Lapatinib Resistant Research Articles

Abstract 2703: Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106

Abstract Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is overexpressed in about 20% of breast cancers and most often in the more aggressive inflammatory breast cancer (IBC). Lapatinib is one of the most potent small molecule inhibitors of HER2. Although Lapatinib has shown to be an effective targeted therapy, development of drug resistance stands as a major problem. Here we show that the anti-cancer activity of the novel fatty acid synthase inhibitor HS-106 can re-sensitize resistant cell lines to Lapatinib. Lapatinib resistant SKBR3 and BT474 breast cancer cell lines were treated with increasing concentrations of HS-106 either alone or in combination with Lapatinib and their effects on cell proliferation were assessed using the DNA dye Hoechst 33342. The ability of HS-106 and Lapatinib drug combination to induce apoptosis were measured in Caspase 3/7 activity assay using the fluorogenic Caspase substrate (DEVD)2 R110. Their effect on apoptosis was further confirmed in Lapatinib resistant BT474 cells using flow cytometry based Annexin V assay. In addition, the effect of HS-106 on the expression levels and phosphorylation of the kinases involved in Her2 pathway including Her2, AKT and ERK were determined. The fatty acid synthase inhibitor HS-106 significantly inhibits the proliferation of Lapatinib resistant cell lines with concentrations. Interestingly, when combined with Lapatinib, the drugs synergize to dramatically affect cell growth. This anti-proliferative activity of HS-106 was found to be the result of induction of apoptosis, Caspase 3/7 activity was found to increase in a dose dependent manner following HS-106 treatment. Low concentrations of Lapatinib and HS-106, which does not induce Caspase activity when administrated separately, were found to significantly induce Caspase activity when combined together. These results were confirmed with the Annexin V assay. By examining the expression levels of HER2 pathway proteins we found a decrease in HER2 levels which was reflected on the levels of the downstream kinases AKT and ERK when treating with high concentrations of HS-106. In a previous work, HS-106 showed a good tolerability and anti-cancer activity in the MMTV neu+ mouse model. By showing potent efficacy against Lapatinib resistant breast cancer cell lines, our data suggest that HS-106 is an excellent drug candidate to advance into translational studies for the treatment of Lapatinib resistant breast cancer. Citation Format: Yazan Alwarawrah, Philip Hughes, Rachid Safi, Donald P. McDonnell, Neil L. Spector, Timothy A. Haystead. Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106. [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 2703. doi:10.1158/1538-7445.AM2015-2703

Abstract 3557: Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib

Abstract Deregulation of erbB-2 and EGFR is frequently detected in breast cancer, which has been associated with poor prognosis. Lapatinib, an orally active small molecule acting as a dual inhibitor of erbB-2 and EGFR, has been used for the treatment of patients with advanced or metastatic erbB-2+ breast cancer. Lapatinib resistance, however, is emerging as a clinical challenge. Understanding the molecular mechanisms of Lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance in breast cancer treatment. Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast and other types of cancers. Through interacting with protein phosphatase 2A (PP2A), CIP2A regulates c-Myc and factors involved in cell proliferation and survival. This study aimed to investigate the role of CIP2A in Lapatinib-mediated inhibition of erbB-2 overexpressing breast cancer cells. Our data showed that Lapatinib downregulated CIP2A in erbB-2 overexpressing SK-BR-3 and 78617 cells, which was correlated with concurrent inactivation of erbB-2, EGFR, Akt, Erk1/2 and mTOR. Overexpression of CIP2A rendered the cells resistant to Lapatinib induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via siRNA sensitized the cells to Lapatinib-mediated effects. We also demonstrated that Lapatinib-induced downregulation of CIP2A can be abolished by LY294002, suggesting that CIP2A downregulation was regulated by Akt. Analysis with cycloheximide (CHX) and MG132 indicated that Lapatinib modulates CIP2A protein stability and promotes its degradation through prosteasome pathway. More importantly, we found that Lapatinib induced differential downregulation of CIP2A between the parental and Lapatinib-resistant (LR) BT-474 cells. Decrease in CIP2A downregulation in BT-474-LR cells suggests its association with Lapatinib resistance. Knockdown of CIP2A via lentiviral siRNA system significantly sensitized Lapatinib induced growth inhibition and apoptosis. Taken together, our results demonstrate that CIP2A is a molecular target of Lapatinib, which plays a critical role in Lapatinib-induced cellular responses. Lapatinib-induced downregulation of CIP2A involves the inhibition of the Akt-CIP2A feedback loop. Correlation between Lapatinib resistance and decreased CIP2A downregulation, as well as CIP2A knockdown-mediated sensitization of BT-474-LR cells indicate that CIP2A is a resistance factor of Lapatinib. Further investigation on Lapatinib-mediated regulation of CIP2A will advance our understanding of Lapatinib-associated antitumor activities and drug resistance. Citation Format: Ming Zhao, Amanda Blackwelder, Harry Lee, Xiaohe Yang. Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib. [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 3557. doi:10.1158/1538-7445.AM2015-3557

