From the Cover: PNAS Plus: Mechanism for activation of mutated epidermal growth factor receptors in lung cancer

The initiation of epidermal growth factor receptor (EGFR) kinase activity proceeds via an asymmetric dimerization mechanism in which a “donor” tyrosine kinase domain (TKD) contacts an “acceptor” TKD, leading to its activation. In the context of a ligand-induced dimer, identical wild-type EGFR TKDs are thought to assume the donor or acceptor roles in a random manner. Here, we present biochemical reconstitution data demonstrating that activated EGFR mutants found in lung cancer preferentially assume the acceptor role when coexpressed with WT EGFR. Mutated EGFRs show enhanced association with WT EGFR, leading to hyperphosphorylation of the WT counterpart. Mutated EGFRs also hyperphosphorylate the related erythroblastic leukemia viral oncogene (ErbB) family member, ErbB-2, in a similar manner. This directional “superacceptor activity” is particularly pronounced in the drug-resistant L834R/T766M mutant. A 4-Å crystal structure of this mutant in the active conformation reveals an asymmetric dimer interface that is essentially the same as that in WT EGFR. Asymmetric dimer formation induces an allosteric conformational change in the acceptor subunit. Thus, superacceptor activity likely arises simply from a lower energetic cost associated with this conformational change in the mutant EGFR compared with WT, rather than from any structural alteration that impairs the donor role of the mutant. Collectively, these findings define a previously unrecognized mode of mutant-specific intermolecular regulation for ErbB receptors, knowledge of which could potentially be exploited for therapeutic benefit.The gene encoding the epidermal growth factor receptor (EGFR) tyrosine kinase is somatically mutated in a substantial fraction of patients with lung cancer. The majority of primary activating EGFR mutations occur within the tyrosine kinase domain (TKD). The most frequent of these, which occur with a combined frequency of 90% (1), are exon 19 deletions that eliminate four amino acids (LREA) from the TKD and exon 21 missense mutations that substitute arginine for leucine at position 834 (L834R) (also identified as L858R in an alternative numbering of the human EGFR sequence that includes the 24 residue signal sequence) (2).Exon 19 deletions and L834R substitutions are associated with increased sensitivity to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, translating to a 70% radiographic response rate in patients (3–5). Unfortunately, all individuals with metastatic disease eventually develop progressive disease after 10–16 mo of treatment with EGFR TKIs. The most common mechanism of acquired resistance is mutation at a second site in the EGFR TKD (the gatekeeper residue), T766M (T790M). This mutation confers resistance by increasing affinity for ATP, with which inhibitors must compete for binding, and also by modestly decreasing intrinsic affinity for TKIs (6).Biochemical and crystallographic studies have shown that activation of the wild-type (WT) EGFR TKD involves formation of an asymmetric dimer in which one molecule allosterically activates its neighbor by promoting the reversal of intramolecular autoinhibitory interactions—acting as a “donor” or “activator” TKD that activates the “acceptor” or “receiver” TKD (7, 8). Crystal structures of individual L834R and T766M EGFR-TKD mutants show that these variants also form asymmetric dimers (6, 9), but whether the double mutant L834R/T766M adheres to the same configuration in the active state is unclear. Biochemical data indicate that the oligomerization potential of mutated EGFRs is enhanced relative to WT. For example, native gel and multiangle light scattering studies showed that the L834R substitution promotes formation of dimers and higher order oligomers of the EGFR TKD (10). Consistent with this observation, cell-based studies have demonstrated a reduced dependence on ligand stimulation for activation of mutated EGFRs. All mutated EGFR TKDs seen in lung cancer show an increase in catalytic efficiency over WT (6, 9, 11, 12). Interestingly, the doubly mutated L834R/T766M EGFR TKD has a two-to fivefold higher catalytic efficiency (k_cat/K_m) than either the singly mutated L834R or T766M mutant TKDs (6).Here, using a biochemical and structural approach, we sought to determine whether mutated EGFRs most commonly associated with primary drug sensitivity and acquired resistance in lung cancer adhere to the well-established asymmetric dimer model. Our biochemical reconstitution data demonstrate that these EGFR mutations do in fact adhere to this model, but with the striking difference that the oncogenic mutants preferentially assume the receiver role when expressed together with WT EGFR. Our crystal structure of active EGFR-L834R/T766M reveals an asymmetric dimer interface essentially the same as that in WT EGFR. We further show that mutated EGFRs can hyperphosphorylate WT EGFR as well as the related family member erythroblastic leukemia viral oncogene (ErbB)2 as a result of their preferential adoption of the acceptor or receiver position in the asymmetric dimer. These findings have important implications for understanding mechanisms of drug sensitivity and resistance in EGFR mutant lung cancers and shed light on the distinct properties of different mutated forms of EGFR.     

