viability assays. The acquisition of an epithelial-to mesenchymal transition (EMT) phenotype was also observed in parallel with the development of resistance. Based on the real-time-based PCR array technology, several genes were altered affecting multiple networks. The most up-regulated genes were TGF-β1 and EGF, and IGFBP-3. These genes are known to have a critical role in Trastuzumab resistance in breast cancer cell lines and/or in the acquisition of EMT. They are also recognized for their role in cancer progression and metastasis. These alterations indicate that the development of Trastuzumab resistance is multifactorial and involves a development of a mesenchymal like phenotype.

Abstract P4-07-06: miR-9 expression, retinoids and their potential role in trastuzumab resistance

Abstract HER2 positive breast cancer accounts for approximately 20-25 % of breast cancers. Trastuzumab, a humanised monoclonal antibody, is approved for the treatment of HER2 positive breast cancer. However, the majority of metastatic HER2 positive breast cancers progress on trastuzumab treatment due to either innate or acquired resistance. The aim of this study is to investigate the role of microRNAs (miRNAs) in a cell line model of acquired trastuzumab resistance. The trastuzumab resistant cell line, SKBR3-T was established by continuous exposure to trastuzumab (10 µg/ml) for 6 months. miRNA extracted from SKBR3 and SKBR3-T was profiled using Taqman low density arrays (TLDA). Differentially regulated miRNAs were selected using >2-fold change and a P-value of <0.05. Individual quantitative RT-PCR (qRT-PCR) was performed to confirm miRNA alterations. Proliferation assays were performed using the acid phosphatase method. Taqman gene expression assays for retinoic acid receptor alpha (RARA) were performed using GAPDH as an endogenous control. Immunodetection of RARA was performed using α-tubulin as a control. Six differentially regulated miRNAs were identified in the SKBR3-T cells. qRT-PCR assays confirmed that four were significantly altered in SKBR3-T compared to SKBR3 cells including miR-9 which was 2.2 fold up-regulated (p=0.04). Utilising miRWalk, we identified RARA as a target for miR-9. We confirmed that RARA mRNA and protein expression are reduced in SKBR3-T cells compared to SKBR3 cells. Treatment with all-trans retinoic acid (ATRA) (0.2 µM–0.025 µM) alone inhibited growth of the SKBR3 cell line in a dose dependent manner but not in the SKBR3-T cell line. Combined treatment with trastuzumab (10 µg/uL) and ATRA (0.2 µM) had a significantly greater growth inhibitory effect on the SKBR3 cell line (90.1±5.2 %) (p=0.0002) than either trastuzumab (39.1±4.0 %) (p= 0.0004) or ATRA alone (57.9±4.2 %) (p=0.001). Interestingly, despite relative insensitivity to ATRA in the SKBR3-T cells (23.7±5.0%) (p=0.86), combined treatment with trastuzumab produced significant growth inhibition in the SKBR3-T cells (74.2±5.1%) compared to either trastuzumab (24.1±4.4%) (p=0.0001) or ATRA (23.7±5.0%) (p=0.86) alone. miR-9 is up-regulated in the acquired trastuzumab resistant SKBR3-T cell line, RARA expression is downregulated, and SKBR3-T cells are resistant to ATRA treatment compared to SKBR3 cells. However, SKBR3-T cells are sensitive to combined treatment with trastuzumab and ATRA. Thus combined treatment with ATRA and trastuzumab may overcome acquired resistance to trastuzumab in HER2 positive breast cancer. Citation Format: Karen Howe, Brigid C Browne, Sinead Aherne, John Crown, Norma O'Donovan. miR-9 expression, retinoids and their potential role in trastuzumab resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-06.

