Biochemical Journal

Research article

HER3 signalling is regulated through a multitude of redundant mechanisms in HER2-driven tumour cells

Dhara N. Amin, Natalia Sergina, Lionel Lim, Andrei Goga, Mark M. Moasser


HER2 (human epidermal growth factor receptor-2)-amplified tumours are characterized by constitutive signalling via the HER2–HER3 co-receptor complex. Although phosphorylation activity is driven entirely by the HER2 kinase, signal volume generated by the complex is under the control of HER3, and a large capacity to increase its signalling output accounts for the resiliency of the HER2–HER3 tumour driver and accounts for the limited efficacies of anti-cancer drugs designed to target it. In the present paper we describe deeper insights into the dynamic nature of HER3 signalling. Signalling output by HER3 is under several modes of regulation, including transcriptional, post-transcriptional, translational, post-translational and localizational control. These redundant mechanisms can each increase HER3 signalling output and are engaged in various degrees depending on how the HER3/PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signalling network is disturbed. The highly dynamic nature of HER3 expression and signalling, and the plurality of downstream elements and redundant mechanisms that function to ensure HER3 signalling throughput identify HER3 as a major signalling hub in HER2-amplified cancers and a highly resourceful guardian of tumorigenic signalling in these tumours.

  • ErbB3
  • human epidermal growth factor receptor 2 (HER2)
  • human epidermal growth factor receptor 3 (HER3)
  • tumorigenesis


The HER [human EGFR (epidermal growth factor receptor)] family of tyrosine kinase receptors consist of four members, EGFR, HER2, HER3 and HER4. Abnormalities in this family underly the pathogenesis of a variety of human cancers. Through transcriptional overexpression, gene amplification, mutational activation or autocrine loop activation, the overactive functions of HER family members are implicated in the pathogenesis of a variety of subtypes of human cancers, designating them as proto-oncogenes [1,2]. These include the amplification and overexpression of HER2 in breast cancers [3], or the mutational activation of EGFR in non-small-cell lung cancers and glioblastomas [4,5], and possibly the mutational activation of HER4 in melanomas [6]. The third member HER3 is not designated as a proto-oncogene, and its amplification or mutational alteration has not been described in human tumours. This may be due to the lack of significant catalytic function in the HER3 kinase domain, a distinguishing characteristic that sets it apart from the other HER family members [79]. The lack of catalytic function, however, does not diminish its importance in signalling. These receptors function predominantly through heterodimerization and their tumorigenic signalling functions frequently involve their family member partners [10]. In particular, HER3 has emerged as a favourite interaction partner in tumorigenic signalling. Although HER3 lacks catalytic function, its tumour-promoting functions may involve the functions of its kinase domain as an allosteric activator of its partners, or the functions of its C-terminal signalling tail, in particular its ability to activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway [11,12]. The HER3 C-terminal signalling tail is uniquely endowed with six consensus binding sites for the p85 subunit of PI3K, and when phosphorylated by its HER family partners, HER3 is highly competent in the activation of the PI3K/Akt signalling pathway [13,14].

The role of HER3 has been highlighted in a number of HER-family-driven cancers. In HER2-amplified breast cancers, HER3 expression is required for tumorigenic growth, and the pharmacological suppression of HER2-amplified tumour growth requires the suppression of both HER2 and HER3 signalling functions [1517]. In non-small cell lung cancers driven by mutationally activated EGFR, suppression of growth correlates best with the suppression of HER3 signalling [18].

Although the role of HER3 as a collateral partner in some types of HER-driven cancers has been described for a number of years, its resiliency and resourcefulness at this function is only recently beginning to be appreciated. In particular, its role as a partner for HER2 in HER2-amplified cancers has been particularly revealing. Although HER2 autophosphorylation is easily inhibited by TKI (tyrosine kinase inhibitor) treatment of HER2-amplified tumours, the inhibition of HER3 phosphorylation by HER2 is not so easily inhibited. This is due to a rapid compensatory restoration of HER3 signalling that functions to preserve tumorigenic HER2–HER3 signalling in the face of continued TKI therapy, an effect that considerably undermines the anti-tumour efficacy of HER2-directed TKIs [17,19].

The resiliency in HER3 signalling forms a major barrier to the effective therapy of cancers driven, in part, through the collateral functions of HER3. In the present study we undertook to better understand the mechanisms that regulate HER3 signalling output in a HER2-amplified cancer cell model. We report in the present paper that HER3 signalling is highly regulated through a variety of mechanisms in a reciprocal link with its downstream signalling throughput. The up-regulation of its signalling output can be triggered through increased transcription, translation, localization or phosphorylation, and through redundant mechanisms engaged by different downstream elements. These data reveal the highly regulated nature of HER3 and its role as a dynamic signalling hub linking HER family oncogenes with downstream PI3K/Akt pathway signalling.


