[Lani]

Abstract
Background. Lapatinib is a HER2 tyrosine kinase inhibitor that has clinical activity in HER2
overexpressing (HER2+) breast cancer. In vitro and clinical studies have shown that lapatinib
enhances the effects of the monoclonal antibody trastuzumab suggesting partially non‐
overlapping mechanisms of action. In order to dissect the differential mechanisms of these
agents, we have studied the effects of lapatinib and trastuzumab on receptor expression
and signaling and have explored a new potential mechanism underlying the profound
antitumor activity of the combination.
Methods. HER2+ breast cancer cells SKBR3 and MCF‐7HER2 were treated with lapatinib,
trastuzumab or both. Assays of receptor expression, phosphorylation, signalling and tumor
growth were performed. Trastuzumab‐dependent cellular cytotoxicity was measured with
different levels of HER2 expression and doses of lapatinib.
Results Lapatinib treatment of HER2+ breast cancer cells inhibited HER2 phosphorylation,
prevented ubiquitination and resulted in a marked accumulation of inactive receptors at the
cell surface. By contrast, trastuzumab caused enhanced HER2 phosphorylation,
ubiquitination and degradation of the receptor. By immunoprecipitation and computational
protein modelling techniques we further demonstrated that the lapatinib‐induced
accumulation of HER2 lead to stabilization of inactive HER2 homo‐ and hetero‐dimers.
Accumulation of HER2 induced by lapatinib and downregulation of HER2 mediated by
trastuzumab were also observed in vivo, where the combination of the two agents triggered
complete tumor regression in all cases after 10 days of treatment. Lapatinib‐induced
accumulation of HER2 at the cell surface markedly enhanced trastuzumab‐mediated ADCC.
Conclusions. Lapatinib results in a marked accumulation of inactive HER2 receptors at the
cell surface both in vitro and in vivo. This increase in receptor numbers at the cell surface
enhances ADCC by trastuzumab. We propose that this is a novel mechanism that may be
clinically relevant and exploitable in the therapy of patients with HER2+ tumors.
Lapatinib induces*accumulation*of*inactive*HER2*at*the*cell* membrane*and*enhances*
antibody‐dependent*cellular*cytotoxicity (ADCC)*mediated*by*trastuzumab:*a*novel*
mechanism*for*the*enhanced*effects*of*combined*ant i‐HER2*therapy******************************#3 594
Lapatinib induces accumulation of HER2 receptors at the cell surface
Lapatinib treatment of the HER2 overexpressing breast cancer cells SKBR‐3 and
MCF‐7HER2 resulted in accumulation of HER2 at the cell surface. Trastuzumab alone
resulted in downregulation of the receptor from the cell membrane. As for the
combined treatment with lapatinib and trastuzumab, the net result was an
accumulation of the receptor at the cell surface of a similar magnitude when
compared to lapatinib alone.
Effects of lapatinib on HER2 ubiquitination, stabilization and dimerization
We transiently expressed HA‐tagged ubiquitin in MCF‐7HER2 cells and analyzed
HER2 ubiquitination in the presence of lapatinib, trastuzumab or the combination of
both. Trastuzumab increased HER2 ubiquitination and degradation whereas, in the
presence of lapatinib, the levels of ubiquitinated receptor were barely detectable.
This was translated to increased stability of the receptor and enhanced formation of
inactive HER2‐containing dimers.
Effects of lapatinib and trastuzumab on BT474 xenografts
Both lapatinib and trastuzumab induced tumor regression of BT474 cell‐derived
xenografts. All the mice receiving the combination of lapatinib and trastuzumab
showed complete tumor regression after 10 days (day 23) of treatment. HER2
staining decreased in tumors treated with trastuzumab but increased in tumors
treated with lapatinib or the combination.
Lapatinib‐induced accumulation of inactive HER2 leads to increased ADCC in vitro
We wanted to test whether the accumulation of HER2 induced by lapatinib could increase
ADCC mediated by trastuzumab. Trastuzumab‐dependent cytotoxicity was significantly
higher in MCF‐7HER2 cells treated with lapatinib compared to untreated cells.
Proposed alternative mechanism of action of lapatinib based on HER2 accumulation
Upon ligand binding or trastuzumab treatment, the HER receptors form dimers and are
phosphorylated (P) in their kinase domains (K). Once phosphorylated, HER2 dimers initiate
signaling and undergo ubiquitination (Ub) and lysosomal degradation. Lapatinib counteracts
receptor phosphorylation, ubiquitination and degradation resulting in HER2 dimer
accumulation at the plasma membrane and rendering the cells more susceptible to the
immune‐mediated action of the anti‐HER antibodies (mAbs).
Conclusions
In conclusion, our results provide a new explanation for the enhanced effects of
the combination of lapatinib and trastuzumab. Lapatinib reduces HER2
ubiquination, prevents HER2 degradation, and induces the formation of inactive
HER2 dimers at the cell surface, which in turn provides an increase in
trastuzumab binding and a greater trastuzumab‐mediated immune response.
This is a therapeutically exploitable mechanism of action that deserves further
study in patients.
Maurizio Scaltriti1,*Chandra Verma2,*Marta*Guzman1,*José*Jimenez1,*Josep*Lluis Parra1,*Kim*Pedersen1,*Stefania Landolfi1,*
Santiago*Ramon y*Cajal1,*Joaquin Arribas1,*José*Baselga1.
1Laboratory*of*Oncology* Research,*Medical*Oncology* Service,*Vall d'Hebron University* Hospital,*Barcelona,*Spain
2Biomolecular*Modeling* &*Design*Group,*Bioinformatics*Institute,*Singapor e.*