Abstract P1-04-04: Activating HER2 mutations promote oncogenesis and resistance to HER2-targeted therapies in breast cancer

Abstract Purpose: Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor2 (HER2) have been reported to lead to resistance to HER2-targeted therapies in HER2-positive breast cancer, while activating mutations of HER2 have been described in HER2-negative breast cancer. The prevalence, clinicopathological characteristics, and phenotypes of HER2 mutations are not well established, thus we sought to describe the HER2 mutation profile of Chinese breast cancer patients. Methods: DNA samples were gathered from breast cancer patients undergoing neoadjuvant (N=102) or adjuvant therapy (N=498) at Fudan University Shanghai Cancer Center between January 1, 2006 and December 31, 2012. Sanger sequencing was performed to analyze all exons of HER2 to identify somatic mutations. To determine the phenotypes of novel HER2 mutations, in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments were conducted. Results: 10 HER2 somatic mutations were observed in 17 patients (17/600, 2.83%). 7 novel HER2 mutations were uncovered, 4 in the transmembrane domain and 3 in the kinase domain. Kinase domain mutations L768S and V773L were detected in HER2-negative tumors, while K753E was found in HER2-positive disease. In vitro kinase assays found that L768S and V773L exhibited a significant increase of tyrosine kinase-specific activity, while Western blots showed that L768S and V773L strongly increased phosphorylation of all signaling proteins in both MCF10A and MCF7cell lines, indicating that they were activating mutations. In Matrigel cultures, L768S and V773L formed acini when seeded in vehicle, but maintained spherical morphology when seeded in culture containing trastuzumab. The addition of lapatinib in Matrigel culture inhibited the growth of all except K753E, which was successfully inhibited by neratinib. Similarly, L768S, V773L and K753E increased the number of cell colonies formed in soft agar, trastuzumab and lapatinib treatment decreased the number of colonies formed by L768S and V773L, but only neratinib could inhibit the colony growth of K753E. Xenograft showed L768S and V773L displayed a more rapid growth, while K753E showed resistance to lapatinib in vivo. MCF10A cells bearing K753E mutation were found to be resistant to lapatinib (IC50>10,000 nmol/L), but could be inhibited by neratinib, though requiring a relatively higher dosage (IC50 of 32 nmol/L) than HER2 WT (IC50 of 480 nmol/L for lapatinib, <2 nmol/L for neratinib) and other HER2 mutations. Meanwhile, clinical follow-up showed that the 2 patients with K753E mutation who received adjuvant trastuzumab treatment presented with either brain or bone metastasis, in their 3rd and 5th year after initial cancer diagnosis, suggesting K753E mutation may have a role in trastuzumab resistance as well. Conclusions: HER2 somatic mutations were found in 2.83% of patients in this study. HER2-positive tumors harboring certain HER2 kinase domain resistance mutations may not benefit from trastuzumab or lapatinib treatment, and neratinib may offer an alternative treatment option for these patients. HER2-negative disease with activating mutations may benefit from HER2-targeted therapies, and may be of interest in prospective clinical trials. Citation Format: Wen-Jia Zuo, Yi-Zhou Jiang, Ke-Da Yu, Zhi-Ming Shao. Activating HER2 mutations promote oncogenesis and resistance to HER2-targeted therapies in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-04-04.

