Survival of HER2-Positive Breast Cancer Cells

Abstract

HER2 is overexpressed in a subset of breast cancers and controls an oncogenic signaling network that inhibits tumor cell death through the specific biochemical regulation of apoptotic pathways. In particular, the mitochondrial pathway for apoptosis is important for death induced by inhibitors of HER2. This review focuses on the connections between this oncogenic signaling network and individual components of the mitochondrial pathway. A comprehensive view of this signaling network is crucial for developing novel drugs in this area and to gain an understanding of how these regulatory interactions are altered in drug-refractory cancers.

Introduction

HER2 is a receptor tyrosine kinase that, when overexpressed, leads to the development of several types of cancer. Although HER2 cannot bind with high affinity to any known ligand, it can heterodimerize with other ligand-bound EGFR family members. Amplification of the HER2 gene, located at 17q12, results in increased mRNA and a functional HER2 receptor.¹ This is observed in approximately 25% of early-stage breast cancers. HER2 signaling and, more generally, receptor tyrosine kinase pathways have been the topic of very intense research. Despite these efforts, a detailed mechanistic picture of these networks has not been fully developed for the HER2-positive breast cancer cell.

The continued dysregulated growth of these cells is due to reinforced signaling connections between the HER2-activated signaling pathways and effector systems of the cell (apoptotic, proliferative, and metabolic). This review will focus on the relationship between known HER2- activated signaling kinases and the mitochondrial pathway of apoptosis. HER2 overexpression confers apoptotic resistance, and several mechanisms have been proposed to underlie this phenomenon. Here, we will review the HER2 signaling network, apoptotic control systems, and several known connections between the 2 that specifically dictate HER2-regulated breast cancer development, treatment, and drug resistance. Specifically, what do we know regarding the causal connection between these signaling events and the ultimate therapeutic end point, apoptosis?

HER2-Targeted Therapies and Resistance

Before the advent of HER2-targeted therapeutics, patients with HER2-positive disease had an associated increase in mortality and recurrence.² At present, there are several EGFR family inhibitors, but only 2 are approved for the treatment of breast cancer. Trastuzumab is the only approved adjuvant treatment specific for early-stage HER2-positive breast cancer. Trastuzumab is a monoclonal antibody that binds HER2 and negatively affects receptor function, thereby exerting an apoptotic signal.³ Several differing mechanisms downstream of trastuzumab inhibition of HER2 activity include the inhibition of PI3K, antigen-dependent cellular cytotoxicity, inhibition of HER2 shedding, G1 arrest, and inhibition of angiogenesis.^4-7

Most advanced-stage HER2-positive breast cancers do not respond to trastuzumab, and the majority of those that do respond progress within 12 months of initiating therapy.^8-13 Resistance to trastuzumab may occur through a variety of reported mechanisms.^5,14-18 First, breast cancer cells can become resistant through activation of the PI3K/AKT pathway. Loss of PTEN (a negative regulator of this pathway) and activation of PIK3CA are associated with resistance.^19,20 A second mechanism of resistance to trastuzumab is the accumulation of a truncated form of HER2, p95-HER2. This is a constitutively active form of HER2 that is not dependent on ligand activation.²¹ A third mechanism of resistance is overexpression of other receptor tyrosine kinases such as Met²² and IGF-1R.^23-27 One further mechanism of resistance is impaired access, or masking, of HER2 through the overexpression of MUC4. This may hinder the binding of trastuzumab and thus contribute to resistance.²⁸

Lapatinib is a dual-specificity small molecule inhibitor of HER2/EGFR that has been approved for the adjuvant treatment of advanced-stage HER2-positive breast cancers.²⁹ While lapatinib is a dual EGFR/HER2 inhibitor in vitro, it appears to selectively inhibit HER2 in vivo. Reduction of HER2 activity leads to decreased signaling through several pathways, including the RAF/MEK/ERK and PI3K/AKT/mTOR pathways and apoptosis.^30-32

Mechanisms of lapatinib resistance are less well understood, as compared to those of trastuzumab. A potential mechanism involving estrogen receptor (ER) activation has been proposed.³³ Lapatinib-mediated FOXO3 induction leads to increases in ER activity, which may cooperate with HER2 to support survival. ER activity may contribute to the induction of AXL. In turn, overexpression of the receptor tyrosine kinase AXL may contribute to resistance.³⁴ Screening of HER2-positive breast cancer cell lines in an assay that combines exogenous growth factors with lapatinib revealed that HGF can drive resistance to this drug in AU565 cells.³⁵ Another possible mechanism of resistance is the engagement of integrin complexes associated with the activation of SRC and FAK.³⁶ Resistance to lapatinib may also involve enhanced signaling through mTOR in a PI3K-independent manner.³⁷ It is very likely that acquired resistance to lapatinib involves a complex series of changes that is distinct from those seen with trastuzumab.

