HER2-negative Breast Cancer Cell Lines Research Articles

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

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

MAP3K1 expression is associated with progression and poor prognosis of hormone receptor-positive, HER2-negative early-stage breast cancer.

In this study, we assessed whether the overexpression of MAP3K1 promotes the proliferation, migration, and invasion of breast cancer cells, which affect the prognosis of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative early stage breast cancer. Two HR-positive, HER2-negative breast cancer cell lines (MCF7 and T-47D) overexpressing MAP3K1 were transfected with two MAP3K1 short hairpin RNA plasmids (shMAP3K1 [#3] and shMAP3K1 [#5]). The proliferation, migration, and invasion of these cells were then examined. We assessed whether shMAP3K1 affects the cell cycle, levels of downstream signaling molecules (ERK, JNK, p38 MAPK, and NF-κB), and sensitivity to chemotherapeutic and hormonal agents. To assess the anti-tumor effect of MAP3K1 knockdown in the breast cancer orthotopic model, MCF7 and T-47D cells treated with or without shMAP3K1 (#3) and shMAP3K1 (#5) were inoculated into the mammary fat pads of mice. In total, 182 patients with HR-positive, HER2-negative T1 and T2 breast cancer and 0-3 nodal metastases were included. Additionally, 73 patients with T1 and T2 breast cancer and negative nodes who received adjuvant endocrine therapy alone were selected as an independent validation cohort. In both cell lines, shMAP3K1 (#3) and shMAP3K1 (#5) significantly reduced cell growth, migration, and invasion by downregulating MMP-9 and by blocking the G2/M phase of the cell cycle and its regulatory molecule cyclin B1. Moreover, both shMAP3K1 (#3) and shMAP3K1 (#5) downregulated ERK-, JNK-, p38 MAPK-, and NF-κB-dependent gene transcription and enhanced the sensitivity of both cell lines to doxorubicin, docetaxel, and tamoxifen. We observed that both shMAP3K1 (#3) and shMAP3K1 (#5) inhibited tumor growth compared with that in the scrambled group of MCF7 and T-47D cell orthotopic tumors. Patients with MAP3K1 overexpression exhibited significantly poorer 10-year disease-free survival (DFS) (70.4% vs. 88.6%, p = 0.003) and overall survival (OS) (81.9% vs. 96.3%, p = 0.001) than those without MAP3K1 overexpression. Furthermore, phospho-ERK (p < 0.001) and phospho-JNK (p < 0.001) expressions were significantly associated with MAP3K1 expression, and both phospho-ERK and phospho-JNK expressions were significantly correlated with poor 10-year DFS and OS. These biological findings, including a significant association between DFS and OS, and the expressions of MAP3K1, phospho-ERK, and phospho-JNK were further validated in an independent cohort. Multivariate analysis identified MAP3K1 expression as an independent poor prognostic factor for DFS and OS. Our results indicate that the overexpression of MAP3K1 plays a major role in the poor prognosis of HR-positive, HER2-negative early stage breast cancer.

Abstract 3978: The growth inhibitory effect of MDV3100 on hormone receptor-positive, HER2-negative breast cancer cell lines with low DYRK2 expression

Abstract Introduction: Tumor progression is the main cause of death in patients with breast cancer. Accumulating evidence suggests that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. Recently, we reported for the first time that DYRK2 inhibits breast cancer stem cells and tumor cell proliferation through transcriptional downregulation of KLF4 and CDK14, respectively.Our previous study showed that Androgen receptor (AR) was a DYRK2-dependent transcriptional activator. In this context, we hypothesized that MDV3100, an inhibitor of AR, may be effective for breast cancer with low DYRK2 expression. Methods: We established stable DYRK2-depleted cells. MCF-7 cells, hormone receptor-positive, HER2-negative breast cancer cell line, were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) along with puromycin to isolate stable cell lines.These manipulated cells were compared with the control cells by various assays. We analyzed the activity of AR by Androgen receptor reporter assay. MTS assays were used to assess growth inhibition after treatment with MDV3100. Results: Androgen receptor reporter assay showed that knocking down DYRK2 induced AR transcription activity in MCF-7 cells.In MCF-7 DYRK2-depleted cells, MTS assays revealed that the treatment by MDV3100 was more effective than that in MCF-7 control cells. Conclusion: These findings demonstrate that reduced DYRK2 expression in hormone receptor-positive, HER2-negative breast cancer increases the activity of AR and significantly inhibits the growth by treatment with MDV3100. Citation Format: Yoshimi Imawari, Rei Mimoto, Kiyotsugu Yoshida. The growth inhibitory effect of MDV3100 on hormone receptor-positive, HER2-negative breast cancer cell lines with low DYRK2 expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3978.