Abstract 1829: Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival

Abstract Human Epidermal Growth Factor Receptor 2 (Her2) is a receptor tyrosine kinase that regulates cell growth and differentiation signaling pathways. The protein Her2 is significantly overexpressed in 20-30% of breast cancer. The available clinical data proves that Her2 amplification in breast cancer is associated with worst overall survival. Therefore, Her2 has become a very important candidate for drug targeting and currently Her2 positive patients are treated with humanized monoclonal antibody, Trastuzumab (Herceptin) and a small molecule, Lapatinib that blocks Her2 tyrosine kinase activity. It is reported that Her2 amplification leads to activation of multiple survival pathways, including PI3K-Akt. However, it is not clearly known how Trastuzumab promotes cell death and what pro-apoptotic pathways are activated downstream of Her2 blockage? It is also reported that patients on Trastuzumab develop subsequent resistance to the therapy. Previously we published that in breast cancer tumors, a pro-apoptotic kinase, Mixed Lineage Kinase-3 (MLK-3) was inhibited, specifically in estrogen receptor (ER) positive tumors. The kinase activity of MLK-3 was down regulated by estrogen and this prevented MLK-3-mediated cell death. Since Her2 serves as a drug target in breast cancer, therefore, we asked whether Her2 plays any role in regulating MLK-3 kinase activation and whether MLK-3 plays any role in Trastuzumab resistance. Our results showed that the endogenous MLK-3 kinase activity was up-regulated in SkBr3 cells upon treatment with Trastuzumab and Lapatinib. Whether the activation of MLK-3 upon Her2 blockage leads to cell death or survival is still not known and experiments are underway to investigate these aspects. We also observed that Her3 was the dimerization partner of Her2 in Trastuzumab- or Lapatinib- mediated MLK-3 activation. Not knowing any role of MLK-3 in Trastuzumab resistance, we used Trastuzumab sensitive and resistant BT474 cell model to investigate the role of MLK-3 in Trastuzumab resistance. Our data clearly showed that knockdown of MLK-3 caused spontaneous cell death in resistant cells. The Trastuzumab treatment further amplified cell death in MLK-3 knockdown resistant cells. The Akt1 activation in these cells was also diminished upon MLK-3 knockdown. Taken together, our data demonstrate that like ER, the Her2 also down regulates MLK-3 kinase activity and perhaps promote cell survival. Furthermore, paradoxically, the MLK-3 knockdown promoted cell death in Trastuzumab resistance BT474 cells, suggesting that MLK-3 could also serve as survival factor in resistant cells. Whether MLK-3 serves as a pro-apoptotic kinase or survival kinase in Trastuzumab mediated pathway needs to be extensively examined in near future. Citation Format: Subhasis Das, Gautam Sondarva, Navin Viswakarma, Rakesh Sathish Nair, Clodia Osipo, Basabi Rana, Ajay Rana. Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival. [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 1829. doi:10.1158/1538-7445.AM2014-1829

Abstract P6-05-08: Nuclear HER3 localisation plays a role in trastuzumab resistance in HER2-positive breast cancer

Abstract Background: HER3 is known to locate in the nucleus. Unlike HER4, nuclear HER3 translocation has not been reported to be due to a proteolytic cleavage process by ADAM17 and gamma-secretase. The mechanisms of nuclear HER3 induction and its role in relation to trastuzumab treatment and resistance for HER2-positive breast cancer is unclear. Methods: Using nuclear fractionation and confocal microscopy, nuclear HER3 localisation was investigated in response to trastuzumab with or without ADAM17 inhibitor and gamma-secretase inhibitor in a panel of HER2 expressing cell lines. We also correlated nuclear HER3 expression by immunohistochemistry with treatment response in patients who underwent window trastuzumab study as well as the survival outcome in a cohort of HER2-positive breast cancer patients using Kaplan–Meier survival curves with Log-rank test. Results: HER3 ligand heregulin and trastuzumab was found to induce nuclear HER3 translocation in HER2-positive breast cancer cell lines, including SKBR3. Nuclear HER3 was also enriched in acquired trastuzumab resistant SKBR3 cells (SKBr3-TR). Trastuzumab treatment induced several HER3 fragments and HER3100kD was found to be responsible for nuclear HER3 enrichment by fractionation. This fragment was confirmed to be a specific band of HER3 as shown by HER3 knockdown. Nuclear HER3 was reduced by inhibiting either gamma-secretase or ADAM17 inhibitor. Gamma-secretase or ADAM17 inhibitor reduced HER3100kD in both SKBr3 and SKBr3-TR cells. In HER2-positive breast cancer patients who underwent window trastuzumab study, baseline nuclear HER3 status was not a predictor of response for trastuzumab monotherapy at day 21. However, nuclear HER3 was enriched after trastuzumab treatment in a poor-responder patient. Total HER3 expression level in cytoplasm positively was correlated with poor response to trastuzumab monotherapy in HER2-positive patients (r = 0.67, p = 0.05). There was no statistically significant difference in disease-free survival between positive and negative nuclear HER3 expression but the number of patients was small (n = 87). Further validation to assess nuclear HER3 expression as a prognostic and predictive biomarker in HER2-positive breast cancer patients undergoing trastuzumab treatment will be assessed in randomized tumour samples from FinHER study. Conclusion: Heregulin and trastuzumab treatment seems to induce nuclear HER3 translocation in some of the HER2 positive breast cancer cells. This may be due to proteolytic cleavage of HER3 as it is reduced by ADAM17 or gamma-secretase inhibitor. Enriched nuclear localisation of HER3 seems to be one possible mechanism of acquired resistance to trastuzumab in HER2-positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-08.

