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(2007) 26, 6577–6592 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW Targeting the function of the HER2 oncogene in human therapeutics

MM Moasser

Department of Medicine, Comprehensive Cancer Center, University of California, San Francisco, CA, USA

The year 2007 marks exactly two decades since human HER3 (erbB3) and HER4 (erbB4). The importance of epidermal -2 (HER2) was func- HER2 in cancer was realized in the early 1980s when a tionally implicated in the pathogenesis of human breast mutationally activated form of its rodent homolog neu cancer (Slamon et al., 1987). This finding established the was identified in a search for in a - HER2 oncogene hypothesis for the development of some induced rat tumorigenesis model(Shih et al., 1981). Its human . An abundance of experimental evidence human homologue, HER2 was simultaneously cloned compiled over the past two decades now solidly supports and found to be amplified in a breast line the HER2 oncogene hypothesis. A direct consequence (King et al., 1985). The relevance of HER2 to human of this hypothesis was the promise that inhibitors of cancer was established when it was discovered that oncogenic HER2 would be highly effective treatments for approximately 25–30% of breast cancers have amplifi- HER2-driven cancers. This treatment hypothesis has led cation and overexpression of HER2 and these cancers to the development and widespread use of anti-HER2 have worse biologic behavior and prognosis (Slamon () in clinical management resulting et al., 1989). This finding established the HER2 in significantly improved clinical antitumor efficacies that oncogene hypothesis that overexpression of HER2 is have transformed the clinical practice of . In the etiologically linked with tumorigenesis in some human shadows of this irrefutable clinical success, scientific cancers. A substantialbody of experimentalevidence studies have not yet been able to mechanistically validate over the past two decades has come to solidly support that trastuzumab inhibits oncogenic HER2 function and it this hypothesis. In numerous in vitro and transgenic remains possible that the current clinical advances are a models, HER2 overexpression by itself is potently consequence of the oncogene hypothesis, but not a transforming. In addition, analysis of human breast translation of it. These looming scientific uncertainties cancers has shown that amplification of the HER2 suggest that the full promise of the treatment hypothesis is an early event in human and along with may not yet have been realized. The coming decade will the experimentalevidence confirming its potently see a second generation of HER2-targeting agents transforming functions, this makes a highly compelling brought into clinical testing and a renewed attempt to case, implicating HER2 overexpression in the genesis of treat HER2-driven cancers through the inactivation of these human cancers. The signaling functions of HER2, HER2. Here, I review the development of treatments that the body of evidence confirming the transforming target HER2 in the context of the HER2 oncogene functions of HER2, the numerous proposed mechan- hypothesis, and where we stand with regards to the clinical isms mediating its transforming functions, and the data translation of the HER2 oncogene hypothesis. establishing the relevance of these findings to human Oncogene (2007) 26, 6577–6592; doi:10.1038/sj.onc.1210478; cancer pathogenesis were discussed in depth previously published online 7 May 2007 (Moasser, 2007). A direct consequence of the HER2 oncogene hypothesis of human cancer was that inhibi- Keywords: HER2; ErbB2; trastuzumab; herceptin; tors of oncogenic HER2 would be highly effective inhibitor; treatment for HER2-driven cancers. Here, I will review where we stand with regards to the testing of this treatment hypothesis and where we currently stand with regards to the therapeutic implications of the HER2 Introduction oncogene hypothesis.

HER2 belongs to the human epidermal (HER) family of consisting of Tumor dependence on HER2 EGFR (HER1, erbB1), HER2 (erbB2, HER2/neu), The tumorigenic potential of HER2 is solidly supported Correspondence: Dr MM Moasser, Department of Medicine, Com- by experimental models (Moasser, 2007). This by itself prehensive Cancer Center, University of California, San Francisco, proposes HER2 as a possible target for anti-cancer UCSF Box 0875, San Francisco, CA 94143-0875, USA, E-mail: [email protected] drugs. However, its suitability as a drug target is Received 15 March 2007; accepted 22 March 2007; published online 7 substantially strengthened by experiments demonstrat- May 2007 ing that HER2-driven tumors are dependent on HER2 HER2 oncogene in human cancer therapeutics MM Moasser 6578 function. This dependency, recently labeled oncogene pathways (Moody et al., 2005). Tetracycline-regulated addiction, identifies oncogenes that are high value NIH3T3-HER2 tumors cells that regress following targets for drug development (Weinstein, 2002). withdrawal of oncogene expression similarly recur following a period of remission despite absence of HER2-dependency of HER2-amplified human cancers oncogene expression, although the molecular features Experimentalmodelsof HER2-overexpressing cancer associated with HER2-independent recurrence in this cells using antisense, ribozyme or short interfering RNA modelare not yet described (Schiffer et al., 2003). The (siRNA) methodologies consistently show that HER2 direct relevance of these models of recurrence to human knockdown induces in cell culture, or tumor tumors is not yet known and awaits analysis of human regression in vivo, in the absence of HER2 expression, tumors that have recurred following a complete remis- while tumor types that do not overexpress HER2 are not sion induced by HER2-targeted therapies. sensitive to HER2 knockdown (Colomer et al., 1994; Juhl et al., 1997; Roh et al., 2000; Choudhury et al., 2004; Faltus et al., 2004). Similar results are seen with Inhibition of HER2 for cancer therapy kinase-dead HER2 and intracellular single-chain anti- HER2 antibodies (Beerli et al., 1994; Messerle et al., The evidence to support the HER2 oncogene hypothesis 1994; Deshane et al., 1996). that HER2 initiates and drives the progression of HER2-overexpressing cancers is almost unimpeachable HER2-dependency in experimental models at this point. The direct consequence of this hypothesis Engineered models of HER2-driven transformation is the treatment hypothesis that inactivation of HER2 using tetracycline-inducible systems confirm that could be highly effective therapy for patients with HER2-induced tumors require HER2 for continued HER2-overexpressing cancers. Due to the large number tumorigenic growth and survival. This has been of patients with this type of cancer, testing of the HER2 demonstrated in an HER2-transformed NIH3T3 tumor treatment hypothesis has been one of the most actively model, wherein tumors regress upon withdrawal of the pursued programs in the cancer therapeutic arena. HER2 oncogene (Baasner et al., 1996; Schiffer et al., Testing in human subjects requires the development of 2003). This has also been corroborated in a tet-inducible safe and effective therapies that inactivate HER2 in transgenic models. Tetracycline-induced expression of patient tumors and in the best scenario are predicted to activated HER2 in squamous epithelia of mice results in produce complete remissions and recapitulate results severe hyperplastic abnormalities of squamous epithelial from preclinical models. Correlative scientific studies of tissues, which reverse upon withdrawalof the HER2 these therapeutic agents in preclinical models and in transgene expression (Xie et al., 1999). Tumors in patients are essentialto determine the validity of the MMTV-neuT mice are also dependent on continued treatment hypothesis. These efforts have led to the oncogene expression. In the MMTV-rtTA/TetO-NeuNT development of the anti-HER2 human monoclonal bitransgenic variant of this modelregulated by doxycy- (mAb) trastuzumab, which has made signifi- cline, when expression of the neuT oncogene is induced cant clinical impact including a reduction in mortality in the mammary tissue of adult mice, the formation of from HER2-overexpressing disease. But mechanistic multiple mammary tumors and metastases is studies have been conflicting and suggest that the induced, and the entire and metastatic treatment hypothesis may not yet have been effectively disease fully regresses when neuT expression is with- tested. If the treatment hypothesis is correct and drawn (Moody et al., 2002). inhibiting oncogenic HER2 function would result in Although each of these models is subject to criticisms complete tumor regression, the clinical impact is relating to their simplicity, when taken in aggregate, predicted to be much larger than currently realized they are highly consistent and collectively make a highly and we may have merely seen the tip of the iceberg. The compelling case that HER2-induced tumors are ad- available data with regards to several anti-HER2- dicted to HER2. This has made HER2 one of the most targeted therapies are reviewed below. The two modali- sought after targets in cancer drug development. ties for which there are considerable data are antibody therapies and small molecule kinase inhibitors. These Potential to escape from HER2 addiction are discussed separately below. The inducible models do suggest the possibility of HER2-independent tumor recurrence that occurs after a Biologic effects of panels of anti-HER2 antibodies period of complete regression. Tumors that are induced Almost immediately following the discovery that the in the MMTV-rtTA/TetO-NeuNT modeland which oncogene HER2 was amplified in many breast and completely regress upon oncogene withdrawal, even- ovarian cancers and linked with disease biology, efforts tually recur after prolonged dormancy without induc- began to develop inhibitors of this oncogene. The tion of the neuT oncogene (Moody et al., 2002). This is technology to develop mAbs had become available at associated with induction of the transcriptionalrepres- this time, and since HER2 functions as a growth factor sor snail, and suggests that second hits may provide receptor, it was a highly rationale hypothesis at the time escape routes for residual tumor cells leading to that a mAb that binds the extracellular domain of tumor recurrence and progression driven by alternative HER2 and interferes with activation would