Abstract P2-03-10: DNA replication licensing is associated with resistance to CDK4/6 inhibitors in ER-positive breast cancer

Abstract Background: The use of CDK4/6 inhibitors (CDK4/6i) has led to a remarkable progress in the treatment of advanced ER-positive (+) breast cancer (BC). The fact that both CDK4/6i and endocrine therapy (ET) target cell-cycle G1-S transition suggests some overlap of resistance mechanisms. However, clinical variability in response to CDK4/6i in patients progressing on ET suggests the involvement of additional or unique resistance mechanisms. Using bioinformatics analyses of clinical and preclinical data, we now identified genes involved in DNA replication licensing, in particular, excess MCM2 as a new mechanism of resistance to CDK4/6i. Methods: Gene expression data from the neoadjuvant NeoPalAna trial of the CDK4/6i palbociclib (Palbo) and the aromatase inhibitor (AI) anastrozole in ER+ BC was downloaded from Gene Expression Omnibus (GSE93204). Response to AI and Palbo was obtained using the published Ki67 immunohistochemistry data and cutoff on biopsies post treatment. Gene Set Enrichment Analysis comparing tumors with different sensitivity to AI vs. Palbo was performed using the Gene Ontology related to DNA replication, DNA repair, and DNA damage checkpoint. The DNA replication gene set was further sub-assigned by their selective role in origin licensing, firing, elongation, replication repair, and checkpoint regulation. A series of bioinformatics analyses was applied on five ER+/HER2-negative BC cell lines with gene expression and DepMap (from CCLE) and Palbo sensitivity data (from GDSC). Two ER+ BC cell models (MCF7 and T47D) and their estrogen deprivation (mimicking AIs)-resistant (EDR) derivatives were used for further studies including RNA-seq, Palbo sensitivity (data from PMID: 33536276), and chromatin fractionation assays. Results: We found that the DNA replication-associated gene set, comprising &amp;gt; 80% genes outside the Rb-loss gene signature, was significantly enriched in baseline biopsies of Palbo-resistant (PalboR) tumors from patients in the NeoPalAna trial. Of the DNA replication genes, the subset of genes involved in origin licensing were preferentially enriched in PalboR vs. AI-resistant tumors. Similarly, the enrichment of genes involved in replication initiation was also seen in ER+/HER2-negative BC cell lines with a decreased response to Palbo. Notably, these PalboR cell lines showed a reduced vitality to shRNA knockdown of the replication initiation genes compared to randomly selected other genes or the Rb-loss gene set. Based on the modeling of DepMap gene dependency score and Palbo sensitivity, we nominated minichromosome maintenance 2 (MCM2), the key origin licensing factor, as the top gene that is essential for PalboR cell survival. Additionally, using our previously reported MCF7-EDR and T47D-EDR cell models with increased or decreased sensitivity to Palbo compared to their parental lines, respectively, we observed a corresponding decrease or increase in the expression of origin licensing genes and MCM2 in EDR vs. parental lines. Decreased Palbo sensitivity in the EDR cells was associated with sustained MCM2 chromatin loading and reduced expression of genes including the cyclin-dependent kinase inhibitor p21. Ongoing studies investigate whether elevated MCM2 levels confer resistance to CDK4/6i by resolving replication stress. Conclusions: Our strategic bioinformatics analyses reveal that excess DNA replication licensing is associated with CDK4/6i resistance in clinical and preclinical settings upon resistance to estrogen deprivation. Among the replication initiation-associated genes, MCM2 plays a potential role in conferring CDK4/6i resistance via sustaining origin licensing and suppressing p21. Our study provides a new avenue via lens of replication licensing to explore novel mechanisms and therapeutic opportunities in CDK4/6i resistance. Citation Format: Sarmistha Nanda, Martin J. Shea, Rachel Schiff, C. Kent Osborne, Mothaffar Rimawi, Xiaoyong Fu. DNA replication licensing is associated with resistance to CDK4/6 inhibitors in ER-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-10.