Abstract 538: IGF2 role in trastuzumab-resistance in JIMT-1 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. Overexpression of HER2 is 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. Dimerization of HER2 activates the MAP kinase and AKT pathways. HER2 receptor also heterodimerizes with the IGF1 receptor in trastuzumab-resistant cell lines. The insulin-like growth factor 2 (IGF2) has an important role in fetal and cancer development signaling through the insulin-like growth factor 1 receptor (IGF1R), the insulin receptor (InsR), and cross-talk with the estrogen receptor alpha and beta (ERα, ERß). We hypothesize that IGF2 mediates trastuzumab-resistance in JIMT-1 cell line. IGF2 mRNA and protein levels were determined in trastuzumab-sensitive cell lines (BT474, SKBR3, and ZR 75-30) and trastuzumab-resistant cell line (JIMT-1) by qPCR and Western blot. We also analyzed the effect of IGF2 inhibition treatment on the phosphorylation levels of HER2. Our study showed that IGF2 increases in trastuzumab-resistant cell line, JIMT-1. Furthermore, inhibition of IGF2 decreases JIMT-1 cell viability compared to control and renders cells sensitive to trastuzumab. These findings provide a potential therapy target for trastuzumab-resistant breast cancers. Citation Format: Xousaen M. Helu, Daisy D. De Leon. IGF2 role in trastuzumab-resistance in JIMT-1 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2013-538

86P The Role of Adam17 in Her2 Moderately Expressing Breast Cancer

ABSTRACT Trastuzumab has been shown to increase the survival of HER2 positive breast cancer (HER2 3+) patients and some clinical trials revealed that patients with moderately expressed HER2 (HER2 2+) breast cancer may also respond. However, there is a need to further understand the mechanisms of resistance to trastuzumab and to explore other targeted therapies. Members of a disintegrin and metalloproteases (ADAMs) are responsible for shedding of a variety of ErbB ligands. The most important members are ADAM10 and ADAM17 and we investigated the role of these ADAMs in trastuzumab treatment and resistance in HER2 2+ breast cancer cell lines. We first assessed the effect of trastuzumab on MDA-MB-453 and MDA-MB-361 cells and confirmed that those are less sensitive than HER2 positive SKBR3 cells but respond better than MCF7 cells. We found that trastuzumab treatment does not decrease pHER2 in HER2 2+ expressing cells. We previously showed that ADAM17 mediated heregulin release maintains HER2 phosphorylation in HER2 3+ breast cancer cells. Therefore we proceeded to investigate the levels of ADAM17 and heregulin and found an increase of both in HER2 2+ cells in comparison to SKBR3 cells. Interestingly, the use of a specific anti ADAM17 antibody leads to a decrease of pPKB as well as pHER3 and pHER2 in MDA-MB-453 cells. Strikingly, in cell viability studies monotherapy with an anti ADAM17 antibody had a significant effect in reducing cell proliferation of these cells and an additive effect in trastuzumab treatment. Furthermore, ADAM10 inhibition had only little effects, indicating that these cells may rely on HER3 activation through heregulin release by ADAM17. Thus, ADAM17 may play a key role in trastuzumab resistance in HER2 moderately expressing breast cancer cells. Our results suggest that in those cells, ADAM17 results in HER ligand sheddase which in turn may lead to the activation of HER receptors and the maintenance of pHER2. We demonstrated that an anti ADAM17 antibody is able to decrease pHER2, pHER3, as well as pPKB and is effective as monotherapy and additive to trastuzumab treatment in HER2 2+ breast cancer cells. We recommend that inhibition of ADAM17 with or without trastuzumab should be considered as a potential targeted therapy strategy in HER2 2+ breast cancer patients. Disclosure G. Murphy: The anti ADAM17 antibody was developed by Prof. Murphy's laboratory. Please see: Tape et al. Cross-domain inhibition of TACE ectodomain. Proc Natl Acad Sci USA 2011; 108(14):5578-83. All other authors have declared no conflicts of interest.

