Dhara N. Amin

HER3 Comes of Age: New Insights into Its Functions and Role in Signaling, Tumor Biology, and Cancer Therapy

The human epidermal growth family (HER) of tyrosine kinase receptors underlies the pathogenesis of many types of human cancer. The oncogenic functions of three of the HER proteins can be unleashed through amplification, overexpression, or mutational activation. This has formed the basis for the development of clinically active targeted therapies. However, the third member HER3 is catalytically inactive, not found to be mutated or amplified in cancers, and its role and functions have remained shrouded in mystery. Recent evidence derived primarily from experimental models now seems to implicate HER3 in the pathogenesis of several types of cancer. Furthermore, the failure to recognize the central role of HER3 seems to underlie resistance to epidermal growth factor receptor (EGFR)- or HER2-targeted therapies in some cancers. Structural and biochemical studies have now greatly enhanced our understanding of signaling in the HER family and revealed the previously unrecognized activating functions embodied in the catalytically impaired kinase domain of HER3. This renewed interest and mechanistic basis has fueled the development of new classes of HER3-targeting agents for cancer therapy. However, identifying HER3-dependent tumors presents a formidable challenge and the success of HER3-targeting approaches depends entirely on the development and power of predictive tools. Clin Cancer Res; 16(5); 1373–83

Resiliency and Vulnerability in the HER2-HER3 Tumorigenic Driver

The ability of certain breast cancers to resist a tyrosine kinase inhibitor drug may be overcome with high intermittent doses. How to Outsmart Breast Cancer Patients with breast cancer enrolled in recent clinical trials of a drug called lapatinib had reason to be optimistic. The growth and metastasis of many breast cancers depend critically on the target of this drug, the Erb receptor human epidermal growth factor 2 (HER2), and it made sense that its inhibition would hobble the cancer’s ability to survive. But some of these patients were ultimately disappointed as only a fraction of cancers responded to the drug, and those responses tended to be partial and transient. New work by Amin et al. in human breast cancer cells tests alternative treatment strategies and suggests that one of these might outwit these cancers. In certain breast tumors, the protein kinase activity of HER2, which is blocked by lapatinib, signals to downstream targets that cause cancer. One of these targets is another member of the same family, HER3, which can bind ligand but does not have catalytic activity of its own, and which in turn activates phosphoinositide 3-kinase (PI3K)–Akt signaling. In previous work, Amin and colleagues showed in human breast cancer cells that drug-induced altered regulation of HER3 through feedback from Akt is responsible for allowing cell to escape the lethal effects of lapatinib. Here, they probe this effect further and try to find a way to bypass the cells’ compensatory mechanism. The first approach was to try to inhibit PI3K at the same time as HER2 tyrosine kinase, but this proved ineffective as these cells were also able to up-regulate the growth signaling pathways and bypass inhibition by this combined treatment. Next, they used much higher doses of lapatinib, which were in fact able to completely and durably extinguish HER2 activity, but which have the disadvantage of being very toxic in vivo. They found a way around this problem by giving these high doses to mice with HER2-dependent tumors on an intermittent schedule, periodically driving blood concentrations high enough to generate a wave of apoptosis in the tumor and effectively preventing growth. The success of these authors in this skirmish with breast cancer marks a reason for renewed optimism in patients with HER2-dependent breast cancer. These second-generation approaches will need to be tested in the clinic, but the HER2-HER3 tumorigenic driver still seems to be an opponent keeping in our sites. About 25% of breast cancers harbor the amplified oncogene human epidermal growth factor receptor 2 (HER2) and are dependent on HER2 kinase function, identifying HER2 as a vulnerable target for therapy. However, HER2-HER3 signaling is buffered so that it is protected against a nearly two-log inhibition of HER2 catalytic activity; this buffering is driven by the negative regulation of HER3 by Akt. We have now further characterized HER2-HER3 signaling activity and have shown that the compensatory buffering prevents apoptotic tumor cell death from occurring as a result of the combined loss of mitogen-activated protein kinase (MAPK) and Akt signaling. To overcome the cancer cells’ compensatory mechanisms, we coadministered a phosphoinositide 3-kinase–mammalian target of rapamycin inhibitor and a HER2 tyrosine kinase inhibitor (TKI). This treatment strategy proved equivocal because it induced both TKI-sensitizing and TKI-desensitizing effects and robust cross-compensation of MAPK and Akt signaling pathways. Noting that HER2-HER3 activity was completely inhibited by higher, fully inactivating doses of TKI, we then attempted to overcome the cells’ compensatory buffering with this higher dose. This treatment crippled all downstream signaling and induced tumor apoptosis. Although such high doses of TKI are toxic in vivo when given continuously, we found that intermittent doses of TKI administered to mice produced sequential cycles of tumor apoptosis and ultimately complete tumor regression in mouse models, with little toxicity. This strategy for inactivation of HER2-HER3 tumorigenic activity is proposed for clinical testing.

