Abstract B43: HER2-amplified gastric and colorectal cancers: Mechanistic insights into HER2 targeting approaches
Authors: Leto Simonetta Maria , Sassi Francesco , Migliardi Giorgia , Zanella Eugenia , Catalano Irene , Bertotti Andrea , Trusolino Livio 
Published: May 2015

Abstract
Only 10% of genetically unselected patients with chemorefractory metastatic colorectal cancer (mCRC) receive clinical benefit from therapy with the anti-EGFR antibodies cetuximab and panitumumab.
Among non-responsive patients, 70% bear tumors harboring at least one alteration in KRAS, NRAS, BRAF and PIK3CA genes; therefore, the remaining 30% of “quadruple negative” cases display still-unidentified features that sustain primary resistance to EGFR-targeted therapies. By performing genotype-response correlations in a preclinical platform of patient-derived mCRC xenografts (xenopatients), our group has recently identified HER2 amplification as a new biomarker of resistance to cetuximab within a quadruple negative population.
We found that the anti-HER2 monoclonal antibody trastuzumab, despite its demonstrated efficacy in patients with HER2-positive breast and gastric cancers, was completely ineffective in HER2-amplified mCRC xenopatients. Importantly, while the dual EGFR/HER2 small molecule inhibitor lapatinib induced disease stabilization, the combination of lapatinib with trastuzumab was synergistic, achieving rapid and long lasting tumor regressions. When the same regimens where applied to HER2-positive gastric cancer cells in vivo, trastuzumab and lapatinib as monotherapies induced disease stabilization, while the combination of both – similar to CRC – had stronger therapeutic effect.
The aim of this study was to better characterize the molecular mechanisms underlying the synergistic effect of the combination therapy in HER2-positive gastric and colorectal cancers.
We first generated three CRC cell lines (NCI-H508, DiFi and HDC-142) stably transfected with the HER2 oncogene and provided proof-of-concept of the causative role of HER2 in installing cetuximab resistance in mCRC. Indeed, while parental controls proved to be extremely sensitive to cetuximab, HER2 overexpressors displayed overt resistance, both in viability assays in vitro and in xenografts experiments in vivo. Moreover, NCI-H508-HER2 cells in vivo were poorly responsive to trastuzumab, while lapatinib induced disease stabilization and the combination of both achieved the strongest therapeutic effect. Overall, these responses were very similar to those observed for HER2-positive mCRC xenopatients, attesting to the reliability of this artificial cellular model for further mechanistic studies.
We next analyzed the activation status of EGFR, HER2 and HER3 upon treatment with trastuzumab, lapatinib and combo and explored the signaling consequences of receptor blockade. Dose-response curves revealed that trastuzumab did not affect activation of EGFR and HER2, but was able to reduce HER3 phosphorylation; at variance, lapatinib and combo potently inhibited phosphorylation of all HERs in a similar manner, with no additive effect by combo. Interestingly, time-course experiments highlighted that treatment with lapatinib resulted in a paradoxical increase of HER3 expression and phosphorylation both in HER2-positive gastric cancer cells and in colorectal cancer cells. Importantly, adding trastuzumab to lapatinib completely neutralized lapatinib-induced HER3 hyper-phosphorylation, without affecting HER3 levels. Possibly, up-regulated HER3 could provide an alternate route to activate a “bypass track” signaling, attenuating the antitumor effects of lapatinib as monotherapy in these tumor settings. Full inhibition of a residual HER2 activity and consequent abolishment of HER3 phosphorylation achieved by the addition of trastuzumab could provide a mechanistic explanation of the synergic effect obtained with the combination therapy.
Overall, these results provide new mechanistic insights into resistance and response to EGFR and HER2 targeted therapies and deserve further investigations to explore the key pathways involved in HER3 feedback up-regulation in the context of HER2-positive cancers.
Citation Format: Simonetta Maria Leto, Francesco Sassi, Giorgia Migliardi, Eugenia Zanella, Irene Catalano, Andrea Bertotti, Livio Trusolino. HER2-amplified gastric and colorectal cancers: Mechanistic insights into HER2 targeting approaches. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B43.