Neratinib is an irreversible kinase inhibitor and a derivative of EKB-569 (EGFR inhibitor) (63–65). Neratinib significantly inhibited EGFR/HER2 kinase after binding to the ATP pocket and blocking downstream signaling pathways (59) and showed anti-cancer bioactivities in HER2-overexpression cell lines and in patients with or without prior trastuzumab treatment (66–68). Neratinib inhibited proliferation and promoted G1-S phase arrest by regulating HER2 and its downstream signaling pathways, specifically through downregulation of pEGFR, pHER2, pAKT, pMEK and pRb levels and cyclin D1 (CCND1) expression and increase of p27 levels in a HER2-dependent manner (69). Some studies also discovered that neratinib improved trastuzumab resistance and restored sensitivity to trastuzumab in HER2⁺ BC (69,70). Many clinical trials investigated the functions of neratinib in treating HER2⁺ BC alone or in combination with trastuzumab, which exhibited great clinical application prospects. A more important role of neratinib was observed when combined with paclitaxel in treating patients who received prior taxane, trastuzumab and lapatinib therapies (71). Neratinib was effective as a single agent or in combination with different chemotherapy drugs in the treatment of HER2+ MBC patients and early BC patients (68,72–74). In a multicenter, randomized, double-blind and placebo-controlled phase III trial (ExteNET), which involved 2,840 women, a total of invasive disease-free survival events of 70 patients in the neratinib group and 109 patients in the placebo group occurred, corresponding to 93.9 and 91.6% 2-year invasive disease-free survival rates, respectively (75). All of these results above show that neratinib is quite effective and is close to clinical application.

Lapatinib showed significant activities in treating HER2⁺ BC either alone or combined with trastuzumab. A considerable portion of HER2⁺ patients were insensitive to lapatinib, which prompted development of a modified drug form. KU004, a derivative of lapatinib, remarkably inhibited pEGFR, pHER2, pAkt and pMAPK in BT474 and NCI-N87 cells, which were treated with lapatinib or not. Treatment with KU004 achieved a G0/G1 phase arrest and corresponding reduction of cells in S and G2/M phases in BT474 cells. KU004 could induce caspase-dependent apoptosis, as caspase-8 and caspase-3 were significantly activated by KU004. In vivo study found that KU004 reduced growth of NCI-N87 ×enograft dramatically in a dose-dependent manner by inducing apoptosis. These in vivo and in vitro results indicate that KU004 has the potential to treat HER2⁺ BC in the future (76).

Trastuzumab, as the first-generation targeted therapy drug, helped many HER2⁺ BC patients to improve their prognosis and quality of life in the future. As we mentioned above, the resistance to trastuzumab limited its application to a large extent. Thus, research and development of new drugs to overcome trastuzumab resistance may contribute significantly to the treatment of HER2+ BC patients.

It has been proven that CCND1 and CDK4 played a vital role in BC (77,78). CDK4/6 inhibitors have shown great potential in preclinical studies and clinical trials, such as palbocilcib and ribociclib which has been applied in clinical (79,80). Though CDK4/6 inhibitors are mainly used to treat estrogen receptor (ER)⁺/HER2⁻ patients, some studies found that they also have potential therapeutic effect on ER⁺/HER2⁺ patients (81). In an exploratory, open-label, phase 2 study (NA-PHER2), the combination of palbociclib, fulvestrant, trastuzumab and pertuzumab was found reduced the expression of Ki67 significantly (82). Palbocilcib could also efficiently inhibit the proliferation of residual HER2⁺ tumor cells that survive T-DM1 in preclinical BC models (83). In a preclinical study, they found that both residual tumor cells and recurrent tumor cells that were present after treatment presented high levels of CCND1and CDK4, which promoted cell proliferation. In addition, abemaciclib, a CDK4/6 inhibitor, inhibited Rb phosphorylation and significantly postponed the process of recurrent tumors, which showed a CDK4/6-dependent increase in expression of CCND1. Strikingly, reduced sensitivity to both lapatinib and trastuzumab was found when CCND1 was over-expressed in trastuzumab/lapatinib-sensitive BC cell lines. Knockdown of CCND1 in resistant cells partially restored the sensitivity to trastuzumab. Phosphorylation of TSC2 and its downstream effectors, P70-S6K and S6RP, was reduced after abemaciclib treatment, which could be enhanced by combined CDK4/6-HER2 inhibition. Furthermore, in vivo study showed that trastuzumab combined with abemaciclib inhibited tumor growth more than either single agent (84). In another preclinical study, abemaciclib with trastuzumab significantly improved tumor growth inhibition and tumor regressions in xenografts progressing on trastuzumab alone (81). In a phase I study, one patient with hormone receptor-negative and HER2⁺ BC experienced an antitumor effect with a 30% decrease in tumor size from baseline. At the same time, some clinical trials are ongoing to prove whether CDK4/6 inhibitors could be applied to ER⁺/HER2⁺ patients (NCT02947685; NCT02774681; NCT02907918; NCT02675231) (81).

