Lapatinib-resistant Cells Research Articles

Abstract B28: Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers

Abstract Although HER2-targeted therapies substantially improve the outcome for patients with HER2-amplified breast cancer, resistance to HER2 targeting is frequently observed. HER2-amplified tumors depend on the HER2-mediated activation of the phosphatidylinositol-3kinase (PI3K)/Akt pathway, which becomes reactivated in many therapeutically resistant HER2-amplified tumors. We investigated the mTOR complexes mTORC1 and mTORC2 as potential therapeutic targets in HER2-amplified breast cancers, based on their roles as activators (mTORC2) or effectors (mTORC1) of Akt. We found that genomic gains in Rictor, encoding a necessary mTORC2 cofactor, correlated with poor survival in breast cancer patients, while genomic gains in Raptor, an mTORC1 cofactor, did not. Rictor protein levels were increased in invasive breast cancers as compared to ductal carcinoma in situ (DCIS). Conditional Rictor gene ablation decreased growth, cell survival and invasion of human HER2-amplified breast cancer cells by blocking Akt signaling. Treatment of HER2 positive cells with the HER2/EGFR inhibitor Lapatinib caused Rictor upregulation. However, Rictor knockdown improved cell killing in Lapatinib-treated cells and impaired Akt signaling and cell survival in Lapatinib-resistant HER2 positive breast cancer cells. Together, these results demonstrate the pivotal role of mTORC2 in HER2-amplified breast cancers and support efforts to develop mTORC2-targeted therapies. Citation Format: Meghan M. Morrison, Bayley A. Jones, Donna J. Hicks, Violeta Sanchez, Valeria M. Estrada, Michelle M. Williams, Dana Brantley-Sieders, Rebecca S. Cook. Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B28.

Abstract P2-07-06: Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive breast cancer

Abstract Background: The Src pathway is known to regulate tumor matastasis and plays a role in epithelial-mesenchymal transition(EMT). Lapatinib, although, has improved clinical outcome for HER2 positive breast cancer patients, acquired resistance to lapatinib remains an important reason influencing its clinical efficacy. Our previous research shows lapatinib acquired resistance HER2 positive breast cancer cell lines, SKBR3-R and BT474-R have EMT phenomenon and multiple pathway activated. Niclosamide is an FDA-approved antihelminthic agent, which is found has cytotoxicity effect on tumor stem cells recently. We wished to determine the effect of niclosamide on EMT and lapatinib resistance cells and investigate niclosamide as a potential therapeutic agent for HER2 positive breast cancer. Methods: SKBR3 and BT474, two HER2 positive breast cancer cell lines, were continuously exposed to increasing doses of lapatinib to establish two stable cell lines resistant to lapatinib, SKBR3-R and BT474-R. Cell proliferation was determined by CCK8 assay. Protein expression was determined by western-blotting. Invasion ability was analyzed by transwell assay. FITC staining flow cytometry (FCM) was conducted to observe the percentage of apoptosis. Results: Both two HER2 positive lapatinib resistant cell lines, SKBR3-R and BT474-R had EMT phenomenon. Niclosamide had a stronger inhibition effect on lapatinib resistant cell lines than parental cell lines, and induced more apoptosis by FCM. Western-blot showed niclosamide could reverse the EMT phenomenon of SKBR3-R and BT474-R with E-cadherin up-regulated and snail,vimentin down-regulated at the concentration of 0.5-1μM. A significant reduction of Src signaling was also confirmed, as well as the downstream Akt and Erk pathway. After adding niclosimide for 48 hours, SKBR3-R and BT474-R’s capability of invasion were inhibited. Conclusion: Our results suggested that niclosamide had a strong cytotoxic effect on HER2 positive lapatinib resistant breast cancer cell lines. The EMT induced by lapatinib resistance could be reversed by niclosamide, associated with the inhibition of Src pathway activation, as well as the downstream pathways. Niclosamide treatment produced reduced levels of invasion, serving as a novel therapeutic way for lapatinib-resistant breast cancer patients. Citation Format: Junjun Liu, Xiaosong Chen, Yan Mao, Kunwei Shen. Niclosamid overcomes epithelial-mesenchymal transition of lapatinib resistance through inhibiting Src activation in HER2 positive 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 P2-07-06.