Recent progress in the discovery of Akt inhibitors as anticancer agents

Akt, also referred to as protein kinase B (PKB) or Related to A and C (RAC), is one of the major direct downstream targets of phosphoinositide 3-kinase (PI3K). As it plays a central role in promoting cancer cell proliferation and survival through a growing list of key substrates, intense efforts are underway to find inhibitors of Akt for the treatment of cancer. Discovery of potent and novel inhibitors of Akt has been facilitated greatly by the availability of the X-ray structure of the active form of Akt and by its structural similarity with other serine/threonine kinases. In this review, new Akt inhibitors for the treatment of cancer are comprehensively reviewed, with emphasis on small molecule inhibitors that bind to the ATP-binding site, allosteric sites and the PH domains. Inhibitors of pseudosubstrates and antisense oligonucleotides, as well as Akt inhibitors with unknown mechanism of actions, are also reviewed. Results of clinical trials of several Akt drug candidates are briefly discussed. A brief summary of Akt structure and regulation and the evidences supporting Akt as a cancer target is provided as well. The patent literature is surveyed through July 2007.     

Pluronic F127 polymeric micelles for co-delivery of paclitaxel and lapatinib against metastatic breast cancer: preparation,optimization and in vitro evaluation

Abstract

The aim of this study was to develop and characterize the paclitaxel (PTX)-lapatinib (LPT) loaded micelles for simultaneous delivery against metastatic breast cancer. Efflux pump-mediated drug resistance influences the efficacy of chemotherapeutic regimens. However, in the newly developed delivery system, LPT was selected to act as chemosensetizer. LPT increases the intracellular level of PTX by inhibition of efflux pumps. Pluronic F127 was selected for the preparation of the micelles, and its critical micelle concentration was determined to be 0.012?mg/ml. D-optimal design was used to analyze the impact of different experimental parameters on PTX and LPT encapsulation ratio. PTX encapsulation ratio was optimized at 68.3%, while LPT encapsulation ratio found to be 70.1%. Transmission electron microscope analyses demonstrate that micelles possess a good core–shell structure without any sharp edge. Laser scattering method results indicated that size of the optimized micelles is 64.81?nm with acceptable polydispersity index (0.309). In vitro release studies showed a sustain release pattern. PTX–LPT-loaded micelles suppressed the proliferation of resistant T-47D cell line (IC₅₀?=?0.6?±?0.1?µg/ml) compared to binary mixture of PTX and LPT (IC₅₀?=?6.7?±?1.2?µg/ml). Therefore, it is concluded that the developed formulation might increase the therapeutic efficacy in drug resistant metastatic breast cancer.     

Impact of ERBB2 mutations on in vitro sensitivity of bladder cancer to lapatinib

Lapatinib, a dual tyrosine kinase inhibitor of ErbB1 and ErbB2, shows a clinical benefit in a subset of patients with advanced urothelial bladder cancer (UBC). We hypothesized that the corresponding gene, ERBB2, is affected by mutations in a subset of UBC and that these mutations impact ErbB2 function, signaling, UBC proliferation, gene expression, and predict response to lapatinib. We found ERBB2 mutations in 5 of 33 UBC cell lines (15%), all of which were derived from invasive or high grade tumors. Phosphorylation and activation of ErbB2 and its downstream pathways were markedly enhanced in mutated cell lines compared with the ERBB2 wild-type. In addition, the gene expression profile was distinct, specifically for genes encoding for proteins of the extracellular matrix. RT112 cells infected with ERBB2 mutants showed a particular growth pattern (“mini-foci”). Upon treatment with lapatinib, 93% of these “mini-foci” were reversed. The sensitivity to lapatinib was greatest among cell lines with ERBB2 mutations. In conclusion, ERBB2 mutations occur in a subset of UBC and impact proliferation, signaling, gene expression and predict a greater response to lapatinib. If confirmed in the clinical setting, this may lead the way toward personalized treatment of a subset of UBC.     