Abstract 5362: The role of adam10 in trastuzumab resistance of her2 positive breast cancer cell lines

Abstract Members of a disintegrin and metalloproteases (ADAMs) are responsible for shedding of a variety of ErbB ligands. One of them, ADAM10, has been shown to cleave several pro-ligands, including betacellulin and HB-EGF. ADAM10 further may play a role in trastuzumab resistance since it is also responsible for HER2 shedding, thereby removing the binding site for trastuzumab. It was reported that ADAM10 inhibitor INCB 7839 could reduce HER2 cleavage in patients with HER2 positive breast cancer. However, a direct correlation of trastuzumab treatment and ADAM10 and the precise role of ADAM10 in trastuzumab resistance have not been elucidated. The aim of this project is therefore to investigate the role of ADAM10 in trastuzumab treatment and resistance in HER2 positive breast cancer cell lines. Using western blot, we showed that one hour of trastuzumab treatment leads to an upregulation of ADAM10 in conditioned medium and cell lysates of BT474 cells, which was correlated with an elevated level of betacellulin. Additionally, the use of a specific ADAM10 inhibitor in turn leads to downregulation of betacellulin levels in culture medium. Whereas one hour of trastuzumab treatment could not decrease pHER2, its combination with ADAM10 inhibitor reduced pHER2 level. To further investigate the role of ADAM10 in relation to trastuzumab treatment, we used cell viability studies to assess the effect of a specific ADAM10 inhibitor with or without trastuzumab treatment. Although monotherapy with ADAM10 inhibitor only had minimal influence, combining it with trastuzumab significantly enhanced trastuzumab in reducing cell proliferation of BT474 cells. Interestingly, the addition of ADAM10 inhibitor to trastuzumab exerts an even greater inhibition of cell proliferation in the BT474 trastuzumab resistant cell line. Since the N-domain of ADAM10 was shown to be cleaved by ADAM9 in other cell lines, we also assessed ADAM9 level in response to trastuzumab treatment. In BT474 lysates, ADAM9 level was increased after one hour of treatment, correlating with increased ADAM10 level in the medium. ADAM9 level was also found to be increased in the trastuzumab-resistant HER2 positive cell line in comparison to the sensitive cell line. Thus, ADAM10 as well as ADAM9 may play a key role in trastuzumab resistance in HER2 positive breast cancer cells. Our results suggest that the increased level of ADAM10 results in HER ligand sheddase which in turn may lead to the activation of HER receptors and the maintenance of pHER2. We demonstrated that ADAM10 inhibitor in combination with trastuzumab is able to decrease pHER2 and potentiate the trastuzumab effect in HER2 positive breast cancer cells. The role of ADAM9 in its regulation of ADAM10 in relation to trastuzumab treatment is currently under investigation. We recommend that inhibition of ADAM10 should be considered as a potential strategy to improve trastuzumab response in HER2 positive breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5362. doi:10.1158/1538-7445.AM2011-5362