Cell lines and reagents used

SkBr3 cells were obtained from A.T.C.C. and cultured at 37°C and 5% CO2 in DMEM (Dulbecco's modified Eagle's medium)/Ham's F12 medium supplemented with 10% FBS (fetal bovine serum), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Sodium orthovanadate and cycloheximide were purchased from Sigma. Lapatinib, gefitinib and erlotinib tablets were purchased from the pharmacy and the drugs were purified as described previously [17,19]. BEZ235 was obtained from Novartis, and rapamycin was obtained from Cell Signaling Technology. PP242 and DG2 were synthesized and provided by Morri Feldman and Kevan Shokat (Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, U.S.A.) as described previously [20,21]. All pharmaceutical drugs were re-constituted in DMSO. For NO donor experiments, cells were treated with or without lapatinib (200 nM) for 48 h. At 8 h before harvest, DETA/NO (diethylenetriamine/NO adduct; Sigma) was added to the cells at a final concentration of 1 mM. For NO inhibition, L-NAME (NG-nitro-L-arginine methyl ester), PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt] and S-MITU (S-methylisothiourea hemisulfate salt) were added to the cells for 24 h.

Western blot analysis

Cell lysates were prepared using modified RIPA (150 mM NaCl, 0.1% SDS, 1% Nonidet P40, 1% sodium deoxycholate and 10 mM sodium phosphate, pH 7.2) buffer supplemented with protease and phosphatase inhibitors. Western blots were performed using antibodies purchased from Santa Cruz Biotechnology [anti-HER3, anti-actin and anti-HA (haemagglutinin)] or Cell Signaling Technology {anti-pS6, anti-phospho-4E-BP1Thr36/47 (4E-BP1 is eukaryotic initiation factor 4E-binding protein 1) and phospho-eNOS [endothelial NOS (NO synthase)]}. The custom-made anti-pTyr1289-HER3 antibody has been described previously [17].

Plasmid and miRNA (microRNA) expression

The pGL3-HER3 promoter construct was a gift from Dr Frederick Domann (University of Iowa, Iowa City, IA, U.S.A.) and has been described previously [22]. The CMV (cytomegalovirus)-Renilla construct was from Promega. The HER3 3′UTR (untranslated region) construct was obtained from Genecopeia. The pBABE-eIF4E (eukaryotic initiation factor 4E) and pBABE-4E-BP1 constructs were a gift from Dr Davide Ruggero (Department of Urology, University of California, San Francisco, CA, U.S.A.). miR-106b MIRIDIAN mimic was obtained from Thermo Fisher. The constructs were transfected into SkBr3 cells using Lipofectamine™ 2000 and standard protocols.

Luciferase reporter assays

Cells transfected with the firefly-luciferase constructs or the CMV-Renilla-luciferase constructs were seeded into 96-well plates 24 h post-transfection. After the cells had attached, the cells were switched to medium containing lapatinib (200 nM) or DMSO (mock) for 48 h. Luciferase activity was measured using standard protocols described in the Dual Glo Luciferase system (Promega). Reporter assays were performed on triplicate wells of cells for luciferase activity on the 3′UTR or promoter construct of HER3, as well as the normalizing control for Renilla for mock- or drug-treated cells for the indicated number of hours. Measurements from each well for the luciferase readout was normalized against the Renilla readout. Ratios for the drug-treated and mock-treated cells were obtained against the average for the mock normalized readout. The mean ratio and S.E.M. was determined on the triplicate values obtained (n=3). Student's t tests were performed on the triplicate values using two-tailed unequal variance parameters.

Quantitative RT (reverse transcription)–PCR

Cells were treated with lapatinib (200 nM), erlotinib (5 μM) or rapamycin (25 nM) for the indicated times. Total cellular RNA was isolated using the Qiagen RNeasy kit following the manufacturer's instructions. RT and PCR amplification of HER3 (official HGNC symbol ERBB3) was performed using primers and methods that have been described previously [17]. Quantitative PCR assays were performed in triplicate for HER3 and the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from cDNA prepared from mock- or drug-treated cells for the indicated number of hours. We used the ΔΔCT methodology to obtain the ratios after normalizing to GAPDH. The ratios were obtained against the average for the mock readout. The mean ratio and S.E.M. was determined on the triplicate values obtained (n=3). Student's t tests was performed on the triplicate values using two-tailed unequal variance parameters.

