Pan-HER-targeted approach for cancer therapy: Mechanisms, recent

The Human Epidermal Growth Factor Receptor family is composed of 4 structurally related receptor tyrosine
kinases that are involved in many human cancers. The efficacy and safety of HER inhibitors have been compared
in a wide range of clinical trials, suggesting the superior inhibitory ability of multiple- HER-targeting blockade
compared with single receptor antagonists. However, many patients are currently resistant to current therapeutic treatment and novel strategies are warranted to conquer the resistance. Thus, we performed a critical
review to summarize the molecular involvement of HER family receptors in tumour progression, recent anti-HER
drug development based on clinical trials, and the potential resistance mechanisms of anti-HER therapy.
1.简介

人表皮生长因子受体（EGFR或HER）家族由4种结构相关的激酶组成（EGFR / HER1，HER2，HER3和HER4）[1]。目前的抗HER剂包括小分子酪氨酸激酶抑制剂（TKIs）和人源化或嵌合单克隆抗体（mAbs）[2-5]。无数癌症(如非小细胞肺癌和乳腺癌)被发现表达HER家族的多个成员，突出了开发多靶抑制剂(如双重HER抑制剂和泛HER抑制剂)的重要性。这些疗法的有效性和安全性已经在官方的临床试验中得到比较,而与单靶点抑制剂相比较以HER家族混合的多靶点抑制剂能够更有效的抑制肿瘤生长.阻断这些受体及其信号传导网络的发现已经在很大程度上改变了许多癌症的治疗策略。 然而，如果不征服抗HER疗法的抗性，就不可能实现临床改善。但是，没有可以在不征服的情况下实现临床改善抗HER治疗的抵抗力。最近涉及的新方法HER抑制剂与靶向其他信号传导的药剂协同作用发现通路更有效地调节肿瘤进展，导致更好地了解HER靶向治疗的作用和抵抗机制。在这里，我们总结了这篇综述HER家族受体在肿瘤进展中的分子参与，最近基于临床试验的抗HER药物开发，以及抗HER治疗的潜在耐药机制。

 

1. Introduction

The Human Epidermal Growth Factor Receptor (EGFR or HER) family is composed of 4 structurally related receptor tyrosine kinases (EGFR/HER1, HER2, HER3 and HER4) [1]. Current anti-HER agents include small molecular tyrosine kinase inhibitors (TKIs) and humanized or chimeric monoclonal antibodies (mAbs) [2–5]. Numerous human cancers, such as non-small-cell lung cancer and breast cancer, were found to express multiple members of the HER family, highlighting the interest in developing multi-target inhibitors such as dual HER inhibitors and pan-HER inhibitors. The efficacy and safety of these therapeutic approaches have been compared in a wide range of clinical trials, and it is becoming increasingly clear that targeting the HER pathway at multiple points offers superior tumour growth inhibitory ability compared with single receptor antagonists in tumour growth. The discovery of the blockade of these receptors and its signalling networks has largely modified the therapeutic strategy of numerous cancers. However, no clinical improvement could have been achieved without conquering the resistance of anti-HER therapy. Recently, novel approaches involving HER inhibitors that synergize with agents that target other signalling

pathways, were found to regulate tumour progression more potently, leading to a better understanding of the action and resistance mechanisms of HER-targeted treatment. Herein, in this review, we summarized the molecular involvement of the HER family receptors in tumour progression, recent anti-HER drug development based on clinical trials, and the potential resistance mechanisms of anti-HER therapy.

2. HER family signaling

2.1. HER ligands and activation of HER receptors

The Human Epidermal Growth Factor Receptor (EGFR or HER) fa[1]mily is composed of 4 structurally related receptor tyrosine kinases (EGFR/HER1, HER2, HER3 and HER4 [1]). HER overexpression has been identified in approximately 15–30% of breast cancers and is also strongly associated with ovarian, stomach, adenocarcinoma of the lung and aggressive uterine cancer [6–8]. All four HER family members are multiple domain proteins that exhibit similar structures, including an extracellular region, a transmembrane region, and an intracellular region of the tyrosine kinase domain [9–11]. The HER receptors with their known ligands, their respective target therapy and dimerization options are presented in Fig. 1. In addition to the common characteristics shared by all HER family receptors, each member owns some distinct features that are discussed as follows.