Abstract 716: Targeting Mek-Erk pathway abrogates hypoxia-mediated lapatinib resistance in ErbB2-positive breast cancer cells

Abstract Overexpression/amplification of the tyrosine kinase ErbB2 is often observed in breast cancers. ErbB2 expression activates EGFR signaling pathway promoting tumorigenesis. Breast cancer ErbB2-positive patients are treated with lapatinib, a dual EGFR/ERBB2 inhibitor. Despite lapatinib clinical efficacy, acquired resistance has been reported and its mechanism is poorly understood. Another factor that is associated with mammary tumors is hypoxia. Hypoxia stabilizes hypoxia inducible factor 1 (HIF-1), a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors are often chemoresistant. Here, we show that hypoxia decreases lapatinib-mediated growth inhibition and apoptosis in three-dimensional cultures. In agreement with this data, hypoxia also promotes activation of signaling pathways downstream from ErbB2/EGFR including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 and can be abrogated by HIF-1 RNAi. Moreover stable overexpression HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to rescue cell proliferation and survival and maintain ERK and AKT signaling in the presence of lapatinib. We also show that activation of ERK signaling is required for hypoxia-mediated lapatinib resistance and inhibition of MEK (a kinase upstream of Erk) reverses hypoxia protective effect on ErbB2-expressing cells. One potential mechanism of how hypoxia/HIF-1 may lead to activation of ERK pathway is via regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of ERK pathway. Here we demonstrate that cells exposed to hypoxia have decreased levels of DUSP2 and it correlate with increased activation of Erk signaling. Moreover, reducing DUSP2 expression alone in ErbB2-positive breast cancer cells is sufficient to reduce lapatinib-mediated effects. And DUSP2 overexpression abrogates hypoxia-mediated protective effect on ErbB2-expressing cells treated with lapatinib. All these data suggests that targeting MEK-Erk signaling pathway may reverse hypoxia mediated lapatinib resistance and might be a viable option for lapatinib resistant ErbB2-posive tumors. Indeed, a combination of lapatinib and an FDA approved MEK inhibitor trametinib abrogates hypoxia protective effect on cell growth inhibition and apoptosis in three dimensional (3D) cultures. Thus targeting MEK/ERK pathway in hypoxic breast cancer may improve an anti-ErbB2 therapy in breast cancer. Citation Format: Sergey Karakashev, Mauricio Reginato. Targeting Mek-Erk pathway abrogates hypoxia-mediated lapatinib resistance in ErbB2-positive breast cancer cells. [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 716. doi:10.1158/1538-7445.AM2015-716

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.

Src mutation induces acquired lapatinib resistance in ERBB2-amplified human gastroesophageal adenocarcinoma models.