Calpain-1 expression is associated with relapse-free survival in breast cancer patients treated with trastuzumab following adjuvant chemotherapy

The calpain family, and their endogenous inhibitor calpastatin, has been implicated in cancer progression, and recent in vitro data have indicated a role in trastuzumab resistance. The aims of our study were to examine expression levels of calpastatin, calpain-1 and calpain-2 in breast tumours from patients treated with trastuzumab following adjuvant chemotherapy to determine their potential as biomarkers to predict therapeutic response. The expression of calpastatin, calpain-1 and calpain-2 was determined, using immunohistochemistry (IHC), in tumours from a series of 93 patients with primary breast cancer treated with surgery and adjuvant chemotherapy with or without trastuzumab followed by trastuzumab to complete 1 year of therapy. IHC was performed using tissue microarrays constructed from cores taken from intratumour and peripheral tumour areas. Expression was correlated with clinicopathologic variables and patient outcome. Calpastatin expression was correlated with Nottingham prognostic index (p = 0.003) and lymph node status (p = 0.007). Trastuzumab resistance was defined as disease relapse during therapy. Calpain-1 expression is associated with relapse-free survival (p = 0.001) and remained significant in multivariate analysis accounting for confounding pathological and treatment variables (hazard ratio 4.60, 95% confidence interval 1.05-20.25; p = 0.043). Calpain-1 may be a useful biomarker to predict relapse-free survival in breast cancer patients treated with adjuvant trastuzumab and chemotherapy. A larger verification study is warranted.

Evolving Strategies for Overcoming Resistance to HER2-Directed Therapy: Targeting the PI3K/Akt/mTOR Pathway

Human epidermal growth factor receptor 2–positive (HER2+) breast cancers, which account for 25%-30% of breast cancers, are characterized by an aggressive course and a high propensity for recurrence in the 4 years following diagnosis. The use of trastuzumab-based chemotherapy in the adjuvant setting has markedly improved the outcome for patients with early stage HER2+ breast cancer. Likewise the use of trastuzumab in combination with chemotherapy in patients with metastatic HER2+ breast cancers has prolonged survival, with current expected median survival of about 3 years. Despite these major improvements in outcome, approximately 10% of patients develop a distant recurrence following adjuvant trastuzumab-based chemotherapy, and all patients with metastatic disease eventually develop disease progression. Known mechanisms of resistance to trastuzumab include increased signaling through upstream growth factors, phosphatase and tensin (PTEN) deficiency and alterations of the HER2 receptor. Many of these mechanisms are being targeted in the clinic in an attempt to improve outcome for patients with HER2+ breast cancers. The phosphatidylinositol 3-kinase (PI3K) pathway plays a key role in trastuzumab-resistance, through these and other mechanisms, and represents a logical target for drug development for trastuzumab-resistant breast cancers. The use of mammalian target of rapamycin (mTOR) inhibition has been demonstrated to potentially reverse resistance to trastuzumab in patients with HER2+, metastatic breast cancers. Phase I and II trials have produced encouraging results when the mTOR inhibitor, everolimus, was combined with trastuzumab with or without chemotherapy, in patients with trastuzumab-resistant HER2+ metastatic breast cancer. These results are being confirmed in ongoing phase III trials in the first-line and trastuzumab-resistant settings. The mechanism of how mTOR inhibitors reverse resistance to trastuzumab remains largely unexplained. Other agents targeting the PI3K pathway in trastuzumab-resistant breast cancers are in early phase clinical trials.