For the miRNA quantification, total RNA was isolated using the Qiagen RNeasy Plus kits. Expression of specific mature miRNAs was confirmed by real-time PCR analysis using a TaqMan Human MicroRNA Assay kit (Applied Biosystems). Samples were analysed in duplicate and CT values were normalized to RNU48 expression.

Surface labelling

Cells were washed with ice-cold PBS (pH 8.0) and then were incubated for 45 min with 0.5 mg/ml EZ-Link sulfo-NHS-biotin (N-hydroxysuccinimide biotin; Thermo Scientific) at 4°C. Cells were washed with PBS (pH 8.0) and lysed with modified RIPA buffer (supplemented with protease and phosphatase inhibitors). Biotinylated proteins were pulled down by adding streptavidin-conjugated agarose (Sigma). The agarose beads, after washes in mRIPA, were subjected to Western blot analysis.


The 5′UTR of HER3 was analysed using Genebee ( Transcription factors binding to the promoter region of HER3 used in the present study was analysed using MatInspector ( and PROMO ( software.

Statistical analyses

Student's t tests were performed using two-tailed tests with unequal variance to obtain P values.


Diversity in HER3 compensation in HER2-amplified cancer cells

First we studied how different HER2-amplified cancer cells respond to the inhibition of HER2. Treatment of a panel of seven HER2-amplified cancer cells with 200 nM lapatinib showed a diversity of responses. Although lapatinib completely inhibited HER3 phosphorylation in the immediate timeframe (1 h), all except for one cancer cell restored HER3 phosphorylation at the new steady state (Figure 1A and Supplementary Figure S1 at In HCC1954 cells, the inhibition of HER3 signalling was complete and durable. This is because of redundancy in receptor tyrosine kinase overexpression. These tumour cells have overexpression of both HER2 and c-Met, and downstream Akt signalling is driven by both HER2 and c-Met [23,24]. As such, the inhibition of HER2 alone failed to inactivate downstream Akt signalling and induce the negative-feedback effects that would lead to restoration of HER3 phosphorylation. HER3 signalling was restored in the other six cell types, but through apparently different mechanisms. In some, the restoration of HER3 signalling was concomitant with a marked increase in HER3 protein expression (SkBr3 and N87), whereas in others there was only a minimal increase in expression, but a marked increase in membrane re-localization (MDA-453 and OE19) (Figure 1B). In some cells HER3 expression was induced in a rapid timeframe (OE19), whereas in most others it required longer latencies. These differences suggest that there are a plurality of mechanisms by which HER3 signalling can be restored in HER2-amplified cancer cells. This may be due either to different signalling circuitries operating in different cancer cell lines, or it may reflect the presence of redundant mechanisms regulating HER3 signalling and which can respond to diminished signalling throughput, such as seen with drug therapy. To study the latter hypothesis, we selected SkBr3 cells for a more in-depth analysis of whether there are in fact a plurality of mechanisms present that can up-regulate HER3 signalling.

Figure 1 Response to TKI therapy in HER2-amplified breast cancer cell lines

(A) A panel of HER2-amplified breast cancer cells were treated with 200 nM lapatinib for up to 72 h to detect the initial down-regulation and subsequent up-regulation of HER2/HER3 and downstream signalling. Immunoblots were performed using the antibodies indicated. (B) The cell-surface expression of HER3 (membrane HER3) was more specifically quantified by labelling cells with an impermeable biotinylation reagent and streptavidin pull-down beads as described in the Experimental section. Total HER3 expression is shown by simple immunoblotting of total cell lysates. The experiments include treatment with 200 nM lapatinib or control for 48 h. MAPK, mitogen-activated protein kinase; p-, phospho-.

HER3 can be transcriptionally induced

Treatment of HER2-driven SkBr3 breast cancer cells with lapatinib increased HER3 transcript levels significantly within 48 h of treatment (Figure 2A and Supplementary Figure S2 at The increase in HER3 transcript was also observed with erlotinib, indicating that it is not a drug-specific mechanism but a drug class effect (Figure 2B and Supplementary Figure S2). To determine whether the increase in HER3 transcript was driven by increased promoter activity, we assayed the activity of a luciferase reporter driven by the HER3 promoter. This revealed that TKI treatment induced an ~4-fold increase in HER3 promoter activity in response to TKI treatment (Figure 2C and Supplementary Figure S3 at This suggests that the TKI-induced increase in HER3 transcript is driven, at least in part, through increased transcriptional activity. Feedback signalling to HER3 transcription is mediated through FOXO (Forkhead box O) transcription factors [25,26]. Analysis of the 1118 bp promoter region included in our HER3 promoter construct using the PROMO database showed no FOXO-binding sites within this sequence.