ERBB2-directed therapy is now a routine component of therapy for ERBB2-amplified metastatic gastroesophageal adenocarcinomas. However, there is little knowledge of the mechanisms by which these tumors develop acquired resistance to ERBB2 inhibition. To investigate this question we sought to characterize cell line models of ERBB2-amplified gastroesophageal adenocarcinoma with acquired resistance to ERBB2 inhibition. We generated lapatinib-resistant (LR) subclones from an initially lapatinib-sensitive ERBB2-amplified esophageal adenocarcinoma cell line, OE19. We subsequently performed genomic characterization and functional analyses of resistant subclones with acquired lapatinib resistance. We identified a novel, acquired Src E527K mutation in a subset of LR OE19 subclones. Cells with this mutant allele harbour increased Src phosphorylation. Genetic and pharmacologic inhibition of Src resensitized these subclones to lapatinib. Biochemically, Src mutations could activate both the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways in the lapatinib-treated LR OE19 cells. Ectopic expression of Src E527K mutation also was sufficient to induce lapatinib resistance in drug-naïve cells. These results indicate that pathologic activation of Src is a potential mechanism of acquired resistance to ERBB2 inhibition in ERBB2-amplified gastroesophageal cancer. Although Src mutation has not been described in primary tumor samples, we propose that the Src hyperactivation should be investigated in the settings of acquired resistance to ERBB2 inhibition in esophageal and gastric adenocarcinoma.

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 950: Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models.

Abstract Background. The dual EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib is approved in metastatic breast cancer patients after trastuzumab failure. Unfortunately, several patients do not respond to treatment due to constitutive resistance, and even in responders the occurrence of disease progression is often observed. Recently, the activation of Src TK has been associated with resistance to anti-HER2 drugs, both trastuzumab and lapatinib, in HER2 overexpressing breast cancer cells. However, the molecular mechanisms by which Src activation may affect response to HER2 antagonists are still partially unclear. Methods. We used a panel of HER2 expressing human breast cancer cell lines with different degree of response to lapatinib: SKBR3, BT474 and MDA-MB-361 (high sensitivity), KPL-4 (moderate sensitivity), JIMT-1 (low sensitivity). Moreover, we generated a cell line with acquired resistance to lapatinib, MDA-MB-361-LR (Lapatinib Resistant). On these models, we investigated the effect of Src silencing via small interference RNA (siRNA) or pharmacological inhibition by saracatinib on lapatinib response, both in vitro and in vivo. Results. The combination of Src inhibition with lapatinib synergistically inhibited survival of human breast cancer cells with both intrinsic or acquired resistance to lapatinib, by interfering with signal transducers involved in cell proliferation and survival. Moreover, both saracatinib and Src specific siRNAs were able to reduce migration and invasion capabilities of breast cancer cells, by affecting Focal Adhesion Kinase (FAK) and paxillin activation. In nude mice xenografted with JIMT-1 cells, the combined treatment with saracatinib and lapatinib produced a cooperative antitumor effect with inhibition of tumor growth, interference with signal transduction and prolongation of mice survival. The combination strongly reduced the incidence of lung metastasis, as revealed through experimental metastasis assays. Interestingly, a significant reduction of EGFR/HER2 co-immunoprecipitation was found in resistant cells, thus suggesting that growth signals could be transduced by other type of TKRs dimers in these cells. Moreover, in breast cancer cell lines with both intrinsic or acquired resistance to lapatinib, Src interacted with EGFR rather than with HER2, in opposition to what was observed in sensitive cells. Consistently, EGFR inhibition through cetuximab or EGFR specific siRNAs was able to partially restore lapatinib sensitivity in resistant cells, by interfering with the reciprocal activation of EGFR and Src. Conclusions. Src plays an important role in the onset of resistance to lapatinib in breast cancer cell lines, and its interaction with EGFR may be involved in this event. Therefore, the combined inhibition of Src and EGFR/HER2 axis may represent a potentially effective therapeutic strategy in order to prevent or overcome resistance to lapatinib. Citation Format: Roberta Rosa, Lucia Nappi, Luigi Formisano, Claudia D'Amato, Valentina D'Amato, Vincenzo Damiano, Roberta Marciano, Ana Paula De Maio, Lucia Raimondo, Umberto Malapelle, Giancarlo Troncone, Sabino De Placido, Roberto Bianco. Src tyrosine kinase contributes to lapatinib resistance in human breast cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2013-950 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

