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Supplementary Table 2 from Activated Phosphoinositide 3-Kinase/AKT Signaling Confers Resistance to Trastuzumab but not Lapatinib

Supplementary Table 1 from Activated Phosphoinositide 3-Kinase/AKT Signaling Confers Resistance to Trastuzumab but not Lapatinib

Trastuzumab and lapatinib provide clinical benefit to women with human epidermal growth factor receptor 2 (HER)–positive breast cancer. However, not all patients whose tumors contain the HER2 alteration respond. Consequently, there is an urgent need to identify new predictive factors for these agents. The aim of this study was to investigate the role of receptor tyrosine kinase signaling and phosphoinositide 3-kinase (PI3K)/AKT pathway activation in conferring resistance to trastuzumab and lapatinib. To address this question, we evaluated response to trastuzumab and lapatinib in a panel of 18 HER2-amplified cell lines, using both two- and three-dimensional culture. The SUM-225, HCC-1419, HCC-1954, UACC-893, HCC-1569, UACC-732, JIMT-1, and MDA-453 cell lines were found to be 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 was active in de novo (SUM-225, HCC-1419, and HCC-1954) and in a BT-474 cell line with acquired resistance to trastuzumab. In these cells, trastuzumab had little effect on AKT phosphorylation, whereas lapatinib retained activity through the dephosphorylation of AKT. Increased phosphorylation of HER2, epidermal growth factor receptor, HER3, and insulin-like growth factor IR correlated with response to lapatinib but not trastuzumab. Loss of PTEN or the presence of activating mutations in PI3K marked resistance to trastuzumab, but lapatinib response was independent of these factors. Thus, increased activation of the PI3K/AKT pathway correlates with resistance to trastuzumab, which can be overcome by lapatinib. In conclusion, pharmacologic targeting of the PI3K/AKT pathway may provide benefit to HER2-positive breast cancer patients who are resistant to trastuzumab therapy. Mol Cancer Ther; 9(6); 1489–502. ©2010 AACR.

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Supplementary Figure 3 from Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

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Supplementary Table 5 from Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

Supplementary Table 3 from Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

Supplementary Figure 1 from Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

Supplementary Figures PDF file - 1342K, Supplementary Figures for Croucher et al. Supplementary Figure 1: SILAC-based (phospho)-proteomic analysis in basal breast cancer cell lines following Lyn knockdown. Supplementary Figure 2: Lyn regulates a small fraction of the SFK signaling network in basal breast cancer cells. Supplementary Figure 3: Specificity of the SgK269 pY635 antibody. Supplementary Figure 4: Effect of Lyn knockdown on protein tyrosine phosphorylation. Supplementary Figure 5: Sub-cellular location of SgK269. Supplementary Figure 6: Knockdown of Src does not affect SgK269 Y635 phosphorylation. Supplementary Figure 7: Functional role of SgK269 in MDA-MB-231 cells.

Supplementary Figure Legends 1-3 from Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

Suppl Material & Suppl. Table S1

Supplementary Figure 2 from Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

Supplementary Table 4 from Tyrosine Phosphorylation Profiling Reveals the Signaling Network Characteristics of Basal Breast Cancer Cells

Intrinsic and acquired resistance to HER-targeting drugs occurs in a significant proportion of HER2-overexpressing breast cancers. Thus, there remains a need to identify predictive biomarkers that could improve patient selection and circumvent these types of drug resistance. Here, we report the identification of neuromedin U (NmU) as an extracellular biomarker in cells resistant to HER-targeted drugs. NmU overexpression occurred in cells with acquired or innate resistance to lapatinib, trastuzumab, neratinib, and afatinib, all of which displayed a similar trend upon short-term exposure, suggesting NmU induction may be an early response. An analysis of 3,489 cases of breast cancer showed NmU to be associated with poor patient outcome, particularly those with HER2-overexpressing tumors independent of established prognostic indicators. Ectopic overexpression of NmU in drug-sensitive cells conferred resistance to all HER-targeting drugs, whereas RNAi-mediated attenuation sensitized cells exhibiting acquired or innate drug resistance. Mechanistic investigations suggested that NmU acted through HSP27 as partner protein to stabilize HER2 protein levels. We also obtained evidence of functional NmU receptors on HER2-overexpressing cells, with the addition of exogenous NmU eliciting an elevation in HER2 and EGFR expression along with drug resistance. Finally, we found that NmU seemed to function in cell motility, invasion, and anoikis resistance. In vivo studies revealed that NmU attenuation impaired tumor growth and metastasis. Taken together, our results defined NmU as a candidate drug response biomarker for HER2-overexpressing cancers and as a candidate therapeutic target to limit metastatic progression and improve the efficacy of HER-targeted drugs. Cancer Res; 74(14); 3821–33. ©2014 AACR.

