A potential mechanism by which resistance to targeted antibodies may develop is via disruption of the interaction between the therapeutic agent and the target protein. Resistance to trastuzumab was associated with increased expression of the membrane-associated glycoprotein MUC4 26. MUC4 was shown to bind and sterically hinder HER2 from binding to trastuzumab 2627. MUC4 has been suggested to contribute to cancer because of its ability to inhibit immune recognition of cancer cells, promote tumor progression and metastasis, suppress apoptosis, and activate HER2 28. MUC4 interacts directly with HER2, an event that is dependent upon an epidermal growth factor (EGF)-like domain on the ASGP-2 subunit of MUC4 26. Through this interaction, it is proposed that MUC4 serves as a ligand for HER2, resulting in increased phosphorylation of HER2 on the residue Tyr1248 26, which is a major phosphorylation site contributing to the transforming ability of the HER2 oncoprotein 29. MUC4 does not affect total HER2 receptor expression levels 2628. The JIMT-1 trastuzumab-resistant cell line described by Nagy and colleagues 27 was established from a breast cancer patient showing her2 gene amplification and primary resistance to trastuzumab 30. Using this model, the authors demonstrated that the level of MUC4 protein was inversely correlated with the trastuzumab binding capacity, and showed that knockdown of MUC4 increased the sensitivity of JIMT-1 cells to trastuzumab 27. Thus, the authors proposed that elevated MUC4 expression masks the trastuzumab binding epitopes of HER2, resulting in steric hindrance of the interaction between this antibody and its therapeutic target, resulting in drug resistance. Interestingly, the authors also reported that HER2 was unable to interact with other proteins, such as EGFR or HER3, because of epitope masking by MUC4.

Trastuzumab resistance has been associated with increased signaling from the insulin-like growth factor-I receptor (IGF-IR). Increased expression of IGF-IR was shown to reduce trastuzumab-mediated growth arrest of HER2-overexpressing breast cancer cells 31. Expression of IGF-binding protein 3, which blocks IGF-I-mediated activation of IGF-IR, restored trastuzumab sensitivity. We recently demonstrated that crosstalk occurs between IGF-IR and HER2, and showed that IGF-IR physically interacts with and phosphorylates HER2 in trastuzumab-resistant cells, but not in trastuzumab-sensitive parental cells 32. Our results showed that resistant cells exhibited more rapid IGF-I stimulation of downstream PI3K/Akt and MAPK pathways relative to parental cells. Inhibition of IGF-IR signaling, either by antibody blockade or IGF-IR tyrosine kinase inhibition, restored trastuzumab sensitivity in our in vitro resistant model, demonstrating the potential importance of this pathway as a therapeutic target in trastuzumab-resistant breast cancer. Similar to Lu and colleagues 33, we observed downregulation of p27^kip1 upon IGF-I stimulation in both parental and resistant cells 32. Importantly, antisense oligonucleotides 34 and small interfering RNA 35 that reduced p27^kip1expression levels also blocked trastuzumab-mediated growth arrest in HER2-overexpressing SKBR3 breast cancer cells. Transfection of p27^kip1or pharmacological induction of p27^kip1 by the proteasome inhibitor MG132 restored trastuzumab sensitivity in our resistant model 36. These results suggest that p27^kip1 is a critical mediator of trastuzumab response, and that its downregulation may occur subsequent to increased signaling from growth factor receptors such as IGF-IR, promoting resistance to trastuzumab.

Growth factor receptor tyrosine kinases, such as HER2 and IGF-IR, activate the PI3K signaling pathway. Constitutive PI3K/Akt activity was previously shown to inhibit cell-cycle arrest and apoptosis mediated by trastuzumab 34. Furthermore, trastuzumab-resistant cells derived from the BT474 HER2-overexpressing breast cancer line demonstrated elevated levels of phosphorylated Akt and Akt kinase activity compared with parental cells 37. These resistant cells also showed increased sensitivity to LY294002, a small molecule inhibitor of PI3K. Nagata and colleagues 11 provided compelling evidence supporting a role for the PI3K/Akt pathway in trastuzumab resistance. They demonstrated that decreased levels of the PTEN phosphatase resulted in increased PI3K/Akt phosphorylation and signaling and blocked trastuzumab-mediated growth arrest of HER2-overexpressing breast cancer cells. Importantly, they showed that patients with PTEN-deficient HER2-overexpressing breast tumors have a much poorer response to trastuzumab-based therapy. Furthermore, they showed that, in PTEN-deficient cells, PI3K inhibitors rescued trastuzumab resistance in vitro and in vivo. These results suggest that PTEN loss may serve as a predictor of trastuzumab resistance, and that PI3K inhibitors should be explored as potential therapies in patients with trastuzumab-resistant tumors expressing low levels of PTEN protein.