Figure 2 Induction of HER3 transcriptional activity

(A and B) HER3 transcript levels were measured by quantitative PCR after treatment of SkBr3 cells with lapatinib (200 nM) or erlotinib (5 μM) for 0, 1 and 48 h. The fold increase in HER3 transcript levels was measured compared with the zero time point. (C) A HER3 promoter-reporter assay was performed using the 1.1 kb HER3-pGL3 promoter construct driving a luciferase reporter, transfected into SkBr3 cells. Shown is the relative fold increase in the HER3 promoter activity upon treatment of the cells for 48 h with lapatinib (200 nM) compared with mock treatment. Results were normalized to a transfected construct reporting Renilla luciferase activity driven by the constitutive CMV promoter. Results are means±S.E.M. for triplicate values. *P<0.05.

HER3 transcript stability can be induced by miRNA down-regulation

Levels of mRNA transcripts can also be regulated by miRNAs which destabilize transcripts by targeting sequences within the 3′UTR of transcripts. To determine whether the 3′UTR of HER3 could mediate the drug-induced increase in HER3 transcripts, we assayed the activity of the luciferase coding sequence fused to the HER3 3′UTR. Lapatinib treatment of SkBr3 cells transfected with the HER3 3′UTR reporter construct showed a 2-fold increase in the firefly-luciferase activity compared with mock cells (Figure 3A and Supplementary Figure S4 at, suggesting that the drug-induced up-regulation of HER3 expression may be due to a decrease in HER3-targeting miRNAs.

Figure 3 Induction of HER3 by miRNA derepression

(A) Luciferase reporter assays were performed using the firefly luciferase coding sequences fused to the 3′UTR of HER3. Shown is the induction of luciferase activity in SkBr3 cells following 48 h of lapatinib (200 nM) treatment. Results are normalized to the constitutive activity of a transfected Renilla luciferase reporter to account for changes due to cell growth inhibition. (B) Quantitative PCR was performed to ascertain the transcript levels of miR-106b after treatment of SkBr3 cells for 48 h with 200 nM lapatinib. Shown is the fold change in miR-106b transcript levels after lapatinib treatment compared with mock treatment. RNU48 was used as a normalization factor. Results are means±S.E.M. for triplicate values. *P<0.05. (C) Western blot analysis of HER3 protein levels after transfection of SkBr3 cells with an miR-106b mimic, and compared with a mock-transfection control. (D) Western blot analysis of HER3 expression following transfection of SkBr3 cells with an miR-106b mimic and 48 h of lapatinib treatment. IB, immunoblot.

In a screen of miRNAs expressed in SkBr3 cells, miR106b emerged as an miRNA that is down-regulated following lapatinib treatment. Quantitative PCR analysis confirmed that miR-106b was down-regulated more than 5-fold within 48 h of lapatinib treatment (Figure 3B and Supplementary Figure S5 at The suppression of HER3 expression by miR-106b was confirmed in a transient transfection assay. Transfection of SkBr3 cells with an miR-106b mimic decreased HER3 expression compared with mock-transfected cells (Figure 3C). Furthermore, if the down-regulation of miR-106b expression was prevented by exogenous expression of miR-106b, the lapatinib-induced up-regulation of HER3 expression was attenuated (Figure 3D).

HER3 expression can be induced through increased protein translation

The up-regulation of HER3 is not just seen when its own signalling is disrupted, such as with TKI treatment. Rather, the expression and signalling activity of HER3 is reciprocally linked in a robust feedback-signalling loop with downstream PI3K/Akt signalling activity in HER2-amplified cancer cells. This was previously shown through the negative and positive perturbation of downstream signalling using downstream inhibitors or an activated Akt construct [17]. Although transcriptional and miRNA mechanisms can be induced to up-regulate HER3 expression when HER2–HER3-driven cancer cells are treated with HER-targeting TKIs, different mechanisms can be induced when the signalling throughput is disturbed at downstream targets. Rapamycin is an allosteric inhibitor of mTORC1 [mTOR (mammalian target of rapamycin) complex 1] and inhibits some, but not all, of the functions of mTORC1. Rapamycin treatment induces a compensatory increase in HER3 protein expression in SkBr3 cells (Figure 4A). This is accompanied by the complete inhibition of the mTORC1 substrate p70S6K1 (p70 S6 kinase 1) (seen through dephosphorylation of its substrate S6), but a compensatory increase in phosphorylation of another mTORC1 substrate 4E-BP1 at Thr37/46 (Figure 4A). In contrast with the TKI-mediated up-regulation of HER3, the rapamycin-induced up-regulation of HER3 is not transcriptionally mediated (Figure 4B). The inhibition of S6K has been shown to induce a compensatory increase in upstream signalling activity in other cellular contexts [2729]. To determine whether the rapamycin-induced up-regulation of HER3 is mediated through an S6K feedback-signalling mechanism, we determined whether a similar induction of HER3 can be seen through directly targeting S6K. Direct inhibition of p70S6K1 with the S6K inhibitor DG2 did not induce an up-regulation of HER3 expression (Figure 4C). Therefore the rapamycin-induced up-regulation of HER3 expression in these cells is not mediated through an S6K-dependent feedback-signalling mechanism. The increase in HER3 observed with rapamycin was not due to a decrease in HER3 protein turnover since HER3 expression is not increased or prolonged by rapamycin in the absence of new protein synthesis (Figure 4D).