Abstract P5-19-22: A phase I clinical trial of entinostat and lapatinib in patients with trastuzumab refractory HER2-positive metastatic breast cancer

Abstract Background: Our preclinical data show that entinostat enhances the efficacy of lapatinib in HER2 positive (HER2+) breast cancer cell lines via FOX-03-mediated Bim1 expression. In-vitro and in- vivo, the combination of entinostat and lapatinib enhanced apoptosis in lapatinib-resistant cells. We conducted a phase I study with the primary objective to determine the RP2D of entinostat plus lapatinib in patients with HER2+ metastatic breast cancer with progressive disease (PD) after trastuzumab treatment. Methods: This was a single-center, open-label study to evaluate the dose limiting toxicity (DLT) and determine the MTD of every-other-week entinostat plus daily lapatinib in 28-day cycles. Patients with locally recurrent or metastatic breast cancer in whom trastuzumab had failed were enrolled. DLT was defined as: any toxicity resulting in 14 or more days of treatment delay, febrile neutropenia (NTP), grade 4 NTP for over 7 days, any grade 3 or higher non-hematologic toxicity, grade 3 nausea, vomiting, diarrhea or electrolyte imbalance lasting over 48 hours despite adequate supportive care, or a platelet count less than 10,000. Grading was assigned according to common toxicity criteria (CTC 4). A standard 3+3 dose escalation design was used. Entinostat was given at 10 mg (level 0), 12 mg (level 1), or 15 mg (level 2) by mouth every 14 days. Lapatinib was given at 1250 mg by mouth daily. Toxicity was evaluated on day 15 and day 28 for C1 and C2, and at the end of each cycle thereafter. Results: Fifteen patients with HER2+ metastatic breast cancer were enrolled. Median age was 52 years (range 26-69), median of all prior systemic treatment was 4 (range 1- 12), and median prior trastuzumab-based regimens was 2 (range 1-6). Eight patients had prior lapatinib exposure. Seven patients had ER+/HER2+ tumors. Median number of cycles completed was 2 (range 1-13). DLT was observed in 0 of 3 patients at level 0, 1 of 6 at level 1, and 1 of 6 at level 2. 3 patients at level 1 had lapatinib dose reductions because of grade 3 rash (n=2) or grade 3 dyspnea (n=1) or grade 3 abdominal pain (n=1). 2 patients at level 2 had entinostat dose reductions because of grade 4 NTP in cycle 1 (n=1) or grade 3 NTP in cycle 5 and grade 4 neutropenia in cycle 6 (n=1). Five patients had SD (defined per 2009 RECIST guidelines): two at dose level 1 for 6 months and 13 months, three at level 2 for 3, 4, and 8 months. Median time to progression was 2 months (range 1-13). The most common treatment related adverse events were fatigue (n=15), myalgia (n=14), nausea (n=14), diarrhea (n=13), anemia (n=11), and rash (n=11). Conclusion: MTD was not reached. Cumulative toxicity of entinostat plus lapatinib was fairly well tolerated. The combination therapy suggests there was clinical activity in at-least 5 patients who had SD in this cohort of heavily pretreated trastuzumab-resistant MBC. Data from the EG104900 clinical trial showed that lapatinib plus trastuzumab significantly improved median overall survival compared with lapatinib alone. Therefore, this study was modified after 15 patients to add trastuzumab to the combination. We are currently conducting a phase 1b trial to determine the MTD of entinostat, trastuzumab and lapatinib. Citation Format: Jie Willey, Ricardo H Alvarez, Daniel J Booser, Sharon H Giordano, Ana M Gonzalez-Angulo, James L Murray, Vincente Valero, Jangsoon Lee, Naoto T Ueno, Summer A Jackson, Fahad A Faruqi. A phase I clinical trial of entinostat and lapatinib in patients with trastuzumab refractory HER2-positive metastatic 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 P5-19-22.

Design and synthesis of piperidine derivatives as novel human heat shock protein 70 inhibitors for the treatment of drug-resistant tumors

HSP70 is a potential target for tumour treatment. HSP70 plays significant roles in several biological processes, including the regulation of apoptosis. In this study, piperidine derivatives were designed as novel HSP70 inhibitors based on virtual fragment screening performed in Dock 4.0, Discovery Studio 2.5 and SYBYL 6.9. A total of 67 novel piperidine derivatives were synthesized. Cell viability assays were performed in 16 cancer cell lines. The emphasis was placed on lapatinib-resistant breast cancer cells (BT/LapR1.0, MDA-MB-361, SK/LapR1.0, and MDA-MB-453). The compounds HSP70-36/37/40/43/46 significantly inhibited the proliferation of human breast cancer cells. Compound HSP70-36 inhibited the growth of BT474 and BT/LapR1.0 cells with IC50 values of 1.41 μM and 1.47 μM, respectively. The binding affinity of HSP70-36/HSP70 was evaluated by surface plasmon resonance and yielded Kd values of 2.46 μM. The LD50 was 869.0 mgkg−1. These data suggest that HSP70-36 may be a potential candidate compound for tumour treatment.