Cell Proliferation Receptor-Enhanced 3D High-Throughput Screening Model for Optimized Drug Efficacy Evaluation in Breast Cancer Cells.

A higher correlation of epidermal growth factor receptor (EGFR)-targeting drugs has been reported with the use of the cell proliferation receptor-enhanced three-dimensional high-throughput screening model (CPRE 3D-HTS model) compared with two-dimensional (2D) cell-based HTS. A greater expression of differential human EGFR 2 (HER2) protein between HER2-positive and HER2-negative cell lines was observed in breast cancer (BC) cell lines cultured using the CPRE 3D-HTS model compared with 2D-cultured cells. When using 2D-cultured cells, properties such as the expression of the cell proliferation receptor are lost as the cells attach to the bottom of the well plate. In an effort to solve this problem, the CPRE 3D-HTS model expressing high cell proliferation receptors was optimized by the selection of alginate as the extracellular matrix. Results from the use of the CPRE 3D-HTS model showed higher drug resistance with increased expression of drug resistance-related proteins. Of particular interest, a higher correlation of HER2-targeted drugs was observed with the use of the CPRE 3D-HTS model. In order to validate this higher correlation of target drugs observed in the CPRE 3D-HTS model, the results of Western blot analysis and high content imaging analysis were analyzed, which confirmed that 3D-cultured BC cell lines showed a greater difference in the expression of HER2-positive and HER2-negative BC cell lines than 2D-cultured cells. Thus, the use of CPRE 3D-HTS using a 384-pillar plate resulted in increased accuracy when screening HER2-targeted drugs in BC, and it is a very useful platform for analyzing the efficacy of targeted drugs by enhancing the expression of HER2.

Trastuzumab effects depend on HER2 phosphorylation in HER2-negative breast cancer cell lines.

The breast cancer (BC) biomarker HER2 (Human Epidermal Receptor 2) is overexpressed in 25% of BC. Only patients with HER2-positive tumors receive HER2-targeting therapies, like trastuzumab (Herceptin). However, some women with a HER2-negative BC could benefit from trastuzumab. This could be explained by the activation/phosphorylation of HER2 that can be recognized by trastuzumab. The aim of this study is to examine trastuzumab effects on HER2 phosphorylation at tyrosine Y877 (pHER2Y877). HER2 and pHER2Y877 status were evaluated in a cohort of BC patients representative of molecular subtypes distribution (n = 497) and in a series of BC cell lines (n = 7). Immunohistochemistry against pHER2Y877 was performed on tissue micro arrays. Cellular proliferation assays were performed on BC cell lines presenting different combinations of HER2 and pHER2Y877 status and treated with increasing doses of trastuzumab (0–150 μg/ml). The prevalence of pHER2Y877 in this cohort was 6%. Nearly 5% of patients with HER2-negative tumors (n = 406, 82%) overexpressed pHER2Y877. Among triple negative BC patients (n = 39, 8%), 7.7% expressed pHER2Y877. Trastuzumab treatment decreased cell proliferation in HER2−/pHER2Y877+ BC cell lines, to an extent comparable to what occurs in HER2+ cell lines, but did not affect HER2−/pHER2Y877− cell lines. Trastuzumab sensitivity in HER2−/pHER2Y877+ cell line is specific to HER2 tyrosine 877 phosphorylation. Hence, with further confirmation in a bigger cohort, trastuzumab treatment could be envisaged as a treatment option to women presenting with HER2−/pHER2+ tumors, representing more than 1000 BC women in Canada in 2019.

ARX788, a novel anti-HER2 antibody-drug conjugate, shows anti-tumor effects in preclinical models of trastuzumab emtansine-resistant HER2-positive breast cancer and gastric cancer.

