- Top of page
- Material and Methods
- Disclosure Statement
- Supporting Information
Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 20% of gastric and gastroesophageal junction cancers in the United States and European Union. Lapatinib, a dual HER2 and epidermal growth factor receptor tyrosine kinase inhibitor, has demonstrated clinical efficacy in HER2-amplified cancer cells. However, several studies have shown that some cytokines can mediate resistance to lapatinib using their receptor tyrosine kinase (RTK) pathways. One of these, Heregulin1 (HRG1), can confer resistance to lapatinib-mediated growth inhibition in HER2-amplified breast cancer cells, but the underlying mechanisms remain unknown. Here, we investigated whether and how HRG1 causes resistance to lapatinib in gastric and gastroesophageal junction cancers in vitro. HER2-amplified gastric and gastroesophageal junction cancer cell lines were highly sensitive to lapatinib. Exposure to HRG1 together with lapatinib rescued cells from lapatinib-induced cell cycle arrest and apoptosis. Downregulation of HER3 with siRNA in the presence of HRG1 re-sensitized HER2-amplified cancer cells to lapatinib. Immunoblotting analysis indicated that HRG1 re-activated HER3 and AKT in the presence of lapatinib, which persisted for at least 72 h. Activation of HER3 and downstream AKT was mediated by residual activity of HER2. HRG1-mediated resistance could be reduced by PI3K/mTOR inhibitors or by complete inhibition of HER2. Thus, we conclude that HRG1 mediates resistance to lapatinib through HER3 and AKT activation, and that this depends on residual HER2 activity. Lapatinib in combination with anti-PI3K therapies or more potent HER2 inhibitors would improve the efficacy and avoid the emergence of resistant cells.
Gastric and gastroesophageal junction cancer is one of the leading causes of cancer-related mortality in the world. Despite the recent reported benefits of combination therapies, the prognosis of advanced gastric or gastroesophageal junction cancer remains poor, and new treatments are sorely needed.[2, 3] Currently, HER2/neu, a member of the human epidermal growth factor receptor (EGFR) family, has attracted particular attention as a druggable target because it is amplified and/or overexpressed in approximately 20% of gastric and gastroesophageal junction cancers and high levels of HER2 expression are associated with poor patient prognosis.[3-7]
Lapatinib (Tykerb, GlaxoSmithKline) is a dual tyrosine kinase inhibitor (TKI) that specifically targets both HER2 and EGFR, thereby inhibiting their activities and their downstream signaling pathways, such as MAPK and AKT.[8, 9] Treatment of gastric cancer patients having HER2-amplified tumors with lapatinib has shown promising results in phase II trials and is currently being tested in phase III trials. However, many patients who initially respond to TKIs eventually develop resistance. Several resistance mechanisms against TKIs, including acquired gene mutation, gene amplification or the activation of non-targeted RTKs by cytokines have been reported.[11-17]
HRG1/Neuregulin-1, known to act as a specific ligand for HER3 and HER4, induces noncovalent heterodimeric complexes of HER3 and HER4 with HER2, and activates multiple biological responses in cells.[18, 19] HRG1 expressed in breast, lung, colon, head and neck, and endometrial cancer cells has been shown to contribute to cancer development by activating HER2 through autocrine and/or paracrine mechanisms.[20-24] Gastric fibroblasts also express HRG1, which influences the proliferation of gastric epithelial cells, leading to the suggestion that HRG1 from gastric fibroblasts contributes to for proliferation of gastric and gastroesophageal junction cancer cells through paracrine mechanisms.[25, 26] Additionally, some reports showed that HRG1 conferred resistance to HER family-targeted therapy in breast cancer cell lines, but the underlying mechanisms are poorly understood.[17, 27]
Here, we investigated whether and how HRG1 confers resistance to lapatinib in gastric and gastroesophageal junction cancers. We found that even with lapatinib treatment there is HER3 and residual HER2 activity, which is needed for HRG1-meditated resistance to lapatinib and re-activation of HER3 and AKT signaling. Our data provide a rationale for developing HER2-targeted therapy with PI3K/mTOR inhibitors, or developing more potent HER2 inhibitors.
- Top of page
- Material and Methods
- Disclosure Statement
- Supporting Information
Gastric and gastroesophageal junction cancer represents a major global health care problem. Despite recent improvements in combination therapies, the prognosis of advanced gastric and gastroesophageal junction cancer remains poor, and new and better treatments are urgently needed. Currently, HER2 has attracted particular attention as a drug target because approximately 20% of gastric and gastroesophageal junction cancer patients have amplified and/or overexpressed HER2, and high levels of HER2 are associated with worse clinical outcome.[3-7] In 2010, the first HER2-targeting drug, trastuzumab, was approved for HER2-positive metastatic gastric and gastroesophageal junction cancer. Following the success of trastuzumab, lapatinib has been assessed in phase III clinical trials comparing first-line therapy with or without this drug in HER2-positive gastric, gastroesophageal junction and esophageal cancer. The results of this trial indicate that Asian may benefit more from the combination with lapatinib than North Americans.
Molecular targeted therapies are effective in patients whose tumors express the appropriate targets. However, the emergence of resistance to molecular targeting therapies is a serious problem. Numerous studies have reported several resistance mechanisms against TKIs, including acquired gene mutation, gene amplification or the activation of different RTKs by cytokines.[11-17] HRG1 has been shown to induce resistance to molecular therapies targeting members of the HER family of RTKs in breast cancer cells, but the underlying mechanisms are unknown.[17, 27] The expression of HRG1 has been shown in breast, lung, colon, head and neck, and endometrial cancer cells and also in gastric fibroblasts.[21-25] Here, we assessed whether HRG1 confers a growth advantage on and resistance to lapatinib treatment in HER2-positive gastric and gastroesophageal junction cancer cells and investigated the underlying mechanisms of such HRG1-mediated resistance.