The Role of Non‐coding RNAs in Breast Cancer Resistance to EGFR Therapy

To achieve the objective of characterizing the miRNA‐mediated mechanisms of resistance to current epidermal growth factor receptor (EGFR) targeted therapies, therapy resistant breast cancer (BC) cell lines were created by exposing therapy sensitive SKBR3 and BT‐474 human BC cells to varying concentrations of gefitinib or lapatinib. The fold resistance, as determined from the IC50s, was 2.5 and 21.5 respectively, for the SKBR3 gefitinib and lapatinib resistant cell lines and 1.9 for the BT‐474 gefitinib resistant cells. The therapy sensitive cells demonstrated higher levels of apoptosis in the presence of the therapeutics compared to the therapy resistant variants. Diferentially expressed miRNAs in therapy resistant and sensitive variants were identified from qRT‐PCR and their targets detected by Western blotting. The oncomiRs miR‐221/222 that promotes the epithelial to mesenchymal (EMT) transition, were upregulated, while miR‐200a, a miRNA that maintains the epithelial phenotype, was downregulated in the therapy resistant cell lines. Accordingly, the miR‐221/222 targets and tumor suppressors PTEN and p27 were downregulated, and the miR‐200 target β‐catenin, a pro‐cancer protein, was upregulated in the therapy resistant variants. In conclusion, this data suggest that downregulation of miR‐200 family, and upregulation of miR‐221/222 is a general mechanism of EGFR therapy resistance in BC.

PI3K-independent mTOR activation promotes lapatinib resistance and IAP expression that can be effectively reversed by mTOR and Hsp90 inhibition

Although HER2 targeted therapies have substantially improved outcomes in HER2 overexpressing (HER2+) breast cancer, resistance to these therapies remains a clinical challenge. To better understand the mechanisms of resistance to lapatinib, a HER2 and EGFR dual kinase inhibitor, we treated HER2+ breast cancer cells with lapatinib for an extended period to generate a lapatinib-resistant (LapR) cell line model and examined cancer-promoting signaling activation in LapR cells. We found that LapR cells possess enhanced mTOR activation, which was independent of PI3K and other known mTOR activators. Lapatinib resistance could be reversed by mTOR kinase inhibition. Intriguingly, LapR cells had constitutive cytosolic cytochrome C, indicating that LapR cells suppress lapatinib-induced apoptosis downstream of cytochrome C release from mitochondria into the cytosol rather than by preventing its release into the cytosol. Consistent with this notion, LapR cells possessed increased levels of 2 of the inhibitors of apoptosis (IAPs), survivin and c-IAP-2, which are reported to block caspase activation downstream of cytosolic cytochrome C release. Further, treatment with the mTOR kinase inhibitor AZD8055 or the Hsp90 inhibitor 17-AAG reversed expression of IAPs and overcame lapatinib resistance in LapR cells. Together, these data suggest that suppression of apoptosis downstream of cytosolic cytochrome C release, possibly through increased expression of IAPs or other caspase-suppressing proteins, may promote lapatinib resistance. Further, PI3K is thought to be the main driver of lapatinib resistance, but our findings indicate that PI3K inhibitors may be ineffective in some lapatinib-resistant HER2+ breast cancers with PI3K-independent activation of mTOR kinase, which may instead benefit from mTOR or Hsp90 inhibitors.

Combining lapatinib and pertuzumab to overcome lapatinib resistance due to NRG1-mediated signalling in HER2-amplified breast cancer.

Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment after their initial dephosphorylation. This was associated with increased expression of NRG1 in cells treated with lapatinib. Exogenous NRG1 partially rescued breast cancer cells from growth inhibition by lapatinib. In addition, both parental and lapatinib-resistant breast cancer cells were sensitive to SGP1, which inhibits binding of NRG1 and other HER3 ligands. Addition of pertuzumab to lapatinib further inhibited NRG1-induced signalling, which was not fully inhibited by either drug alone. In animal model, a combination of pertuzumab to lapatinib induced a greater tumor regression than either lapatinib or pertuzumab monotherapy. This novel combination treatment may provide a promising strategy in clinical HER2-targeted therapy and may inhibit a subset of lapatinib-resistant breast cancer, although the group of patients that will respond to this therapy requires further stratification.

