Leihua Liu

Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways.

Dual EGFR/erbB2 inhibition is an attractive therapeutic strategy for epithelial tumors, as ligand-induced erbB2/EGFR heterodimerization triggers potent proliferative and survival signals. Here we show that a small molecule, GW572016, potently inhibits both EGFR and erbB2 tyrosine kinases leading to growth arrest and/or apoptosis in EGFR and erbB2-dependent tumor cell lines. GW572016 markedly reduced tyrosine phosphorylation of EGFR and erbB2, and inhibited activation of Erk1/2 and AKT, downstream effectors of proliferation and cell survival, respectively. Complete inhibition of activated AKT in erbB2 overexpressing cells correlated with a 23-fold increase in apoptosis compared with vehicle controls. EGF, often elevated in cancer patients, did not reverse the inhibitory effects of GW572016. These observations were reproduced in vivo, where GW572016 treatment inhibited activation of EGFR, erbB2, Erk1/2 and AKT in human tumor xenografts. Erk1/2 and AKT represent potential biomarkers to assess the clinical activity of GW572016. Inhibition of activated AKT in EGFR or erbB2-dependent tumors by GW572016 may lead to tumor regressions when used as a monotherapy, or may enhance the anti-tumor activity of chemotherapeutics, since constitutive activation of AKT has been linked to chemo-resistance.

Study of the Biologic Effects of Lapatinib, a Reversible Inhibitor of ErbB1 and ErbB2 Tyrosine Kinases, on Tumor Growth and Survival Pathways in Patients With Advanced Malignancies

PURPOSE This was a pilot study to assess the biologic effects of lapatinib on various tumor growth/survival pathways in patients with advanced ErbB1 and/or ErbB2-overexpressing solid malignancies. PATIENTS AND METHODS Heavily pretreated patients with metastatic cancers overexpressing ErbB2 and/or expressing ErbB1 were randomly assigned to one of five dose cohorts of lapatinib (GW572016) administered orally once daily continuously. The biologic effects of lapatinib on tumor growth and survival pathways were assessed in tumor biopsies obtained before and after 21 days of therapy. Clinical response was determined at 8 weeks. RESULTS Sequential tumor biopsies from 33 patients were examined. Partial responses occurred in four patients with breast cancer, and disease stabilization occurred in 11 others with various malignancies. Responders exhibited variable levels of inhibition of p-ErbB1, p-ErbB2, p-Erk1/2, p-Akt, cyclin D1, and transforming growth factor alpha. Even some nonresponders demonstrated varying degrees of biomarker inhibition. Increased tumor cell apoptosis (TUNEL) occurred in patients with evidence of tumor regression but not in nonresponders (progressive disease). Clinical response was associated with a pretreatment TUNEL score > 0 and increased pretreatment expression of ErbB2, p-ErbB2, Erk1/2, p-Erk1/2, insulin-like growth factor receptor-1, p70 S6 kinase, and transforming growth factor alpha compared with nonresponders. CONCLUSION Lapatinib exhibited preliminary evidence of biologic and clinical activity in ErbB1 and/or ErbB2-overexpressing tumors. However, the limited sample size of this study and the variability of the biologic endpoints suggest that further work is needed to prioritize biomarkers for disease-directed studies, and underscores the need for improved trial design strategies in early clinical studies of targeted agents.

A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer

The development of acquired resistance to ErbB2 tyrosine kinase inhibitors limits the clinical efficacy of this class of cancer therapeutics. Little is known about the mechanism(s) of acquired resistance to these agents. Here we establish a model of acquired resistance to N-{3-chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2 (methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (lapatinib), an inhibitor of ErbB2 and ErbB1 tyrosine kinases by chronically exposing lapatinib-sensitive ErbB2-overexpressing breast cancer cells to lapatinib, simulating the clinic where lapatinib is administered on a daily chronic basis. Analysis of baseline gene expression in acquired lapatinib-resistant and parental cells indicates estrogen receptor (ER) signaling involvement in the development of resistance. Using gene interference, we confirm that acquired resistance to lapatinib is mediated by a switch in cell survival dependence and regulation of a key antiapoptotic mediator from ErbB2 alone to codependence upon ER and ErbB2 rather than loss of ErbB2 expression or insensitivity of ErbB2 signaling to lapatinib. Increased ER signaling in response to lapatinib is enhanced by the activation of factors facilitating the transcriptional activity of ER, notably FOXO3a and caveolin-1. Importantly, we confirm that lapatinib induces ER signaling in tumor biopsies from patients with ErbB2-overexpressing breast cancers receiving lapatinib therapy. These findings provided the rationale for preventing the development of acquired resistance by simultaneously inhibiting both ER and ErbB2 signaling pathways. Establishing clinically relevant models of acquired resistance to ErbB2 kinase inhibitors will enhance therapeutic strategies to improve clinical outcomes for patients with ErbB2-overexpressing breast cancers.