Oncogene HER2 oncogene in human cancer therapeutics MM Moasser 6579 inhibit tumorigenic HER2 function. The proof-of- principle experiment was initially conducted in the neuT transformation model. In this model, it was found that anti-Neu mAbs downregulate NeuT expression, supress , inhibit transformation and tumor growth in mice (Drebin et al., 1985, 1988). This suggested that HER2-overexpressing human cancers could also poten- tially be treated with mAbs. Over 100 mAbs were developed by numerous groups against the extracellular domain of human HER2. The effects of these mAbs on HER2-overexpressing human cancers turned out to be much more complicated than predicted from the more simplistic neuT model. The activities of some of these is inactive panels of mAbs against HER2-overexpressing tumor Growth inhibition correlates with homodimerization and endocytic degradation Engineered TM domain No correlation between HER2and turnover growth inhibition No correlation between HER2 downregulation and growth inhibition, Fab fragment also inhibits growth cell lines have been characterized and published and are summarized in Table 1. The results from these studies revealthat anti-HER2 mAbs can produce highlydiverse outcomes. These include both growth inhibitory or in vivo . 4D5 inhibits growth stimulatory effects, differentiating effects and and pro-apoptotic effects. Some mAbs induce HER2 phos- phorylation and others do not, some induce HER2 in vitro downregulation and others do not, some inhibit in vitro tumorigenic growth in vivo and others do not. The results of all these studies taken together do not formulate a clear picture of the mechanism by which in vivo an anti-HER2 mAb can inhibit tumor growth. Specifi- cally, cell growth inhibition or tumor growth inhibition growth Growth inhibition Cause growth inhibition (Calu-3) Internalization requires bivalency and Fab induces apoptosis Some are growth inhibitorydifferentiating, and some are growth stimulatory Only mutant HER2 was(soft growth agar) inhibited 1 of 4 inhibitstumor cell growth growth, 2 of 4 inhibit inhibit growth in agar of SkBr3 does not correlate with the mAb ability to downregulate HER2. In addition, anti-HER2 mAbs downregulate mutationally activated HER2 much more effectively than wild-type HER2, reproducing the effects seen with anti-Neu mAbs in the NeuT model(van Leeuween et al., 1990). Adding complexity to the picture is that even growth inhibition in vitro does not correlate with tumor growth inhibition in vivo such that some mAbs are

growth stimulatory in cell-culture models yet inhibit Published monoclonal anti-HER2 antibodies tumor growth in mice (Stancovski et al., 1991; Harwerth et al., 1993). The mechanistic principles underlying the diversity of findings from anti-HER2mAbs remain Table 1 unclear to this day. But the compelling data regarding downregulation internalize the role of HER2 in human tumorigenesis and evidence Biochemical findings (in Her2++ tumors) Biological findings (in Her2++ tumors)induce endocytic degradation Notes downregulation of mutant ErbB2 is strong induce downregulation of antitumor efficacy of some anti-HER2 mAbs in preclinical models drove the clinical development of at least one such agent.