Abstract 1828: A genomic-transcriptomic-phosphoproteomic study to interrogate lapatinib resistance in an HER2 over expressing SKBR3 human breast cancer cell line model

Abstract Background: Lapatinib is a tyrosine kinase inhibitor that inhibits EGFR and HER2 and is licensed to treat HER2-amplified breast cancer. Understanding mechanisms of acquired resistance is an unmet clinical need. Methods: SKBR3 cells were continuously exposed to increasing concentrations of lapatinib over a 12-month period. The parent cell line (SKBR3) and resulting lapatinib-resistant cell line (SKBR3LR) then underwent a set of studies including: a) western blot analysis for PGP, b) whole exome sequencing, c) whole genome expression microarray, d) quantification of 587 phosphoproteins. All data were integrated using Metacore software. Sensitivity of SKBR3 and SKBR3LR to a panel of drugs based on the findings was carried out by 96-hr sulforhodamine assays. Results: The GI50 values for lapatinib in SKBR3LR and SKBR3 cells were 3582 nM (SD 241.6) and 10.3 nM (SD 1.1). There was no induction of PGP protein or mRNA expression levels in SKBR3LR compared to SKBR3. Somatic mutations in KIT and MEK3 which were not present in SKBR3 were detected in SKBR3LR. There was a reduction in the levels of p-HER2 and p-EGFR in SKBR3LR; however, no differences in the levels of p-HER3, p-HER4, and p-KIT between the cell lines were observed. Analysis of proteins downstream in the RAS-RAF-MEK and PI3K-AKT-m-TOR showed increased levels of p-MEK but no significant changes in p38MAPK and no differences in p-AKT levels, but an increase in p-P70S6K in SKBR3LR compared to SKBR3. The phosphorylation network changes were integrated with transcription factors that showed significant enrichment for binding sites in the genes that were differentially expressed in SKBR3LR. Deregulation of the MAPK signalling network was one of the proposed mechanisms of resistance of SKBR3LR cells to lapatinib. Differential sensitivity to small-molecule agents was used to interrogate the mechanism of lapatinib resistance further. There was no significant differential sensitivity of SKBR3 and SKBR3LR to imatinib, GI50 values 16.2 uM (SD 1.2) and 13.64 uM (SD1.1), respectively. SKBR3LR was more resistant than SKBR3 to an AKT inhibitor AKT1/2i; GI50 values 8 uM (SD 0.45) vs 17.3 uM (SD 1.1) respectively. Interestingly, SKBR3LR was more sensitive to the HSP90 inhibitor, AUY922; GI50 values 3.2 nM (SD 1.1) and 11.2 nM (SD 0.42) in SKBR3LR and SKBR3 cells, respectively. Experiments are ongoing using targeted agents to understand the mechanism of lapatinib resistance and suggest potential therapeutic strategies to overcome these, including novel combinations, particularly the use of HSP90 inhibitors. Conclusions: Integrating genomic-transcriptomic and phosphoproteomic data provides useful insights into the mechanism of resistance to lapatinib. The lapatinib-resistant cell line model was sensitive to the HSP90 inhibitor, AUY922, suggesting HSP90 inhibitors could be clinically effective in this setting. Citation Format: Parames Thavasu, Robert Te Poele, Mark Hughes, Adam Stewart, Paul Workman, Udai Banerji. A genomic-transcriptomic-phosphoproteomic study to interrogate lapatinib resistance in an HER2 over expressing SKBR3 human breast cancer cell line model. [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 1828. doi:10.1158/1538-7445.AM2014-1828

Sorafenib/regorafenib and lapatinib interact to kill CNS tumor cells.

The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3-GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, BCL-XL, or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy.

Epidermal growth factor-receptor activation modulates Src-dependent resistance to lapatinib in breast cancer models.

IntroductionSrc tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib.MethodsTo determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses.ResultsSrc activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling.ConclusionsComplete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance.