The majority of HER2-positive breast or gastric cancers treated with T-DM1 eventually show resistance to this agent. We compared the effects of T-DM1 and ARX788, a novel anti-HER2 antibody-drug conjugate, on cell growth and apoptosis in HER2-positive breast cancer and gastric cancer cell lines sensitive to T-DM1, gastric cancer cell lines resistant to T-DM1, HER2-negative breast cancer cell lines, and T-DM1-resistant xenograft models. ARX788 was effective in T-DM1-resistant in vitro and in vivo models of HER2-positive breast cancer and gastric cancer. ARX788 showed a pronounced growth inhibitory effect on all five HER2-positive cell lines tested, of which two gastric cancer cell lines had acquired resistance to T-DM1. ARX788 evoked more apoptotic events compared to T-DM1. While JIMT-1 and RN-87 xenograft tumors progressed on T-DM1 treatment, all such tumors responded to ARX788, and four out of the six JIMT-1 tumors and nine out of the twelve RN-87 tumors disappeared during the ARX788 treatment. Mice treated with ARX788 survived longer than those treated with T-DM1. The data support evaluation of ARX788 in patients with HER2-positive breast cancer or gastric cancer including cancers that progress during T-DM1 therapy.

Differential altered expression of let-7a and miR-205 tumor-suppressor miRNAs in different subtypes of breast cancer under treatment with Taxol.

MicroRNAs (miRNAs) are small non-coding RNAs which have been considered as major players in the process of carcinogenesis and drug responsiveness of breast cancer. In this study, we aimed to investigate the expression pattern of let-7a and miR-205 tumorsuppressor miRNAs in breast cancer cell lines under treatment with paclitaxel. The half maximal inhibitory concentration (IC50) of paclitaxel was determined for 4 breast cancer cell lines, including MCF-7, MDA-MB-231, SKBR-3, and BT-474 by an MTT assay. The expression level of let-7a and miR-205, and their targets, K-RAS and HER3, was determined before and after treatment with paclitaxel, using quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). After treatment, the expression level of both let-7a and miR-205 was significantly increased in HER2- overexpressing cell line BT-474 (26.4- and 7.2-fold, respectively). In contrast, the HER2-negative cell lines MCF-7 and MDA-MB-231 showed a significantly decreased expression of both let-7a (30.3- and 13.5-fold, respectively) and miR-205 (20- and 18.1-fold, respectively). Controversially, SKBR-3 revealed a significantly decreased expression of both let-7a (1.3-fold) and miR-205 (1.3-fold). The expression level of K-RAS as a target of let-7a decreased in all cell lines significantly, but the pattern of alteration in the expression level of HER3 as a target of miR-205 in all cell lines was the reverse of the pattern of alteration in the expression level of miR-205. Our results confirmed a better response of HER2-overexpressing breast cancer to paclitaxel at the miRNA level. One putative reason could be the upregulation of tumor-suppressor miRNAs after treatment with paclitaxel. On the other hand, HER2-negative breast cancer cell lines showed a significantly decreased expression of tumor-suppressor miRNAs, a putative mechanism of resisting the therapy.

Biological indicators of response and resistance to PARP inhibition in BRCA wild-type breast cancer.

125 Background: Although PARP inhibitors are currently being tested in clinical trials in breast cancer patients, it is not well understood which subgroup of patients will best respond to such therapy or how to combine anti-PARP therapy to improve therapeutic response. Methods: We assessed the response of a panel of 8 HER2-negative breast cancer cell lines against three PARP inhibitors: veliparib, olaparib, and BMN 673. Efficacy of carboplatin as a single agent and in combination with PARP inhibitors was determined. Therapeutic response and cellular phenotype were elucidated using live-cell imaging, high-content imaging and immunofluorescence. We computed IC50 as the measure of sensitivity to each PARP inhibitor across the panel of cell lines. Cellular phenotype was ascertained by quantifying endpoints for apoptosis and DNA damage. Results: BMN 673 demonstrated the highest potency, with IC50 values in the nanomolar range, while the IC50 values of olaparib and veliparib were in the micromolar range. Drug sensitivity was independent of BRCA mutational status. Biomarkers of apoptosis and DNA damage varied with the PARP inhibitor and across different cell lines. The resistant cell lines could be classified into two groups that varied in DNA damage response and cell death due to DNA damage. A combined DNA damage – cell death (DDD) Response Score was developed and correlated strongly with IC50 values in BRCA wild-type breast cancer cell lines. The DDD Response Score identified cell lines that were highly sensitive to single-agent PARP inhibitors or carboplatin. A Response Score that identified intermediate sensitivity may predict which cell lines may benefit from the combination of anti-PARP therapy and carboplatin. Conclusions: Overall, a spectrum of response to three different PARP inhibitors was identified in a panel of eight breast cancer lines. Cellular phenotype can help classify resistant cancer cell lines. We have developed an experimental approach that may help to inform which breast cancer subtypes may benefit from PARP inhibitors and carboplatin, both as single agents, and in combination.