Abstract A104: Involvement of FOXO1 in E2/ER pathway and tamoxifen resistance in MCF-7 cells

Abstract Introduction: Estrogen receptor positive breast tumors frequently become resistant to Tamoxifen therapy. Elucidating mechanisms that contribute to resistance are critical for developing therapeutic targets. Previously, we have shown in our lab that FOXO1, a pro-apoptotic transcription factor, plays a role in Trastuzumab resistance, and is inactivated in Her2-overexpressing tumors associated with increased AKT activation. Restoration of FOXO1, both by inhibiting the AKT pathway and by FOXO1 transfection, restored cell-sensitivity to Trastuzumab. Currently, we are exploring whether FOXO1 may play a similar role in resistance to Tamoxifen in ER positive breast cancer cells. We hypothesize that loss of function of FOXO1 may contribute to Tamoxifen resistance and reactivation may restore sensitivity to tamoxifen. Methods: We have generated two tamoxifen resistant clones and in addition have studied a Tamoxifen resistant clone called MCF-7 TAMR/HER2 (HER2-transfected). tamoxifen resistant clones were generated by passaging wildtype MCF-7 (WT) cells through sphere cultures in the presence of tamoxifen. These clones are termed MCF-7 TAMR7 and MCF-7 TAMR26, representing 7 and 26 passages in sphere culture respectively. Cell line characteristics were assayed by immunohistochemistry (IHC) and RT-Q-PCR. Furthermore, MTT assays were used to test for E2 response in WT and TAMR cells. Results: Tamoxifen resistance was acquired by utilizing the sphere assay in the presence of Tamoxifen. IHC results revealed a significant loss of ER receptor in TAMR7 and TAMR26 clones. RT-Q-PCR demonstrated 85% loss of ER receptor mRNA in the TAMR7 and TAMR26 compared to WT cells. There was no difference in ER expression in WT and TAMR/HER2 cells. KI67 and CyclinD1 expression were lower in TAMR (TAMR7 and TAMR26) cells compared to WT and TAMR/HER2 cells. TAMR26 cells had lower proliferative response to E2 compared to WT cells. There was increased expression of CK5 in TAMR26 cells. FOXO1 was phosphorylated and located in the cytoplasm of the TAMR cells (TAMR7 and TAMR26) compared to WT and TAMR/HER2 cells. Conclusions: This study demonstrates that one mechanism of Tamoxifen resistance may be through the loss of the Tamoxifen target, the estrogen receptor (ER). TAMR clones isolated from mammosphere cultures with long term Tamoxifen treatment acquire an ER negative phenotype. These cells demonstrate loss of function of FOXO1. In addition, TAMR cells with high HER2 and normal ER, also acquire Tamoxifen resistance, by mechanisms not clearly understood. Further studies are currently underway in our lab to explore these mechanisms. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A104.

PTEN, PIK3CA, p-AKT, and p-p70S6K Status: Association with Trastuzumab Response and Survival in Patients with HER2-Positive Metastatic Breast Cancer

Phosphatase and tensin homolog (PTEN) is a key modulator of trastuzumab sensitivity in HER2-overexpressing breast cancer. Because PTEN opposes the downstream signaling of phosphoinositide 3-kinase (PI3K), we investigated the role of PTEN and other components of the PI3K pathway in trastuzumab resistance. We analyzed the status of PTEN, p-AKT-Ser473, and p-p70S6K-Thr389 using immunohistochemistry. PIK3CA mutation status was analyzed by direct sequencing. Primary tumor tissue was available from 137 patients with HER2-overexpressing metastatic breast cancer who had received trastuzumab-based chemotherapy. We observed that each of the four biomarkers alone did not significantly correlate with trastuzumab response, whereas PTEN loss alone significantly correlated with shorter survival times (P = 0.023). PI3K pathway activation, defined as PTEN loss and/or PIK3CA mutation, was associated with a poor response to trastuzumab (P = 0.047) and a shorter survival time (P = 0.015). PTEN loss was significantly associated with a poor response to trastuzumab (P = 0.028) and shorter survival time (P = 0.008) in patients who had received first-line trastuzumab and in patients with estrogen receptor- (P = 0.029) and progesterone receptor-negative tumors (P = 0.033). p-AKT-Ser473 and p-p70S6K-Thr389 each had a limited correlation with trastuzumab response. When these markers were combined with PTEN loss, an increased correlation with patient outcome was observed. In conclusion, PI3K pathway activation plays a pivotal role in trastuzumab resistance. Our findings may facilitate the evaluation of tumor response to trastuzumab-based and targeted therapies.