Highlights of This Issue

Espin (ESPN) is a multifunctional actin-bundling protein and is an important regulator of cytoskeleton dynamics. At present, there have been no reports showing the expression and function of Espin in cancers, including melanoma. Yanagishita and colleagues reveal that Espin is strongly expressed in human melanomas as well as in RET-transgenic mice. Moreover, depletion of Espin has a dramatic impact on the metastatic activity of melanoma cells in vitro and in vivo. These findings reveal Espin not only as a novel metastatic regulator in melanoma but also as a potential biomarker for disease progression.The p53 tumor suppressor family member, p63 (TP63), plays a pivotal role in epidermal development, aging, and tumorigenesis. In an effort to understand the underlying mechanism by which p63 expression is controlled, Xu and colleagues identify the RNA-binding protein RBM24 as a novel regulator of p63 status via mRNA stability. Specifically, overexpression of RBM24 decreases, whereas knockdown of RBM24 increases, p63 expression. Mechanistically, RBM24 is able to bind to multiple regions in the 3′UTR of p63 and destabilize the p63 transcript. Together, these observations provide significant insight into the understanding of how p63 expression is regulated through a posttranscriptional mechanism.Management of epithelial ovarian cancer (EOC) that harbors wild-type BRCA remains a major challenge due to acquired chemoresistance and poor overall survival. Development of mechanism-based and/or targeted combination therapy approaches is critically needed for improved clinical outcome. Lin and colleagues demonstrate that triapine, a small molecule inhibitor of ribonucleotide reductase, sensitizes BRCA wild-type EOC cells to PARP and topoisomerase II inhibitors. Mechanistic studies demonstrate that triapine impairs homologous recombination repair (HRR) by obliterating CtIP phosphorylation, DNA double-strand break (DSB) resection, BRCA1 and Rad51 foci formation, and endonuclease-induced DSB repair. These findings provide a strong rationale for targeting HRR and using triapine to augment the efficacy of PARP and topoisomerase inhibitors for the treatment of BRCA wild-type EOC.HER2/Neu (ERBB2) is a tyrosine kinase and is a major contributor to disease progression in various types of breast cancer. Consequently, its biomarker status is fundamentally important and its activity is a key target for therapeutics. Despite initial efficacy, HER2-targeted treatment approaches eventually result in resistance. Here, Bailey and colleagues report that NF-κB activity renders HER2-positive cancers resistant to HER2-directed therapy. Interestingly, Lapatinib-resistant cells had altered NF-κB and were more sensitive to simultaneous HER2 and NF-κB inhibitor treatment than either therapy alone. Through the use of genomic analyses, a NF-κB signature was deduced that correlated with clinical outcome. Thus, this study implicates NF-κB in HER2 therapy resistance and suggests that combinatorial NF-κB and HER2 inhibition has therapeutic benefit.

NF-κB Activation-Induced Anti-apoptosis Renders HER2-Positive Cells Drug Resistant and Accelerates Tumor Growth

Breast cancers with HER2 overexpression are sensitive to drugs targeting the receptor or its kinase activity. HER2-targeting drugs are initially effective against HER2-positive breast cancer, but resistance inevitably occurs. We previously found that NF-κB is hyperactivated in a subset of HER2-positive breast cancer cells and tissue specimens. In this study, we report that constitutively active NF-κB rendered HER2-positive cancer cells resistant to anti-HER2 drugs and cells selected for lapatinib resistance upregulated NF-κB. In both circumstances, cells were antiapoptotic and grew rapidly as xenografts. Lapatinib-resistant cells were refractory to HER2 and NF-κB inhibitors alone but were sensitive to their combination, suggesting a novel therapeutic strategy. A subset of NF-κB-responsive genes was overexpressed in HER2-positive and triple-negative breast cancers, and patients with this NF-κB signature had poor clinical outcome. Anti-HER2 drug resistance may be a consequence of NF-κB activation, and selection for resistance results in NF-κB activation, suggesting that this transcription factor is central to oncogenesis and drug resistance. Clinically, the combined targeting of HER2 and NF-κB suggests a potential treatment paradigm for patients who relapse after anti-HER2 therapy. Patients with these cancers may be treated by simultaneously suppressing HER2 signaling and NF-κB activation. The combination of an inhibitor of IκB kinase (IKK) inhibitor and anti-HER2 drugs may be a novel treatment strategy for drug-resistant human breast cancers.

Evaluating HER2 amplification status and acquired drug resistance in breast cancer cells using Raman spectroscopy

The overexpression of human epidermal growth factor receptor 2 (HER2) is associated with increased breast cancer recurrence and worse prognosis. Effective treatments such as trastuzumab and lapatinib for patients with HER2 overexpression target the blockade of HER2 signaling activities but are often limited by the emergence of acquired drug resistance. This study applied Raman spectroscopy to differentially identify the amplification status of HER2 in cells and to characterize the biochemical composition of lapatinib resistant and sensitive HER2+ breast cancer cells in response to the drug. Raman spectra from BT474 (HER2+ breast cancer cell), MCF-10A (HER2- control), and HER2+ MCF-10A (HER2+ control) were analyzed using lasso and elastic-net regularized generalized linear models (glmnet) for multivariate statistical analysis and were discriminated to groups of different HER2 expression status with an overall 99% sensitivity and specificity. Enhanced lipid content and decreased proteome were observed in HER2+ cells. With lapatinib treatment, lapatinib-resistant breast cancer cells demonstrated sustained lipogenesis compared with the sensitive cells.