144 Alterations in Apoptosis-related Genes in Cell Line Models of Acquired Lapatinib Resistance, Identifies Potential Therapeutic Targets for the Treatment of Lapatinib Resistant HER2-positive Breast Cancer

144 Alterations in Apoptosis-related Genes in Cell Line Models of Acquired Lapatinib Resistance, Identifies Potential Therapeutic Targets for the Treatment of Lapatinib Resistant HER2-positive Breast Cancer

Abstract 1887: Targeting oncoprotein stability overcomes lapatinib-resistance due to ERBB2 kinase domain mutations

Abstract ERBB2 kinase domain mutations were recently reported in some solid cancers. Moreover, certain ERBB2 mutations were shown to cause lapatinib resistance in vitro thus predicting their emergence in treated patients. We have recently shown that ERBB2-L755S, ERBB2-L755P and ERBB2-T798M mutants cause lapatinib resistance by stabilizing the active kinase conformation (DFG-in), which is incompatible with lapatinib binding (DFG-out). Using an in vitro cell-based drug resistance screen we also showed that these lapatinib resistant mutations might cause secondary resistance in patients treated with lapatinb. Thus, it is important to search for alternate treatment strategies to overcome lapatinib resistance. ERBB2 kinase is a client for HSP90 and is degraded by HSP90 inhibitor treatment. We thus tested if targeting ERBB2 mutant stability by inhibiting HSP90 overcomes lapatinib resistance. Since the kinase domain is important for the ERBB2 interaction with HSP90, we tested whether ERBB2 kinase domain mutants retained their interaction with the chaperone. Co-immunoprecipitation analysis showed that the interaction of ERBB2 mutants with HSP90 is intact. Importantly, HSP90 inhibitor treatment resulted in the degradation of lapatinib-resistant ERBB2 mutants as observed with the wild type ERBB2 kinase. Thus, HSP90 inhibitors may offer an alternative treatment option to overcome primary or secondary lapatinib resistance in patients harbouring ERBB2 mutations. Moreover, combined targeting of different physiological aspects (enzyme activity and protein stability) may prevent the emergence of secondary drug resistance due to kinase domain mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1887. doi:1538-7445.AM2012-1887

Abstract 1910: Acquired resistance to HER2 targeted therapy: A common mechanism

Abstract There are currently two FDA approved targeted therapies being utilized in the clinic to treat Her2+ breast cancer, trastuzumab and lapatinib. Acquired resistance has become a major concern and several mechanisms for resistance have been proposed. The upregulation of a truncated transcriptional variant of Darpp-32, known as t-Darpp, is a characteristic shared by several independently developed trastuzumab-resistant cell lines. Moreover, exogenous t-Darpp overexpression is sufficient for conferring trastuzumab resistance. No consistent mechanism, however, has been identified for lapatinib resistant cells. By inhibiting signaling through Her2+ pathways such as PI3K/Akt and Erk, lapatinib induces a strong apoptotic response by down-regulating survivin and inducing BIM. Darpp-32 and t-Darpp both have been linked to apoptosis, with t-Darpp identified as having anti-apoptotic properties through its regulation of Bcl-2. The purpose of the current study was to determine if t-Darpp or other dysregulated apoptosis proteins play a role in lapatinib resistance. Lapatinib-resistant SKBR3 cells (Her2+, ER-) were selected by continuous exposure to increasing concentrations of lapatinib (0.3-2.0µM) over the course of one year. Using Sulforhodamine B and colony forming assays, we found that individual clones and pools of SK/LapR cells were, on average, 50-fold resistant to lapatinib, relative to parental SKBR3 cells. This resistance phenotype was stable even after cells were maintained for 1 month outside of drug. As observed in trastuzumab resistance and similar to previous reports of lapatinib-resistant cells, we found that SK/LapR cells had sustained PI3K/Akt signaling in the presence of lapatinib, whereas parental cells did not. Additionally, survivin inactivation and BIM induction by lapatinib seemed to be partially impaired in SK/LapR cells. Finally, SK/LapR cells appeared to down-regulate Darpp-32 and up-regulate t-Darpp, similar to what we have reported in BT474 cells selected for resistance to trastuzumab. Although t-Darpp overexpression is sufficient to confer resistance to trastuzumab, this does not appear to be the case for lapatinib resistance – SK.tDp cells transfected with a t-Darpp cDNA are not resistant to lapatinib. Experiments are currently underway to determine if the t-Darpp overexpression that we observe in SK/LapR cells is required for lapatinib resistance. Identifying a shared mechanism of resistance to lapatinib and trastuzumab would provide a novel therapeutic target that could be widely used among Her2+ breast cancer patients to ameliorate acquired drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1910. doi:1538-7445.AM2012-1910