Antibody-drug conjugates (ADCs) are constructed by covalently attaching small-molecule toxins to antibodies (85–87). T-DM1 is a ADCs that has shown significant activity in treating HER2⁺ BC (86,87). A new biparatopic ADC was constructed, consisting of the trastuzumab scFv unit, the 39S Fv unit, and AZ13599185, which inhibits microtubule polymerization during mitosis. The new biparatopic ADC retained HER2 binding specificity and could effectively deliver cytotoxic agents into the targeted tumors. Compared to T-DM1, the new biparatopic ADC demonstrated at least 10-fold cell killing activity in HER2-expressing cancer cell lines in a HER2-dependent manner and showed activity in cancer cells that were intrinsically resistant to T-DM1. In vivo study suggested the possibility of clinical application of the new biparatopic ADC, as it induced full tumor degradation and inhibited tumor growth in a primary BC xenograft model and an intrinsically or acquired T-DM1-resistant xenograft model, which was not observed after treatment with T- DM1 alone or in combination with pertuzumab. These valuable results show that development of the new biparatopic ADC was necessary to explore a more effective drug for the treatment of HER2⁺ BC (88).

Due to the putative role of EGFR in trastuzumab resistance, blocking EGFR and its corresponding downstream signaling pathways may produce better prognosis. The src homology 2 (SH2) containing tyrosine phosphatase-1 (SHP-1) regulated the intracellular phosphotyrosine level and was reduced or absent in many kinds of cancer cell lines and tissues (89–91). Overexpression of wild-type SHP-1 sensitized trastuzumab-resistant cells to trastuzumab treatment. Furthermore, overexpression of wild-type SHP-1 also significantly upregulated apoptosis and decreased EGFR and HER2, as well as their phosphorylation levels, in trastuzumab-resistant cells. Wild-type SHP-1 could bind to pEGFR and pHER2, and thus reduced both pEGFR and pHER2 protein levels. In vivo study demonstrated that SHP-1-overexpression mice showed delayed tumor progression and growth and achieved a better OS after treatment with trastuzumab compared with the control (92).

Fatty acid synthase (FASN) showed activity in promoting tumorigenesis by activating HER2/PI3K/AKT/mTOR and MAPK signaling pathways (93–95). EGFR and pEGFR increased in trastuzumab resistant cells noticeably, which was consistent with the study of Wu et al (92). Pertuzumab combined with EGCG, a classical FASN inhibitor, was more effective than pertuzumab alone against resistant HER2⁺ BC cells and increased the apoptosis of cancer cells in a trastuzumab plus lapatinib-resistant HER2⁺ PDX model compared with pertuzumab alone. In vivo study showed that dual FASN and HER2 blockade achieved more effective tumor reduction compared with EGCG or pertuzumab as single agent (50).

The occurrence of all tumors is due to genetic mutations, so gene therapy is the most effective treatment approach for all cancers. Using an effective vector to accurately deliver drugs to the tumors is a very valid treatment method to stop the tumor progression.