Enhanced PI3K p110α Signaling Confers Acquired Lapatinib Resistance That Can Be Effectively Reversed by a p110α-Selective PI3K Inhibitor

Although the HER2-targeting agents trastuzumab and lapatinib have improved the survival of patients with HER2-positive breast cancer, resistance to these targeted therapies is a major challenge. To investigate mechanisms of acquired lapatinib resistance, we generated acquired lapatinib resistance cell models by extended exposure of two HER2-positive breast cancer cell lines to lapatinib. Genomic and proteomic analyses revealed that lapatinib-resistant breast cancer cells gained additional phosphoinositide 3-kinase (PI3K) activation through activating mutation in PI3K p110α and/or increasing protein expression of existing mutant p110α. p110α protein upregulation in lapatinib-resistant cells occurred through gene amplification or posttranscriptional upregulation. Knockdown of p110α, but not p110β, the other PI3K catalytic subunit present in epithelial cells, inhibited proliferation of lapatinib-resistant cells, especially when combined with lapatinib. Lapatinib-resistant xenograft growth was inhibited persistently by combination treatment with the p110α-selective PI3K inhibitor BYL719 and lapatinib; the drug combination was also well tolerated in mice. Mechanistically, the combination of lapatinib plus BYL719 more effectively inhibited Akt phosphorylation and, surprisingly, Erk phosphorylation, than either drug alone in the resistance model. These findings indicate that lapatinib resistance can occur through p110α protein upregulation-mediated, and/or mutation-induced, PI3K activation. Moreover, a combinatorial targeted therapy, lapatinib plus BYL719, effectively overcame lapatinib resistance in vivo and could be further tested in clinical trials. Finally, our findings indicate that p110β may be dispensable for lapatinib resistance in some cases. This allows the usage of p110α-specific PI3K inhibitors and thus may spare patients the toxicities of pan-PI3K inhibition to allow maximal dosage and efficacy.

Abstract P5-08-03: In vitro resistance to ERBB2-targeted therapies does not bestow cross-resistance to antibody dependent cellular cytotoxicity

Abstract Background: The advent of ERBB2-directed therapies, including trastuzumab and lapatinib, has changed the clinical management of ERBB2+ breast cancer and improved patient outcomes. However, both de novo and acquired resistance to ERBB2-targeting agents remain significant clinical problems; thus, it is important to understand the mechanisms that tumor cells use to resist and/ or escape ERBB2-directed therapeutic agents. A growing body of evidence supports the role of immune effector cells in trastuzumab-induced tumor cell lysis in vivo, which is hypothesized to occur through antibody dependent cellular cytotoxicity (ADCC) mediated by natural killer cells bearing FC gamma receptors. Existing in vitro models of trastuzumab and lapatinib resistance are limited in that they are most often generated by incubating tumor cells with drug alone, outside the context of the host immune system. Consequently, the impact of in vitro generated drug resistance on ADCC susceptibility has not been defined. In this study, we tested existing trastuzumab and lapatinib resistant breast cancer cell lines for ADCC sensitivity. Materials and Methods: The ERBB2 amplified breast cancer cell lines BT474 and SKBR3, which are sensitive to trastuzumab-mediated ADCC, were selected for trastuzumab and lapatinib resistance. Drug resistant cells were generated by serial culture in the presence of escalating doses of trastuzumab and/or lapatinib until complete tolerance was acquired at clinically achievable concentrations (100μg/ml trastuzumab and 2.6μM lapatinib). Drug sensitivity was assessed by Alamar Blue assay. Cell resistance to trastuzumab-mediated ADCC was measured by Calcein-AM release assay, using 1μg/ml antibody concentration and four different effector target ratios (20:1, 10:1, 5:1, and 1:1), with a minimum of five replicates. Human peripheral blood lymphocytes freshly isolated from buffy coat were used as effector cells. Results and Discussion: Neither BT474 nor SKBR3 cells with acquired resistance to single agent trastuzumab (HerR), lapatinib (LapR), or both in combination (DualR) demonstrated cross-resistance to effector cell-mediated ADCC at any of the E:T ratios tested. At the highest E:T ratio tested (20:1), trastuzumab-induced ADCC of control BT474 cells resulted in 61.92% cell lysis (95% CI = 3.97), compared to 64.97% lysis for BT-HerR cells (95% CI = 2.87, Student's t-test p > 0.1). Similarly, BT-LapR and DualR cell lysis was 58.13% and 63.66%, respectively (95% CI = 2.62 and 6.57, Student's t-test p > 0.1). Additionally, trastuzumab-induced ADCC of control SKBR3 cells resulted in 89.98% cell lysis (95% CI = 4.29), compared to 93.53% lysis for SK-HerR cells (95% CI = 0.81, Student's t-test p > 0.1). SK-LapR and DualR cell lysis was 94.81% and 92.09%, respectively (95% CI = 0.6 and 2.66, Student's t-test p > 0.1). Our results indicate the importance of incorporating immune effector cells to in vitro models of trastuzumab resistance. Accordingly, we have begun modeling ADCC resistance in vitro to further investigate the relationship between targeted antibody therapies, effector cell-mediated cytolysis, and mechanisms of tumor immune escape. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-03.

Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO2) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/α-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.

Abstract A60: Activation of compensatory receptor tyrosine kinases after the acquisition of resistance to anti-Her2 therapies.

Abstract Background: Acquired resistance to therapy is a main obstacle in the success of cancer treatment. In HER2+ breast cancer (BC) patient survival has raised considerably after the introduction of the anti-target therapies Trastuzumab and Lapatinib. However, a considerable number of patients develop resistance to anti-HER2 therapies by HER2-related and unrelated mechanisms. Cancer cells displaying HER2 overexpression initially rely on the oncogenic addiction for HER2-controlled survival signaling pathways. However, the fact that some tumors resistant to anti-HER2 therapies still present HER2 amplification suggest the activation of compensatory pathways for the control of cell survival. Objectives: We aimed to identify compensatory survival pathways activated after the acquisition of resistance to Trastuzumab and Lapatinib that could be therapeutically targeted to overcome resistance to anti-HER2 therapies. We focus our efforts in identifying activated RTKs given that they are easily targetable proteins. Experimental Procedures: To identify compensatory pathways related to tumor escape to anti-HER2 therapies we developed an in vitro model of drug resistance by continuous exposure of the MDA-MB-453, BT474, and SKBR3 cell lines to increased concentrations of Trastuzumab or Lapatinib for over 6 months. Activated RTKs were identified by a phosphoproteome array, and their role in cell survival in cell lines resistant to anti-HER2 therapies was validated by siRNA and chemical inhibition experiments. Results: The resistant phenotype of the cell lines was confirmed by determining cell proliferation and apoptosis upon drug treatment. Moreover, the resistant cell lines displayed the more aggressive biological behavior in vitro and in vivo, as evidenced by the increased clonogenic ability and their tumorigenicity in immmunosupressed mice. We did not observed changes in the copy number of 17q12 (HER2) or 7p12 (EGFR) by FISH or in the mRNA levels by qPCR in any of the resistant cell lines, although there was a significant increase in the protein levels as determined by immunohistochemistry. By phosphoproteome array we identified the activation of several RTKs. The INGENUITY pathway analysis of the receptors identified indicated a main relevance for the processes related to cell signaling and cell cycle, cellular growth and proliferation, and more interestingly with axonal guidance signaling and human embryonic stem cell pluripotency. To confirm the involvement of those receptors in the activation of compensatory pathways in Trastuzumab and Lapatinib resistant cell lines, we performed a chemical screening. We further validated the involvement of several receptors in cell survival by siRNA. Conclusion: Several compensatory pathways for cell survival are activated after the acquisition of resistance to Trastuzumab and Lapatinib. The inhibition of such pathways could be used a new therapeutic approach for the treatment of resistant tumors, or to avoid the development of resistant cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A60. Citation Format: Patricia Fernandez Nogueira, Anke Bill, Inga Rye, Domiziana Costamagna, Hege Russnes, Pedro Gascón, Alex Gaither, Vanessa Almendro. Activation of compensatory receptor tyrosine kinases after the acquisition of resistance to anti-Her2 therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A60.

Src and CXCR4 are involved in the invasiveness of breast cancer cells with acquired resistance to lapatinib

Lapatinib is a dual EGFR and ErbB-2 tyrosine kinase inhibitor that has significantly improved the clinical outcome of ErbB-2-overexpressing breast cancer patients. However, patients inexorably develop mechanisms of resistance that limit the efficacy of the drug. In order to identify potential targets for therapeutic intervention in lapatinib-resistant patients, we isolated, from ErbB-2-overexpressing SK-Br-3 breast cancer cells, the SK-Br-3 Lap-R-resistant subclone, which is able to routinely grow in 1 µM lapatinib. Resistant cells have a more aggressive phenotype compared with parental cells, as they show a higher ability to invade through a matrigel-coated membrane. Lapatinib-resistant cells have an increased Src kinase activity and persistent levels of activation of ERK1/2 and AKT compared with parental cells. Treatment with the Src inhibitor saracatinib in combination with lapatinib reduces AKT and ERK1/2 phosphorylation and restores the sensitivity of resistant cells to lapatinib. SK-Br-3 Lap-R cells also show levels of expression of CXCR4 that are higher compared with parental cells and are not affected by Src inhibition. Treatment with saracatinib or a specific CXCR4 antibody reduces the invasive ability of SK-Br-3 Lap-R cells, with the two drugs showing cooperative effects. Finally, blockade of Src signaling significantly increases TRAIL-induced cell death in SK-Br-3 Lap-R cells. Taken together, our results demonstrate that breast cancer cells with acquired resistance to lapatinib have a more aggressive phenotype compared with their parental counterpart, and that Src signaling and CXCR4 play an important role in this phenomenon, thus representing potential targets for therapeutic intervention in lapatinib-resistant breast cancer patients.