Truncated ErbB2 receptor (p95ErbB2) is regulated by heregulin through heterodimer formation with ErbB3 yet remains sensitive to the dual EGFR/ErbB2 kinase inhibitor GW572016

The expression of the NH2 terminally truncated ErbB2 receptor (p95ErbB2) in breast cancer correlates with metastatic disease progression compared with the expression of full-length p185ErbB2. We now show that heregulin (HRG), but not EGF, stimulates p95ErbB2 phosphorylation in BT474 breast cancer cells. Furthermore, phospho-p95ErbB2 forms heterodimers with ErbB3, but not EGFR, while p185ErbB2 heterodimerizes with both EGFR and ErbB3. The predilection of p95ErbB2 to heterodimerize with ErbB3 provides an explanation for its regulation by HRG, an ErbB3 ligand. GW572016, a reversible small molecule inhibitor of EGFR and ErbB2 tyrosine kinases, inhibits baseline p95ErbB2 phosphorylation in BT474 cells and tumor xenografts. Inhibition of p95ErbB2, p185ErbB2, and EGFR phosphorylation by GW572016 resulted in the inhibition of downstream phospho-Erk1/2, phospho-AKT, and cyclin D steady-state protein levels. Increased phosphorylation of p95ErbB2 and AKT in response to HRG was abrogated to varying degrees by GW572016. In contrast, trastuzumab did not inhibit p95ErbB2 phosphorylation or the expression of downstream phospho-Erk1/2, phospho-AKT, or cyclin D. It is tempting to speculate that trastuzumab resistance may be mediated in part by the selection of p95ErbB2-expressing breast cancer cells capable of exerting potent growth and prosurvival signals through p95ErbB2-ErbB3 heterodimers. Thus, p95ErbB2 represents a target for therapeutic intervention, and one that is sensitive to GW572016 therapy.

Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells.

Antibodies and small molecule tyrosine kinase inhibitors targeting ErbB2 exhibit distinct, noncross resistant mechanisms of action. Here, apoptosis of ErbB2-overexpressing breast cancer cells was enhanced by combining lapatinib, an inhibitor of ErbB1 and ErbB2 tyrosine kinases, with anti-ErbB2 antibodies, including (i) trastuzumab, a humanized monoclonal antibody, and (ii) pAb, rabbit polyclonal antisera generated by vaccination with a human ErbB2 fusion protein. Treating ErbB2-overexpressing breast cancer cell lines with a relatively low concentration of lapatinib alone resulted in a minimal increase in tumor cell apoptosis with an associated decrease in steady-state protein levels of p-ErbB2, p-Akt, p-Erk1/2, and notably survivin, compared to baseline. Exposure to pAb alone reduced total ErbB2 protein, disrupting ErbB3 transactivation, leading to a marked inhibition of p-Akt; however, survivin protein levels remained unchanged and apoptosis only increased slightly. Treatment with trastuzumab alone had relatively little effect on survivin and apoptosis was unaffected. Combining lapatinib with either pAb or trastuzumab markedly downregulated survivin protein and enhanced tumor cell apoptosis. The association between the inhibition of survivin and enhanced apoptosis following the combination of ErbB2-targeted therapies provides a biological effect in order to identify therapeutic strategies that promote tumor cell apoptosis and might improve clinical response.

Lapatinib Antitumor Activity Is Not Dependent upon Phosphatase and Tensin Homologue Deleted on Chromosome 10 in ErbB2-Overexpressing Breast Cancers

Trastuzumab antitumor activity in ErbB2-overexpressing breast cancers seems to be dependent upon the presence of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphatase that dampens phosphatidylinositol 3-kinase-Akt signaling. Consequently, PTEN deficiency, which occurs in 50% of breast cancers, predicts for resistance to trastuzumab monotherapy. Here, we show that lapatinib, a small-molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, exerts its antitumor activity in a PTEN-independent manner. Steady-state phosphorylated ErbB2 (p-ErbB2) and p-Akt (S473) protein levels were inhibited within 30 min following lapatinib but not in response to trastuzumab in BT474 and Au565 cells (two ErbB2-overexpressing breast cancer cell lines that are sensitive to the proapoptotic effects of lapatinib). Whereas trastuzumab reportedly inhibits SRC phosphorylation (Y416), which in turn reduced SRC-ErbB2 protein interactions, lapatinib had no effect on either variable. To assess the potential functional role that PTEN might play in lapatinib antitumor activity, we selectively knocked down PTEN in BT474 and Au565 cells using small interfering RNA transfection. Loss of PTEN did not affect induction of tumor cell apoptosis by lapatinib in either cell line. In addition, lapatinib inhibited Akt phosphorylation in MDA-MB-468 cells, an ErbB1-expressing/ErbB2 non-overexpressing breast cancer line, despite their PTEN-null status. Moreover, patients with ErbB2-overexpressing inflammatory breast cancers responded to lapatinib monotherapy regardless of PTEN status. Thus, lapatinib seems to exert its antitumor activity in ErbB2-overexpressing breast cancers in a PTEN-independent manner. These data emphasize the importance of assessing PTEN status in tumors when selecting ErbB2-targeted therapies in patients with breast cancer.