Recent studies by 2 different groups utilized PI3K and AKT inhibitors and profiled receptor tyrosine kinase changes using antibody arrays.^38,39 PI3K inhibition increased the levels of phosphorylated IGF-1R, insulin receptor (IR), EGFR, HER3, HER4, FGFR1, FGFR2, FGFR3, FGFR4, EPHA1, TIE2, TRKA, FLT3, MER, and MST1R. Several of these receptors were subsequently found to be up-regulated at the transcriptional level. Silencing of the IR and IGF-1R sensitized these cells to PI3K inhibition. The same group showed that lapatinib induces the expression of HER3, which also protects cells from apoptosis.⁴⁰ One explanation for these findings is that when the cell perceives death, it attempts to express as many growth factor receptors as it can. This increases the probability that local growth factors can save the cell before apoptosis is initiated.

How relevant is this phenomenon to lapatinib treatment? Microarray analysis of lapatinib-treated BT474 cells has been published, and mining of this dataset revealed that several receptor tyrosine kinases are transcriptionally regulated after 24 hours of lapatinib treatment.^34,41 This list includes HER3, HER4, IR, IGF-1R, and MER. Additionally, several other receptors are up-regulated including EPOR, LEPR, PTGER4, and NPY1R. Several signal transducers that can mediate these signals are also up-regulated, including JAK1 and PIK3CA. The importance of many of these receptors and transducers to resistance still remains unexplored.

It appears that FOXO-mediated transcription is involved in the decision-making process that breast cancer cells employ after drug exposure.^38,40 Both FOXO1 and FOXO3 are increased in the microarray dataset that analyzed lapatinib treatment of BT474 cells.³⁴ Another study has shown that nuclear FOXO3a levels are increased after 4 hours of lapatinib exposure in BT474 cells, indicating that this transcription factor might be inducing several of its target genes fairly early. FOXO family members can induce apoptotic factors such as BIM and KLF6.⁴² It is also likely that FOXO family members simultaneously induce genes associated with survival. Which genes are turned on in response to lapatinib-activated FOXO and the balance of death/survival genes are not known. Because of the potential global regulatory role that this transcription factor plays in this effect, further research using a systems approach (ChIP-Chip and ChIP-Seq) is warranted in this area.

HER2 Signaling Pathways

The EGFR family of receptors consists of 4 members: EGFR, HER2, HER3, and HER4.^43,44 These receptors contain an extracellular ligand–binding domain, transmembrane domain, and intracellular domain involved in signal transduction. Heterodimerization of HER2 and HER3 is important for the proliferation of HER2-positive breast cancer cell lines.⁴⁵ Upon tyrosine phosphorylation of HER2 and HER3, various adapter proteins are recruited to the receptor and are themselves phosphorylated. These adapter proteins activate multiple signal transduction pathways. The 2 that will be discussed here are the RAF/MEK/ERK (ERK cascade) and PI3K/AKT/mTOR pathways. The ERK cascade is initiated by the formation of a complex comprising HER2, GRB2, and SOS. SOS is a guanine nucleotide exchange factor for RAS and thus leads to its activation. Active RAS then recruits RAF to the membrane, where it is phosphorylated and activated.⁴⁶ The specific RAF isoforms (A-RAF, B-RAF, and RAF1) that are involved in HER2-positive breast cancers have not been identified. RAF activates the dual-specificity kinase MEK1/2, and this regulates ERK1/2 by phosphorylation on highly conserved residues. ERK can then phosphorylate numerous substrates to stimulate proliferation and survival.⁴⁷

ERK activity is also regulated by scaffolding proteins that are specific for individual components of this pathway. Several scaffolds have been shown to control ERK signaling and include KSR, paxillin, β-arrestin, PEA-15, and MP1.⁴⁸ Very little is known about how these scaffolds control breast cancer cell signaling and whether specific complexes are more central then others for tumor growth.