2.1.1. EGFR

EGFR is also referred to as HER1. EGFR overexpression plays an important role in numerous cancers, including squamous-cell carcinoma of the lung, anal cancers, glioblastoma and epithelial tumours of the head and neck [12,13], which we will discuss in more details.

Known ligands that bind to the EGFR include epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), TGF-α, amphiregulin (AR), betacellulin (BTC), epigen (EPG) and epiregulin (EPR) [14]. These proteins are transmembrane precursors and are separated by proteases to initiate the release downstream growth factors [15]. Upon activation by its specific ligands, the monomeric form of EGFR/HER1 undergoes a transition to create an active homodimer [1]. The homo-dimerization (e.g., EGFR–EGFR) or heterodimerization (e.g., EGFR–HER2) of HER family receptors subsequently activates intracellular kinase domains, transphosphorylates tyrosine residues, and ultimately stimulates multiple downstream effector molecules [1].

2.1.2. HER2

In contrast to the other family members (HER1, HER3 and HER4),HER2 has not yet been identified with any ligands [16,17] and can be dimerized without ligand binding [18]. HER2 can also be dimerized without ligand binding [18] and is activated via the heterotypic inter[1]action with other EGFR receptors, making it a preferred dimerization partner of other members of the HER family [19]. The overexpression or amplification of HER2 has been observed in up to 30% of breast cancer patients and the HER2-positive subtype is significantly correlated with worse prognosis than HER2-negative breast cancer [20]. Numerous studies were performed to unveil the association between HER2 expression and survival outcome of other cancer types [21–23]. Moreover, many HER2-targeted drugs such as trastuzumab, have been developed or under clinical trials for cancer therapies, which we will describe more below.

2.1.3. HER3

EGFR, HER2, and HER4 exhibit intrinsic TK activity, whereas HER3 is a kinase-defective receptor [24] that is transactivated by other HER family members [25]. A class of ligands, known as neuregulins (NRG), with various isoforms bind directly to HER3 and HER4 [26]. In contrast to other HER family members which are activated through autophosphorylation upon ligand binding, the unbound HER3 acts as a kinase[1]impaired protein that is unable to phosphorylate other proteins and requires transphosphorylation by its binding to other HER receptors [27,28]. As a heterodimerization partner of HER2, HER3 is widely correlated with tumour growth, metastasis and drug resistance [29,30].

2.1.4. HER4

HER4 shares structural similarities with other HER members and binds to ligands such as neuregulins-2 and -3. Unlike other HER receptors, HER4 is characterized by its antiproliferative and proapoptotic activity [31]. HER4 expression is correlated with favorable prognostic factors such as low histological grade and better prognosis in patients with breast cancer [32–34].

2.2. Mechanism of HER signalling hyperactivation in human cancers

HER ligands are believed to be one of the key targets in cancer therapy based on the observation that over-expressed HER ligands were significantly correlated with human tumour progression, increased tumour microvessel density and enhanced chemotaxis and invasive potential of cancer cells [35,36]. HB-EGF expression is elevated in various types of cancer types, including ovarian, gastric and breast cancer, melanoma and glioblastoma; in addition, amphiregulin is increased in pancreatic, colon and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells [37].