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

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.

Effective treatment of HER2-amplified breast cancer by targeting HER3 and β1 integrin

Abstract The central role of HER2 as the disease driver and HER3 as its essential partner has made them rational targets for the treatment of HER2-amplifed breast cancers, and there is considerable interest in developing highly effective treatment regimens for this disease that consist of targeted therapies alone. Much of these efforts are focused on dual targeting approaches, particularly dual targeting of the HER2-HER3 tumor driver complex itself, or vertical combinations that target downstream PI3K or Akt in addition to HER2. There is also potential in lateral combinations based on evidence implicating cross-talk with other membrane receptor systems, particularly integrins, and such lateral combinations can potentially involve either HER2 or HER3. We established a preclinical model of targeting HER3 using doxycycline-inducible shRNA and determined the efficacy of a β1 integrin inhibitor in combination with targeting HER3. We report that targeting HER3 and β1 integrin provides a particularly effective combination therapy approach for HER2-amplified cancers, surpassing the combination of HER2 and β1 integrin targeting, and evading some of the safety concerns associated with direct HER2-targeting. This further validates HER3 as a major hub mediating the tumorigenic functions of HER2 and identifies it as a high value target for lateral combination therapy strategies.

Disentangling multidimensional spatio-temporal data into their common and aberrant responses

With the advent of high-throughput measurement techniques, scientists and engineers are starting to grapple with massive data sets and encountering challenges with how to organize, process and extract information into meaningful structures. Multidimensional spatio-temporal biological data sets such as time series gene expression with various perturbations over different cell lines, or neural spike trains across many experimental trials, have the potential to acquire insight about the dynamic behavior of the system. For this potential to be realized, we need a suitable representation to understand the data. A general question is how to organize the observed data into meaningful structures and how to find an appropriate similarity measure. A natural way of viewing these complex high dimensional data sets is to examine and analyze the large-scale features and then to focus on the interesting details. Since the wide range of experiments and unknown complexity of the underlying system contribute to the heterogeneity of biological data, we develop a new method by proposing an extension of Robust Principal Component Analysis (RPCA), which models common variations across multiple experiments as the lowrank component and anomalies across these experiments as the sparse component. We show that the proposed method is able to find distinct subtypes and classify data sets in a robust way without any prior knowledge by separating these common responses and abnormal responses. Thus, the proposed method provides us a new representation of these data sets which has the potential to help users acquire new insight from data.

Chemical probing of HER2-amplified cancer cells identifies TORC2 as a particularly effective secondary target for combination with lapatinib.

The clinical impact of HER2 inhibitors in the treatment of HER2-amplified breast cancers has been largely confined to chemotherapy combination regimens, since HER2 inhibitors appear to have very modest efficacies by themselves. This is due to the resilient nature of the functionally relevant HER2-HER3 tumor driver, bidirectionally linked with downstream PI3K/Akt pathway signaling, which can break through the inhibitory effects of most current HER2 or HER3 targeting therapies. A vertical combination approach targeting HER2 and a downstream pathway is a highly rational strategy for much more effective targeted therapy of this disease. However the importance of these downstream pathways in many human tissues and cells significant limits their usefulness as secondary targets by narrowing the therapeutic index of such combination therapies. The secondary target that can afford the highest potential for clinical translation is the one with the highest synergy against tumor cells in combination with HER2-inhibition, allowing the widest therapeutic index for clinical translation. We conducted a comparative analysis of such secondary targets in combination with the HER2 inhibitor lapatinib and find that the inhibition of mTor affords the highest degree of synergy. In further dissecting the individual roles of TORC1 and TORC2 complexes using pharmacologic and genetic tools, we find that it is specifically the inactivation of TORC2 that most synergistically enhances the efficacy of lapatinib. Although inhibitors that selectively target TORC2 are not currently available, these data make a compelling case for their development.