Lapatinib induces autophagic cell death and inhibits growth of human hepatocellular carcinoma

Lapatinib, an orally adminstered small-molecule tyrosine kinase inhibitor targeting epidermal growth factor receptors (EGFR) and Her2/Neu, has been widely accepted in the treatment of breast cancer. In this study, we found that lapatinib induced cytotoxicity in human hepatoma Huh7, HepG2 and HA22T cells. For the mode of cell death, we found lapatinib induced a higher percent of dead cells and a lower percent of hypodiploid cells, suggesting non-apoptotic cell death in lapatinib-treated hepatoma cells. Moreover, lapatinib-induced autophagy in hepatoma cells was confirmed by the detection of autophagic LC3-II conversion, the up-regulation of autophagy-related proteins, and the down-regulation of p62 by immunoblotting. Autophagic cell death was demonstrated by images of punctuated LC3 patterns, a higher percent of acridine orange positive cells, as well as a partial rescue of cell death by autophagy inhibitor 3-methyladenine or chloroquine. We also found massive vacuoles in lapatinib-treated hepatoma cells by electronic microscopy. In addition, the shRNA of knocked-down autophagy-related proteins rescued the hepatoma cells from lapatinib-induced growth inhibition. We also demonstrated a reduction of tumorigenesis by lapatinib in vivo. In conclusion, lapatinib induced autophagic cell death and the growth of human hepatoma cells. Our study provides potential cancer therapies by using lapatinib as a treatment for hepatoma.     

Beyond trastuzumab: novel therapeutic strategies in HER2-positive metastatic breast cancer

The use of trastuzumab, a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2) alteration present in 25 to 30% of breast cancers, has been associated with improved survival outcomes in both the adjuvant and metastatic settings. However, despite the robust clinical efficacy of trastuzumab in HER2-positive metastatic breast cancer (MBC), primary and secondary resistance remains a clinical challenge. Although lapatinib has demonstrated modest activity in this setting, trials reported to date have yet to demonstrate improvements in overall survival with its use. Novel therapeutic strategies to circumvent trastuzumab resistance are warranted, and agents targeting the HER, vascular endothelial growth factor, heat shock protein 90, phosphoinositide 3 kinase/Akt/mammalian target of rapamycin, and insulin-like growth factor-1 receptor pathways represent rational approaches in the management of HER2-positive disease. In this review, early-phase and emerging trial data surrounding the use of these promising agents in HER2-positive MBC will be discussed.     

Long-term management of patients with metastatic renal cell carcinoma on targeted agents

Trastuzumab, a humanized monoclonal antibody directed against HER2, used alone or in combination with chemotherapy, has shown significant clinical benefit in improving survival in the metastatic setting, as well as halving the recurrence rate and improving survival in HER2-positive early breast cancer. Lapatinib is an orally active, reversible, small-molecule tyrosine kinase inhibitor that potently inhibits both HER1 and HER2 tyrosine kinase activity. This agent is the most advanced in terms of clinical trials and has been shown to have a favorable safety profile. Owing to the promising activity seen in advanced breast cancer, lapatinib is the ideal candidate for testing in the adjuvant setting. Adjuvant Lapatinib and/or Trastuzumab Treatment Optimisation (ALTTO) is a four-arm randomized trial designed to compare trastuzumab and lapatinib in women with early-stage HER2-positive breast cancer. Specifically, ALTTO will examine which anti-HER2 agent is more effective and which is their best schedule of administration, namely, what benefit will be derived by taking the drugs separately, in tandem order or in combination. Overall, 8000 patients will be enrolled worldwide.