Development of trastuzumab (Herceptin) Panelof 2 They induce and N Panelof 17 Most induce phosphorylation,some Out of those over 100 anti-HER2 mAbs generated in the 1980 and 1990s, one was developed for clinical testing. . The mAb 4D5 was selected among a panel of mouse . . (1991) Panelof 11 4D5 HER2 downregulates Some inhibit growth et al

anti-HER2 antibodies at Genentech Inc. (South San et al

Francisco, CA, USA) for development because of its et al antitumor effects in vitro and in mouse models (Hudziak et al., 1989; Shepard et al., 1991). Mouse mAb 4D5 was . (1995); Klapper humanized for clinical use yielding several humanized . (1990) Panelof 4 of Downregulation ErbB2 is poor, . (2005) One mAb Induces ErbB2 phosphorylation and et al . (1991); Bacus

variants. Some of these engineered variants lost in vitro et al . (1989) Panelof 10 Not reported Not reported . (1991); Srinivas . (1992, 1993) Panelof 4 2 of 4 induce phosphorylation,3 of 4 . (1989); Sarup . (2006) One mAb Internalizes Inhibits growth antiproliferative efficacy despite high affinity binding to et al et al et al et al et al . (2001) Panel of 2 They internalize (both as scFv or IgG) No growth inhibition Internalization does not require bivalency . (1996) Panelof 12 Most induce phosphorylation Growth data not reported, one mAb et al

HER2, but others retained antiproliferative efficacy and et al . (1993) Panel of 11 Some internalize and downregulate HER2 2 inhibit monolayer growth of SkBr3, 7

one such clone was selected for further clinical develop- et al et al . (1997); Yarden (1990) ment (named trastuzumab, Herceptin) (Carter et al., et al Hudziak (1993) De Lorenzo Belimezi Tagliabue Neve Reference McKenzie 1992). The constant regions of these humanized mAbs Stancovski (1992); Hurwitz et al Van Leeuween Harwerth Xu Kita

Oncogene HER2 oncogene in human cancer therapeutics MM Moasser 6580 were engineered for optimum participation in antibody- et al., 2001; Burstein et al., 2003; Marty et al., 2005). The dependent cellular cytotoxicity (ADCC) or complement- biggest impact of trastuzumab has been in the treatment dependent cytotoxicity (CDC), and indeed trastuzumab of patients with potentially curable early-stage breast is much more efficient than its murine counterpart in cancer. In early-stage HER2-amplified breast cancer mediating ADCC (Carter et al., 1992; Lewis et al., 1993; patients who receive after surgicalresec- Tokuda et al., 1996). Trastuzumab has reduced cell tion, the addition of trastuzumab to their chemotherapy culture antiproliferative activity compared with the regimens significantly prolongs disease-free survival and murine 4D5, but equally potent antitumor efficacy in reduces the chances of disease recurrence (Figure 1) mouse xenograft models (Tokuda et al., 1996; Baselga (Piccart-Gebhart et al., 2005; Romond et al., 2005). et al., 1998; Clynes et al., 2000). Although these adjuvant therapy studies are still in their early years of follow-up, the powerful effects seen in the early follow-up period is widely believed to translate to a Clinical antitumor activity of trastuzumab significant reduction in mortality from HER2-amplified The clinical antitumor activity of trastuzumab has now breast cancer and the use of trastuzumab has rapidly been extensively characterized in numerous clinical become the standard management of early-stage breast studies spanning the past decade and a half. Initial cancer patients. The clinical antitumor activity of difficulties in identifying the subset of patients with trastuzumab is limited to tumors with HER2 over- HER2-overexpressing tumors by clinically available expression and trastuzumab has no significant clinical immunohistochemical methods were finally overcome activity against breast cancers without HER2 over- by clinical implementation of a fluorescence in situ expression (Vogel et al., 2002; Mass et al., 2005). At this hybridization (FISH) assay to detect HER2 time, its single-agent activity appears to be limited to amplification and it is now evident that trastuzumab breast cancers and it has much less clinical antitumor induces tumor regression in approximately 30–35% of activity against ovarian or endometrialcancers with patients with HER2-amplified metastatic breast cancer if HER2 overexpression (Bookman et al., 2003; Fleming used as upfront therapy (Vogel et al., 2002; Mass et al., et al., 2003) and continues to be investigated in other 2005), and has much less activity if used after other types of cancer. (Baselga et al., 1996). In patients with metastatic disease, trastuzumab is not curative and disease progression resumes after a median duration of Implications of trastuzumab to the HER2 approximately 5 months despite continuous trastuzu- oncogene hypothesis mab therapy (Vogel et al., 2002). The most beneficial These improvements in the clinical management of clinical use of trastuzumab has been in combination patients with HER2-amplified breast cancer afforded by with various cytotoxic chemotherapies. The addition of trastuzumab are a direct consequence of the HER2 trastuzumab to multiple chemotherapy regimens sig- oncogene hypothesis of breast cancer initially proposed nificantly increases their antitumor efficacy (Slamon two decades ago and are a testament to the potentialof

Figure 1 Outcome results with a median follow-up time of 2 years in 3351 patients with early-stage breast cancer treated with chemotherapy and randomized to the addition of trastuzumab or control. (a) The proportion of patients in each arm that remains cancer-free at the indicated years of follow-up. Many more patients from the trastuzumab-treated arm (solid line) are cancer-free compared with the controlarm (broken line).( b) The proportion of patients in each arm that is alive at the indicated years. More patients from the trastuzumab-treated arm are alive compared with the control arm. The effect of trastuzumab on disease-free and overall survival is highly statistically significant. (Romond et al., 2005) Copyright 2005 Massachusetts MedicalSociety, Allrights reserved.