ERK directly phosphorylates and activates the RSK family of kinases (RSK1-RSK4).^49,50 RSK activity is important for the survival of several breast cancer cell types, including the HER2-positive BT474 cell line.^51-53 Stimulation of HER2-positive breast cancer cell lines with heregulin leads to the activation of RSK.⁵⁴ A mechanistic understanding of how RSK controls cell survival in these cells has not been established, but the answers to these questions are pressing, as several pharmacological RSK inhibitors are under development.⁵⁵

The AKT pathway is stimulated by the recruitment of PI3K to active growth factor receptors.⁵⁶ Once recruited, PI3K isoforms can phosphorylate membrane phospholipids at the 3′ position to generate PIP₃. This leads to the recruitment of AKT to the membrane, where it is phosphorylated by PDK-1 and TORC2 on residues T308 and S473, respectively. This pathway is negatively regulated by PTEN dephosphorylation of PIP₃. AKT can phosphorylate several substrates related to the proliferation and survival of HER2-positive breast cancer cells. FOXO1 inactivation by AKT signaling is blocked by trastuzumab and has been reported to be crucial for the drug’s effects.⁵⁷

In addition to the many substrates of AKT that go on to directly influence cell physiology, this kinase also activates the mTOR/p70S6K pathway.^58,59 AKT can phosphorylate TSC2, a GTPase-activating protein specific for the small GTPase RHEB at several residues, thereby inhibiting its activity.^60,61 Increased RHEB activity, due to loss of its upstream inhibitor, stimulates mTOR activity.⁶²

While a comprehensive view of HER2 signaling pathways is not yet entirely clear for any single system or breast cancer cell line, many of the above-mentioned signaling components are integral parts of most HER2-positive model systems. We next consider how these pathways control the mitochondrial pathway for apoptosis.

Apoptotic Pathways

Apoptosis is a normal developmental process that results in cell death and is characterized by nuclear condensation and cleavage of critical cellular proteins.⁶³ There are 2 major signaling mechanisms that lead to apoptosis, termed the extrinsic pathway (mediated by death receptors) and the mitochondrial pathway. The focus here will be on how HER2 signaling regulates the latter.

The BCL-2 family of proteins comprises a network of related proteins that are divided into 3 classes: the antiapoptotic BCL-2 proteins (BCL-2, BCL-xL, BCL-w, and MCL-1), the proapoptotic BCL-2 effectors (BAX and BAK), and the proapoptotic BH3-only proteins (BAD, BID, BIM, BMF, NOXA, and PUMA).⁶³ The network of physical interactions between these proteins directly controls their activity. The overexpression of antiapoptotic proteins allows for resistance to proapoptotic stimulation despite cellular stress and subsequent proapoptotic signaling. BAK and BAX are responsible for inducing mitochondrial outer membrane permeabilization and cytochrome c release, and their activity is regulated by the BH3-only proteins by direct (i.e., binding to BAK/BAX) and indirect (i.e., inhibiting the antiapoptotic proteins) mechanisms.⁶⁴

Permeabilization of the mitochondrial outer membrane leads to the release of cytochrome c and subsequent binding of cytochrome c to APAF-1. The complex of cytochrome c, APAF-1, and caspase-9 is referred to as the apoptosome.^65,66 The apoptosome cleaves and activates caspase-3. Caspase-3 is an executioner caspase and directly cleaves essential proteins, thus triggering an irreversible step in the apoptotic process. The inhibitor of apoptosis proteins serve as a last check on apoptosis signaling. They bind and inactivate caspase-3.⁶⁷ They are in turn inhibited by several proteins that are released from the mitochondria, such as SMAC/DIABLO and HTRA2.

This system is complex with multiple points of control. In the next sections, we will discuss known and potential regulatory mechanisms by which HER2-activated signaling pathways can inhibit apoptosis in breast cancer cells.