Figure 4 Induction of HER3 protein by translational up-regulation

(A) SkBr3 cells were treated with the mTORC1 inhibitor rapamycin (25 nM) for 0, 1, 48 and 72 h and the lysates were analysed for total levels of HER3, phosphorylation of HER3, phosphorylation of S6 or phosphorylation of 4E-BP1 at the Thr37/46 site. (B) Quantitative PCR was performed to measure HER3 transcript levels after treatment with rapamycin (25 nM) for 0, 1 or 48 h. Shown is the fold change in HER3 transcript compared with time zero. (C) SkBr3 cells were treated with the S6K inhibitor DG2 for 0, 1, 48 and 72 h and lysates were analysed as in (A). (D) Western blot analysis of SkBr3 cell lysates after treatment for the indicated time points with 25 nM rapamycin in the presence or absence of cycloheximide (100 μg/ml). (E) Diagram of the secondary structure of the 5′UTR regions of HER3 and actin assembled by Genebee. (F) Schematic representation of the effects of rapamycin and DG2 on mTORC1 functions and how this affects HER3 signalling. (G) Western blotting for HER3 protein expression after transfection of SkBr3 cells with HA-tagged eIF4E or 4E-BP1, or vector controls. Blotting for the HA tag was performed to confirm expression of the transfected genes. (H) SkBr3 cells treated with rapamycin after transfection with the 4E-BP1-expressing vector. p-, phospho-; pY, phospho-tyrosine.

Since rapamycin induces a compensatory up-regulation of 4E-BP1 phosphorylation, we thought that the up-regulation of HER3 protein expression may be mediated through increased protein translation driven by increased 4E-BP1 phosphorylation. HER3 has a highly structured 5′UTR with a free energy of −61.8 kcal/mol (1 kcal=4.184 kJ) (Figure 4E, shown in comparison with actin with a free energy of −14.1 kcal/mol), rendering it more dependent on eIF4E, which is negatively regulated by 4E-BP1 (Figure 4F). We investigated the role of 4E-BP1/eIF4E function in the regulation of HER3 expression through transient transfection. The overexpression of eIF4E increased HER3 expression and, conversely, overexpression of 4E-BP1 decreased HER3 levels (Figures 4G and 4H). These data show that HER3 protein expression can be up-regulated in response to drug inhibition through a translational mechanism involving the regulation of 5′cap-binding complexes.

HER3 signalling can be induced without an induction of expression

Since rapamycin treatment induces a compensatory increase in 4E-BP1-mediated HER3 protein translation, the complete inhibition of mTORC1 functions with a mTOR kinase inhibitor should avert this mechanism of HER3 up-regulation. Treatment of SkBr3 cells with the mTOR kinase inhibitors PP242 or BEZ235 inhibited all mTORC1 functions, including the inhibition of both S6 and 4E-BP1 phosphorylation (Figure 5A). Consequently there was no up-regulation of HER3 protein expression with these mTOR kinase inhibitors (Figure 5A). Despite this, HER3 signalling activity was up-regulated without an increase in its total expression (Figure 5A). The addition of an mTOR kinase inhibitor also blocked the lapatinib-induced up-regulation of HER3 expression, as shown previously [17]. The up-regulation of HER3 signalling activity occured within 1 h of mTOR kinase inhibitor treatment. The increase in HER3 phosphorylation induced by mTOR kinase inhibitors was not due to an increase in membrane localization of HER3 as there was no increase in plasma-membrane-bound HER3 following treatment with these inhibitors (Figure 5B). The increase in HER3 phosphorylation is likely to be mediated through decreased dephosphorylation, probably through inhibition of phosphatases that regulate HER3. We have shown previously that this can occur through an inhibition of HER3 tyrosine phosphatases by increased reactive oxygen species [19]. Alternatively, HER3 phosphorylation could be influenced by changes in PTP (protein tyrosine phosphatase) expression and/or activity. The dephosphorylation of HER3 in SkBr3 cells is a rate-limiting step in HER3 signalling and potentially subject to regulation by feedback signalling mechanisms. This is evident in the sodium vanadate treatment of these cells, which inhibits PTPs and induces an increase in HER3 phosphorylation (Figure 5C). The specific PTPs that regulate HER3 are not currently known.