Abstract 737: Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance

Abstract Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a subset of pre-treatment specimens from TBCRC006. Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (&amp;gt;100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed. Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ∼30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ∼30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC50: Afa 0.02μM vs. L 3 μM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation. Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura Heiser, Nicholas Wang, Kyle Covington, Edward Chen, Alexander Renwick, Tamika Mitchell, Marty Shea, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne Fuqua, Gary Chamness, Chad Shaw, Marilyn Li, David Wheeler, Susan Hilsenbeck, Mothaffar Fahed Rimawi, Joe Gray, C.Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy 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 737. doi:10.1158/1538-7445.AM2015-737

Abstract P5-05-03: Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib

Abstract Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [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 P5-05-03.

Trogocytosis-mediated expression of HER2 on immune cells may be associated with a pathological complete response to trastuzumab-based primary systemic therapy in HER2-overexpressing breast cancer patients

BackgroundTrogocytosis is defined as the transfer of cell-surface membrane proteins and membrane patches from one cell to another through contact. It is reported that human epidermal growth factor receptor 2 (HER2) could be transferred from cancer cells to monocytes via trogocytosis; however, the clinical significance of this is unknown. The aim of this study is to demonstrate the presence and evaluate the clinical significance of HER2+ tumor-infiltrated immune cells (arising through HER2 trogocytosis) in HER2-overexpressing (HER2+) breast cancer patients receiving trastuzumab-based primary systemic therapy (PST).MethodsTo assess the trogocytosis of HER2 from cancer cells to immune cells, and to evaluate the up- and down-regulation of HER2 on immune and cancer cells, peripheral blood mononuclear cells from healthy volunteers and breast cancer patients were co-cultured with HER2+ and HER2-negative breast cancer cell lines with and without trastuzumab, respectively. The correlation between HER2 expression on tumor-infiltrated immune cells and a pathological complete response (pCR) in HER2+ breast cancer patients treated with trastuzumab-based PST was analyzed.ResultsHER2 was transferred from HER2+ breast cancer cells to monocytes and natural killer cells by trogocytosis. Trastuzumab-mediated trogocytosed-HER2+ effector cells exhibited greater CD107a expression than non-HER2-trogocytosed effector cells. In breast cancer patients, HER2 expression on tumor-infiltrated immune cells in treatment naïve HER2+ tumors was associated with a pCR to trastuzumab-based PST.ConclusionsHER2-trogocytosis is visible evidence of tumor microenvironment interaction between cancer cells and immune cells. Given that effective contact between these cells is critical for immune destruction of target cancer cells, this interaction is of great significance. It is possible that HER2 trogocytosis could be used as a predictive biomarker for trastuzumab-based PST efficacy in HER2+ breast cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1041-3) contains supplementary material, which is available to authorized users.

Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1)