Antiapoptotic BCL-2 Proteins in HER2-Positive Breast Cancer Cells

Several antiapoptotic proteins are expressed in HER2- positive breast cancer cell lines. Using an siRNA-based approach, it has recently been shown that BT474 and SKBR3 cells are dependent on MCL-1 for the inhibition of apoptosis.⁶⁸ Silencing of MCL-1, but not BCL-2 or BCL-xL, results in an increase in apoptosis. One potential caveat that should be considered when interpreting this study is that an N-terminally deleted MCL-1 isoform has a role in several mitochondrial processes that are not related to apoptosis.⁶⁹ Therefore, the death observed⁶⁸ may be attributed to the indirect stimulation of apoptosis. Recently, co-inhibition of MCL-1 and HER2 was found to drive apoptosis through a mechanism dependent on ER stress and the generation of mitochondrial reactive oxygen species.⁷⁰

A more prominent role for MCL-1 versus BCL-2 or BCL-xL is further supported by studies using small molecule inhibitors of antiapoptotic BCL2 family members. ABT-737, a small molecule BH3 mimetic that inhibits BCL-2 and BCL-xL, but not MCL-1, does not increase apoptosis, indicating that MCL-1 is the predominant antiapoptotic protein.^71,72 Studies utilizing obatoclax (GX15-070), an inhibitor of all 3 antiapoptotic BCL2 family members, have demonstrated that it can modestly increase apoptosis in SKBR3 and BT474 cells.⁷³ Upon co-treatment with lapatinib and obatoclax, the authors observed a synergistic apoptotic response.

In response to lapatinib, apoptosis is most likely not dependent on the down-regulation of MCL-1 (total protein) because its protein expression levels are not altered in response to lapatinib treatment.⁷² ERK can phosphorylate and activate MCL-1 through phosphorylation at 2 sites.⁷⁴ However, the role of ERK in regulating MCL-1 has not been explored in HER2-positive breast cancer cells. In addition to activation through posttranslational modifications, MCL-1 may also be regulated through altered protein-protein interactions with other BCL-2 proteins (see below).

In response to lapatinib, BT474 and SKBR3 cells increase protein levels of BCL-xL (but not BCL-2), which may be a resistance response.⁷⁵ Interestingly, silencing of BCL-xL acts synergistically with trastuzumab to induce apoptosis in BT474 cells.⁷⁶ Therefore, while MCL-1 might be the major antiapoptotic protein, others may be involved when MCL-1 is inhibited or unable to engage.

Regulation of BAD

BAD (BCL-2 antagonist of cell death) is a BH3-only protein that acts as a molecular convergence point of several protein kinases. BAD was identified through yeast 2-hybrid assays as a BCL-2–interacting protein.⁷⁷ When in a dephosphorylated state, it can bind and neutralize antiapoptotic proteins (BCL-2 and BCL-xL). Growth factors and cytokines phosphorylate several residues, thereby inhibiting its activity. The PI3K/AKT pathway results in BAD phosphorylation at S136,⁷⁸ while the ERK pathway is responsible for the phosphorylation of BAD at S112.^79,80 Phosphorylation at S112 is mediated by another kinase, RSK.^80-82 Additionally, phosphorylation of S155 by PKA can inhibit BAD function.^83,84 Phosphorylation at either of these sites generates a 14-3-3 binding site on BAD. This can sequester BAD and limit its access to the mitochondrial outer membrane. Additionally, phosphorylation at S112 triggers polyubiquitination and the decreased stability of BAD. BAD acts as an OR switch; if the AKT or ERK pathway is active, BAD is turned off.⁸⁵

In one study, treatment of the HER2-positive cell line SKBR3 with lapatinib abolished ERK activation but did not attenuate S112 phosphorylation levels after a 72-hour incubation.⁸⁶ This study did not assess earlier time points at which the inhibition of HER2 may have activated BAD and triggered an initial wave of apoptosis. Importantly, lapatinib treatment strongly decreased BAD S112 levels in BT474 cells.³⁴ Further study using siRNA to silence BAD expression can be used to determine whether lapatinib- and trastuzumab-induced apoptosis is dependent on BAD activity.