Oncogene HER2 oncogene in human cancer therapeutics MM Moasser 6581 scientific research to impact human health and disease extracellular domain of HER2 constitutively exists in an mortality. But, while the success of trastuzumab is a active conformation resembling the ligand-bound state consequence of the HER2 oncogene hypothesis, it is not of the other HER family , precluding any sufficient to validate it. Validation of the oncogene potential activating role for ligands (Cho et al., 2003; hypothesis requires mechanistic evidence that trastuzu- Garrett et al., 2003). Therefore, the hypothesis that mab treats patients through inactivation of tumor trastuzumab inhibits direct ligand binding and activa- HER2. This evidence is currently lacking and more tion of HER2 is all but dismissed at this point. An than a decades work in trying to determine the alternative hypothesis that has been proposed is that mechanism of action of trastuzumab has produced trastuzumab inhibits the interaction of HER2 with its largely conflicting and inconclusive results and a HER family partners or possibly other interacting compelling mechanistic model of how and whether proteins. But convincing evidence in support of this trastuzumab inhibits oncogenic HER2 function has not hypothesis has not yet emerged. In pulldown assays, emerged. trastuzumab does not inhibit HER2–HER3 interaction (Agus et al., 2002), and in fluorescence resonance energy transfer (FRET)-based assays trastuzumab also does Mechanism of action of trastuzumab – HER2 not inhibit HER2 interaction with EGFR or HER3 downregulation (Diermeier et al., 2005). In a different modelusing Extensive studies over the past decade have attempted to truncated HER proteins fusing them to b-galactosidase determine the molecular mechanism underlying the fragments in an complementation assay, trastu- clinical antitumor activity of trastuzumab. The simplest zumab was reported to inhibit EGFR–HER2 interac- hypothesis derives from the previously established anti- tion but not HER2–HER3 interactions (Wehrman et al., NeuT mAb and anti-HER2 4D5 mAb data showing that 2006). The artificialnature of the truncated receptors these mAbs induce the degradation of the targeted used in the latter study makes it less reliable, specially in surface receptors NeuT or HER2 (discussed above). light of FRET evidence to the contrary. Although this seems like a fairly simple hypothesis to test conclusively, extensive analyses by numerous investigators studying the effect of trastuzumab on Mechanism of action of trastuzumab – inhibition of tumor cell HER2 expression have produced conflicting HER2 cleavage results, even in similar cell types and assays. While some Trastuzumab binding inhibits the proteolytic cleavage studies show that trastuzumab downregulates HER2 in and shedding of HER2 by ADAM proteases (Molina HER2-overexpressing tumor cells (Cuello et al., 2001; et al., 2001; Liu et al., 2006). This may, in part, inhibit Citri et al., 2002; Lee et al., 2002; Marches and Uhr, the invasive properties of HER2-transformed cells, since 2004), other studies clearly show that it does not (Austin truncated HER2 induces a more invasive morpho- et al., 2004; Longva et al., 2005). Part of the complexity logy and is associated with increased kinase activity, in this field was resolved when it was determined that increased transforming efficiency, and is increased in trastuzumab binds and internalizes with surface HER2 patients with metastatic disease (Segatto et al., 1988; but re-emerges with HER2 at the surface, merely Christianson et al., 1998; Egeblad et al., 2001; Molina accompanying HER2 passively along its normal endo- et al., 2002). Therefore, trastuzumab may inhibit this cytic recycling route (Austin et al., 2004). The most aspect of HER2 function although the transforming compelling evidence at this point seems to support the function of HER2 is not known to require truncation position that trastuzumab does not induce the down- and many HER2-overexpressing breast cancers do not regulation of HER2 in tumor cells. Consistent with this, have significant truncation of HER2. three clinical studies show no reduction in tumor HER2 expression in patients undergoing treatment with Mechanism of action of trastuzumab – other findings trastuzumab (Gennari et al., 2004; Mohsin et al., 2005; While the therapeutically important effect of trastuzu- Arnould et al., 2006). Therefore, it appears unlikely that mab on the function of its direct target HER2 remains the clinical antitumor activity of trastuzumab is to be defined, numerous reports have emerged describ- mediated through downregulation of tumor HER2. ing the effects of trastuzumab on downstream signaling pathways. The antiproliferative effect of mAb 4D5 or Mechanism of action of trastuzumab – HER2 signaling trastuzumab in cell-culture models is associated with the The principal prevailing hypothesis that rationalized the induction of p27 and G1 block (Lane et al., 2000; development of trastuzumab and other anti-HER2 Marches and Uhr, 2004; Le et al., 2005). Trastuzumab mAbs, for most of the 90s was that it inhibits the affects the expression of tumor angiogenic factors and activation of HER2 by yet undiscovered ligands. exhibits certain anti-angiogenic properties in mouse However, the hypothesized HER2 ligand was never models (Izumi et al., 2002). Trastuzumab suppresses Akt discovered, and biochemicalscreens, post-genome com- signaling in some tumor cell types but not others putionalstudies and crystalstructure revelations have (Normanno et al., 2002; Yakes et al., 2002; Longva made it clear that HER2 has no physiologic ligand, and et al., 2005), increases plasma and tensin that its ligand responsive functions are mediated homolog (PTEN) localization and activity in cells through heterodimerization with its ligand-activated (Nagata et al., 2004; Longva et al., 2005), and its HER family partners (Sliwkowski, 2003). In fact, the antiproliferative and antitumor effects are attenuated by