Although HER2 targeted therapies have improved prognosis for HER2 positive breast cancer, HER2 positive cancers which co-express ER have poorer response rates to standard HER2 targeted therapies, combined with chemotherapy, than HER2 positive/ER negative breast cancer. Administration of hormone therapy concurrently with chemotherapy and HER2 targeted therapy is generally not recommended. Using publically available gene expression datasets we found that high expression of IGF1R is associated with shorter disease-free survival in patients whose tumors are ER positive and HER2 positive. IGF1R is frequently expressed in HER2 positive breast cancer and there is significant evidence for crosstalk between IGF1R and both HER2 and ER. Therefore, we evaluated the therapeutic potential of targeting ER and IGF1R in cell line models of HER2/ER/IGF1R positive breast cancer, using tamoxifen and two IGF1R targeted tyrosine kinase inhibitors (NVP-AEW541 and BMS-536924). Dual inhibition of ER and IGF1R enhanced growth inhibition in the four HER2 positive cell lines tested and caused an increase in cell cycle arrest in G1 in BT474 cells. In addition, combined treatment with trastuzumab, tamoxifen and either of the IGF1R TKIs enhanced response compared to dual targeting strategies in three of the four HER2 positive breast cancer cell lines tested. Furthermore, in a cell line model of trastuzumab-resistant HER2 positive breast cancer (BT474/Tr), tamoxifen combined with an IGF1R TKI produced a similar enhanced response as observed in the parental BT474 cells suggesting that this combination may overcome acquired trastuzumab resistance in this model. Combining ER and IGF1R targeting with HER2 targeted therapies may be an alternative to HER2 targeted therapy and chemotherapy for patients with HER2/ER/IGF1R positive breast cancer.

Prolonged treatment of HER2 positive breast cancer cells with tyrosine kinase inhibitors (TKIs) leads to the emergence of acquired resistance. However, the effects of continuous TKI exposure on cell fate, and the steps leading to the acquisition of a resistant phenotype are poorly understood. To explore this, we exposed five HER2 positive cells lines to HER2 targeted therapies for periods of up to 4 weeks and examined senescence associated &beta, galactosidase (SA-&beta, gal) activity together with additional markers of senescence. We found that lapatinib treatment resulted in phenotypic alterations consistent with a senescent phenotype and strong SA-&beta, gal activity in HER2-positive cell lines. Lapatinib-induced senescence was associated with elevated levels of p15 and p27 but was not dependent on the expression of p16 or p21. Restoring wild type p53 activity either by transfection or by treatment with APR-246, a molecule which reactivates mutant p53, blocked lapatinib-induced senescence and caused increased cell death. In contrast to lapatinib, SA-&beta, gal activity was not induced by exposing the cells to trastuzumab as a single agent but co-administration of lapatinib and trastuzumab induced senescence, as did treatment of the cells with the irreversible HER2 TKIs neratinib and afatinib. Neratinib- and afatinib-induced senescence was not reversed by removing the drug whereas lapatinib-induced senescence was reversible. In summary, therapy-induced senescence represents a novel mechanism of action of HER2 targeting agents and may be a potential pathway for the emergence of resistance.

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

Background: HER2-positive breast cancer (BC) accounts for approximately 15% of all BC. HER2-targeted therapies, such as trastuzumab and lapatinib, have significantly improved the outcome for these patients. However, HER2-targeted therapy resistance is a common clinical issue. We have previously shown that protein phosphatase 2A (PP2A) plays a role in mediating acquired lapatinib resistance in HER2-positive BC and that response to lapatinib is enhancedin vitroby the lab-grade PP2A inhibitor, okadaic acid. The aim of this study was to examine the in vitro and in vivo efficacy of LB-100, a PP2A inhibitor that has completed phase I clinical testing (NCT01837667), in models of HER2-positive BC with acquired resistance to lapatinib. Methods: HER2-positiveSKBR3 and HCC1954 BC cell lines were treated with 250 nM or 1 μM lapatinib, respectively, for 6 months to generate lapatinib-resistant SKBR3-L and HCC1954-L cell lines. In vitro sensitivity to lapatinib and LB-100 was assessed by 2D acid phosphatase assay. Combination index (CI) values were generated to identify synergistic combinations. Propidium iodide staining was used to determine cell cycle arrest and apoptosis. In order to examine the in vivo efficacy of LB-100, HCC1954-L cells were implanted into the mammary fat pad of BALB/c nude mice and treated with vehicle, lapatinib, LB-100, or lapatinib plus LB-100. To examine the prevention of the development of lapatinib resistance, SKBR3 and HCC1954 cells were treated twice weekly with lapatinib, LB-100 or the combination and stained with crystal violet when confluent. Results: SKBR3-L and HCC1954-L cells were resistant to lapatinib at clinically relevant concentrations (IC50values = 2.37 ± 0.58 μM and 1.67 ± 0.34 μM). This represents a 46- and 5.2-fold decrease in lapatinib sensitivity. LB-100 had a greater anti-proliferative effect in the lapatinib-resistant SKBR3-L and HCC1954-L cell lines compared to their respective parental cell lines (IC50values = 2.12 ± 0.2 μM v 5.38 ± 0.6 μM, and 2.31 ± 0.19 μM v 5.32 ± 0.82 μM, respectively). LB-100 overcame lapatinib resistance in both models, as lapatinib plus LB-100 was synergistic in both cell lines (CI values = 0.56 ± 0.13 and 0.68 ±0.26). LB-100 caused cell death through the induction of apoptosis in SKBR3- L (p = 0.019) and HCC1954-L (p = 0.046) and the addition of lapatinib to LB-100 increased apoptotic induction in HCC1954-L cells (p=0.046).Lapatinib plus LB-100 was well tolerated in vivo. The HCC1954-L cell line maintained resistance to lapatinib in vivo and the combination of lapatinib and LB-100 significantly reduced HCC1954-L tumour volume compared to all other treatment arms (p = 0.0006). Interestingly, in vitro short-term resistance assays showed that the addition of LB-100 to lapatinib could also block the emergence of lapatinib resistance in both parental SKBR3 and HCC1954 cell lines. Conclusions: This study indicates that LB-100 has in vitro and in vivo efficacy against lapatinib-resistant HER2-positive BC cell line models and justifies further investigation into its potential to circumvent or prevent lapatinib resistance in HER2-positive BC. Citation Format: Conlon N, McDermott M, Browne B, Roche S, O'Neill F, Meiller J, Browne A, Eustace A, Collins DM, O'Donovan N, Crown J. Pre-clinical investigation of PP2A inhibitor LB-100 in overcoming and preventing lapatinib resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-02.

