since standard preclinical models have notoriously overestimated the clinical potential of her tkis , we challenged the traditional approach to evaluating tkis in these models .
traditionally , signaling inhibitors are thought to have a continuous suppressive effect through rapid and sustained inhibition of their direct molecular targets and downstream signaling events .
this notion of drug therapy may be too simplistic . clearly , continuous exposure to a growth factor stimulus does not produce continuous high output downstream signaling .
rather , it leads to a sequelae of signaling events , programmed by negative and positive feedback signaling , until establishment of a new steady state in the presence of continued stimulus .
we find here that continuous exposure to tkis similarly leads to a sequence of signaling events that manifest over time , until a new steady state is reached . with this new perspective
, we report that her3/pi3k / akt signaling is not effectively inhibited by current tkis .
in particular , the allocation of kinase and signaling functions to different members within the her family allows the signaling substrate her3 to restore signaling activity despite significant inhibition of her2 kinase , in effect buffering her3/pi3k / akt signaling against an incomplete loss of her2 kinase function .
this inherent signal buffering capacity allows tumor cells to evade the pro - apoptotic effects of tkis resulting in a significant loss of their anti - tumor activity .
the highly effective treatment of her - driven cancers may require drugs with much higher potency or drugs that completely inactivate her kinase function .
irreversible tkis , although more potent , are subject to similar limitations . due to their reactive groups and reduced selectivity ,
future highly selective irreversible inhibitors may turn out to be more effective . until highly specific and fully inactivating drugs can be designed , combination treatment strategies designed to undermine the resiliency of her family signaling may offer the most promising approach in the near future .
in addition , inhibition of autophosphorylation activity deceptively overstates the efficacy of tkis and is a poor in vivo biologic marker .
the signal buffering capacity endowed by the separation of kinase and signaling functions to different family members in the her kinase family attests to an evolutionary advantage conferred by the loss of catalytic activity in the her3 protein kinase .
this can shed light on why approximately 10% of the human kinome appears to be catalytically inactive 28 .
reagent sources are detailed in supplementary materials . for immunofluorescence studies , cells grown on fibronectin coated cover slips
were treated as indicated , fixed in 4% paraformaldehyde , permeabilized , and stained with the indicated primary antibodies and fitc conjugated secondary antibodies .
apoptotic cells were identified and quantified by analysis of annexin v binding using the annexin v - fitc apoptosis detection kit ( calbiochem ) according to the manufacturer s instructions , or by their sub - g1 dna content and quantified by facs analysis as previously described 30 .
all experimental arms were done in duplicate and displayed as averages with standard of deviation error bars .
cells were seeded at a density of 300,000 cells per well in 12-well plates and transfected the following day . for sirna transfections 100300nmol of sirna ( dharmacon )
was premixed with lipofectemine2000 in opti - mem media and then added to each well .
for plasmid transfections , 2 ug of plasmid dna was premixed with lipofectamine2000 in opti - mem media and added to wells for 6 hours .
freshly prepared sulfo - nhs - ss - biotin was added to the final concentration of 0.5 mg / ml in pbs . following 45min incubation at 4c cells
cells were rinsed twice with pbs and incubated with 10 m of freshly prepared h2dcfda in phenol - red free media for 45 min at 37c .
reagent sources are detailed in supplementary materials . for immunofluorescence studies , cells grown on fibronectin coated cover slips
were treated as indicated , fixed in 4% paraformaldehyde , permeabilized , and stained with the indicated primary antibodies and fitc conjugated secondary antibodies .
apoptotic cells were identified and quantified by analysis of annexin v binding using the annexin v - fitc apoptosis detection kit ( calbiochem ) according to the manufacturer s instructions , or by their sub - g1 dna content and quantified by facs analysis as previously described 30 .
all experimental arms were done in duplicate and displayed as averages with standard of deviation error bars .
cells were seeded at a density of 300,000 cells per well in 12-well plates and transfected the following day . for sirna transfections 100300nmol of sirna ( dharmacon ) was premixed with lipofectemine2000 in opti - mem media and then added to each well . for plasmid transfections ,
2 ug of plasmid dna was premixed with lipofectamine2000 in opti - mem media and added to wells for 6 hours .
freshly prepared sulfo - nhs - ss - biotin was added to the final concentration of 0.5 mg / ml in pbs . following 45min incubation at 4c cells
cells were rinsed twice with pbs and incubated with 10 m of freshly prepared h2dcfda in phenol - red free media for 45 min at 37c .
 oncogenic tyrosine kinases have proven to be promising targets for the development of highly effective anticancer drugs . 
 however her family tyrosine kinase inhibitors ( tkis ) show only limited activity against her2-driven cancers despite effective inhibition of egfr and her2 in vivo 
 18 . 
 the reasons for this are unclear . 
 signaling in trans is a key feature of this multimember family and the critically important pi3k / akt pathway is driven predominantly through transphosphorylation of the kinase - inactive her3 9,10 . 
 we report that her3 and consequently pi3k / akt signaling evade inhibition by current her family tkis in vitro and in tumors in vivo . 
 this is due to a compensatory shift in her3 phosphorylation - dephosphorylation equilibrium driven by increased membrane her3 expression driving the phosphorylation reaction and reduced her3 phosphatase activity impeding the dephosphorylation reaction . 
 these compensatory changes are driven by akt mediated negative feedback signaling . 
 although her3 is not a direct target of tkis , her3 substrate resistance undermines their efficacy and has thus far gone undetected . 
 the experimental abbrogation of her3 resistance by sirna knockdown restores potent pro - apoptotic effects to otherwise cytostatic her tkis , re - affirming the oncogene - addicted nature of her2-driven tumors and the therapeutic promise of this oncoprotein target . 
 however , since her3 signaling is buffered against an incomplete inhibition of her2 kinase , much more potent tkis or combination strategies are required to effectively silence oncogenic her2 signaling . 
 the biologic marker to guide her tkis should be the transphosphorylation of her3 . 
Discussion
Brief Methods
Cell culture and reagents
Apoptosis
Transfections
Cell surface biotinylation
Reactive Oxidation Species Assay
Supplementary Material