A new delivery platform, which was able to escape from lysosomal degradation and distribute symmetrical in the cytoplasm, was established to send mature tumor suppressor microRNA125a-5p to treat HER2⁺ MBC. miR125a-5p significantly decreased cellular proliferation and migration in comparison with control cells, which might have been largely caused by knockdown of the HER2 mRNA levels. HA-LNP-miR125a-5p reduced the levels of HER2, PI3K, pAKT, Ki67 and pERK1/2 by over 30, 35, 40, ~40 and 20%, respectively, which indicated that HA-LNP-miR125a-5p could treat BC through inhibiting HER2 and its downstream signaling pathways (96).

Vaccines that target HER2⁺ BC may become the best way to prevent tumorigenesis. Campbell et al (97) had previously shown that the immunostimulatory peptide Hp91 had potent immune effects, and subsequently, Hp91 was encapsulated inside poly (D,L-lactic-co-glycolic) acid nanoparticles (PLGA-NPs) to construct a possible BC vaccine. The new vaccine significantly increased DCs when compared to free Hp91 in vitro. Furthermore, in vivo study showed that injection of the new vaccine noticeably increased HER2-specific IFN-γ spot-forming cells compared to the control group and the free Hp91 group (whether administered at the same concentration or higher). Hp91 encapsulated in PLGA-NPs delayed the tumor development by approximately three months compared to mice that were injected with free Hp91 and delayed the tumor development by approximately 5 months compared to control mice, and it prolonged the OS. These results proved that Hp91 encapsulated inside PLGA-NPs could be developed into an effective BC vaccine (97).

Natural anti-cancer substances played an important role in the treatment of BC and some new natural anti-cancer substances will play a role in the treatment of HER2⁺ BC. Paclitaxel is the most successful natural anti-cancer drug, which was extracted first from the bark and wood of Pacific Yew tree (98). Hesperetin (HP) and naringenin (NG) belong to flavonoids, which have shown anti-cancer and pro-apoptotic activity (99). An anthraquinone compound, aloe-emodin (AE), effectively suppressed HER2 expression and cell proliferation in a dose-dependent manner consistent with promotion of apoptosis and G1 cell cycle arrest in HER2⁺ BC cells. Twist and Y-box binding protein 1 (YB-1) have been investigated for promoting cell proliferation, tumor metastasis, invasion, angiogenesis and anticancer-drug resistance (100,101). In vitro study showed that AE observably inhibited cancer cell migration and invasion rates through promoting E-cadherin levels, which restored the epithelial cell adhesion, and inhibiting factors, which activated cancer cell metastasis. These results had important clinical significance and were confirmed by in vivo study in a SkBr3 cell xenograft model (102). Irisin is newly discovered, secreted from muscle tissue and adipose tissue, and recognized as an adipokine, which has been proven to participate in breast carcinogenesis (103,104). Irisin or its structural analogues may become new therapeutic drugs (105). Several studies have investigated the use of apigenin decreasing the risk of a variety of cancers, including BC (106–108). NAX014, a derivative of berberine (BBR), was effective in inhibiting a variety of cancer cells (109,110) tended to reduce HER2 expression in tumor tissues (111). Acetyl tanshinone IIA (ATA), a tanshinone IIA derivative (112), had shown activity against BC (113). In vivo study showed that injection of ATA reduced tumor volume and tumor weight remarkably (112).

IFN-γ and TNF-α had shown antitumor effects (114). This study emphasized the exciting roles of TNF-α and IFN-γ in treating HER2⁺ BC. No HER2⁺ BC cell increase due to induction of cell apoptosis was observed in the dual cytokine-treated group in comparison with the untreated or single cytokine-treated group. To investigate the mechanism of whether TNF-α and IFN-γ induced cell apoptosis, they treated TUBO and 4T1 cells with either dual Th1 cytokines, actinomycin D (positive control), or no treatment (negative control). They found that dual TNF-α and IFN-γ treatment decreased pro-caspase-3 levels significantly, corresponding with marked increase in activated caspase-3 levels. Treatment of Th1 cytokines and selective caspase-3 agonist (PAC-1) induced HER2 loss observably and showed a strong correlation with cancer cell apoptosis. Interestingly, downregulated HER2 expression could be eliminated by caspase-3/7 inhibitor in TUBO cells (115).