Insulin-like growth factor-1 receptor (IGF1R) signalling is implicated in resistance to trastuzumab. However, the benefit of co-targeting HER2 and IGF1R has not been extensively studied, and the relationship between activated IGF1R and clinical response to trastuzumab has not been reported. This study aimed to evaluate the combination of trastuzumab with IGF1R tyrosine kinase inhibitors (TKIs) in a panel of HER2-positive breast cancer cell lines, and to examine the relationship between IGF1R expression and activation and response to trastuzumab in HER2-positive breast cancer patients. The anti-proliferative effects of trastuzumab combined with IGF1R TKIs BMS-536924 or NVP-AEW541 were measured in nine HER2-positive cell lines. IGF1R and phosphorylated IGF1R/insulin receptor (pIGF1R/IR) were measured by immunohistochemistry in 160 tumour samples from trastuzumab-treated patients (ICORG 06-22). The HER2-positive cell lines displayed varying sensitivity to IGF1R TKIs alone (IC(50)s: 0.7 to10 μM). However, when combined with trastuzumab, a significantly enhanced effect was observed in five cell lines treated with BMS-536924, and three with NVP-AEW541. While IGF1R levels correlated with reduced response to NVP-AEW541 alone, neither IGF1R nor pIGF1R were predictive of response to BMS-536924 or NVP-AEW541 in combination with trastuzumab. Low HER2 levels correlated with response to BMS-536924 in combination with trastuzumab. Akt levels correlated with improved response to trastuzumab and NVP-AEW541 (P = 0.039). Cytoplasmic IGF1R staining was observed in all tumours, membrane IGF1R was detected in 13.8 %, and pIGF1R/IR was detected in 48.8 %. Although membrane IGF1R staining was associated with larger tumour size (P = 0.041), and lower tumour grade (P = 0.024), no association between IGF1R or pIGF1R/IR and patient survival was observed. In conclusion, while neither IGF1R expression nor activation was predictive of response to trastuzumab, these pre-clinical data provide evidence that co-targeting HER2 and IGF1R may be beneficial in some HER2-amplified breast cancers.

Grb2-associated binder 1 (Gab1) is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the epidermal growth factor receptor (EGFR). Although the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this paper we describe the functional characterization of these Gab1 mutants. MCF-10A immortalized mammary epithelial cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared with wild-type Gab1 controls. Expression of Gab1 or the mutants promoted epidermal growth factor (EGF)-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. The two mutants also enhanced branching morphogenesis in a different mammary epithelial cell line, HC11. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but ;not Y83. Cellular stimulation with EGF or hepatocyte growth factor (HGF) led to a transient, or sustained, induction of T387 phosphorylation, respectively. As T387 corresponds in position to Gab2 T391, which suppresses Gab2 signaling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative-feedback regulation of Gab1. Interrogation of publically-available databases revealed additional cancer-associated mutations at, or in close proximity to, identified serine/threonine phosphorylation sites in other docking proteins. These data indicate that aberrant Gab1 signaling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.