Oncogene HER2 oncogene in human cancer therapeutics MM Moasser 6582 PTEN knockdown (Nagata et al., 2004; Fujita et al., that trastuzumab works through an ADCC-mediated 2006). Consistent with a functionalrolefor PTEN in mechanism has led some investigators to determine clinical antitumor efficacy, tumors with reduced or whether this activity can be further enhanced by absent PTEN are relatively resistant to trastuzumab- additionalimmunostimulatoryapproaches. Consistent containing chemotherapy regimens (Nagata et al., 2004; with this hypothesis, IL-2 enhances the in vitro ADCC Fujita et al., 2006). Although these data sets are activity of trastuzumab (Carson et al., 2001; Kubo et al., complicated by the concomitant use of cytotoxic 2003). However, a phase I trialof this combination chemotherapy regimens, they are the only currently modality found no evidence of increased clinical activity existing evidence linking intracellular signaling with the associated with IL-2-induced NK cell expansion (Repka clinical antitumor activity of trastuzumab. An associa- et al., 2003). In another approach, a bispecific antibody tion between trastuzumab resistance and PTEN loss by that targets HER2 as well as CD3 antigen on T cells was itself does not necessarily imply that trastuzumab developed to recruit T cells to tumor cells. This agent inhibits tumors through direct effects on tumor signal- showed promising evidence of activity in its phase I ing, since PTEN loss has also been shown to mediate study and further studies are underway to determine its immunoresistance (Parsa et al., 2007). antitumor efficacy (Kiewe et al., 2006). In another approach, the in vitro ADCC activity of trastuzumab appears to be augmented by defucosylation, and the Mechanism of action of trastuzumab – immunological fucosyl-negative version of trastuzumab has been targeting proposed for clinical testing (Suzuki et al., 2007). An increasing body of evidence suggests that the in vivo These preclinical experimental models and clinical antitumor effects of the anti-HER2 mAb 4D5 and the observational studies have provided compelling evi- humanized trastuzumab may be mediated, at least in dence that much of the antitumor activity of trastuzu- part, if not entirely, through immunological targeting mab is mediated through immunological targeting mechanisms. mAb 4D5 activates ADCC in vitro (Carter mechanisms. Although these data sets are still relatively et al., 1992; Clynes et al., 2000). This activity was greatly few, the failure of the signaling field to confirm that the enhanced during the process to engineer the humanized clinical activities of trastuzumab are mediated through version and trastuzumab is indeed highly efficient at inhibition of HER2 signaling has led to more and more activating ADCC in vitro (Carter et al., 1992; Cooley attention being focused on the pursuit of the immuno- et al., 1999; Carson et al., 2001; Kubo et al., 2003; logical targeting hypothesis and additional studies to Repka et al., 2003). Mouse genetic models that further explore this hypothesis are underway. experimentally manipulate Fc receptor function posi- tively or negatively clearly demonstrate the role of host immunologic mechanisms in the antitumor efficacy of Targeting HER2 with these agents. The antitumor activity of both mAb 4D5 A second anti-HER2 mAb is currently undergoing and trastuzumab are almost entirely abolished in the clinical testing. From the initial panel of anti-HER2 loss-of-function FcRgÀ/À mice, while the antitumor mAbs developed at Genentech Inc. (South San Fran- activity of subtherapeutic doses of mAb 4D5 and cisco, CA, USA) the mAb 2C4 was also selected for trastuzumab are greatly enhanced in the gain-of-func- further characterization and development and has tion FcRIIÀ/À mouse model(Clynes et al., 2000). properties distinct from mAb 4D5. mAb 2C4 has been Furthermore, when the Fc region of the mAb 4D5 is modified for human clinical use by recombinant mutated at a single position to eliminate engagement of engineering to generate the humanized version pertuzu- host Fcg receptors, the mutant mAb 4D5 retains all its mab (Adams et al., 2006). Pertuzumab is currently in vitro antiproliferative activity but loses its in vitro undergoing clinical trials and preliminary evidence ADCC activity and loses its in vivo antitumor efficacy suggests that it can enhance the efficacy of trastuzumab, (Clynes et al., 2000). This modelhas provided highly but its single agent activity in HER2 overexpressing compelling evidence that the antitumor activity of breast cancer is not yet known (Baselga et al., 2007), trastuzumab is mediated, in large part, through immu- Pertuzumab has little activity in breast cancers without nological targeting of tumor cells. HER2 overexpression and in unselected ovarian cancers Investigators have also recently begun to look for (Cortes et al., 2005; Fleming et al., 2005). clinical evidence of immunological targeting by trastu- The activities of 2C4 or pertuzumab on cell-signaling zumab. In a clinical study of a trastuzumab-containing pathways have only been reported by selected investi- chemotherapy regimen compared with case-matched gators, since these agents are not yet available for study controls, trastuzumab treatment was associated with to the wider scientific community. Compared with mAb significantly increased numbers of activated cytolytic 4D5 or trastuzumab, mAb 2C4 has much less anti- natural killer (NK) cells within tumors (Arnould et al., proliferative activity in vitro (Hudziak et al., 1989; Takai 2006). In a second study of trastuzumab monotherapy, et al., 2005), but has in vivo antitumor activity in a the treatment induced varying degrees of tumor infiltra- number of tumor types including tumors without HER2 tion with lymphoid cells and the patients that responded overexpression (Agus et al., 2002; Takai et al., 2005). to trastuzumab had the highest degree of tumor 2C4 is reported to inhibit heregulin-mediated HER2 infiltration and higher ADCC activity receptor complex formation, phosphorylation, and measured ex vivo (Gennari et al., 2004). The evidence MAPK and Akt activation in breast cancer cells without

Oncogene HER2 oncogene in human cancer therapeutics MM Moasser 6583 HER2 overexpression (Agus et al., 2002). Determina- using purified EGFR, HER2 or HER4 kinases, and tion of the effects of pertuzumab on HER2-overexpres- these are listed in Table 2. However, the biological sing cancers awaits much more preclinical and clinical relevance of these in vitro characteristics is not evident in studies. cell-based assays. The EGFR selective agent gefitinib inhibits the phosphorylation of all of the HER proteins in cells, as does the HER2-selective agent CP-654577 Targeting HER2 with HER kinase inhibitors (Moasser et al., 2001; Shepherd et al., 2001; Normanno The technology to develop selective et al., 2002). In cell proliferation assays, HER2-over- inhibitors (TKIs) for human use succeeded antibody expressing tumors are particularly sensitive to the highly therapeutics by almost a decade. These agents, at least in EGFR-selective TKIs gefitinib, AG1478, CGP-59326A theory, have certain advantages over antibody therapies and EKB-659 (see Table 2). In fact, in an engineered for the treatment of HER2-amplified cancers. Antibody HER2-overexpressing tumor model, the level of HER2 therapies are cell impermeable agents that bind the overexpression directly correlates with sensitivity to the extracellular domain of HER2 and till today it remains EGFR-selective TKI AG1478 (Emlet et al., 2006). The unclear whether or how this binding activity can mechanisms underlying the activities of EGFR-selective suppress oncogenic HER2 function, although they TKIs against HER2 signaling and HER2-driven growth may induce the clinically important immunological are not currently understood, and may be due to the targeting of HER2-overexpressing cancer cells. TKIs direct inhibition of HER2 kinase by their weaker anti- are cell-permeable agents and can potentially inhibit the HER2 activity, high intracellular concentrations of these ligand-dependent and -independent kinase activity of agents precluding their target selectivities, or a necessary HER2 residing within the intracellular domain. This role for EGFR in HER2-driven growth. The latter strategy has a solid rational basis since kinase activity is appears unlikely, since at least in fibroblast models essentialfor the oncogenic function of neu or HER2 HER2 is transforming even in the absence of EGFR (Weiner et al., 1989; Qian et al., 1994). At least in expression (Chazin et al., 1992). These uncertainties with theory, these agents offer the opportunity to inactivate respect to the biologic importance of target specificity HER2 kinase function in patients with HER2-over- are somewhat academic and virtually all HER TKIs expressing cancer, and for the first time directly test the show activity against HER2-driven tumors in mouse HER2 oncogene hypothesis in patients. However, TKIs models regardless of their in vitro target selectivities (see don’t have the singular target specificity of antibodies Table 2) and they are all potentially candidate agents to and their off-target effects potentially limits their test the validity of the HER2 treatment hypothesis in therapeutic index compared with antibodies. patients with HER2-overexpressing cancers.