The full-length 185 kDa HER2 protein has been reported to be cleaved by matrix metalloproteases into a 110 kDa extracellular domain (ECD), which is released into cell culture media 383940 or circulating in serum in vivo 41424344, and a 95 kDa amino-terminally truncated membrane-associated fragment with increased kinase activity 45. Elevated serum levels of HER2 ECD correlate with poor prognosis in patients with advanced breast cancer 4142434446. Of potential importance, trastuzumab blocked HER2 ECD proteolytic cleavage in vitro 47, and patients with elevated pre-treatment ECD levels had higher response rates to trastuzumab 4849. HER2 overexpression in breast cancers correlated with elevated pre-treatment levels of circulating HER2 ECD in patients treated with trastuzumab and paclitaxel, and among these patients, responses correlated with a decline in ECD levels over 12 weeks of therapy versus lower responses in those whose ECD levels remained high post-treatment 50.

Zabrecky and colleagues 40 first described the presence of cleaved ECD in the culture medium of HER2-overexpressing SKBR3 breast cancer cells. The authors showed that HER2-targeted monoclonal antibodies bound to circulating ECD, competing away binding to membrane-bound HER2. Hence, signaling from the receptor form of HER2 continued in the presence of HER2 antibodies, indicating that HER2 ECD promoted resistance to HER2-targeted antibody therapy. However, the predictive role of elevated baseline ECD prior to treatment is not well defined. In one study, elevated HER2 ECD levels predicted favorably for response to trastuzumab and docetaxel 24, but other studies showed limited predictive value in this setting. Interestingly, declining levels of circulating HER2 ECD correlate with improved disease-free survival in several studies 2449. A meta-analysis of 8 clinical trials revealed that patients whose HER2 ECD levels declined by at least 20% in the first few weeks after initiation of trastuzumab-based therapy had improved disease-free and overall survival compared with patients whose HER2 ECD levels did not drop 51. Hence, circulating ECD of HER2 may be a serum marker useful for predicting response to trastuzumab. In contrast to these studies, a recent study by Anido and colleagues 52 suggests that truncated forms of HER2 are actually the result of alternative initiation of translation from different methionines within the her2 sequence, which are referred to as C-terminal fragments of HER2. The authors present compelling in vivo data showing that trastuzumab does not inhibit growth of mammary xenografts of the T47D breast cancer cell line stably transfected with the truncated form of HER2, but does inhibit growth of T47D HER2 stable transfectant xenografts. Hence, this study suggests that the presence of truncated forms of HER2 may promote resistance to trastuzumab.

The recombinant humanized HER2 monoclonal antibody pertuzumab (Omnitarg™, 2C4, Genentech) represents a new class of drugs called dimerization inhibitors; these have the potential to block signaling by other HER family receptors, as well as inhibiting signaling in cells that express normal levels of HER2. Pertuzumab sterically blocks dimerization of HER2 with EGFR and HER3, inhibiting signaling from HER2/HER3 and HER2/EGFR heterodimers 53. Interestingly, we also observed that pertuzumab disrupted interaction between HER2 and IGF-IR in trastuzumab-resistant cells 32. Trastuzumab and pertuzumab bind to different epitopes in the extracellular domain of HER2, with trastuzumab binding domain IV of the extracellular domain 54 and pertuzumab binding near the junction of domains I, II, and III of the HER2 extracellular domain 55. Thus, pertuzumab could theoretically be effective in trastuzumab-resistant tumors. However, while combining trastuzumab with pertuzumab produced synergistic apoptosis in HER2-overexpressing trastuzumab-naïve breast cancer cells 56, this agent failed to demonstrate statistically significant differences on the viability of trastuzumab-resistant breast cancer cells 3032. The mechanisms by which trastuzumab-resistant cells develop cross-resistance to alternative HER2-targeted antibodies are unclear, but may reflect aberrations in downstream signaling pathways resulting in resistance to a variety of HER2-targeted agents. Clearly, additional preclinical studies are required to determine the potential efficacy of novel HER2-targeted antibodies in trastuzumab-resistant breast cancers.

Lapatinib (Tykerb™, GSK572016, formerly GW572016; GlaxoSmithKline, Research Triangle Park, NC, USA) is a dual tyrosine kinase inhibitor targeted against both EGFR and HER2. In comparison to other tyrosine kinase inhibitors in clinical trials (for example, gefitinib, erlotinib), interaction of lapatinib with EGFR and HER2 is reversible, similar to other agents, but dissociation is much slower, allowing for prolonged downregulation of receptor tyrosine phosphorylation in tumor cells. Differences in enzyme-inhibitor structures could account for differences in dissociation off-rate, as EGFR is in a closed conformation when lapatinib binds versus a more open conformation when gefitinib binds 57. However, effects on HER2 appear to be more critical to efficacy of lapatinib than effects on EGFR, and the HER2 status is a determinant of lapatinib activity while EGFR status is apparently not. Pre-clinically, lapatinib induced potent growth arrest and/or apoptosis in EGFR- and HER2-dependent tumor cell lines and xenograft models, and blocked downstream MAPK and Akt activation 58. In vitro studies demonstrated that the combination of lapatinib with anti-HER2 antibodies enhanced apoptosis of HER2-overexpressing breast cancer cells, and that lapatinib-mediated apoptosis was associated with downregulation of survivin 59. Interestingly, resistance to lapatinib was recently shown to be mediated by increased signaling from the estrogen receptor (ER) in ER-positive HER2-overexpressing breast cancers, suggesting that co-targeting of ER and HER2 may be beneficial in this population 60.