BACKGROUND: Although trastuzumab has improved the prognosis for HER-2 positive breast cancer, not all HER-2 positive breast tumours respond to trastuzumab treatment and those that initially respond frequently develop resistance. Insulin-like growth factor-1 receptor (IGF1R) signalling has been previously implicated in trastuzumab resistance. We tested IGF1R inhibition to determine if dual targeting of HER-2 and IGF1R improves response in cell line models of acquired trastuzumab resistance. METHODS: HER-2, IGF1R, phospho-HER-2, and phospho-IGF1R levels were measured by ELISA in parental and trastuzumab-resistant SKBR3 and BT474 cells. IGF1R signalling was targeted in these cells using siRNA and the tyrosine kinase inhibitor NVP-AEW541. RESULTS: IGF1R levels were significantly increased in the trastuzumab resistant model, SKBR3/Tr, compared to the parental SKBR3 cell line. In both the SKBR3/Tr and BT474/Tr cell lines, inhibition of IGF1R expression with siRNA or inhibition of tyrosine kinase activity by NVP-AEW541 significantly increased response to trastuzumab. The dual targeting approach also improved response in the parental SKBR3 cells but not in the BT474 parental cells. CONCLUSIONS: Our results confirm that IGF1R inhibition improves response to trastuzumab in resistant HER-2 positive breast cancer cells, and also suggest that dual targeting of IGF1R and HER-2 may improve response in some trastuzumab-sensitive cells.

Trastuzumab and lapatinib provide clinical benefit to women with human epidermal growth factor receptor 2 (HER)–positive breast cancer. However, not all patients whose tumors contain the HER2 alteration respond. Consequently, there is an urgent need to identify new predictive factors for these agents. The aim of this study was to investigate the role of receptor tyrosine kinase signaling and phosphoinositide 3-kinase (PI3K)/AKT pathway activation in conferring resistance to trastuzumab and lapatinib. To address this question, we evaluated response to trastuzumab and lapatinib in a panel of 18 HER2-amplified cell lines, using both two- and three-dimensional culture. The SUM-225, HCC-1419, HCC-1954, UACC-893, HCC-1569, UACC-732, JIMT-1, and MDA-453 cell lines were found to be 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 was active in de novo (SUM-225, HCC-1419, and HCC-1954) and in a BT-474 cell line with acquired resistance to trastuzumab. In these cells, trastuzumab had little effect on AKT phosphorylation, whereas lapatinib retained activity through the dephosphorylation of AKT. Increased phosphorylation of HER2, epidermal growth factor receptor, HER3, and insulin-like growth factor IR correlated with response to lapatinib but not trastuzumab. Loss of PTEN or the presence of activating mutations in PI3K marked resistance to trastuzumab, but lapatinib response was independent of these factors. Thus, increased activation of the PI3K/AKT pathway correlates with resistance to trastuzumab, which can be overcome by lapatinib. In conclusion, pharmacologic targeting of the PI3K/AKT pathway may provide benefit to HER2-positive breast cancer patients who are resistant to trastuzumab therapy. Mol Cancer Ther; 9(6); 1489–502. ©2010 AACR.

e13021Background: We have previously shown that protein phosphatase 2A (PP2A) may mediate acquired lapatinib resistance and inhibition of PP2A with okadaic acid enhances lapatinib response in vitro...

Alternative splicing of survivin mRNA gives rise to multiple isoforms, that is, survivin and 3 splice variants, survivin-2B, survivin-3B and survivin-DeltaEx3. The aim of this study was to compare the expression of survivin, survivin-2B and survivin-DeltaEx3 in normal breast tissue, fibroadenomas, primary breast cancer and axillary nodal metastases. Survivin, survivin-2B and survivin-DeltaEx3 mRNA were measured using semiquantitative RT-PCR. In the primary carcinomas, we related mRNA for each form of survivin to both survivin protein and apoptosis. For each type of breast tissue, survivin was the predominant form detected, being present in 146 out of 156 (93.6%) primary breast carcinomas, 11 out of 11 (100%) axillary nodal metastases, 21 out of 31 (67.7%) fibroadenomas and five out of 22 (22.7%) specimens of normal breast tissue. Levels of the three forms of survivin were significantly higher in the carcinomas compared to normal breast tissue (P0.0001). Levels of both survivin-2B and survivin-DeltaEx3 but not survivin were significantly higher in nodal metastases than primary carcinomas. Survivin mRNA levels correlated significantly with survivin protein. Finally, both survivin and survivin-DeltaEx3 but not survivin-2B correlated positively with apoptosis. Although survivin, survivin-2B and survivin-DeltaEx3 were all detected in both malignant and nonmalignant breast tissue, the predominant form was survivin. Our results suggest that the different forms of survivin may have different roles in apoptosis in breast cancer.

Background: HER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling. Results: Long-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L. Conclusions: Our data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies.