PD01-09: Identifying Novel Mechanisms of Resistance to Lapatinib in ERBB2+ Breast Cancer Cells through Whole Genome Mutational Analysis.

Abstract Background: Overexpression of the epidermal growth factor receptor ERBB2 (HER2) is found in 20% of human breast cancers. Therapies targeting ERBB2 including trastuzumab and lapatinib have significantly improved the outlook for women with ERBB2+ breast cancer. However, resistance to these agents occurs frequently and remains a significant clinical problem. In the case of lapatinib resistance, the mechanism(s) of resistance remain poorly understood, since the current proposed rationale thought to limit lapatinib's anti-tumor effects has been difficult to reconcile with clinical data. Therefore, we hypothesize that novel mechanisms of resistance could be identified by mapping genomic variations in ERBB2+ cells with acquired resistance to lapatinib. The identification of such mutations may provide insights into mechanisms of resistance and may indicate therapeutic strategies to overcome lapatinib resistance in ERBB2+ breast cancer. Material and Methods: SKBR3 breast cancer cells resistant to lapatinib were generated through serial passage by exposure of drug sensitive parental SKBR3 cells to increasing concentrations of lapatinib up to the peak plasma concentration observed in human subjects (2.6 uM (SK-lapR)). Multiple signaling pathways in lapatinib sensitive and resistant cells were interrogated by Reverse Phase Protein Array (RPPA) and western blot analysis. To identify genome wide somatic mutations, the Exome of lapatinib resistant and sensitive SKBR3 cells was sequenced utilizing next generation deep sequencing. Following exclusion of germline variants, the acquired gene mutations in lapatinib resistant SKBR3 cells were confirmed by DNA re-sequencing of PCR amplified DNA segments. Results and Discussion: Analysis of activated signaling pathways in lapatinib resistant and sensitive SKBR3 cells did not confirm any of the previously proposed mechanisms of resistance. In particular, these cells show no activation of AKT or alternative receptor tyrosine kinases such as IGF-IR, ERBB3 or c-Met. However they exhibit sustained activation of mTORC1 and ERK1/2, as well as phosphorylation of STAT3, STAT5, rpS6 and CREB. Initial sequence analysis of exome and transcriptome reveals the presence of 76 single nucleotide variants/Indels differing between sensitive and resistant cells with 34/76 validated as true mutations present in the genome of lapatinib resistant SKBR3 cells, including mutations in LATS2, MAP3K5, SMAD3 and PDGFRA. This is the first exome sequence analysis to be reported which defines a drug resistant phenotype in ERBB2+ breast cancer. Ongoing work includes investigation of mutations as drug resistance mediators and analysis of copy number variations and gene fusions/translocations to systematically search for molecular alterations, with the goal of providing a rationale for the design of new combination therapies aimed at lapatinib resistance for ERBB2+ breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-09.

P5-06-09: Acquired Sensitivity to TRAIL Mediated Apoptosis in Lapatinib Resistant SKBR3 Cells.