Our results showed that gastric cancer-associated fibroblasts expressed HRG1 at higher levels than HER2-amplifed gastric and gastroesophageal junction cancer cell lines. HRG1 reversed lapatinib-mediated growth inhibition in the cancer cell lines, with the exception of the HER3-negative line SK-GT-2. Cell cycle arrest and apoptosis induced by lapatinib were reduced by HRG1 treatment of both NCI-N87 and OE19, albeit apoptosis was more obvious in the former. The different mechanisms of action of lapatinib and HRG1 might depend on cell type or protein expression levels of HER2 and HER3.
Downregulation of HER3 expression with siRNA abrogated the rescue effect of HRG1 and completely restored lapatinib-mediated growth inhibition. These results indicated that HER3 is responsible for HRG1-mediated resistance to lapatinib. HER3 overexpression is significantly associated with advanced TNM stage and strongly associated with tumor progression and poor prognosis in gastric cancer. This suggests that HER3 overexpression might correlate with lapatinib resistance in cancer patients.
Recently, Garrett et al. found that lapatinib induces transcriptional and posttranslational upregulation of HER3 and pHER3 was recovered after 24 h of lapatinib treatment in breast cancer cell lines.[15, 35] Although HER3 was upregulated in gastric and gastroesophageal junction cancer cell lines by lapatinib, pHER3 was not recovered without HRG1. This discrepancy might depend on cell type and derivation. Additionally, exposure to HRG1 and lapatinib together induced less upregulation of HER3 than lapatinib alone. Because inhibition of AKT activity induces HER3 expression via feedback mechanisms, HRG1-mediated AKT activation might suppress this feedback. Interestingly, pHER2 was slightly restored by 24 h of lapatinib treatment regardless of the presence of HRG1. Lapatinib treatment results in the accumulation of HER2 on the cell surface; thus, this phosphorylation effect might depend on homodimerization of surface HER2.[37, 38] We also discovered that lapatinib reduced EGFR expression in a time-dependent manner. Epidermal growth factor receptor activity is necessary for its src-mediated degradation but EGFR is one of the targets of lapatinib and was dephosphorylated in our experiments. Dephosphorylated EGFR might be degraded by some other unknown mechanism.
pAKT, but not MAPK, recovered under the influence of HRG1 in the presence of lapatinib. LY294002 and everolimus, PI3K and mTOR inhibitors, respectively, reduced the rescue effects. This result indicates that the PI3K/AKT/mTOR pathway is a pathway for HRG1-mediated resistance to lapatinib. Everolimus already finished testing in phase III clinical trials for advanced gastric cancer in 2012, but failed to meet the primary endpoint of improved overall survival. Our data suggest that combination therapy with lapatinib and everolimus in HER2-amplified gastric and gastroesophageal junction cancer may improve efficacy and prevent the emergence of resistant cells.
pHER3 was recovered by HRG1 treatment even in the presence of lapatinib. Although EGFR, Src, c-MET, FGFR2 and PYK2, are known to associate with HER3, none of the inhibitors of these kinases suppressed the HRG1-mediated phosphorylation of HER3 and AKT, and did not restore lapatinib-mediated growth inhibition. We therefore hypothesized that residual HER2 activity despite the presence of lapatinib is responsible for HRG1-mediated HER3 phosphorylation. Although we could not clearly demonstrate direct cross-talk through HER2/HER3 heterodimers by HRG1 in the presence of lapatinib by coimmunoprecipitation experiments (data not shown), higher concentrations of lapatinib or a combination of lapatinib with BIBW2992 (but not trastuzumab), further suppressed phosphorylation of HER2, pHER3 and pAKT, and reduced HRG1-mediated rescue effects. HER3 is thought to be an inactive pseudokinase, although Shi et al. recently reported that it can have low level kinase activity and can trans-autophosphorylate its intracellular region. These investigators also found that lapatinib was unable to inhibit trans-phosphorylation of HER3 in vitro. Our results and theirs support the notion that residual HER2 phosphorylates HER3 in the presence of HRG1. Higher concentrations than we used in vitro are not achievable in patients, at least not in breast cancer, using the current daily schedule. Although we have not formally confirmed the lapatinib concentration achievable in gastric and gastroesophageal junction cancer patients, it is unlikely that higher concentrations could be achieved in these patients either.
PI3K/mTOR inhibition and complete HER2 inhibition significantly reduced HRG1-mediated rescue effects, but neither treatment completely reversed growth inhibition. HER3 siRNA experiments indicate that interactions between HER3 and unknown targets may be involved in these rescue effects. Recently, anti-HER3 therapies such as antibody treatment and use of antisense oligonucleotides were reported to improve the efficacy of lapatinib in vitro and in vivo.[33, 42] Combinations of anti-HER2 and anti-HER3 therapies with lapatinib might be effective for overcoming HRG1-mediated resistance.
In summary, HRG1 induces resistance to lapatinib through interactions between HRG1 and HER3, residual HER2 activity and the PI3K/AKT/mTOR pathway. PI3K/mTOR inhibition or more complete HER2 inhibition abrogated the HRG1-mediated rescue effect. These results indicate that lapatinib in combination with anti-PI3K therapies or more potent HER2 inhibitors might be more potent strategies for treating HER2-positive gastric and gastroesophageal junction cancer patients.