The antibody trastuzumab (Herceptin) has substantially improved overall survival for patients with aggressive HER2-positive breast cancer. However, about 70% of all treated patients will experience relapse or disease progression. This may be related to an insufficient targeting of the CD44highCD24low breast cancer stem cell subset, which is not only highly resistant to chemotherapy and radiotherapy but also a poor target for trastuzumab due to low HER2 surface expression. Hence, we explored whether the new antibody-drug conjugate T-DM1, which consists of the potent chemotherapeutic DM1 coupled to trastuzumab, could improve the targeting of these tumor-initiating or metastasis-initiating cells. To this aim, primary HER2-overexpressing tumor cells as well as HER2-positive and HER2-negative breast cancer cell lines were treated with T-DM1, and effects on survival, colony formation, gene and protein expression as well as antibody internalization were assessed. This revealed that CD44highCD24lowHER2low stem cell-like breast cancer cells show high endocytic activity and are thus particularly sensitive towards the antibody-drug conjugate T-DM1. Consequently, preexisting CD44highCD24low cancer stem cells were depleted by concentrations of T-DM1 that did not affect the bulk of the tumor cells. Likewise, colony formation was efficiently suppressed. Moreover, when tumor cells were cocultured with natural killer cells, antibody-dependent cell-mediated cytotoxicity was enhanced, and EMT-mediated induction of stem cell-like properties was prevented in differentiated tumor cells. Thus our study reveals an unanticipated targeting of stem cell-like breast cancer cells by T-DM1 that may contribute to the clinical efficacy of this recently approved antibody-drug conjugate.

Abstract P5-08-06: PI3K blockade enhances the antitumor activity of eribulin in PIK3CA-mutant eribulin-resistant tumor xenografts

Abstract Eribulin is a recently approved microtubule-targeting agent (MTA) for the management of heavily pretreated metastatic breast cancer (BC) patients. Constitutive PI3K/Akt/mTOR survival pathway activation, either via mutational activation of the PI3K catalytic subunit (PIK3CA) or via inactivation of the tumor suppressor PTEN, may confer resistance to MTAs. Hence, we hypothesized that PI3K-pathway activation limits the antitumor activity of eribulin in HER2-negative BC and that PI3K inhibition enhances the efficacy of this chemotherapeutic agent. The predictive value of PIK3CA mutation or PTEN loss towards eribulin response was interrogated in vitro, using a panel of fourteen HER2-negative BC cell lines, and in vivo using six tumor models from cell-line or patient-derived tumors. While PIK3CA mutation did not seem to be predictive in vitro, the PIK3CA-mutated xenograft models underwent tumor progression upon single-agent eribulin therapy (Table 1). In the absence of concomitant PIK3CA mutation, PTEN loss was neither predictive in vitro nor in vivo. Moreover, eribulin induced PI3K-pathway activation in tumor xenografts, a potential escape mechanism to MTA-therapy. To validate the negative predictive value of PIK3CA mutation, BC tumors from patients treated with eribulin in the neoadjuvant and in the metastatic setting are being queried, as the pharmacodynamic activation of the PI3K-pathway upon eribulin treatment. Class I pan-PI3K (BKM120) or PI3K-a-specific (BYL719) inhibitors were used in vitro to block the PI3K-pathway concomitantly with eribulin treatment, resulting in enhanced antiproliferative and proapoptotic activity. Strikingly, in PIK3CA mutant xenograft models, while eribulin alone exhibited limited antitumor activity compared to PIK3CA-wild type models, co-administration of a PI3K inhibitor induced marked tumor regression (BKM120 data in Table 1, BYL719 data will be reported). Moreover, addition of the PI3K inhibitor at progression with eribulin single-agent also resulted in tumor regression. Of note, PIK3CA-wild type models also exhibited increased antitumor activity with the combined therapy compared to single-agent treatments. The precise mechanism by which the combination of eribulin and a PI3K-targeting agent results in tumor regression is currently under investigation, embracing both the induction of mitotic catastrophe in tumor cells and the regularization of the tumor vasculature. These results support the clinical development of therapeutic regimens combining PI3K-inhibitors to the approved MTA eribulin and might be predictive of clinical benefit both in the PIK3CA-mutant and -wild type breast cancer population. Table 1. Percentage change in tumor volume.StatusPIK3CA mutantPIK3CA mutant and PTEN-lowPTEN-lowWTModel/ TreatmentMCF7LPDX44CAL51MDA-MB-468PDX88PDX98Eribulin39 ± 9683 ± 51364 ± 127-47 ± 14-26 ± 34-36 ± 33BKM120501 ± 169116 ± 111119 ± 73-9 ± 23128 ± 3787 ± 112Eribulin + BKM120-70 ± 27-80 ± 1051 ± 46-75 ± 12-94 ± 5-65 ± 13Patient- (PDX) and cell line-derived tumor xenografts were treated with eribulin mesylate (0.1mg/kg, 3IW) and/or BKM120 (27.5 mg/kg, 6IW) for 26-31 days.. IW, in week. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-06.