Figure 5 Induction of HER3 signalling by enhanced phosphorylation

(A) SkBr3 cells were treated with the mTOR kinase inhibitor PP242 (1 μM) or BEZ235 (250 nM) for the indicated time points, and the expression of total and phosphorylated HER3 was assayed by immunoblotting. (B) Cell-surface HER3 (mHER3) expression was determined by biotinylation of the surface proteome using cell-impermeable reagents and subsequent streptavidin pulldowns followed by HER3 immunoblotting. Results are shown for control or PP242 (1 μM) treatment for 48 h. (C) SkBr3 cells were treated with 1 mM sodium vanadate for 0, 1 or 4 h. Cell lysates were analysed by Western blotting as indicated. p-, phospho-; pY, phospho-tyrosine.

HER3 signalling can be induced by membrane trafficking

Another potential means of modulation of HER3 signalling is through the regulation of its membrane localization. HER3 protein is found in cytoplasmic and membrane pools, but HER3 signalling in HER2-amplified cancer cells is generated through HER2–HER3 interactions occurring at the plasma membrane. As such, co-localization with membrane HER2 is potentially subject to regulation by membrane trafficking of HER3.

Since HER3 signalling in HER2-amplified tumours is tightly coupled to Akt activity, its membrane expression may be linked to trafficking mechanisms known to be regulated by Akt. Akt is capable of either positive or negative regulation of protein translocation to the membrane. One mechanism for the negative regulation of membrane translocation is through the regulation of NO signalling (Figure 6A). Akt phosphorylates and activates NOS leading to increased NO production [3032]. NO is known to inhibit vesicular trafficking and inhibit exocytosis through the nitrosylation of NSF (N-ethylmaleimide-sensitive factor) [33,34]. NSF is a cytoplasmic protein that, through its ATPase activity, dissassembles vesicular SNARE (soluble NSF-attachment protein receptor) complexes leading to membrane fusion and exocytosis [35,36]. Therefore Akt can negatively regulate membrane trafficking through NO signalling. This is in contrast with other mechanisms whereby Akt can positively regulate membrane trafficking, such as seen in the insulin-induced membrane localization of GLUT4 (glutamate transporter 4) [37,38].

Figure 6 Induction of HER3 membrane trafficking

(A) Schematic diagram depicting how Akt can regulate protein exocytosis through NO signalling. (B) SkBr3 cells were treated with the NO scavenger PTIO (10 μM), or the NOS inhibitors S-MITU (0.5 mM) or L-NAME (5 mM) for 24 h and surface membrane HER3 expression was assayed by streptavidin pulldowns of the biotinylated surface proteome as shown. Total cellular HER3 levels are shown for comparison. (C) SkBr3 cells were treated with 200 nM lapatinib for 48 h or the NO donor DETA/NO for 8 h. The surface HER3 (mHER3) and total cellular HER3 expression were assayed as described above. (D) The phosphorylation of eNOS was assayed by immunoblotting following lapatinib treatment (200 nM). p-, phospho-.

The negative regulation of HER3 localization by NO signalling is apparent in experiments designed to interfere with NO signalling. Treatment of SkBr3 cells with the NOS inhibitors L-NAME or S-MITU, or the NO scavenger PTIO induces an increase in HER3 plasma membrane expression without an increase in total cellular HER3 protein expression (Figure 6B). The TKI-induced up-regulation of total cellular HER3 expression is accompanied by a parallel increase in membrane HER3 expression. This increase in membrane HER3 induced by TKI treatment can be inhibited by NO donor treatment without reducing total cellular HER3 expression (Figure 6C). The effects of TKI treatment on Akt activity are transient and the compensatory up-regulation of HER3 eventually restores Akt activity [17,19]. These effects are also seen in the Akt substrate eNOS, which is similarly dephosphorylated and inhibited following TKI treatment, and its phosphorylation partially restored with prolonged TKI therapy at the new steady state (Figure 6D).