The RTK (receptor tyrosine kinase) family is considered another potential target in cancer therapy given that mutation or over  expression of RTK family members has been observed in numerous diseases marked by abnormal proliferation including breast cancer, ovarian cancer, lung cancer, stomach cancers, and oral squamous cell carcinoma [2–4,38,39]. Among them, the HER family is one of the most comprehensively studied RTKs in cancer development. The HER family receptors, especially EGFR and HER2, appear to participate in pulmonary carcinogenesis [40]. In numerous studies, HER2-positive tumours are associated with an inferior prognosis in breast cancer patients [18,41,42]. Recurring HER-2 mutations have also been identified in patients with lung cancer [43–45]. A comprehensive analysis of 81 studies, including 27,161 patients and spanning sixteen years, demonstrated that HER2 overexpression could predict worse survival out[1]comes in breast cancer patients [46]. A breadth of literature suggests that EGFR overexpression indicates worse survival [47–50]. Khelwatty et al. analysed the expression of HER family members in tumour specimens from 86 patients with metastatic colon cancer and found that the disease-free survival was significantly poorer in patients with EGFR expression in 50% of tumour cells and co-expression of EGFR/HER-4 in greater than 10% of tumour cells [51]. Similarly, HER-3 expression was found that significantly correlate with head and neck squamous cell carcinoma mortality and therefore represents a potential target for cancer immunotherapy [52,53].

3. Mechanisms of current anti-HER pathway therapies

3.1. Single target agents

Current therapeutic development targeting the HER family mainly focused on small molecule tyrosine kinase inhibitors (TKIs) and humanized or chimeric monoclonal antibodies (mAbs) [5]. TKIs such as gefitinib and erlotinib are now employed in the treatment of patients with various cancer types such as non-small cell lung cancer (NSCLC) [54–58]. Both inhibitors bind reversibly to the intracellular functional tyrosine kinase domain of the HER receptor to inhibit corresponding auto-phosphorylation and downstream signalling [59–63]. Gefitinib is

effective as a first-line therapy in advanced NSCLC patients carrying EGFR mutations [64,65]. Exon 19 deletion mutations and the L858 mutation of EGFR genes cause enhanced gefitinib sensitivity in NSCLC patients [66,67], whereas only 10%–20% of wild type EGFR patients respond to gefitinib [68,69]. Compared with wild type EGFR tumours, patients with EGFR mutations exhibit increased progression-free survival after EGFR TKI treatment [70]. The ability to enter cells and act on intracellular domains of receptors such as EGFRvIII and truncated HER2 receptor is crucial for the target specificity and the clinical toxicity of TKIs. In addition, some breast cancers were found to express truncated forms of HER2 to which trastuzumab could not bind and resulting in drug resistance to mAb [71], but sensitivity to TKIs. The HER-directed TKIs reviewed in this study are presented in Table 1.

However, mAbs are more target-specific than the TKIs and cause much less off-target side-effects [72]. Monoclonal antibodies that target HER[1]family receptors that are currently approved or being investigated are presented in Table 2.

that specifically targeted the HER2 pathway in HER2-positive diseases[73]. Trastuzumab is a humanized mAb that directly targets the domain IV of the extracellular segment of the HER2 receptor [74]. This domain enhances the antitumor activity of paclitaxel and doxorubicin in xenografts with HER2 overexpression [75] and stimulates clinical responses in approximately 12–34% of HER2-overexpressing metastatic breast cancers (MBC) [76]. The precise mechanism of trastuzumab efficacy is not fully understood. It is possible that trastuzumab: i) inhibits the ligand-independent receptor dimerization [77]; ii) promotes the

ligand-dependent cell-mediated cytotoxicity (ADCC) [78,79]; iii) blocks the ADAM-mediated proteolytic cleavage and the generation of truncated active p95HER2 by blocking metalloprotease cleavage sites on HER2 ECD [73] [80]; iv) the downregulates certain signalling pathways such as ras-Raf-MAPK and PI3K/Akt, NF- κ β, VEGF, and IL-8 and blocks

cycle progression by forming p27/Cdk2 complexes [81,82]. Another potential mechanism involves HER2 internalization and degradation mediated by trastuzumab [83], whereas some studies suggested that receptor levels were unaffected after trastuzumab treatment [84].