Intrinsic and acquired resistance to HER-targeting drugs occurs in a significant proportion of HER2-overexpressing breast cancers. Thus, there remains a need to identify predictive biomarkers that could improve patient selection and circumvent these types of drug resistance. Here, we report the identification of neuromedin U (NmU) as an extracellular biomarker in cells resistant to HER-targeted drugs. NmU overexpression occurred in cells with acquired or innate resistance to lapatinib, trastuzumab, neratinib, and afatinib, all of which displayed a similar trend upon short-term exposure, suggesting NmU induction may be an early response. An analysis of 3,489 cases of breast cancer showed NmU to be associated with poor patient outcome, particularly those with HER2-overexpressing tumors independent of established prognostic indicators. Ectopic overexpression of NmU in drug-sensitive cells conferred resistance to all HER-targeting drugs, whereas RNAi-mediated attenuation sensitized cells exhibiting acquired or innate drug resistance. Mechanistic investigations suggested that NmU acted through HSP27 as partner protein to stabilize HER2 protein levels. We also obtained evidence of functional NmU receptors on HER2-overexpressing cells, with the addition of exogenous NmU eliciting an elevation in HER2 and EGFR expression along with drug resistance. Finally, we found that NmU seemed to function in cell motility, invasion, and anoikis resistance. In vivo studies revealed that NmU attenuation impaired tumor growth and metastasis. Taken together, our results defined NmU as a candidate drug response biomarker for HER2-overexpressing cancers and as a candidate therapeutic target to limit metastatic progression and improve the efficacy of HER-targeted drugs. Cancer Res; 74(14); 3821–33. ©2014 AACR.

Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal–regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interference–mediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention. National Health and Medical Research Council of Australia, Cancer Council New South Wales (NSW) and Science Foundation Ireland (Grant No. 06/CE/B1129). DRC and DS were supported by Fellowships from Cancer Institute (CI) NSW. FH is supported by The Ministry of Education and Research, Bundesministerium für Bildung und Forschung (03Z1CN21). CMT is the recipient of a Research Scholarship from CINSW, and LZ an Australian Postgraduate Award. DGO is supported by a National Breast Cancer Foundation (NBCF) and Cure Cancer Australia Foundation Postdoctoral Fellowship. AS is supported by an Early Career Fellowship from the NBCF. Deposited by bulk import

e12092Background: We have previously reported that lapatinib (L) induces reversible senescence in HCC1419 HER2 positive breast cancer cells (McDermott et al, ASCO 2011). In this study we examined i...

To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry–based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e.g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup. Cancer Res; 70(22); 9391–401. ©2010 AACR.

Amplification of the HER-2 gene occurs in approximately 25% of breast cancers, causing up-regulation of key signaling pathways which control cell growth and survival. In breast cancer patients, HER-2 overexpression correlates with an aggressive phenotype and poor prognosis. HER-2, therefore, has become the focus of many anti-cancer therapeutic approaches. Trastuzumab (Herceptin), a humanized monoclonal antibody directed against the extracellular domain of HER-2, was the first FDA-approved HER-2-targeted therapy for the treatment of metastatic breast cancer. However, not all HER-2-overexpressing patients respond to trastuzumab and most that initially respond develop resistance within one year of treatment. Trastuzumab resistance has been studied in cell line models of resistance and several mechanisms of resistance have been proposed. More recent anti-HER-2 strategies involve targeting its tyrosine kinase domain; for example, lapatinib (Tykerb) is a dual HER-2 and EGFR tyrosine kinase inhibitor and has shown efficacy as a single agent and in combination with other therapeutics. A number of novel HER-2 antagonists are currently in preclinical or clinical development, including both monoclonal antibodies and small molecule inhibitors. Increased understanding of HER-2 signaling in breast cancer, and of response and resistance to HER-2 antagonists, will aid the development of strategies to overcome resistance to HER-2 targeted therapies.

HER2 positive breast cancer accounts for approximately 20-25 % of breast cancers. Trastuzumab, a humanised monoclonal antibody, is approved for the treatment of HER2 positive breast cancer. However, patients with metastatic HER2 positive breast cancer frequently develop resistance to trastuzumab. The aim of this study is to investigate microRNAs (miRNAs) in cell line models of acquired trastuzumab resistance to identifty altered miRNAs which may be biomarkers of resistance. The trastuzumab resistant cell line, SKBR3-T was established by continuous exposure to trastuzumab (10 µg/ml) for 6 months. MiRNA extracted from SKBR3, and SKBR3-T was profiled using Taqman low density arrays (TLDA). Six differentially regulated miRNAs were identified in the SKBR3-T cells. qRT-PCR assays confirmed that four were significantly altered in the SKBR3-T compared to SKBR3 cells: miR-224 1.6 fold down-regulated (p=0.01), miR-221 5.8 fold up-regulated (p=0.04), miR-222 was 3.8 fold up-regulated (p=0.01) and miR-9 was 2.2 fold up-regulated (p=0.04). In another model of acquired trastuzumab resistance, BT474-Tr, miR-221 and -222 were also up-regulated, miR224 expression was switched off. No significant change in miR-9 was observed in BT474-Tr. We analysed expression of the four miRNAs in a panel of HER2 positive cell lines which included 6 innate trastuzumab resistant cell lines and 3 innately sensitive cell lines. miR-224 was significantly down-regulated in the 6 resistant cell lines and miR-221 was significantly up-regulated in 3 of the 6 resistant cell lines, compared to the sensitive cell lines. miR-222 and miR-9 were not consistently altered in the resistant cell lines compared to the sensitive cell lines. We analysed expression of these miRNAs in a small cohort of patients who showed durable complete response (pCR) (9 %) following treatment with trastuzumab and chemotherapy [1], and a control group of patients whose disease progressed following treatment. miRNA was extracted from FFPE tumor samples (n=12) and analysed by qRT-PCR. The mean levels of expression of miR-9, -221, and -222 were higher in non-responders (n=6) than responders (n=6) and the mean levels of miR-224 were lower in the non-responders compared to responders. These results suggest that miR-9, -221, -222 and -224 may be potential biomarkers of trastuzumab resistance. 1. Gullo, G., et al., Durable complete response following chemotherapy and trastuzumab for metastatic HER2-positive breast cancer. Annals of Oncology, 2012. Citation Format: Karen Howe, Alex J. Eustace, Giuseppe Gullo, Brigid Browne, Sinead Aherne, John Crown, Norma O'Donovan. Altered expression of miRNAs in acquired trastuzumab resistance. [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 1826. doi:10.1158/1538-7445.AM2014-1826