Regulation of BIM

BIM (BCL-2–interacting mediator of cell death) is a BH3-only protein that, like BAD, is exquisitely sensitive to growth factor treatment. Importantly, and in contrast to BAD, BIM is a “direct activator” of BAX/BAK.⁸⁷ It can physically interact with BAX/BAK and promote their ability to induce mitochondrial outer membrane permeabilization and apoptosis. BIM is regulated through both transcriptional and posttranslational mechanisms. The FOXO family of transcription factors can be phosphorylated by AKT and, in response, is excluded from the nucleus. Upon removal of receptor stimulation, FOXO transcription factors translocate to the nucleus and activate the transcription of several genes involved in apoptosis. BIM transcription is stimulated by FOXO activity.⁸⁸ BIM is inhibited by growth factor stimulation at the posttranslational level. Phosphorylation at several sites by ERK blocks binding to BAX⁸⁹ and stimulates degradation through the ubiquitin proteasome system.^75,90 RSK phosphorylation of BIM has been reported to act in concert with ERK to stimulate ubiquitination by βTRCP.⁹¹

BIM seems not to play a major role in paclitaxel-induced apoptosis of HER2-positive breast cancer cells.⁹² BIM appears to be a crucial apoptotic trigger for mammary epithelial cells during the clearance of luminal cells in duct formation.⁹³ Most importantly, silencing of BIM attenuates lapatinib-induced apoptosis in HER2-positive cells.⁷² These studies clearly demonstrated a strong up-regulation of BIM after 24 and 48 hours of drug treatment in BT474 cells. The levels of BIM in untreated cells were low to undetectable. In contrast to these studies, levels of BIM were clearly present in untreated cells, although lapatinib-mediated regulation was not assayed.⁶⁸ These studies were utilizing the same cell line, and the underlying experimental differences that might explain this discrepancy are not clear. However, it is clear that a detailed molecular understanding of the protein interactions and biochemical modifications of BIM in HER2-positive breast cancer will pave the road for the development of therapeutics to this target.

Regulation of BMF

BMF (BCL-2–modifying factor) is a BH3-only apoptotic protein that has some functional similarities to BIM.⁹⁴ Interestingly, in human mammary epithelial cells, BMF induction is important for apoptotic induction after anoikis.⁹⁵ In response to paclitaxel, BMF can displace BIM from antiapoptotic proteins, and this results in the activation of BAX/BAK.⁹⁶ This may be occurring through JNK-mediated phosphorylation of BMF/BIM.⁹⁷ Hypoxia suppresses BIM and BMF expression.⁹⁸ There is little known regarding the relationship of BMF and HER2 in breast cancer cells. BMF protein expression is reduced after the overexpression of HER2 in MCF7 cells.⁹⁹ It would be interesting to see whether BMF levels are increased after the addition of lapatinib in HER2-positive cell lines.

Putting the Pieces Together: A Global View of Antiapoptotic Signaling in HER2-Positive Breast Cancer Cells

The number of signaling components that can potentially influence apoptotic decisions in cells is overwhelming.⁶⁴ A movement towards developing cell type–specific models describing these networks is necessary for a comprehensive understanding of these processes. This review has described the role and regulation of specific signaling components and apoptotic regulators and identified features allowing HER2-dependent survival in human breast cancer cells overexpressing this oncogene (Fig. 1).

Figure 1.

Interface between the HER2 signaling network and apoptotic control centers. HER2 activates 2 major signaling pathways (ERK and AKT) that utilize transcriptional and posttranscriptional mechanisms to inhibit the mitochondrial pathway of apoptosis. The network dynamics are illustrated before the addition of lapatinib (A) and after the addition of the drug (B). These interactions are supported by the literature, but the true complexity of this interface is likely much greater.

In these cancer cells, signaling through the ERK and AKT pathways drives an active suppression mechanism of BIM and BAD (Fig. 1A). Upon inhibition of HER2, with lapatinib, the activity of these kinases is reduced (Fig. 1B). BIM is transcriptionally up-regulated by active FOXO and stabilized in a dephosphorylated state (ERK can no longer phosphorylate BIM).

This model is limited to the HER2-positive breast cancer cell in isolation and does not accurately account for apoptosis via paracrine and stromal factors. Nonetheless, this model is particularly useful for understanding the effects of a HER2 antagonist, such as lapatinib, on cell signaling and apoptosis.