Trastuzumab also binds to the EGFR/HER2 interaction sites [85] and therefore reduces the formation of EGFR/HER2 complexes. Tradition[1]ally only breast cancer patients classified as HER2-positive are considered eligible for HER2-targeted therapy. However, IHC and FISH

testing can occasionally be equivocal, and some HER2-negative patients also responded to trastuzumab [86,87]. A second humanized mAb, pertuzumab binds to a different site of

the HER2 ECD (extracellular domain II) compared with trastuzumab (domain IV), and inhibits ligand-induced heterodimerization with HER3. Pertuzumab sterically inhibits NRG-induced dimerization of HER2 with HER3 [88], which consequently blocks the signaling pathway activated by HER2/HER3 heterodimers [89]. In early pre[1]clinical and clinical trials, pertuzumab was found to inhibit proliferation of in multiple human cancer cell lines overexpressing HER2 including breast, prostate and lung cancer cells [90,91] and the growth of both high- and low-expressing HER2-positive breast xenografts in vivo [92]. Despite its benefits, the use of pertuzumab is limited in some patients because it selectively binds to the dimerization site of HER2 with HER3, not to the EGFR, leading to the limited inhibition effect on EGFR/HER2 dimerization [85]. Current observations suggest the use of pertuzumab in combination with trastuzumab and chemotherapy rather than alone. Pertuzumab has also been approved for clinical application when administered together with trastuzumab in the neoadjuvant set[1]ting after docetaxe treatment [93], producing synergistic apoptosis in HER2-overexpressing breast cancer cells [90]. A more recent phase III trial [94] investigated the same combination as adjuvant treatment for patients with early breast cancer and observed no improvement in OS. Therefore, for early HER-2 positive breast cancer, long-term analysis is still needed to determine the clinical benefit of pertuzumab. Cetuximab is a chimeric human-murine monoclonal anti-EGFR antibody that competes with ligands to bind EGFR ECD and ultimately inhibits the tyrosine kinase activation of EGFR [95–97]. The efficacy of cetuximab has been demonstrated in multiple clinical trials, and cetuximab offers improved survival outcomes in patients with NSCLC,SCCHN (squamous-cell carcinoma of head and neck), and mCRC (metastatic carcinoma of colon and rectum) [98]. A phase II trial found that the cetuximab maintenance therapy following radiotherapy contributed to superior locoregional control (LCR) at the 1 year follow-up in patients with advanced oropharyngeal cancer [99].

 

3.2. Dual and pan- HER inhibitors

Pan-HER inhibitors are either TKIs or mixture of antibodies that target non-overlapping epitopes on EGFR, HER2 and HER3 [100], such as inhibitors of the two (dual-HER inhibitors), or four receptors of the HER family (pan-HER inhibitors). Given that many cancers express multiple HER family receptors, it is becoming increasingly clear that targeting the HER pathway at multiple points exhibits superior inhibitory tumour growth efficacy compared with single receptor inhibitors in tu[1]mour growth. Pan-HER treatment was sustainably effective in cell lines harbouring mutations or amplification of genes, including TP53, KRAS,

EGFR and ERBB2 genes. Similar efficacy could also be observed in xenograft models derived from ovarian, colorectal, pulmonary, and pancreatic cancer patients resistant to previous targeted therapies [101].

Similar promising antitumor effects were reported, demonstrating that pan-HER could delay tumour growth in NSCLC and HNSCC cell lines and xenografts intrinsically resistant to cetuximab [98]. Additionally, pan[1]HERtreatment also exhibited superiority in delaying both primary and acquired resistance compared with single agents, given that targeting a single HER family member could result in the up-regulation of other members, thereby resulting in tumour heterogeneity and plasticity [101].

An in vitro study [100] of HER dependent cell lines demonstrated that, compared with single mAbs, pan-HER mAb mixtures were more effective at inhibiting proliferation and reducing EGFR, HER2 and HER3 levels. Furthermore, pan-HER mAb mixtures could reduce the level of basal EGFR homo- and hetero-dimerization regardless of the presence of ligands, which is not be observed with single mAbs. Im[1]portant ongoing clinical trials with pan-/dual HER-targeted therapies are provided in Table 3.