The development of HER-selective TKIs Synthetic and naturalproduct inhibitors of HER Clinical antitumor activity of HER TKIs – current data kinases from diverse structures were initially studied in Numerous TKIs are currently in various stages of the early 1990s, but their limited potencies and preclinical and clinical development. The clinical deve- specificities precluded their usefullness as antitumor lopment of each of these is prioritized towards specific agents. The field was revolutionized by the discovery of cancer subtypes by their sponsors, therefore the modified quinazoline compounds as highly specific and activities of some of them against HER2-driven cancer potent inhibitors of the EGFR (Ward et al., 1994; may not be tested early in their course of development. Rewcastle et al., 1995). Extensive structure–activity But a limited amount of clinical data is now available to relationships were determined and numerous improved give a preliminary impression of the antitumor efficacy quinazoline compounds have subsequently been deve- of this class of TKIs in patients with HER2-over- loped with varying selectivity characteristics for indivi- expressing cancers. The currently reported phase II dual HER family members. In addition to quinazolines, efficacy data is summarized in Table 3. In addition to severalother structures have now been found to the drugs listed here, numerous multi-targeting TKIs potently and selectively inhibit HER kinases. Table 2 that target other kinase families in addition to HER lists a number of HER TKIs that have been publicly family kinases are in preclinical and clinical testing, and disclosed, and for which preclinical data have been although such drugs may be found to be active in the presented. In addition to these, numerous other agents treatment of HER2-driven cancers, their multi-targeting are in development, which are not yet disclosed at the characteristics make them less suited to test the HER2 time of this review. Almost all of these agents are ATP oncogene hypothesis. Completed phase II studies of analogs and inhibit kinase activity by binding within the gefitinib and have been reported in breast ATP pocket of the catalytic domain (Stamos et al., 2002; cancer patients. Although these were not conducted Wood et al., 2004). Some of these compounds bind specifically in patients with HER2-overexpressing can- reversibly within the ATP pocket and are competitive cer, the cohorts include patients with HER2-overexpres- with ATP, while others bind irreversibly and are sing disease. Overall response rates of 0–10% were seen noncompetitive with ATP. in these studies. The most informative data currently Although HER family kinases are highly homolo- comes from clinical studies of . Lapatinib has gous, many TKIs show selectivity among the individual specifically been developed for the treatment of HER2- family members when assayed by in vitro kinase assays overexpressing breast cancer and numerous clinical

Oncogene 6584 Oncogene

Table 2 List of disclosed HER family TKIs in preclinical and clinical development

Developer Compound Class Type Selectivity in vitro IC50 (nM) Activity demonstrated against these HER2+++ References tumor models

EGFR HER2 HER4 In vitro In vivo

AstraZeneca ZD1839, Quinazoline Reversible 27 3700 BT474, SKBr3, SKOv3, BT474, Wakeling et al. (2002); Moasser gefitinib MCF-7HER2 MCF-7HER2, et al. (2001); Moulder et al. GLM-1, GLM-4 (2001); Ghossein and Bhatta- charya (2001); Warburton et al. (2004); Yokoyama et al. (2006) therapeutics cancer human in oncogene HER2 OSI-Genentech-Roche OSI-774, Quinazoline Reversible 2 350 KPL-4, Calu-3 Moyer et al. (1997); Akita and erlotinib Sliwkowski (2003); Friess et al. (2005) GlaxoSmithKline GW572016, Thio-quinazoline Reversible 11 9 367 BT474, Calu3, N87, BT474 Rusnak et al. (2001); Konecny lapatinib HB4aHER2, SUM225, et al. (2006) H16N2HER2, SUM190, SKBr3, UACC893, UACC812 ParkeDavis-Pfizer CI-1033, Quinazoline Irreversible 0.8 19 7 Rabindran (2005); Slichenmyer PD183805, et al. (2001)

Moasser MM OSI-Pfizer CP-724714 Quinazoline Reversible 4300 8 SKBr3, another panel of 9 FREV664E-HER2 Jani et al. (2004a, b); Finn et al. (2004) OSI-Pfizer CP-654577 Quinazoline Reversible 670 11 BT474, SKBr3 FREV664E-HER2 Rabindran (2005); Barbacci et al. (2003) Wyeth-Ayerst CL-387785, Quinazoline Irreversible 0.4 SKBr3, 3T3HER2 Discafani et al. (1999); Torrance EKI-785 et al. (2000) Academic AG1478 Quinazoline Reversible 3 2000 SKBr3, BT474 MMTV-neu Levitzki and Gazit (1995); Kurokawa et al. (2000); Lenferink et al. (2000, 2001) Arry Biopharma Arry- Quinazoline Reversible 7 2 10 BT474 BT474, Calu3, Miknis et al. (2005); Pheneger 334543 MDA-453 et al. (2005) BIBW-2992 Quinazoline Irreversible 0.5 14 BT474, N87 MDA-453, N87, Solca et al. (2006) SKOv3 AVEO AV-412, Quinazoline Irreversible 1 18 BT474, KPL-4, Robinson and Lin (2006); Fujii Pharm.-Misubishi MP-412 BH248 et al. (2005) Novartis AEE-788 Pyrrolopyrimi- Reversible 6 6 160 BT474, SKBr3 HC11neuT Traxler et al. (2004) dine Novartis CGP- Pyrrolopyrimi- Reversible 70 >10 uM SKBr3, ZR75-30 Lydon et al. (1998) 59326A dine Novartis PKI-166, Pyrrolopyrimi- Reversible 1 11 430 BT474, SKBr3 HC11neuT Traxler et al. (2001); Brandt et al. CGP-75166 dine (2001) Wyeth-Ayerst EKB-569, Cyanoquinoline Irreversible 39 1200 SKBr3 Wissner et al. (2003); Torrance pelitinib et al. (2000) Wyeth-Ayerst HKI-272 Cyanoquinoline Irreversible 92 59 3T3neu, SKBr3, BT474 3T3neu,BT474, Rabindran et al. (2004); Tsou SKOv3 et al. (2005) Wyeth-Ayerst HKI-357 Cyanoquinoline Irreversible 33 34 SKBr3 3T3neu,BT474, Kwak et al. (2005); Tsou et al. SKOv3 (2005) BristolMyers Squibb BMS- Pyrrolotriazine Reversible 22 32 190 BT474,N87,KPL4, KPL4, Sal2 Wong et al. (2006) 599626 HCC1419,HCC1954, Sal2 Takeda TAK-165, Triazole >25 uM 6 BT474 BT474 Naito et al. (2002); Yoshida et al. (2002); Pubchem CID#6444692) HER2 oncogene in human cancer therapeutics MM Moasser 6585 Table 3 Results of currently presented phase II efficacy studies of HER TKIs in HER2 overexpressing cancers Reference Drug Phase Selection criteria N Response Comments rate (%)