A TORC2–Akt Feed-Forward Topology Underlies HER3 Resiliency in HER2-Amplified Cancers

The requisite role of HER3 in HER2-amplified cancers is beyond what would be expected as a dimerization partner or effector substrate and it exhibits a substantial degree of resiliency that mitigates the effects of HER2-inhibitor therapies. To better understand the roots of this resiliency, we conducted an in-depth chemical-genetic interrogation of the signaling network downstream of HER3. A unique attribute of these tumors is the deregulation of TORC2. The upstream signals that ordinarily maintain TORC2 signaling are lost in these tumors, and instead TORC2 is driven by Akt. We find that in these cancers HER3 functions as a buffering arm of an Akt–TORC2 feed-forward loop that functions as a self-perpetuating module. This network topology alters the role of HER3 from a conditionally engaged ligand-driven upstream physiologic signaling input to an essential component of a concentric signaling throughput highly competent at preservation of homeostasis. The competence of this signaling topology is evident in its response to perturbation at any of its nodes. Thus, a critical pathophysiologic event in the evolution of HER2-amplified cancers is the loss of the input signals that normally drive TORC2 signaling, repositioning it under Akt dependency, and fundamentally altering the role of HER3. This reprogramming of the downstream network topology is a key aspect in the pathogenesis of HER2-amplified cancers and constitutes a formidable barrier in the targeted therapy of these cancers. Mol Cancer Ther; 14(12); 2805–17. ©2015 AACR.

Abstract 5438: Resistance to β1 integrin inhibition in HER2 amplified cancers is mediated by HER3

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Our previous work focused on investigating β1 integrin as a target for BC, alone and with radiation therapy. It is well recognized that there is significant cross talk between the EGFR family of receptors and β1 integrin signaling, and a relative resistance of HER2+ BC to β1 integrin inhibitory agents. In 2006, we published our findings that β1 integrin inhibition enhanced the effect of trastuzumab in HER2+ BC. Subsequently, others and we reported that β1 integrin inhibition enhanced the cytostatic effect of multiple HER2-targeted therapies in HER2+ BC. However, the nature of cross talk between β1 integrins and HER2, and the role of β1 integrin in HER2+BC resistance remains unknown. In addition, HER2+BCs have higher rates of local recurrence after radiotherapy, consistent with previous work showing relative radioresistance in HER2+BC. The most potent mitogenic HER2 signaling in HER2+BC occurs upon heterodimerization with HER3. We hypothesized that HER3 could mediate significant resistance to β1 integrin targeting in HER2+BC. To test this, we correlated β1 integrin and HER3 expression levels in SKBR3 cells over several time points using WB. Inhibitions of β1 integrin activity or shRNA knockdown of HER3 lead to upregulation of HER3 and β1 integrin, respectively. To test whether HER3 knockdown enhanced β1 integrin inhibition, we estimated proliferation by quantitating Ki-67 expression in SKBR3 cells in response to treatment. β1 integrin inhibition lead to a ∼50% reduction in Ki-67 positive cells (48%-22%), which was further enhanced by 50% with HER3 knockdown (12%), p<0.05. In addition, HER3 knockdown enhanced the cytostatic effect of ionizing radiation (IR) by ∼50% (32% to 14%), p<0.05. Enhanced cytostasis was correlated with a significant down modulation of p-HER2 and p-473 Akt on WB. In conclusion, our data indicate that HER3 plays a role in mediating resistance to β1 integrin inhibition in HER2+BC. Further mechanistic and in vivo validation are on going. Citation Format: Catherine C. Park, Dhara Amin, Hui Zhang, Mark Moasser. Resistance to β1 integrin inhibition in HER2 amplified cancers is mediated by HER3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5438. doi:10.1158/1538-7445.AM2014-5438