HER2-specific T lymphocytes kill both trastuzumab-resistant and trastuzumab-sensitive breast cell lines in vitro

Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, the resistance to anti-HER2 therapy is a growing clinical dilemma. We aim to determine whether HER2-specific T cells generated from dendritic cells (DCs) modified with HER2 gene could effectively kill the HER2-positive breast cancer cells, especially the trastuzumab-resistant cells. The peripheral blood mononuclear cells (PBMCs) from healthy donors, whose HLA haplotypes were compatible with the tumor cell lines, were transfected with reconstructive human adeno-association virus (rhAAV/HER2) to obtain the specific killing activities of T cells, and were evaluated by lactate dehydrogenase (LDH) releasing assay. Trastuzumab produced a significant inhibiting effect on SK-BR-3, the IC50 was 100ng/ml. MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro. HER2-specific T lymphocytes killed effectively SK-BR-3 [(69.86±13.41)%] and MDA-MB-453 [(78.36±10.68)%] at 40:1 (effector:target ratio, E:T), but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 (less than 10%). The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner, and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3. These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.

Antitumor and anticancer stem cell activity of a poly ADP-ribose polymerase inhibitor olaparib in breast cancer cells

Although the poly adenosine diphosphate (ADP)-ribose polymerase (PARP) inhibitor olaparib is known to have potent antitumor activity in BRCA-related breast cancer cells, a limited number of preclinical and clinical studies have shown antitumor activity of olaparib in non-BRCA-related breast cancer. We investigated antitumor activity of olaparib in breast cancer cell lines derived from patients with nonfamilial sporadic breast cancer. Effects of olaparib alone or in combination with five different chemotherapeutic agents on cell growth, cell cycle progression, apoptosis, and proportion of cancer stem cells using the mammosphere assay and CD44/CD24/ESA cell surface marker assay were investigated in a panel of six sporadic breast cancer cell lines. Extracellular-signal-regulated kinase (ERK) phosphorylation was also investigated to elucidate action mechanisms of olaparib. Olaparib inhibited the growth of two estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell lines and two ER-negative and HER2-negative breast cancer cell lines (50% growth inhibitory concentrations 1.3-3.0 μM) associated with G2/M accumulation and induction of apoptosis. In contrast, two HER2-positive cell lines were resistant to olaparib. Interestingly, olaparib significantly decreased the proportion of putative cancer stem cells in either sensitive or resistant cell lines. In addition, olaparib increased expression of p-ERK. Combined treatments of olaparib with a mitogen-activated protein kinase kinase (MEK) inhibitor U0126 completely suppressed expression of p-ERK. These treatments also inhibited the G2/M accumulation and apoptosis induction by olaparib. Among five chemotherapeutic agents commonly used for breast cancer treatment, only an irinotecan metabolite SN38 showed additive antitumor activity with olaparib. Importantly, the combined treatment enhanced the increase in G2/M accumulation and apoptosis induction as well as a decrease in the proportion of cancer stem cells. This study has indicated for the first time that the PARP inhibitor olaparib has substantial antitumor and anticancer stem cell activity in breast cancer cell lines of nonfamilial origin. Upregulation of p-ERK might explain, at least in part, antitumor and anticancer stem cell activity of olaparib. Combined treatment of olaparib with irinotecan might be effective in treatment of non-BRCA-related breast cancer.

183 An Italian Cost-effectiveness Analysis of Paclitaxel Albumin (nab Paclitaxel) Vs Solvent-based Paclitaxel for Metastatic Breast Cancer Patients – the CONSTANZA Study