HER2 positive breast cancer accounts for approximately 25 % of all breast cancer cases. Trastuzumab, a humanised monoclonal antibody, is an approved established treatment for HER2 positive breast cancer; however, patients that initially respond frequently develop resistance. The aim of this study is to investigate microRNAs in cell line models of acquired and innate trastuzumab resistance. MicroRNA was extracted from the HER2 positive cells; SKBR3, BT474, and the acquired trastuzumab resistant variants SKBR3-T and BT474-T, and from a panel of innate trastuzumab sensitive (BT474, EFM-192A, SKBR3 and MDA-MB-361) or resistant cell lines (UACC-732, JIMT-1, HCC-202, HCC-1954, HCC1569 and MDA-MB-453), in triplicate. MicroRNA profiling was performed on SKBR3 and SKBR3-T using Taqman Low Density Arrays (TLDA). Differentially regulated miRNAs were selected using > 2-fold change and a P-value of Conclusions This is the first report of the involvement of miR-9 and miR-224 in trastuzumab resistance in HER2 positive breast cancer. Preliminary functional studies suggest that miR-224 may play a role in regulating cell growth in HER2 positive breast cancer cells. Disclosure All authors have declared no conflicts of interest.

632 Background: HER-2, a member of the transmembrane receptor tyrosine kinase ErbB family, is over-expressed in approximately 25% of BC. HER-2 targeted therapies, in particular, T, a monoclonal antibody targeting HER-2, and lapatinib (L), a reversible HER-2 tyrosine kinase inhibitor, have been shown to significantly improve the prognosis for HER-2 positive BC patients. However, resistance to T and/or L is a significant clinical problem. The aim of this study is to assess the activity of N (HKI-272), an irreversible HER-2 tyrosine kinase inhibitor, in HER-2 overexpressing BC cell lines, including T and/or L resistant cells. Methods: Using proliferation assays, the effect of N was assessed alone and in combination with T in HER-2 positive BC cell lines, including T and/or L resistant cell lines. The effect of N on HER-2 and downstream signalling molecules, Erk and Akt, was determined by immunoblotting. Results: HER-2 positive BC cell lines, including T and/or L resistant cells, are sensitive to N alone with IC50 values (concentration which inhibits 50% of growth) ranging from 1 to 280 nM. The combination of N and T has additive effects in SkBR3 and BT474 which are sensitive to T and also in SKBR3-Lwhich are resistant to L. In the cell lines HCC1954, HCC1954-L, MDA-MB-453, JIMT1 and SKBR3-HL which are resistant to T, combined treatment with T and N showed no enhancement compared to N alone. Finally, N decreased phosphorylation of HER-2, Erk and Akt in all cell lines tested. Conclusions: Our results suggest that N should be studied in patients with HER-2 positive BC, including patients with T and/or L resistant BC. We also demonstrate that N in combination with T may be more effective than either agent alone in T sensitive cells.