Anti-ErbB2 mAb therapy failed to reduce tumor growth in IL21R-/- mice; however, anti-ErbB2 mAb therapy succeeded in WT mice, which indicated that the IL21 signaling pathway had a strong correlation with trastuzumab resistance. Furthermore, IL21R expressed by CD8⁺ T-cells was required for mice that transferred with WT CD8⁺ T-cells to achieve anti-ErbB2 mAb therapy success, compared with the mice treated with IL21R^−/− CD8⁺ T-cells. Recombinant IL21 combined with anti-ErbB2 mAb therapy significantly inhibited tumor growth and showed activity in lowering metastatic tumors (116).

Recombinant DNA-derived viral capsid protein-1 (rVP1) has been proven to induce apoptosis and suppress invasion in several cancers. They investigated that both in vivo and in vitro rVP1 treatment significantly inhibited cancer cell metastasis and invasion, consistent with increased E-cadherin and decreased a-vimentin levels in vitro. Furthermore, the mRNA and protein expression of HER2 were inhibited by rVP1 in a dose-dependent manner (117).

Valproic acid (VPA) showed activity in cell apoptosis (118) and controversial effects in inhibiting BC cells (119,120). During HER2-overexpression in SKBR3 BC cells, cell growth was inhibited by VPA in a dose- and time-dependent manner consistent with observably increased p21 WAF1 expression, which could inhibit tumor cell differentiation and growth (121,122). Cleaved caspase-3 levels were upregulated by ~2-fold after the treatment with VPA, corresponding with a higher number of TUNEL positive cells in the SKBR3 cell line compared with the control group (123).

EZN4150, an antisense oligonucleotide, is an inhibitor of the PIK3CA gene that encodes the p110α type I PI3K catalytic subunit. EZN4150 combined with lapatinib or BKM120 (a type I PI3K inhibitor) decreased pAkt and increased cleaved caspase-3 levels, achieving a greater effect in combination than either compound alone. Autophagy is a process that could inhibit tumor initiation but support tumor progression (124,125). The role of autophagy in promoting cell survival was blocked by EZN4150 instead of BKM120 in a p110α-independent manner, which might be mediated by Vps34 ablation, as EZN4150 downregulated both p110α and Vps34 expression. The combined knockdown of p110α and Vps34 significantly decreased cell numbers, increased the level of cleaved caspase-3 and increased lapatinib-mediated growth inhibition in BT474 and SKBR3 cell lines. Although both lapatinib and BYL719 (a p110α-specific PI3K inhibitor (126) treatment robustly induced caspase 3/7 activity, SAR405, an inhibitor of Vps34, more significantly increased caspase 3/7 activity than lapatinib, BYL719 or their combination. These results established Vps34 as a new therapeutic target, and EZN4150 was able to improve clinical prognosis by increasing tumor cell killing (127).

Programmed death ligand 1 (PD-L1) is the ligand of programmed death 1 (PD-1). Anti-PD-1/PD-L1 therapy is a novel immune-checkpoint inhibition therapy and anti-PD-1/PD-L1 agents, such as nivolumab, pembrolizumab, atezolizumab, durvalumab and avelumab have been widely applied to treat various types of cancer (128–130). Anti-PD-1/PD-L1 agents have shown antitumor activity in BC, especially in the triple-negative subtypes of breast cancer (TNBC) (131,132). But PD-L1 expression is associated with HER2⁺ status and there is an independent poor prognostic impact of PD-L1 in HER2⁺ BCs (132–134). In a phase 1b trial which 168 patients with MBC received avelumab, avelumab showed an acceptable clinical activity (135). Currently, many clinical trials in HER2⁺ cohort are ongoing, such as NCT02648477 and NCT02129556 for pembrolizumab, NCT02605915 for atezolizumab and NCT02649686 for durvalumab (131). Anti-PD-1/PD-L1 agents will benefit the patients with HER2⁺ BC in the future.