The role of HER3 as a signalling partner for EGFR and HER2 has been known for many years. Its signalling functions are regulated through its ligand-mediated conformational activation, leading to dimerization with its kinase-competent partners and consequent phosphorylation of its C-terminal signalling tail [11,12]. In physiological signalling, HER3 can initiate and regulate the signalling process by virtue of its direct recognition of and activation by ligand stimuli.

HER3 is important in tumorigenic signalling as well, although it does not appear to initiate the signal generation in these scenarios. Its particular importance as a collateral partner in HER2-induced tumorigenesis has also been recognized for a number of years. Although HER3 is not transforming by itself, it is synergistically transforming with HER2 [39]. Furthermore, the functions of HER3 are required for tumorigenic growth in HER2-amplified cancers, as shown in knockdown experiments [15,16]. Since HER3 is catalytically inactive, its functions in HER2-driven tumorigenic signalling had been presumed to be a slave function, transmitting signals generated by HER2 and dictated entirely by the activities of the HER2 kinase. This paradigm was dismissed when it became apparent that the inhibition of HER2 kinase activity in HER2-driven cancer cells fails to silence HER3 signalling, revealing that HER3 signalling activity is not entirely dictated by its partner HER2 [19]. The signalling activity of HER3 is tightly coupled in a reciprocal relationship with downstream PI3K/Akt signalling in a way that ensures that signalling throughput is maintained, despite inhibition of HER2 functions by inhibitors [17]. As such, the expression of HER3 is a potential point of impact in tumorigenic signalling, and in drug resistance.

In the present study we show that the expression of HER3 can be increased through a multitude of redundant mechanisms in HER2-amplified cancers, all of which ultimately function to preserve tumorigenic signalling downstream of HER3. The specific mechanism or mechanisms engaged to increase HER3 signalling output depend on the specific manner in which the network topology is disturbed, and the specific upstream or downstream signalling proteins that are targeted by drugs. The plurality of mechanisms that can be engaged to increase HER3 signalling output reveal the highly dynamic nature of this HER family member, and attest to its role as a critical signalling hub that links its kinase-competent HER partners with the important cellular functions regulated by the Akt/mTOR network.

One mode of induction of HER3 expression is through increased transcriptional activity. We show in the present study that HER3 promoter activity is indeed induced in response to HER TKI treatment. This, at least in part, accounts for increased HER3 mRNA transcripts seen with HER TKI therapy. The transcriptional regulation of HER3 has been shown to be in part regulated through FOXO1 and FOXO3a transcription factors through binding sites >2.5 kb upstream of the transcription start site [25,40]. The HER3 promoter used in our experiments encompasses only 1.1 kb of upstream sequences, excluding the putative FOXO1- and FOXO3-binding sites, revealing a role for additional transcription factors in the transcriptional induction of HER3 following TKI treatment. Transcription factors described to regulate HER3 via more proximal elements include AP-2 (activating protein-2), ZNF217 (zinc-finger protein 217) and CtBP2 (C-terminal binding protein 2) [22,41].

Another mode of induction of HER3 expression is through the down-regulation of repressive miRNAs. miRNAs can interfere with expression through destabilization of the mRNA transcript or suppression of its translation. miRNAs typically act through target sequences in the 3′UTR of mRNA transcripts, and the fact that the 3′UTR of HER3 confers lapatinib inducibility to the luciferase-coding sequences suggests a component of regulation mediated through miRNAs. From an miRNA microarray screen we identified miR-106b levels as a HER3-targeting miRNA species that is reduced upon TKI treatment. The role of miR-106b was confirmed more specifically, as shown in Figures 3(B) and 3(C). Many other miRNAs may also regulate HER3 expression. In particular miR-205 has been described to regulate HER3, and miR-125a and miR-125b have been described to regulate both HER2 and HER3 [42,43]. In the present study we specifically looked for TKI-induced changes in miRNA expression, and the one miRNA we have been able to confirm to be affected by TKI treatment is miR-106b.

Another mode of regulation of HER3 is through increased translation. We have previously shown that an activated Akt construct represses HER3 protein levels without an associated down-regulation of HER3 mRNA transcripts [17]. However, the induction of HER3 transcription was suppressed by the activated Akt construct. This suggested that Akt can negatively regulate HER3 expression through both transcriptional and post-transcriptional mechanisms. We show in the present study that rapamycin also induces an increase in HER3 protein expression without an induction of HER3 transcript levels, in agreement with previous reports [25]. The mechanism of translational up-regulation induced by rapamycin is through the regulation of cap-binding proteins. Although rapamycin effectively inhibits mTORC1 phosphorylation of p70S6K, it fails to inhibit the mTORC1 phosphorylation of 4E-BP1, and in fact induces a compensatory increase in 4E-BP1 phosphorylation [44]. We show that the induction of HER3 protein expression is specifically a consequence of the induction of 4E-BP1, not the inhibition of S6K. The results from lapatinib treatment are also consistent with this, since lapatinib treatment does effectively inhibit S6 phosphorylation, yet the induction of HER3 in this scenario is mediated predominantly through mechanisms other than translational up-regulation. Although in other cellular contexts a negative-feedback signalling loop puts IRS-1 (insulin receptor substrate-1) expression under feedback from S6K and regulates insulin sensitivity [2729], the circuitry is different in these HER2-amplified tumour cells. The 5′UTR of HER3 is highly structured with multiple hairpins and loops typical of transcripts highly regulated by 5′cap-binding proteins.