Purpose: Although the poly ADP-ribose polymerase (PARP) inhibitor olaparib is known to have a potent anti-tumor activity in BRCA-related breast cancer cells, a limited number of preclinical studies have shown anti-tumor activity of olaparib in BRCA-naive breast cancer cell lines. We have investigated the anti-tumor activity of olaparib in breast cancer cell lines derived from patients with non-familial sporadic breast cancer. Methods: Effects of olaparib (AstraZeneca) alone or in combination with five different chemotherapeutic agents (cisplatin, doxorubicin, etoposide, paclitaxel and SN38) on cell growth, cell cycle progression, apoptosis and proportion of cancer stem cells using the mammosphere assay and CD44/CD24/ESA cell surface marker assay were investigated in a panel of six sporadic breast cancer cell lines. The ERK phosphorylation was also investigated to elucidate mechanisms of action of olaparib. Results: Olaparib significantly inhibited the growth of two estrogen receptor (ER)-positive and HER2-negative breast cancer cell lines and two ER−negative and HER2-negative breast cancer cell lines (the 50% growth inhibitory concentrations were 1.3-3.0mM) associated with the G2/M accumulation and induction of apoptosis. In contrast, two HER2-positive cell lines were resistant to olaparib. Interestingly, olaparib significantly decreased the proportion of putative cancer stem cells in either sensitive or resistant cell lines. In addition, olaparib increased the expression of p-ERK. Combined treatments of olaparib with a MEK inhibitor U0126 completely suppressed the expression of p-ERK. These treatments also inhibited the G2/M accumulation and apoptosis induction by olaparib. Among five chemotherapeutic agents commonly used for breast cancer treatment, only an irinotecan metabolite SN38 showed an additive anti-tumor activity with olaparib. Importantly, the combined treatment enhanced an increase in the G2/M accumulation and apoptosis induction as well as a decrease in the proportion of cancer stem cells. Conclusions: This study confirms that the PARP inhibitor olaparib has substantial anti-tumor and anti-cancer stem cell activity in breast cancer cell lines of a non-familial origin. Up-regulation of p-ERK might explain, at least in part, anti-tumor and anti-cancer stem cell activity of olaparib. A combined treatment of olaparib with irinotecan might be effective in the treatment of non-BRCA-related breast cancer.

Preclinical evaluation of sunitinib, alone and in combination with trastuzumab, in HER2 positive breast cancer (BC)

14689 Background: There is a potentially therapeutically relevant interaction between the HER2 and VEGF systems. In addition to targeting VEGFR, the multi-target kinase inhibitor (TKI) sunitinib malate targets c-Kit, PDGFR, RET, FLT-3 and CSF-1R. The aim of this study was to assess the direct anti-tumour effects of sunitinib, alone and in combination with trastuzumab in HER2 positive breast cancer. Methods: A panel of HER-2 positive and HER-2 negative BC cell lines were tested with sunitinib malate and compared with imatinib mesylate, which targets Bcr-Abl, PDGFR and c-Kit. IC50s were determined using the acid phosphatase assay. HER-2 positive cell lines were treated with a combination of sunitinib and trastuzumab. Results: IC50 values were similar in the HER-2 positive and negative cell lines for both imatinib and sunitinib. All of the cell lines showed greater sensitivity to sunitinib than to imatinib. Sunitinib was tested in combination with trastuzumab in two trastuzumab sensitive cell lines (SKBR3 and BT474) and two trastuzumab-resistant cell lines (JIMT-1 and HCC-1419). Combined treatment with sunitinib and trastuzumab showed improved response compared to either drug alone, in all of the HER-2 positive cell lines tested (Table). Conclusions: Our findings suggest that sunitinib alone shows modest anti-tumour activity against breast cancer cells but shows a positive interaction with trastuzumab in HER-2 positive BC cell lines in vitro. Together with the anit-angiogenic effects of sunitinib, this combination of targeted therapies may improve response to trastuzumab in HER2 positive breast cancer. Cell Line IC50s (+ std dev) % Growth (+ std dev) Imatinib (μM) Sunitinib (μM) Sunitinib (7.5 μΜ) Trastuzumab (7.5 nΜ) Sunitinib (7.5 μΜ) + trastuzumab (7.5 nΜ) SKBR-3 14.9+2.3 5.4+0.9 45.4+5.1 65.2+5.2 33.3+4.3 JIMT-1 23.4+4.1 7.3+1.9 60.9+11.3 99.5+1.9 50.4+10.9 BT-474 17.7+3.6 5.0+1.3 57.6+6.1 39.6+6.2 20.1+10.1 HCC-1419 32.3+3.7 8.4+2.9 90.9+16.3 86.1+5.6 74.5+7.3 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Pfizer Oncology, Roche