3.2.1. Lapatinib

Lapatinib (GW572016) is a dual reversible TKI that inhibits both

EGFR and HER2 in both ligand-dependent and -independent signalling pathways [102]. In contrast to trastuzumab, lapatinib targets the intracellular domain [103], which leads to the application of lapatinib in trastuzumab-resistant settings [104]. Experimental evidence suggests that lapatinib better reduced tyrosine phosphorylation of EGFR and HER2 compared with EGFR-specific TKI's, which lead to the inhibition of PI3K and Akt both in vitro and in vivo [105]. Completely blocking Akt activation in HER2 overexpressing cells strongly induced apoptosis [26]. A phase IB study [106] evaluated the effect of lapatinib in patients

with pre-treated metastatic solid tumours (breast cancer 33%) that exhibited EGFR or HER-2 overexpression via immunohistochemistry. In this study, patients were randomized to receive lapatinib at doses of 500, 650, 900, 1200, or 1600 mg once daily. On day 28, a biopsy from a

breast cancer patient with a remarkable partial response exhibited a reduced phospho-EGFR and phospho-HER2, phospho-MAPK index, cy[1]clin D, and TGF and increased tumour cell apoptosis. On the contrary, a biopsy in a patient with progressive disease revealed no increase in apoptosis after lapatinib treatment.

This agent was approved by the FDA in the treatment of patients with HER2 overexpressing breast cancer in combination with capecitabine [107]. In a randomized phase III study, patients with advanced HER2-positive breast cancer were randomized to the capecitabine or the lapatinib plus capecitabine group [108,109]. Patients receiving the com[1]bination therapy revealed a prolonged median TTP (time to progression) from 4.3 to 6.2 months and improved ORR (objective response rate) from 14 to 24% [108]. Another phase III study examined the efficacy of adding lapatinib to paclitaxel chemotherapy in patients with both HER2-normal and HER-overexpressing breast cancer [109]. Similar ORR improvement in lapatinib-added group was observed in a more recent phase III study with greater than 400 previously untreated patients with HER2-positive breast cancer [110]. Prolonged progression-free survival (PFS) and OS were also be observed. The addition of lapatinib was associated with a longer median TTP of 36.4 versus 25.1 weeks (HR 0.53, 95% CI 0.31–0.89, P = 0.005), but HER2 normal patients did not present any clinical improvement compared with placebo. However, in these two studies and consistent with early clinical trials [111], lapatinib was associated with a significantly increased rate of adverse events. Diarrhoea was identified as a notable (60%) toxic event in the lapatinib-based therapy group, with 12% of cases classified as grade III [108], and other toxicities included rash, mucositis, and vomiting, etc.

In addition to the concomitant use of lapatinib and chemotherapy, some studies have assessed the effectiveness of lapatinib combined with tamoxifen. The combination restored tamoxifen sensitivity in EGFR- or HER-2-expressing cells and induced maximal regression of HER-2 overexpressing, tamoxifen-resistant MCF-7 xenografts in vivo [112].

Finally, a randomized phase III trial compared lapatinib plus letrozole therapy with letrozole alone in patients with advanced metastaticbreast cancer. These studies mostly investigated the role of lapatinib in advanced breast cancer; however, its role was also clarified in a wide

range of tumour types such as cancers of the urothelial tract [113].

Recent studies have indicated that single-agent lapatinib only demonstrated a marginal benefit in DFS compared with placebo for patients with HER2-positive early breast cancer who previously received adjuvant chemotherapy [94,114]. In a 5-year follow-up [115], trastuzumab plus lapatinib either in combination or in sequence failed to improve DFS or OS compared with single agent trastuzumab. Currently lapatinib is not recommended in the adjuvant setting, as a single agent or in combination with trastuzumab. This limitation led to the development of sapitinib, which displays similar potent inhibition of both EGFR and HER2 but is relatively more active in a ligand-dependent pathway [116]. Although sapitinib did not appear to improve clinical outcomes in low-HER2-expressing breast cancer in combination with either paclitaxel [117] or anastrozole [118], whether this agent is effective in other tumour types remains uncharacterized.