Although in HER2-amplified tumour cells the level of HER3 phosphorylation and signalling can be regulated through its level of expression or through its membrane localization, its signalling activity can also be regulated directly through regulation of its phosphorylation. This is most apparent upon treatment with mTOR kinase inhibitors which suppress the total cellular or membrane up-regulation of HER3 expression, yet they still elicit a compensatory up-regulation of HER3 phosphorylation. This is highly unlikely to be due to phosphorylation by tyrosine kinases outside the HER family, as we have previously exhaustively investigated this hypothesis with no evidence to support it [17,19]. The observed increase in HER3 phosphorylation seen with mTOR kinase inhibitors is probably mediated through a down-regulation of HER3 dephosphorylation. This could be either through mechanisms specific for certain PTPs, or it could be through a more general inhibition of PTPs through an increase in reactive oxidative species, as is seen following TKI treatment of these cells. It is difficult to speculate on these mechanisms, since the specific PTPs that regulate the phosphorylation of HER3 are not yet known.

Another mode of regulation of HER3 is through the regulation of its membrane localization. In particular, its membrane expression has been shown to be up-regulated by the transmembrane mucin MUC4 [45]. We did not observe drug-induced changes in MUC4, but it remains possible that some drugs can elicit such a compensatory cellular response. We did observe Akt-driven NO-mediated membrane trafficking as one of the compensatory mechanisms induced by drug treatment.

The functions of HER3 as a collateral partner in tumorigenic growth driven by its HER family partners is now well established, in particular in HER2-driven breast cancers where its functions are essential. The tumour cell requirement for continued HER3 signalling is evident in the feedback-signalling relationship we previously demonstrated between upstream HER3 signalling and downstream Akt signalling activities [17]. Determining the precise circuitry that protects HER3 against drug-induced loss of its signal can lead to ideas about more effective combination therapy approaches that can potentially disrupt cellular mechanisms involved in the compensatory up-regulation of HER3. However, we now find that the feedback to HER3 is not a simple circuit and can be engaged through a variety of mechanisms. There appears to be a multitude of redundant mechanisms regulating HER3 signalling in HER2-amplified tumours, which are rapidly inducible by drugs that disrupt the flow of signal along the HER3/PI3K/Akt/mTOR signalling pathway. The highly dynamic nature of HER3 expression and signalling, and the plurality of downstream mechanisms ultimately regulating its signalling functions identify HER3 as a major signalling hub in HER2-amplified cancers. This only attests further to the central role of HER3 in HER2-driven tumorigenesis.


The present study was conceived by Mark Moasser and designed by Dhara Amin, Mark Moasser and Andrei Goga. The experiments were performed by Dhara Amin, Natalia Sergina and Lionel Lim. The data was prepared by Dhara Amin and Mark Moasser.


This work was supported by the National Institutes of Health [grant numbers CA122216, CA112970].


We thank Morri Feldman and Kevan Shokat for providing PP242 and DG2, and Frederick Domann and Davide Ruggero for plasmid reagents as detailed in the Experimental section.

Abbreviations: CMV, cytomegalovirus; DETA/NO, diethylenetriamine/NO adduct; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; EGFR, epidermal growth factor receptor; eIF4E, eukaryotic initiation factor 4E; eNOS, endothelial NO synthase; FOXO, Forkhead box O; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, haemagglutinin; HER, human epidermal growth factor receptor; L-NAME, NG-nitro-L-arginine methyl ester; miRNA, microRNA; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; NOS, NO synthase; NSF, N-ethylmaleimide-sensitive factor; PI3K, phosphoinositide 3-kinase; p70S6K1, p70 S6 kinase 1; PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt; PTP, protein tyrosine phosphatase; RT, reverse transcription; S-MITU, S-methylisothiourea hemisulfate salt; TKI, tyrosine kinase inhibitor; UTR, untranslated region


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