Regulation of Survivin by ErbB2 Signaling: Therapeutic Implications for ErbB2-Overexpressing Breast Cancers

In breast cancer, overexpression of ErbB2 or aberrant regulation of survivin, a member of the inhibitor of apoptosis family, is associated with resistance to chemo/hormone therapy and predicts for a poor clinical outcome. A functional link between the two predictive factors has not been previously shown. Here, using genetic and pharmacologic approaches to block ErbB2 signaling, we show that ErbB2 regulates survivin protein expression in ErbB2-overexpressing breast cancer cells. Selective knockdown of ErbB2 using small interfering RNA markedly reduced survivin protein, resulting in apoptosis of ErbB2-overexpressing breast cancer cell lines such as BT474. Alternatively, inhibition of ErbB2 signaling using lapatinib (GW572016), a reversible small-molecule inhibitor of ErbB1/ErbB2 tyrosine kinases, at pharmacologically relevant concentrations, leads to marked inhibition of survivin protein with subsequent apoptosis. The effect of lapatinib on survivin seems to be predominantly posttranslational, mediated by ubiquitin-proteosome degradation as lactacystin, a proteosome inhibitor, reverses these effects. Furthermore, lapatinib down-regulated the expression of His-tagged survivin, which was under the transcriptional control of a heterologous promoter, providing additional evidence supporting a posttranslational mechanism of regulation. In contrast, trastuzumab and gefitinib failed to down-regulate survivin in ErbB2-overexpressing breast cancer cells. Importantly, the clinical relevance of these findings was illustrated in patients with ErbB2-overexpressing breast cancer whose clinical response to lapatinib was associated with marked inhibition of survivin in their tumors. These findings shed new light on the mechanism by which ErbB2 overexpression protects against apoptotic stimuli in breast cancer and identifies therapeutic interventions to improve clinical outcomes in these aggressive tumors.

Resistance to ErbB2 Tyrosine Kinase Inhibitors in Breast Cancer Is Mediated by Calcium-Dependent Activation of RelA

The widespread clinical use of therapies targeting the ErbB2 receptor tyrosine kinase oncogene represents a significant advance in breast cancer treatment. However, the development of therapeutic resistance represents a dilemma limiting their clinical efficacy, particularly small-molecule tyrosine kinase inhibitors that block ErbB2 autophosphorylation and activation. Here, we show that lapatinib (GW572016), a highly selective, small-molecule inhibitor of the ErbB2 and epidermal growth factor receptor tyrosine kinases, which was recently approved for the treatment of advanced-stage ErbB2+ breast cancer, unexpectedly triggered a cytoprotective stress response in ErbB2+ breast cancer cell lines, which was mediated by the calcium-dependent activation of RelA, the prosurvival subunit of NF-κB. Abrogation of lapatinib-induced RelA activation using either small interfering RNA constructs or an intracellular calcium chelator enhanced the apoptotic effects of lapatinib in parental ErbB2+ breast cancer cells and overcame therapeutic resistance to lapatinib in ErbB2+ breast cancer lines that had been rendered resistant to lapatinib through chronic exposure to the drug, mimicking the clinical setting. In addition, analysis of changes in phospho-RelA expression in sequential clinical biopsies from ErbB2+ breast cancers treated with lapatinib monotherapy revealed marginally statistically significant differences between responders and nonresponders, which was consistent with our preclinical findings. Elucidating the regulation of RelA by lapatinib in ErbB2+ breast cancers, and showing its role in the development of therapeutic resistance to lapatinib, identifies another therapeutic target to overcome or prevent the onset of resistance to lapatinib in some women with ErbB2+ breast cancers. Mol Cancer Ther; 9(2); 292–9

Truncated ErbB2 Expressed in Tumor Cell Nuclei Contributes to Acquired Therapeutic Resistance to ErbB2 Kinase Inhibitors

ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185ErbB2). In addition to p185ErbB2, truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2+ breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2+ breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2+ breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs. Mol Cancer Ther; 10(8); 1367–74. ©2011 AACR.

Photo-Activated Psoralen Binds the ErbB2 Catalytic Kinase Domain, Blocking ErbB2 Signaling and Triggering Tumor Cell Apoptosis

Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85ErbB2) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85ErbB2. Here we show that PUVA reduced p85ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.