3.2.2. Neratinib

Neratinib is an irreversible HER tyrosine kinase inhibitor derived from modification of EKB-569 and is a more HER1-selective agent [119].

Neratinib selectively interacts with cysteine residues, including Cys-773 in EGFR and Cys-805 in HER2 and also with HER4 as previously reported [120]. Low doses of neratinib reduce HER2 and EGFR phosphorylation in HER2-overexpressing BT474 and EGFR-amplified A431 cells [121], and these effects were still observed 5 h after acute treatment. This study suggested a potential mechanism involving G1-S cell cycle arrest and an increased sub-G1 population in vitro that correlated with tumour cell apoptosis. As discussed previously, neratinib, which is similar to other multi-target tyrosine kinases, acts against the intracellular domain of EGFR, HER2, and HER4, leading to synergic effects with trastuzumab, which targets extracellular domains [122,123]. This action also ex[1]plained why neratinib effectively overcomes trastuzumab resistance invitro [124]. At the 5-year follow-up of an on-going randomized phase III trial, 1 year of adjuvant treatment with neratinib significantly reduced relapse in early HER2-positive breast cancer patients [125].

Several studies have evaluated the combined use of neratinib with chemotherapy, such as the promising antitumor activity of neratinib together with vinorelbine in HER2 positive metastatic breast cancer patients [126]. Another phase II study evaluated the concomitant use of neratinib and paclitaxel. An overall response rate (ORR) of 73%, and a complete response (CRR) of 7% were reported, and 9% of patients achieved stable disease for greated than 24 weeks [127]. In addition, the combination of neratinib with capecitabine exhibited an overall response rate of 64% for lapatinib-naive patients and 57% for lapatinib pre-treated patients [128].

Previous research reported that neratinib offers increased benefit in HER2-positive breast cancers compared with other breast cancer sub[1]types. In vivo experiments in mice indicated the efficacy of neratinib in tumour growth inhibition of EGFR-amplified and HER2-overexpressing cells compared with low EGFR and HER2-expressing cells [121]. An in

vitro study demonstrated that the response to neratinib was correlated with baseline levels of HER2 and phosphorylated HER2 [124]. However, some studies demonstrated that HER2 negative patients might also benefit from the addition of neratinib to chemotherapy [129]. Whether neratinib is effective in breast tumours with lower HER expression, the threshold of which was based on therapeutic reaction to trastuzumab, is now of great research interest. The most common adverse event observed with neratinib treatment is diarrhoea of any grade, and other toxic effects include nausea, fatigue, vomiting, neutropenia, and anorexia [127,130].

However, based on previous evidence, diarrhoea could be controlled with antidiarrheal medication. Therefore, in recent clinical trials, antidiarrheal agents are recommended for all patients administered neratinib to lower the risk of diarrhoea.

Another potential interest is the role of neratinib in central nervous system (CNS) progression. A randomized phase II trial in patients with HER2-positive metastatic breast cancer was presented at the 2015 ASCO meeting. The cumulative incidence of CNS recurrence for neratinib plus paclitaxel was 14.4% versus 32.1% for trastuzumab plus paclitaxel [131]. Another trial evaluated the efficacy of neratinib in patients with brain metastasis after CNS-directed therapy including radiotherapy and radiosurgery. Although the results suggested a relatively low objective response rate of 8%, only 12.5% of patients received six or more cycles of neratinib-combined chemotherapy and studies are on-going to form a firm conclusion [132]. Of note, although neratinib has long been considered a pan-HER inhibitor, it can also target non-HER receptor kinases with homologous ATP kinase domains at certain concentrations [133].

3.2.3. Afatinib