Abstract 4268: Breast cancer stem cells play a role in trastuzumab resistance in mouse xenograph models

Abstract One of the most significant advances in breast cancer therapeutics has been the development of HER2 targeted therapies for treatment of HER2 overexpressing breast cancer. The use of HER2 inhibitors such as trastuzumab have improved patient outcome in both the adjuvant and advanced setting. However, one-third of HER2-positive tumors do not respond to HER2 targeted agents and resistance may develop in patients with chronic exposure. Nearly 50% of patients who respond to HER2 targeted agents relapse within a year. The mechanism of resistance to HER2 targeted agents is not entirely clear. However, increasing evidence indicates that this resistance may be associated with loss of PTEN (phosphotase and tensin homolog), the gain of function of somatic mutations of PI3KA or truncation of the extracellular domain of HER2. We have recently demonstrated that trastuzumab reduces the cancer stem cell (CSC) population in trastuzumab sensitive but not trastuzumab resistant cell lines. In order to elucidate the mechanisms of trastuzumab resistance we engineered MCF7 cells to overexpress HER2, knockdown PTEN or both. HER2 overexpression or PTEN knockdown increased the proportion of CSC's as assessed by the Aldefluor assay and tumor initiation in NOD/SCID mice. Cell lines with both HER2 overexpression and PTEN knockdown contained a significantly higher proportion of Aldefluor positive (3.2 %) compared to either alteration alone (1.7%, 1.9% respectively and 0.5 % in parental cells). In addition, cells with both PTEN deletion and HER2 overexpression showed several fold increase in their invasive properties in vitro as compared cells with either alteration alone. Furthermore, cells with the combined alterations but not those with either alone were highly metastatic in NOD/SCID mice consistent with the demonstrated role of CSC's in mediating tumor metastasis. Trastuzumab treatment significantly reduced the Aldefluor positive population in HER2 overexpressing cells. In contrasts trastuzumab had no effect on the Aldefluor positive population in cells with both HER2 overexpression and PTEN knockdown. These studies suggest that PTEN deletion renders Her 2 overexpressing MCF7 breast cancers resistant to trastuzumab by regulating the CSC population. Strategies aimed at blocking downstream signaling through PI3K or Akt inhibition may effectively target this important cell population in trastuzumab resistant tumors. 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 4268.

Heterotrimerization of the Growth Factor Receptors erbB2, erbB3, and Insulin-like Growth Factor-I Receptor in Breast Cancer Cells Resistant to Herceptin

Primary and acquired resistance to the breast cancer drug trastuzumab (Herceptin) is a significant clinical problem. Here, we report enhanced activation of downstream signaling pathways emanating from the growth factor receptors erbB2, erbB3, and insulin-like growth factor-I receptor (IGF-IR) in trastuzumab-resistant breast cancer cells. Interactions between IGF-IR and erbB2 or erbB3 occur exclusively in trastuzumab-resistant cells, where enhanced erbB2-erbB3 interactions are also observed. Moreover, these three receptors form a heterotrimeric complex in resistant cells. erbB3 or IGF-IR knockdown by short hairpin RNA-mediated strategies upregulates p27(kip1), inactivates downstream receptor signaling, and resensitizes resistant cells to trastuzumab. Our findings reveal a heterotrimer complex with a key role in trastuzumab resistance. On the basis of our results, we propose that trastuzumab resistance in breast cancer might be overcome by therapeutic strategies that jointly target erbB3, erbB2, and IGF-IR.