Abstract Introduction Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor is approved for the treatment of trastuzumab-refractory HER2 positive metastatic breast cancer. However, not all HER2 positive tumors respond to lapatinib and patients who initially respond frequently relapse. Understanding the molecular alterations associated with acquisition of lapatinib resistance may lead to the identification of targets to overcome resistance. Thus, the aim of this study was to examine changes in apoptosis in a model of acquired lapatinib resistance. Methods SKBR3 cells were treated with 250 nM lapatinib twice weekly for 6 months to establish the lapatinib resistant cell line SKBR3-L. The effects of TNF-related apoptosis-inducing ligand (TRAIL) and tumour necrosis factor-α (TNF-α) on cell survival and apoptosis induction were examined in SKBR3 and SKBR3-L cells, using the acid phosphatase proliferation assay and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Cleavage of poly ADP ribose polymerase (PARP) was used to confirm apoptosis. TRAIL-1 and TRAIL-2 receptor expression were measured by flow cytometry (FACS). Results In SKBR3-L cells, TRAIL treatment (25 ng/ml) significantly inhibited cell survival (94.0 ± 12.0 %) compared to the SKBR3 cells (11.0 ± 2.2%). Treatment with TNF-α (0.5 ng/ml) also resulted in significant inhibition of cell survival (68.2 ± 10.1 %) in SKBR3-L cells compared to SKBR3 cells (9.4 ± 9.0 %). TRAIL (25 ng/ml) induced a low level of apoptosis in SKBR3 cells (7.0 ± 4.0%), whereas in SKBR3-L cells, TRAIL (25 ng/ml) induced significant apoptosis (54.8 ± 2.6%, p < 0.001), as determined by the TUNEL assay. We confirmed by western blotting that TRAIL treatment for 72 hours resulted in higher levels of PARP cleavage in SKBR3-L cells compared to SKBR3 cells. Both western blotting and FACS analysis of TRAIL receptor expression showed that neither TRAIL-1 nor TRAIL-2 receptor expression was significantly elevated in SKBR3-L cells compared to SKBR3 cells. Conclusions SKBR3-L cells, a model of acquired lapatinib resistance, display significant sensitivity to TRAIL induced apoptosis compared to the parental cell line SKBR3. Thus, targeting TRAIL may represent a novel therapeutic strategy to overcome acquired lapatinib resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-09.

P1-12-06: The Role of MAPK and PI3K/AKT/mTOR Signaling in Innate Lapatinib Resistance.

Abstract Background: Lapatinib is a tyrosine kinase inhibitor which blocks downstream MAPK and P13K/AKT/mTOR proliferation and survival signaling pathways in HER2 positive breast cancer cell lines, tumor xenografts and HER2 positive breast cancer patients. However, pre-clinical and clinical studies have shown that not all HER2 positive patients respond to lapatinib and thus innate resistance to lapatinib exists. The identification of biomarkers of lapatinib response is therefore critical and would enable individual therapeutic decisions to be based on tumor biology rather than basic histopathology data alone. The aim of this study was to examine the role of MAPK and PI3K/AKT/mTOR signaling in a panel of lapatinib sensitive and resistant HER2−amplified breast cancer cell lines to identify pharmacodynamic markers of response to lapatinib treatment. Methods: Dose response curves were generated to determine sensitivity to lapatinib in a panel of 17 HER2−amplified breast cancer cell lines. Total and phosphorylated levels of HER2, HER3, EGFR, AKT, ERK, S6K and eEF2 were determined following 24 hours lapatinib treatment in each of the cell lines. Results: Twelve of the cell lines were sensitive to lapatinib with IC50g < 1 μM while 5 of the cell lines were innately resistant to lapatinib with IC50g > 1 μM. Levels of pHER2 and pHER3 were decreased in response to lapatinib in all cell lines independent of sensitivity to lapatinib. This suggests that inhibition of HER2/HER3 activation is not indicative of response to lapatinib treatment. There was also no correlation between the levels of HER2, HER3, and EGFR and sensitivity to lapatinib in the cell line panel. In lapatinib sensitive cell lines, lapatinib decreased PI3K (pAKT), mTOR (pS6K) and MAPK (pERK) signaling and increased peEF2 levels. In contrast the levels of pAKT, pS6K, peEF2 and pERK were maintained following lapatinib treatment in lapatinib resistant cells. The continued activation of these proteins in the presence of lapatinib suggests a possible feedback mechanism that warrants further investigation. These data confirm that maintained signaling through either the P13K/AKT/mTOR pathway or the MAPK pathways in the presence of lapatinib can be an early pharmacodynamic biomarker of response. Conclusions: Maintenance of pAKT, pS6K, peEF2 and pERK levels, in response to lapatinib treatment correlates with lapatinib resistance. These data suggest that alterations in the P13K/AKT/mTOR and MAPK pathways play an important role in innate lapatinib resistance and pharmacologically targeting these pathways is a rational therapeutic approach for overcoming innate lapatinib resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-06.