617 Background: Not all HER2-positive breast cancer patients respond to HER2-targeted drugs and some, who initially respond, subsequently relapse. There is a need to identify biomarkers for improved patient selection. Here we aimed to identify gene expression changes associated with response to HER2-targeted drugs. Methods: To identify mRNA changes associated with resistance, whole genome microarrays were used to profile conditioned medium (CM) from HER2-positive cell lines (SKBR3, HCC1954) and their lapatinib (L)-resistant variants (SKBR3-LR, HCC1954-LR). Neuromedin U (NmU) over-expression, identified in this way, was confirmed in the L-resistant cells and corresponding CM by qPCR and ELISA. Pooled data from 21 published expression datasets (n=3489 patients) was mined to relate NmU mRNA to tumour subtype and patients' outcome. NmU cDNA and siRNAs were used to over-express and knock-down expression of NmU, respectively, prior to assessing it’s association with response to L, Trastuzumab (T) and Neratinib (N). Results: Analysis of the tumour data showed NmU expression to be particularly associated with poor outcome for patients with HER2-positive tumours (p=0.000005). In cell lines, we identified NmU mRNA and protein to be at significantly higher levels in L-resistant cells and corresponding CM, compared to that of L-sensitive SKBR3 and HCC1954. This trend was observed for T-resistant and N-resistant SKBR3 cells (SKBR3T, SKBR3N) and their CM, compared to sensitive parent SKBR3 cells and CM. NmU cDNA over-expression in SKBR3 and HCC1954 increased resistance to L, T and N. Knock-down of NmU endogenous levels in SKBR3-LR and innately L-resistant MDA-MB-361 and T47D sensitised these cells to L, T and N. Further analysis of our NmU over-expressing and knock-down cells indicated NmU to also be associated with increased motility, invasion and anoikis resistance. Conclusions: NmU expression is prognostic of poor outcome in HER2-positive breast tumours. In cell line models, NmU over-expression is associated with resistance to L, T and N. Taken together, we propose NmU as a possible prognostic and predictive biomarker for HER2-positive cancers, with potential also as a co-target to help circumvent resistance to these drugs. [SFI: 08/SRC/B1410]

633 Background: Lapatinib (L) is approved for the treatment of trastuzumab (T) resistant HER2 positive metastatic breast cancer. Not all HER2 positive tumors respond to L and patients who initially respond frequently relapse, due to the development of resistance to L. Understanding the molecular changes associated with L resistance may lead to the identification of targets to overcome resistance. Methods: SKBR3 cells were treated with either 250 nM L, or 125nM L and 5 µg/ml T twice weekly for 6 months to establish the resistant cell lines SKBR3-L and -TL. We measured by TUNEL assay the effect of L on apoptotic induction in SKBR3, -L and -TL cells. Gene array analysis of the SKBR3, -L and -TL cells identified differences in the expression of apoptosis-related genes, which were validated by immunoblotting. Finally using combinations of obatoclax (O) and L were tested in L resistant cells. Results: In SKBR3 cells, L (500 nM) induces apoptosis (15.8 ± 2.0%) compared to untreated controls (4.6±2.7%), whilst in SKBR3-L and -TL cells L did not induce significant apoptosis compared to controls. Gene array analysis showed that BAX mRNA is down regulated 3.2 fold in SKBR3-L cells compared to the parental cells. In SKBR3-TL cells, MCL-1 mRNA expression is increased 2.1 fold, whilst BAX mRNA is down regulated 2.1 fold compared to the parental cells. Immunoblotting confirmed that BAX protein expression was reduced in the SKBR3-L (1.5 fold, p=0.058) and significantly reduced in SKBR3-TL cells (2.0 fold, p=0.039) compared to SKBR3. MCL-1 protein expression was significantly increased in the SKBR3-L (1.6 fold, p=0.035) and -TL cells (2.3 fold, p=0.031) compared to SKBR3. Combining O (200 nM) and L (500 nM) in both SKBR3 and SKBR3-L cells produced greater growth inhibition than either drug on its own (SKBR3: 86.9±1.0% vs 73.5±3.1% for L (p

583 Background: Resistance to HER2 targeted therapies is a significant clinical problem. To study acquired resistance to the dual EGFR/HER2 tyrosine kinase inhibitor lapatinib (L) in breast cancer,...

Basal-type breast cancers account for up to 25 % of breast cancer cases, depending on the patient population. These cancers usually lack estrogen, progesterone and erbB2 receptors and are therefore resistant to endocrine or trastuzumab therapy, and markers that stratify this subset according to clinical behaviour have yet to be identified. Consequently, basal breast cancers present a major treatment dilemma. In order to identify potential therapeutic targets and prognostic markers for this disease subgroup, we are currently undertaking a detailed characterization of tyrosine kinase signaling networks in basal breast cancer cells. Mass spectrometry (MS)-based global profiling of the tyrosine phosphoproteome of basal breast cancer cells identified a prominent Src family kinase (SFK) signaling network that featured high expression of the SFK Lyn (Hochgräfe et al, Cancer Res 2010 doi: 10.1158/0008-5472.CAN-10-0911). In order to identify Lyn substrates, we used stable isotope labelling with amino acids in cell culture (SILAC) in combination with immunoaffinity-based phosphotyrosine enrichment and LC-MS/MS to profile cellular tyrosine phosphorylation following transient siRNA-mediated Lyn knockdown in MDA-MB-231 and BT-549 basal breast cancer cells. This identified several Lyn substrates characteristic of both cell lines, which included annexin A2 (Y42), 40S ribosomal protein S10 (Y12) and SgK269 (Y635). The latter is a relatively understudied kinase that contains several tyrosine phosphorylation sites that are likely to act as recruitment sites for SH2/PTB domain-containing signalling proteins, and a C-terminal kinase-like domain that lacks the consensus DFG motif usually regarded as essential for kinase activity. Protein expression of SgK269 was markedly higher in basal, versus luminal, breast cancer cells, while mRNA transcript levels in the two cell types were similar, indicating post-transcriptional regulation of SgK269 expression. Phosphorylation of SgK269 on Y635 could be detected readily in basal breast cancer cells. Overexpression of SgK269 in MCF-10A immortalized mammary epithelial cells led to a more elongated morphology in monolayer and perturbed acinar morphogenesis in Matrigel, resulting in large, multilobular acini that failed to undergo proliferative suppression in long-term culture. SgK269-overexpressing cells also exhibited enhanced tyrosine phosphorylation of Stat3. In addition, we undertook stable shRNA-mediated knockdown of SgK269 in BT-549 cells. This reduced soft agar growth and Stat3 activation. These studies identify a novel oncogenic signalling pathway in basal breast cancer involving the kinases Lyn and SgK269, which may represent a target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2011-3131