Inhibition of oncogenic signaling pathways leads to a reduction in the enzymatic activity of multiple protein kinases. This is a fairly rapid process that likely occurs over the first 20 to 30 minutes of inhibitor treatment and for most kinases within the first minutes. Dephosphorylation of proapoptotic proteins should follow soon after. Formation of mitochondrial pores and subsequent release of cytochrome c should follow soon after these posttranslational changes in BCL-2 family members. It is very likely that cells will not commit to apoptosis with such haste. An irreversible decision of life or death may be expected to have a subswitch that can only be fully activated after several hours. In the HER2-positive breast cancer cell, this subswitch is likely the FOXO-dependent transcriptional up-regulation of BIM.⁸⁸ To increase BIM mRNA levels, FOXO factors must first be dephosphorylated, dissociate from 14-3-3 proteins, translocate to the nucleus, and directly stimulate transcription.

To determine which of the antiapoptotic BH3 proteins are required for HER2-mediated survival, focused siRNA screens can be performed. Large effects on apoptosis may only be seen when inhibiting combinations of antiapoptotic BH3 proteins. Another aspect of the system that should be explored further is the protein-protein interactions of MCL-1. One of the most significant findings in this system is that MCL-1 is a crucial antiapoptotic factor. A bottom-up approach may be useful for connecting MCL-1 regulation to HER2 signaling.

Immunoprecipitation of antiapoptotic BH3 proteins may isolate specific binding partners that form complexes during apoptotic induction. This approach can also be used in combination with mass spectrometry to find interactors that are differentially induced or decreased by lapatinib.

Systems biology and its associated global and computational approaches to dissecting signaling networks have already been applied to certain aspects of HER2-positive breast cancer. Proteomic analysis of growth factor–treated mammary epithelial cells engineered to overexpress EGFR family members has revealed complex tyrosine phosphorylation patterns.^100,101 A SILAC-based proteomics analysis has recently been performed on EGF-stimulated BT474 cells in the absence or presence of lapatinib.¹⁰² Computational studies have used differential equations to simulate transduction through EGFR family receptors.¹⁰³ Experiments utilizing combinatorial drug combinations in breast cancer cells have led to predictive models for therapeutic application.^104,105 Powerful siRNA and shRNA screens have been used to interrogate the crucial components of individual breast cancer cell lines.^106-108 In particular, global shRNA screens previously performed^107,108 identified MCL-1 as being significantly essential in both the BT474 and SKBR3 cells. Applying both the approaches and findings from the studies described above to apoptosis in HER2-positive breast cancer cells may lead to mechanistic insights.

Perspectives

Evidence accumulated from genomic and systems-level analyses of breast cancers and derived cell lines shows a tremendous diversity that reflects 1) cells that evolved from different layers or compartments of the mammary epithelium and 2) differential addiction to oncogenes or oncogenic networks. It is now firmly established that different breast cancer subtypes respond differentially to treatments. A central component of therapy for HER2-positive breast cancer is the use of HER2-selective inhibitors such as lapatinib. Therefore, a comprehensive understanding of how HER2 controls this subset of breast cancer cells and, more importantly, how these cells respond to these drugs will be particularly useful for advances in treatment.

In this review, the molecular circuitry connecting HER2 signaling pathways and the apoptotic machinery involved in the mitochondrial pathway of apoptosis were explored. The signaling system controlling survival that is best supported by the literature centers on MCL-1. Inhibition of HER2 leads to the accumulation of BIM and subsequent association of BIM with MCL-1 (Fig. 1). Additionally, BIM may also directly associate with BAX/BAK to increase their ability to form mitochondrial pores. The mechanistic detail of these events in HER2-positive breast cancer cell lines has not yet been worked out. Other pathways for survival may also be in place. The BH3-only protein BAD is regulated in response to lapatinib treatment (Fig. 1). However, its importance in inducing cell death is still not clear. It would induce death by forming complexes with proapoptotic proteins other than MCL-1. Which targets are relevant to HER2-positive breast cancer cells? These issues are currently being investigated.

A significant issue is how these cells respond to treatment. For example, is the apoptotic signaling network constantly changing in response to chemotherapeutics and HER2 inhibitors? Further research in the area of HER2-positive breast cancer apoptosis using a global or systems-level approach may shed light on new mechanisms and molecular players.