Autophagy facilitates the Lapatinib resistance of HER2 positive breast cancer cells

ErbB2 receptor (HER2) tyrosine kinase was overexpressed in about 25% breast cancers, and was correlated with extremely aggressive phenotype and poor prognosis. Lapatinib, an oral, reversible inhibitor of both ErbB2 and EGFR tyrosine kinases, was approved in combination with capecitabine for treating advanced stage ErbB2 positive breast cancers. However, the clinical response of Lapatinib was seriously limited by the drug resistance. We established the Lapatinib resistant breast cancer cell lines and the preliminary data demonstrated the increased autophagosome formation in the stable resistant cells. The resistant cells were re-sensitized to Lapatinib after treated with autophagy inhibitor. According to our preliminary data and related reference, we hypothesized that autophagy could facilitate the ErbB2 positive breast cancer cells to be Lapatinib resistant and promoted the survival of the resistant cells. The abrogation of autophagy might restore the drug sensitivity. Autophagy might be one of the targets to overcome the Lapatinib resistance.

Abstract P4-01-05: Lapatinib Resistance Confers Cross-Resistance to Microtubule Inhibitors in ErbB2-Overexpressing Breast Cancer Cells

Abstract ErbB2 gene amplification occurs in approximately 20% of all breast cancers and is associated with an aggressive subtype with poor clinical prognosis. Acquired resistance to erbB2-targeted therapies is a common occurance even in patients who show an initial clinical response to trastuzumab or lapatinib-based regimens. Optimal treatment for relapsed disease following erbB2 targeted therapies has not been defined. Therefore, we sought to evaluate whether cytotoxic agents have cross-resistance to lapatinib in cell lines that have acquired resistance to lapatinib. Here we established a model of acquired lapatinib resistance derived from erbB2 overexpressing breast cancer cell lines, BT474 and SKBR3. Lapatinib resistant cell lines, BTLapR and SKLapR, were generated by growth in the continuous presence of 2.6 uM lapatinib (peak plasma concentration in human subjects) resulting in IC50 of 4.72 uM (BTLapR) and 0.083 uM (BT474) (P<0.0001). IC50 of SKLapR and SKBR3 were 17.72 uM and 0.1637 uM (P<0.0001), respectively. Next, resulting cells were treated with different classes of chemotherapeutics (2 taxanes, 2 vinca-alkaloids, 1 epothilone, 1 anthracycline, 1 antimetabolite, 2 platinums, and 1 alkylating agent) in order to address cross-resistance to lapatinib. BT474 and BTLapR as well as SKBR3 and SKLapR were exposed to each chemotherapeutic agent and cell viability was evaluated at 72h using a cell proliferation assay (AlamarBlue®). We found that BTLapR was significantly cross-resistant to 5 microtubule inhibitors (paclitaxel, docetaxel, vinorelbine, vinblastine, and ixabepilone), but SKLapR was not (shown in table below). IC 50 of Significant Drugs on Lapatinib Resistant BT474 cells Previous studies have shown that resistance to lapatinib can switch cell survival signaling from the erbB2 to ER pathway, and that ER signaling can stimulate beta III tubulin expression. We therefore hypothesize the differential responses to microtubule inhibitors in BTLapR and SKLapR might be explained by the fact that BT474 has both estrogen (ER) and progesterone receptor (PR), but SKBR3 express neither. Moreover, beta-III tubulin is known to be a key resistance mechanism of microtubule inhibition. Western blot analysis of baseline protein expression in both acquired lapatinib-resistant and parental cells indicates that ER signaling is involved in stimulating beta III tubulin and is linked to the development of resistance to microtubule inhibitors. These findings point to a potential mechanism to address cross resistance between lapatinib and microtubule-interacting agents, which could have potential clinical implications when considering salvage therapies following lapatinib treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-05.

