Brigid C. Browne

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.

Tyrosine phosphorylation profiling reveals the signaling network characteristics of basal breast cancer cells

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.

HER-2 Signaling and Inhibition in Breast Cancer

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.

Inhibition of IGF1R activity enhances response to trastuzumab in HER-2-positive breast cancer cells

BACKGROUND although trastuzumab has improved the prognosis for HER-2-positive breast cancer patients, 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. MATERIALS AND METHODS HER-2, IGF1R, phospho-HER-2, and phospho-IGF1R levels were measured by enzyme-linked immunosorbent assays in parental and trastuzumab-resistant SKBR3 and BT474 cells. IGF1R signalling was targeted in these cells using both small interfering RNA (siRNA) and the tyrosine kinase inhibitor, NVP-AEW541. RESULTS IGF1R levels were significantly increased in the trastuzumab-resistant model, SKBR3/Tr, compared with 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 HER-2-positive breast cancer cells and suggest that dual targeting of IGF1R and HER-2 may improve response in HER-2-positive tumours.

Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

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.

Functional characterization of cancer-associated Gab1 mutations.

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.

PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

BackgroundHER2 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.ResultsLong-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.ConclusionsOur 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.

Global characterization of signalling networks associated with tamoxifen resistance in breast cancer

Acquired resistance to the anti‐estrogen tamoxifen remains a significant challenge in breast cancer management. In this study, we used an integrative approach to characterize global protein expression and tyrosine phosphorylation events in tamoxifen‐resistant MCF7 breast cancer cells (TamR) compared with parental controls. Quantitative mass spectrometry and computational approaches were combined to identify perturbed signalling networks, and candidate regulatory proteins were functionally interrogated by siRNA‐mediated knockdown. Network analysis revealed that cellular metabolism was perturbed in TamR cells, together with pathways enriched for proteins associated with growth factor, cell–cell and cell matrix‐initiated signalling. Consistent with known roles for Ras/MAPK and PI3‐kinase signalling in tamoxifen resistance, tyrosine‐phosphorylated MAPK1, SHC1 and PIK3R2 were elevated in TamR cells. Phosphorylation of the tyrosine kinase Yes and expression of the actin‐binding protein myristoylated alanine‐rich C‐kinase substrate (MARCKS) were increased two‐ and eightfold in TamR cells respectively, and these proteins were selected for further analysis. Knockdown of either protein in TamR cells had no effect on anti‐estrogen sensitivity, but significantly decreased cell motility. MARCKS expression was significantly higher in breast cancer cell lines than normal mammary epithelial cells and in ER‐negative versus ER‐positive breast cancer cell lines. In primary breast cancers, cytoplasmic MARCKS staining was significantly higher in basal‐like and HER2 cancers than in luminal cancers, and was independently predictive of poor survival in multivariate analyses of the whole cohort (P < 0.0001) and in ER‐positive patients (P = 0.0005). These findings provide network‐level insights into the molecular alterations associated with the tamoxifen‐resistant phenotype, and identify MARCKS as a potential biomarker of therapeutic responsiveness that may assist in stratification of patients for optimal therapy.

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation

The oncogenic signal transducer Gab2 mediates altered cytoskeletal organization and enhanced cell migration of mammary epithelial cells via down-regulation of RhoA activity. This sheds new light on the role of Gab2 in cancer cell metastasis.

Neuromedin U: A Candidate Biomarker and Therapeutic Target to Predict and Overcome Resistance to HER-Tyrosine Kinase Inhibitors

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.