Abstract 3043: Akt-mTorc2 signaling underlies cellular addiction to HER3 signaling in HER2-amplified cancers.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC HER2 amplified breast cancers are characterized by constitutive HER3 phosphorylation. Efforts to target the functionally relevant HER2-HER3 signaling complex in these tumor cells has proven difficult due to robust feedback signaling mechanisms that restore its signaling in the face of anything short of the complete inactivation of HER2 kinase activity. To study this in more depth, we used a combination of genetic and chemical genetic approaches designed to study the cellular adaptational responses to network perturbation. We find that positive or negative perturbation of any signaling node from upstream HER2 or HER3 down to the two mTor complexes results in compensatory responses that function to maintain homeostasis. The responses to various perturbations are mechanistically distinct, but taken together reveal an uncompromising core topology driven by a concentric Akt-mTorc2 positive-feedback loop buffered by HER3 and PHLPP nodes. While mTorc1 signaling is also subject to feedback regulation, it is not involved in the core topology and its output is not always protected in these tumors. The central role of mTor is consistent with its known functions as a master regulator of cellular homeostasis highly conserved across eukaryotes. But the inclusion of HER3 in this central homeostatic network is a tumor cell anomaly. To better delineate how this homeostatic network is altered in tumor cells, we compared and contrasted the network topology in a panel of HER2-amplified cancer cells with a panel of immortalized breast epithelial cells. Much of the feedback circuitries are preserved across tumor and untransformed cells. But the driving Akt-mTorc2 positive-feedback loop is lacking in untransformed cells. This is due to the fact that Akt drives mTorc2 signaling in tumor cells but not in untransformed cells. Consistent with a central role of mTorc2 in these tumors, knockdown of Rictor, but not Raptor, is lethal in HER2-amplified SkBr3 tumor cells. These studies reveal a key alteration of Akt-mTor signaling in HER2 driven tumors that underlies the pathological incorporation of HER3 into an mTor-driven network, thus putting the HER2-HER3 tumor driver under the protection of this evolutionarily ancient pathway, highly competent at homeostasis. Citation Format: Dhara N. Amin, Deepika Ahuja, Gregory S. Ducker, Paul Yaswen, Kevan Shokat, Mark Moasser. Akt-mTorc2 signaling underlies cellular addiction to HER3 signaling in HER2-amplified cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3043. doi:10.1158/1538-7445.AM2013-3043

Abstract 1653: Highly effective treatment of HER2-amplified breast cancers through the complete inactivation of the HER2-HER3 tumorigenic driver

Approximately 25% of breast cancers are driven by amplification of the HER2 oncogene and over-activity of the HER2-HER3 signaling complex. It is a promising hypothesis that the pharmacologic inactivation of HER2-HER3 signaling with tyrosine kinase inhibitors (TKIs) in patients will be a highly effective treatment for this disease. Treatment of HER2 amplified breast cancers with HER2 targeting agents shows that the HER2-HER3 complex is endowed with a two-log signal buffering capacity, making drug potency a critical and potentially limiting variable. The buffering capacity is mediated through Akt driven negative feedback signaling, which has the ability to amplify HER2-HER3 signaling and protect it against incomplete inhibition of HER2 kinase. The concomitant use of downstream inhibitors induces compound sensitizing and desensitizing mechanisms and incremental anti-tumor effects. The HER2-HER3 signal buffering mechanisms cannot ultimately compensate for the total loss of HER2 catalytic activity, and at fully inactivating doses of TKI, the failure to phosphorylate HER3 and the dual loss of MAPK and Akt pathway signaling leads to apoptotic tumor cell death. Such HER2-inactivating dosing is not tolerable in vivo using continuous schedules but is feasible and highly effective using intermittent dosing. We show that continuous dosing of the HER2 TKI, lapatinib, at its maximum tolerated dose in mice bearing HER2-amplified tumors produces modest growth inhibitory effects. However, a five day repeated intermittent schedule allows eight fold higher dosing and produces much more profound tumor regression and durable anti-tumor effects. Therefore high dose intermittent TKI therapy is an imminently testable clinical treatment hypothesis that can fully inactivate HER2-HER3 signaling by overpowering its signal buffering capacity, revealing a monotherapy potential in HER2-targeting TKIs that may surpass many of the combination therapy approaches currently being explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1653.