Baselga et al. (2005) Gefitinib/ZD1839 Phase II Advanced breast ca 31 3 Robertson et al. (2003) Gefitinib/ZD1839 Phase II Advanced breast 19 10 ca – TAM-resistant Albain et al. (2002) Gefitinib/ZD1839 Phase II Advanced breast ca 63 2 28 HER2+++ patients Winer et al. (2002) Erlotinib/OSI-774 Phase II Advanced breast ca 69 3 many HER2+++ patients Tan et al. (2004) Erlotinib/OSI-774 Phase II Advanced breast ca 18 0 2 HER2+++ patients Blackwell et al. Lapatinib/GW572016 Phase II HER2+++ breast 81 8 (2004, 2006) ca – after trastuzumab Burstein et al. (2004) Lapatinib/GW572016 Phase II HER2+++ breast 140 4 ca – after chemo Iwata et al. (2006) Lapatinib/GW572016 Phase II HER2+++ breast 45 24 Preliminary ca – after chemo Gomez et al. (2005) Lapatinib/GW572016 Phase II HER2+++ breast 40 30 Preliminary ca – first line Campos et al. (2005) Canertinib/CI-1033 Phase II ovarian ca – after 105 0 2 HER2+++ patients chemo efficacy studies as well as correlative scientific studies while on therapy to determine the suppression of tumor have been conducted and are ongoing to determine the EGFR/HER2 signaling by immunohistochemical stain- activity of this agent in patients with HER2-over- ing (Spector et al., 2005). This study showed mixed expressing breast cancer. The phase II efficacy studies results with variable degrees of suppression of targets, completed and confirmed to date show a response rate partly because this was a phase I dose-escalation study of 4–8% in patients with HER2-overexpressing breast involving patients with different types of cancers, cancer. Two additionalongoing, but unconfirmed including cancers not known to be HER2 dependent, studies, have reported higher response rates in the 24– and starting dose levels that are likely less effective at 30% range. A number of additionalphase II studies are target suppression. The data does however show a underway to test the efficacy of other investigational reduction in EGFR and HER2 phosphorylation in most HER TKIs in patients with HER2-overexpressing breast patients, and a reduction in MAP kinase signaling. A cancer and there will be much more data emerging in the reduction in Akt signaling was less evident in this data next few years. The data so far shows evidence of weak set. In a phase II study of gefitinib in breast cancer clinical antitumor activity in this disease. In addition to patients, biopsies and tumor biopsies were obtained the studies reviewed here, numerous clinical studies are in many patients before and while on therapy for underway to determine whether the addition of TKIs immunohistochemicalanalysisof target suppression to cytotoxic chemotherapeutics, to trastuzumab, or to (Baselga et al., 2005). This study showed effective hormonaltherapies produces new combinations with suppression of EGFR phosphorylation and MAPK increased clinical activity and prolongation of survival signaling in skin and tumor from drug therapy, but no in patients. These studies may lead to better treatment suppression of Akt signaling. HER2 phosphory- options for patients, but they are not a direct test of the lation was not assayed on this study and the three HER2 oncogene hypothesis, and a detailed discussion of patients with HER2-overexpressing tumors that were these studies is beyond the scope of this review. enrolled on this study did not have on-treatment tumor biopsies for analysis. It should be noted that the use of immunohistochemicaltechniques on paraffin-embedded Evidence that HER TKIs inhibit HER2 function tissues using phosphospecific antibodies is fraught with in patients technical problems that limits their reliability, and until The HER2 oncogene hypothesis would predict that the newer techniques emerge, such studies should be vast majority of HER2-driven tumors would initially interpreted with caution (Baker et al., 2005). Despite respond to treatments that suppress HER2 kinase the technicalproblems associated with phosphoprotein function. Correlative studies of tumor material from immunostaining and the fact that these two studies were patients on treatment are essentialfor understanding not designed to specifically determine target inactivation whether HER2 function and signaling has effectively in HER2-overexpressing tumors at maximaldrug been suppressed by these treatments. Such correlative dosage, they do appear to suggest that the drugs do studies require purely research interventions in consent- reach their tumor targets and at least partially inactivate ing patients by performing biopsies of their tumors just them. Tumor biodistribution does not seem to be a before and while on therapy, and these studies are limiting step since, at least with regards to gefitinib, extremely difficult to accomplish for a myriad of tumor and tissue concentrations have been measured practicaland ethicalreasons. At leasttwo groups thus and are much higher than serum concentrations far have succeeded in generating clinical scientific data and well above concentrations that fully suppress in patients on HER TKIs. In a phase I clinical study of EGFR and HER2 signaling in cell-culture models lapatinib, tumor biopsies were obtained before and (McKillop et al., 2005).