Important to the issue of trastuzumab resistance, lapatinib was shown to inhibit growth of HER2-overexpressing breast cancer cells maintained long-term on trastuzumab 61. We have observed that lapatinib induces significant apoptosis in trastuzumab-resistant cells to the same degree as in parental, trastuzumab-sensitive cells. Furthermore, lapatinib appears to have inhibitory effects on IGF-I signaling in the resistant cells, suggesting that its growth inhibitory activity may be due not only to anti-EGFR/HER2 activities but also to IGF-IR inhibition (Nahta R, Yuan LX, Yu D, Esteva FJ, submitted).

Exciting clinical data have strongly positioned lapatinib for FDA approval against HER2-overexpressing breast cancers. The phase I study EGF10004 examined heavily pretreated patients with EGFR-expressing and/or HER2-overexpressing MBC who were randomly assigned to one of five dose cohorts of lapatinib 62. Four patients with trastuzumab-resistant MBC, two of whom were classified as having inflammatory breast cancer, had partial responses. A recent phase III trial of HER2-overexpressing MBC patients who were heavily pretreated and trastuzumab-refractory demonstrated that combination lapatinib and capecitabine resulted in a doubling of median time to progression and median progression-free survival (both 36.9 weeks) compared with capecitabine alone (median time to progression 19.7 weeks and progression-free survival 17.9 weeks) 63. Such results are rarely if ever seen in this patient population, and support lapatinib as a promising new agent for patients who have progressed on trastuzumab-based therapy.

Based on preclinical evidence suggesting a role for IGF-IR signaling in the development of trastuzumab resistance 313233, novel IGF-IR-targeted agents have been introduced into pharmaceutical testing and are being assessed in preclinical trastuzumab-resistant models. In vitro studies demonstrated that inhibition of HER2 signaling using trastuzumab, and inhibition of IGF-IR signaling using a dominant negative construct produced synergistic growth inhibition of HER2-overexpressing breast cancer cells 64. Triple combination treatment of BT474 ER-positive HER2-overxpressing breast cancer cells or MCF7 ER-positive IGF-IR-elevated breast cancer cells with ER, HER2, and IGF-IR antagonists further augmented apoptotic effects of single agents or dual combinations 65. In addition, our data demonstrate increased apoptosis when lapatinib and the IGF-IR monoclonal antibody alpha IR3 are combined in trastuzumab-resistant cells (Nahta R, Yuan LX, Yu D, Esteva FJ, submitted). Therapeutic strategies that target both the HER2 and IGF-I signaling pathways should be studied further for potential use in cancers that progress on trastuzumab.

Inhibitors of pathways downstream of the HER2 receptor may combat trastuzumab resistance. Perifosine is an Akt inhibitor undergoing clinical testing in patients with solid tumors and hematological malignancies 6667. As most Akt inhibitors have not achieved clinical development due to excessive toxicity in preclinical models, an alternative approach to blocking PI3K/Akt signaling is the use of small molecules that inactivate the kinase mTOR, which functions downstream of Akt. Three mTOR inhibitors being tested in clinical trials for patients with breast cancer and other solid tumors are CCI-779 (temsirolimus; Wyeth-Ayerst, Madison, NJ, USA), RAD001 (everolimus; Novartis, New York, NY, USA), and AP23573 (Ariad; Cambridge, MA, USA) 6869. Based on the results of Nagata and colleagues 11, in which low PTEN-expressing breast tumors were found to have reduced response to trastuzumab, our group launched a clinical trial of trastuzumab in combination with the mTOR inhibitor RAD001 in patients with HER2-overexpressing MBC resistant to trastuzumab-based therapy. Additionally, drug discovery programs are focusing on developing more effective, less toxic, direct inhibitors of the Akt kinase family.

Another class of agents called histone deacetylase inhibitors is being explored in the setting of HER2-overexpressing MBC. Preclinical work demonstrated that the histone deacetylase inhibitor hydroxamic acid analogue, LAQ824, significantly reduced HER2 levels in SKBR3 and BT474 breast cancer cells by promoting proteasome-dependent degradation and reduced transcription of HER2 70. These effects on HER2 were associated with induction of p27kip1 and inhibition of Akt and MAPK signaling. In addition, the combination of LAQ824 with trastuzumab induced marked apoptosis in vitro. Another hydroxamate-based histone deacetylase inhibitor called suberoylanilide hydroxamic acid similarly reduced HER2 protein levels 71. It was found that suberoylanilide hydroxamic acid induced acetylation of the HER2 chaperone protein heat shock protein 90 (hsp90), reducing interaction between the proteins and promoting ubiquitination and degradation of HER2. Suberoylanilide hydroxamic acid and trastuzumab combined resulted in synergistic induction of apoptosis in BT474 and SKBR3 breast cancer cells. These in vitro studies paved the way for clinical trials examining the combination of histone deacetylase inhibitors with trastuzumab.