Abstract B007: c-Src and PRC2 activity in ErbB2-driven mammary tumorigenesis and acquired resistance to ErbB2-targeted therapy

Abstract Elevated expression and activity of the non-receptor tyrosine kinase c-Src are associated with a poor prognosis in breast cancer (1). In this context c-Src, which has not been found to be mutated in breast cancer, is thought to be activated primarily through oncogenic signaling by upstream receptors. This frequently occurs in tumors driven by the overexpression of ErbB2 (2), a receptor tyrosine kinase (RTK) amplified in approximately 20-30% of all breast cancer cases (3). To investigate the mechanisms by which c-Src co-operates with activated RTK signaling to promote mammary tumorigenesis, we have developed transgenic mouse models allowing conditional c-Src ablation specifically in the mammary epithelium. In contrast to germline knockout of c-Src (4), mammary epithelial-specific deletion of c-Src does not affect ductal outgrowth. However, mammary epithelial c-Src knockout significantly delays tumorigenesis driven by ErbB2 or PyV mT, a viral oncogene that acts as an RTK mimic. The tumors that eventually develop exhibit an inflammatory and necrotic phenotype with severely impaired growth compared to wild-type controls. This phenotype correlates with alterations in the expression and activity of key metabolic regulators and with activation of the retinoblastoma tumor suppressor pathway. The expression of a known target of the Rb-E2F1 pathway, the lysine methyltransferase EZH2 (5), is also dependent on c-Src activity in transgenic mouse tumours and in human ErbB2-amplified breast cancer cell lines. A component of the Polycomb repressor complex 2 (PRC2) that di/tri-methylates histone H3 on lysine 27 (H3K27me2/me3), EZH2 is also a crucial promoter of ErbB2-driven tumour progression in vivo. Previous studies of cell line models have demonstrated that activation of Src family kinases (SFKs) is associated with the acquisition of resistance to targeted therapies directed against ErbB2 (6, 7). We demonstrate here that, in addition to elevated activity of SFKs, ErbB2+ breast cancer cell lines with acquired resistance to the ErbB2 tyrosine kinase inhibitor Lapatinib also overexpress EZH2 and have significantly increased H3K27me3 compared to parental cells. Chemical inhibition or RNAi-mediated silencing of either EZH2 or c-Src significantly attenuates the growth of Lapatinib-resistant cells, indicating a possible reversal of the drug-resistant phenotype. Overall, our data indicate that control of PRC2 expression and activity by c-Src may be an important factor in the progression of ErbB2-driven breast cancer and the acquisition of resistance to ErbB2-targeted therapies. 1. Elsberger B, Fullerton R, Zino S, Jordan F, Mitchell TJ, Brunton VG, et al. Breast cancer patients' clinical outcome measures are associated with Src kinase family member expression. Br J Cancer.103:899-909. 2. Wilson GR, Cramer A, Welman A, Knox F, Swindell R, Kawakatsu H, et al. Activated c-SRC in ductal carcinoma in situ correlates with high tumour grade, high proliferation and HER2 positivity. Br J Cancer. 2006;95:1410-4. 3. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177-82. 4. Kim H, Laing M, Muller W. c-Src-null mice exhibit defects in normal mammary gland development and ERalpha signaling. Oncogene. 2005;24:5629-36. 5. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. Embo J. 2003;22:5323-35. 6. Rexer BN, Ham AJL, Rinehart C, Hill S, de Matos Granja-Ingram N, Gonzalez-Angulo AM, et al. Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition. Oncogene. 2011. 7. Zhang S, Huang WC, Li P, Guo H, Poh SB, Brady SW, et al. Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways. Nat Med. 2011;17:461-9. Citation Format: Harvey W. Smith, Alison Hirukawa, Virginie Sanguin-Gendreau, Dongmei Zuo, William J. Muller. c-Src and PRC2 activity in ErbB2-driven mammary tumorigenesis and acquired resistance to ErbB2-targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B007.