Oncogene HER2 oncogene in human cancer therapeutics MM Moasser 6586 Evidence that TKIs do not fully inhibit oncogenic consequence of the HER2 oncogene hypothesis, their HER2 functions nonspecific mechanisms of action limits their usefullness Significant mechanistic insight into the effective sup- in validating the hypothesis and are not reviewed here. pression of oncogenic HER2 signaling by TKIs was Readers are referred to several excellent recent reviews recently afforded by the analysis of steady-state HER3 for a comprehensive coverage of these HER2-targeting and downstream Akt signaling. While TKI treatment approaches (Chen et al., 2003; Menard et al., 2003; effectively suppresses EGFR and HER2 autophosphor- Brand et al., 2006; Kumar and Pegram, 2006). ylation and downstream MAP kinase signaling in Numerous immunological modalities are also being HER2-amplified tumors, HER3 appears to escape TKI pursued to enable host defense mechanisms to target therapy at conventionaldoses and concentrations HER2-overexpressing cancer cells, and these are also (Sergina et al., 2007). This is due to Akt-driven negative beyond the scope of this review. In addition, numerous feebdack signaling, which restores HER3-signaling approaches to specifically suppress HER2 expression activity despite significant suppression of HER2 kinase have been postulated over the years and continue to function, and thereby maintaining downstream Akt- be developed. These include antisense, ribozyme and signaling and numerous Akt-driven pathways important siRNA approaches to inhibit HER2 expression. These for tumor survival(Sergina et al., 2007). This feedback modalities have not yet translated to clinically effective loop essentially buffers HER3 signaling against the products that can test the validity of the HER2 incomplete loss of HER2 kinase function and under- oncogene hypothesis in patients. Future development scores the tumor cells critical need to maintain Akt- of these technologies, in particular siRNA approaches, signaling and numerous Akt-driven pathways important may lead to a new class of agents that can specifically for tumor survival. The fact that HER3 signaling is and effectively inactivate oncogenic HER2 function. buffered against an incomplete inactivation of HER2 kinase raises the bar for drug development, because it suggests that proper testing of the HER2 oncogene Resistance to inhibitors of HER2 hypothesis will require drugs that can completely Numerous mutationalevents downstream of growth inactivate HER2 kinase function. Testing this principle factor receptors have now been described in cancers. in cell-culture models with much higher concentrations These include activating in BRAF, Ras, of TKI or with the addition of anti-HER3 siRNA PIK3CA and inactivating mutations or deletions of approaches shows that HER2-overexpressing tumor PTEN. Since these function downstream of HER2 cells will undergo apoptosis if HER2 function, including and since each of these mutations induces constitutive its of HER3 and Akt signaling, is signaling activity, at least in theory these mutations can interrupted for 48 h or longer (Sergina et al., 2007). uncouple downstream pathways from HER2 rendering This reaffirms the oncogene-addicted nature of HER2- tumor growth independent of HER2 and resistant to driven tumors and suggests that if HER2 can be inhibitors of HER2. In breast cancers, Ras and BRAF effectively inactivated in patient tumors, this could mutations are rare, but HER2 overexpression occurs produce highly significant and rapid antitumor res- frequently with PIK3CA mutations, but rarely with ponses. But the doses required to completely inactivate PTEN mutation (Saal et al., 2005). There is currently no HER2 and effectively suppress HER3 signaling will data to determine whether the coexistence of PIK3CA likely produce significant toxicities in patients due to mutation confers resistance to TKIs in HER2-over- their off-target effects and may not be safely achievable. expressing breast cancers. Of note, the commonly used The effective suppression of oncogenic HER2 function BT474 cell line commonly used as a model of HER2- in patients may require drugs that are much more amplified breast cancer, and which is sensitive to TKIs potent than the current generation of compounds and at and trastuzumab, harbors an unusualmutation in the same time are highly selective with a wide thera- exon I of PIK3CA ((Saal et al., 2005) and cosmic peutic index. database). PTEN mutations are rare in breast cancers, but reduced PTEN expression may have biological significance and is often seen in breast cancers (Bose Other approaches to treat HER2-overexpressing cancers et al., 2002). PTEN loss has been shown to be induce Numerous other pharmacologic approaches are being TKI resistance in an EGFR-amplified breast cancer cell pursued to develop effective therapies for patients with line (Bianco et al., 2003; She et al., 2003), but the HER2-overexpressing cancers. These include anti- analogous effect has not been shown in a HER2- HER2 antibodies conjugated to a number of cellular amplified model. Future studies will establish whether toxins or anti-HER2 antibodies placed on chemothe- PIK3CA and PTEN are important determinants of TKI rapy containing immunoliposomes that can deliver sensitivity in HER2-overexpressing tumors. However, cytotoxics more effectively to HER2-overexpressing the analysis of clinical resistance will only be meaningful cancer cells. A number of agents can interfere with if drugs have been developed that fully inactivate cellular mechanisms that regulate or HER2 function. As discussed above in this review, expression and these can reduce HER2 expres- trastuzumab does not seem to inactivate HER2 and its sion in tumor cells. These include histone deacetylase mechanism of action remains unclear, and TKIs studied inhibitors, inhibitors and COX-2 inhibitors. to date seem to be partialinhibitors of oncogenic HER2 While these promising treatment approaches are a signaling in vivo.

Oncogene HER2 oncogene in human cancer therapeutics MM Moasser 6587 Current status of the HER2 oncogene hypothesis tested in patients. Much more data will be forthcoming The preliminary data with lapatinib is a promising hint in the coming years with regards to numerous other that at least a minority of HER2-overexpressing tumors HER TKIs with distinct structuraland biochemical may be dependent on HER2 kinase function. But why properties, including their antitumor efficacies and their the majority of patients failto respond to this therapy biochemicaltarget effects in patients tumors. If these remains to be determined. It is possible that the HER2 agents are unable to completely inactivate HER2, then oncogene hypothesis is wrong, and that despite the we may have to await a newer generation of more potent abundant and highly compelling evidence from experi- agents. Alternatively, HER2 oncogenic function can be mentalmodelsthat HER2-overexpressing breast cancers inactivated by the combination of a HER TKI and an are HER2 driven and HER2 dependent, that this inhibitor of the HER3/PI3k/Akt-signaling pathway. hypothesis does not hold true for patients with breast Inhibitors of this pathway are also in preclinical cancer and the experimental models are too simplistic to development and the combination treatment hypothesis predict the behavior of naturally occurring cancers. will likely also be tested in the coming years as these However, the HER2 oncogene hypothesis can only be agents enter clinical testing phases. discounted if clinical studies show lack of antitumor The scientific evidence implicates a role for HER2 in efficacy in spite of effective inactivation of tumor HER2 breast cancer similar to - in chronic myelogenous function and this has certainly not been shown. The (CML). Since effective inhibition of bcr-abl alternative possibility is that current therapies do not produces complete remissions in nearly all patients in effectively suppress oncogenic HER2 function in tu- chronic phase CML, it is imperative that the analogous mors. The recent revelation that current TKIs are not treatment hypothesis regarding HER2 be effectively effectively suppressing HER3/PI3K/Akt signaling tested in breast cancer. The potentialinherent in this strongly supports this position, and opens the way for hypothesis is enormous and includes the scenario of the a future generation of TKIs. first instance of an epithelial cancer being eradicated Therefore, it appears almost surely the case that the even in advanced stages. Such an outcome would have HER2 oncogene hypothesis has not yet been effectively historic significance.

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