ErbB2T798I Mediated Drug Resistance To Lapatinib In Breast Cancers And The Tumor Suppressor Role Of PHLDA1/2in Breast And Lung Cancers

Objective:ErbB2is a cell membrane surface-bound receptor tyrosine kinase and is involved in thesignal transduction pathways leading to cell growth and proliferation. This gene is amplifiedin10–20%of breast cancers, and amplification results in protein overexpression, whichdenotes an aggressive phenotype. Overexpression, amplification and occasionally activatingmutations of ErbB2also occur in other cancers, including non-small cell lung cancer. Further,somatic mutations in the kinase domain of ErbB2were discovered in various solid cancersincluding lung and breast carcinomas. A dual, reversible EGFR/ErbB2inhibitor, lapatinib（Tykerb）in particular has demonstrated significant activity in ErbB2-positive breast cancersand now is approved to be used in this indication. Inhibition of ErbB2tyrosineautophosphorylation by lapatinib abrogates downstream Ras-Raf-ERK1/2and PI3K-AKTgrowth/survival signaling in ErbB2overexpressing breast cancer cell lines and in patientswith ErbB2-overexpressing breast cancers. The uniform development of acquired resistanceto lapatinib unfortunately limits its clinical efficacy. Therefore, a better understanding of themechanisms of drug resistance and the entire array of downstream changes should allow thedevelopment of alternative treatment, the identification of novel actionable targets and thedevelopment of more effective and durable strategies for the treatment of lung and breastcancers.Methods:1. We investigate the lapatinib sensitivity of ErbB2-positive lung/breast cancer cellsthrough MTS assay and apoptosis assay. We identified an array of putative acquiredresistance mutations through the random mutagenesis assay and tested the irreversibleinhibitors overcoming thiese resistances.2. Transcriptional profiling study was done to identify the early ErbB2target genesincluding PHLDA1/2. We studied the function of PHLDA1and PHLDA2on the anchorage dependent and independent cell growth in breast cancer and lung cancercells. We studied the expression of PHLDA1in24primary breast cancer samples.3. We constructed the immunofluorescent protein expression vector for PHLDA2andstudied the influence of PHLDA2on the translocation of Akt to cell membranethrough the immunoflurecence and confocal imaging. We expressed and purifiedPHLDA2protein in vitro and studied the influence of PHLDA2on the binding abilityof p-Akt to PIPs and the further impct of PHLDA2on the activity of Akt. Moreimportantly, we investigated the correlation of PHLDA2and p-Akt in a tissuemicroarray study.Results:1. We identified several mutations, including the gatekeeper ErbB2mutation ErbB2-T798I, as mediating resistance.2. ErbB2-T798I engineered cell models indeed show resistance to lapatinib but remainsensitive to the irreversible EGFR/ErbB2inhibitor, PD168393suggestive of potentialalternative treatment strategies to overcome resistance.3. PHLDA1and PHLDA2were identified as immediately downregulated genes upononcogenic EGFR/ErbB2signaling inhibition.4. We find significant down-regulation of PHLDA1in primary breast cancer andPHLDA1is statistically significantly less expressed in ErbB2negative compared withErbB2positive tumors consistent with its regulation by ErbB2.5. PHLDA1overexpression blocks AKT signaling, inhibits cell growth and enhanceslapatinib sensitivity further supporting an important negative growth regulatorfunction.6. We find that PHLDA2competes with the PH domain of Akt for binding of membranelipids, thereby inhibiting Akt translocation to the cellular membrane and activation.7. We demonstrate the suppression of anchorage dependent and independent cell growthby PHLDA2. 8. We show that PHLDA2overexpression inhibits AKT phosphorylation that results inreduced cell proliferation and decreased PHLDA2expression results in reducedsensitivity of lung cancer cells to lapatinib.9. In an immunohistochemical study of primary lung tumors, we find PHLDA2proteinexpression correlates positively with Akt activation in human lung cancers.Conclusion:We showed the functional relevance of the T798I ErbB2mutation as a potentialresistance mechanism against reversible ErbB2inhibitors, modeled by lapatinib, inErbB2-positive cancer cells. Our studies suggest that irreversible ErbB2inhibitors might beable to overcome or prevent the development of potentially key resistance mechanisms suchas the gatekeeper secondary T798mutation. PHLDA1and PHLDA2might have keyinhibitory functions in lung and breast cancer cells and a better understanding of theirfunctions might point at novel therapeutic options. In summary, our studies define novelways of modulating sensitivity and resistance to EGFR/ErbB2inhibition in lung and breastcancers.