Abstract 613: Increased PI3K/AKT activity, which confers resistance to trastuzumab, can be overcome by lapatinib

Abstract Trastuzumab and lapatinib provide clinical benefit to women with HER2 positive breast cancer. However, not all patients whose tumors contain the HER2 alteration respond. There are no clinically validated biomarkers of resistance to therapy. We evaluated responses to trastuzumab and lapatinib in a panel of 17 HER2-amplified cell lines in both two and three dimensional culture and investigated the role of receptor tyrosine kinase (RTK) signaling and PI3K/AKT pathway activation in resistance. We also conditioned four cell lines to acquire trastuzumab resistance, and two additional cell lines to acquire lapatinib resistance. The SUM-225, HCC-1419, HCC-1954, UACC-893, HCC-1569, UACC-732, JIMT-1 and MDA-453 cell lines are innately resistant to trastuzumab, whereas the MDA-361, MDA-453, HCC-1569, UACC-732, JIMT-1, HCC-202 and UACC-893 cells are innately lapatinib resistant. Lapatinib is active in both de novo (SUM-225, HCC-1419 and HCC-1954) and acquired trastuzumab resistant cell lines. Increased phosphorylation of HER2, EGFR, HER3 and IGF-1R correlated with response to lapatinib but not trastuzumab. p95HER2 levels correlated with response to lapatinib, however p95HER2 levels also correlated with levels of full length HER2, indicating that p95HER2 may not be an independent marker of lapatinib response. A decrease in AKT phosphorylation in response to trastuzumab or lapatinib treatment correlated significantly with response to either agent, indicating that loss of pAKT is a biomarker of response to both HER2-targeted therapeutics. Accordingly, trastuzumab treatment of trastuzumab resistant cells had little effect on AKT phosphorylation, whereas treatment of these cells by lapatinib significantly reduced pAKT levels. Thus, lapatinib retains its activity in trastuzumab resistance via continued dephosphorylation of AKT. We also observed that loss of PTEN or the presence of activating mutations in PI3K marked resistance to trastuzumab yet lapatinib response was independent of these factors. Thus, increased activation of the PI3K/AKT pathway confers resistance to trastuzumab which can be overcome by lapatinib. In conclusion, pharmacological targeting of the PI3K/AKT pathway may provide benefit to HER2 positive breast cancer patients who are resistant to trastuzumab therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 613.

Intracellular Src Family Kinases Mediate PI3K-Akt Pathway Activation and Resistance to Lapatinib in HER2-Overexpressing Human Breast Cancer Cells.

Abstract We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, and SUM190) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots was undetectable in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein subtrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in BT-474 and HCC1954 LR cells compared to drug-sensitive cells. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 sensitive cells with AZD0530 and Lap for 14 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 707.

Abstract PR-6: Src family kinases mediate PI3K-Akt pathway activation and resistance to lapatinib in HER2-overexpressing human breast cancer cells

Abstract We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, SUM190, and UACC893) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots remained inhibited in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein substrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in four of the LR lines: BT-474, MDA-MB-361, HCC1954, and UACC893. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases, whereas Yes was the predominant SFK in MDA-MB-361 and UACC893 cells. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 or MDA-MB-361 sensitive cells with Src inhibitors and Lap for 21 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-6.