The identification of minimally-invasive biomarkers, in efforts to aid in patient-tailored disease management, has become a key focus of cancer research. Recent studies by ourselves and others have reported the existence of circulating miRNAs associated with breast cancer. Despite this progress, resistance to anti-cancer treatment remains a major obstacle in the treatment of this disease and unfortunately there are no reliable methods of predicting sensitivity/resistance to anti-cancer agents. This study aimed both to (i) identify miRNAs differentially-expressed in the breast cancer cell line SKBR3 and its Trastuzumab-resistant variant, SKBR3-H, developed by exposure to Trastuzumab for 6 months; and to (ii) investigate if corresponding differential levels of miRNAs were detectable in conditioned media (CM) from these cells, reflecting the relative sensitive/resistance profile of the SKBR3-H comparing to age-matched control SKBR3 cells. Global analysis of miRNAs was successfully performed on biological triplicate RNA specimens isolated from SKBR3 cells, SKBR3-H cells and on their respective CM, using TaqMan low density arrays representing 667 human miRNAs. 71.5% (477/667) miRNAs were detected in SKBR3; most (85%; 406/477) of which were detected in its CM. For SKBR3-H, 75.7% (505/667) miRNA analyzed were present in the cells, with 70% (354/505) of these also detected in corresponding CM. Of the 187 miRNAs up-regulated by at least 2-fold in SKBR3-H compared to SKBR3 cells, 6.42% (12/187) were up-regulated in corresponding CM. Of the 62 miRNAs down-regulated by at least 2 fold in SKBR3-H compared to SKBR3 cells, more than half (53.2%; 33/62) were down-regulated in corresponding CM. Several differentially regulated miRNAs were selected from TLDA results and subsequently validated in triplicate by qRT-PCR. Overall, the many hundred (∼500) miRNAs detected in CM further supports the existence of extracellular miRNAs and so their potential as minimally-invasive biomarkers. Furthermore, we identified a number of miRNAs that may help predict response to Trastuzumab. Our current research involves investigating the relative expression of miRNAs in SKBR3 variants resistant to other HER2-targeted agents and also examining the potential target genes for miRNAs identified as of most relevance here. Exploring the occurrence of these miRNAs in relevant patients’ serum specimens is now warranted to investigate clinical application. Acknowledgements: Science Foundation Ireland's SRC award to MTCI (08/SRC/B1410). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4938. doi:10.1158/1538-7445.AM2011-4938

Approximately 25% of breast cancers overexpress HER2. Although HER2 targeted therapies, trastuzumab and more recently lapatinib, have improved prognosis for HER2 breast cancer patients not all HER2 positive tumors respond to HER2 targeted therapies. Using phosphoproteomic profiling, we examined mechanisms of resistance in an in vitro model of acquired lapatinib resistance. SKBR3 cells were conditioned in lapatinib containing media (200 nM) for six months, after which time the resulting SKBR3-L cells exhibited significantly reduced growth inhibition (27.7 ± 0.1 %) when treated with lapatinib (1 µM) treatment compared to parental SKBR3 (90.7 ± 0.2 %) (p To examine the role of eEF2 in lapatinib resistance, levels of phospho-eEF2, eEF2, phospho-p70S6K and p70S6K, were examined in a panel of HER2 positive breast cancer cell lines following 24 hour treatment with lapatinib (1 µM). Treatment with lapatinib resulted in a decrease in phospho-p70S6K and an increase in phospho-eEF2, with no change in the levels of total protein, in lapatinib sensitive cell lines. In order to further examine the role the mTOR pathway and eEF2, plays in acquired resistance to lapatinib, the effect of rapamycin, alone and in combination with lapatinib, was investigated in SKBR3 par and SKBR3-L cells. Combined treatment with lapatinib (200 nM) and rapamycin (2 nM) results in similar inhibition of growth (64.5 ± 3.3 %) in SKBR3-L cells as lapatinib alone in SKBR3 parental cells (62.5 ± 0.6 %). In conclusion, response to lapatinib is associated with decreased eEF2 activity in HER2 positive cell lines. Furthermore, inhibition of mTOR signaling, which causes inhibition of eEF2 activity, appears to restore sensitivity to lapatinib in our model of acquired lapatinib resistance. 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 3633.

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.