Therapeutic strategies for afatinib‐resistant lung cancer harboring HER2 alterations

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non‐small‐cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan‐HER inhibitor afatinib could be a useful therapeutic agent as HER2‐targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib‐resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib‐resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial‐to‐mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)‐like features. The afatinib‐resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC‐like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.

marked antitumor activity in patients with non-small cell lung cancer (NSCLC) carrying activating EGFR mutations. 1 Other oncogenic alterations, such as ALK, KRAS, NRAS, BRAF, MET and FGFR, have also been identified in some subsets of NSCLC patients as possible treatment targets. [2][3][4] Human epidermal growth factor receptor 2 (HER2) is a member of the HER family, which is composed of 4 receptor tyrosine kinases (RTK). It is frequently overexpressed in various human cancers, and many preclinical studies have demonstrated that overexpression of HER2 or mutations of the HER2 kinase domain play an important role in oncogenic transformation and tumorigenesis. [5][6][7][8] In the case of breast and gastric cancers, targeted therapies in patients with HER2positive tumors have been clinically proven to be effective. 9,10 Among NSCLC patients, the reported frequency of HER2 overexpression and HER2 amplification are 11%-32% and 2%-23%, respectively. [11][12][13][14] HER2 mutations are identified in approximately 2%-4% of NSCLC and are usually mutually exclusive of other driver mutations. 15,16 However, it remains to be established whether HER2-targeted therapy is of clinical benefit in patients with HER2-altered NSCLC. Previous clinical trials have failed to show any clinical benefit of trastuzumab monotherapy or any additional effect of trastuzumab over conventional first-line chemotherapy in HER2-positive NSCLC patients. [17][18][19] However, subsets of patients with high HER2 expression levels, HER2 gene amplification or HER2 mutations have been demonstrated to show a good response to HER2-targeted treatment. 17,20,21 In addition, a recent retrospective study showed that HER2-targeted therapy was beneficial in combination with chemotherapy for patients with HER2mutant NSCLC. 22 These findings suggest the potential usefulness of HER2-targeted therapy in HER2-altered NSCLC patients.
Afatinib, also known as BIBW2992, is an irreversible pan-HER (EGFR, HER2, HER3 and HER4) inhibitor, and has recently been approved for the treatment of NSCLC patients carrying EGFR mutations in the tumor. We previously reported that afatinib could inhibit cell growth in both HER2-amplified and HER2-mutant NSCLC cells, just as in EGFR-mutant NSCLC cells, through G1 arrest and apoptosis. 23 In clinical settings, afatinib monotherapy was effective in 3 out of 3 assessable patients with an HER2-mutated adenocarcinoma in an exploratory study of 5 patients. 21 In addition, the recent retrospective studies showed the potential benefit of afatinib for patients with HER2-mutant lung cancers. 22,24 While further prospective studies including randomized trials are required, afatinib could be approved as one of the therapeutic options for HER2-altered NSCLC patients.
However, development of acquired resistance to afatinib, just like the case for other HER inhibitors, has been reported in most EGFRmutant NSCLC patients treated with afatinib, 25,26 and the situation is considered to be similar for HER2-altered NSCLC patients. Therefore, overcoming acquired resistance to afatinib is a critical clinical issue.
In this study, we experimentally established various afatinib-resistant cell lines from HER2-altered NSCLC cell lines as the parental cell lines, and examined the molecular profiles of these cells to uncover the mechanisms of acquisition of the drug resistance, and investigated the treatment strategies for these cells with acquired resistance to afatinib. Three NSCLC cell lines (Calu3, NCI-H2170 [H2170] and NCI-H1781 [H1781]), purchased from ATCC (Manassas, VA, USA), were used as the patent cell lines for this study. These cell lines were authenticated by ATCC using short-tandem-repeat polymorphism analysis, and used within 6 months of receipt. All the lung cancer cells were cultured in RPMI-1640 media supplemented with 10% FBS, in a humidified incubator with 5% CO 2 at 37°C. Afatinib-resistant sublines were established by different methods: the parental cells were cultured with stepwise escalation of the concentration of afatinib in the culture medium (Calu3 and H2170: from 5 nmol/L to 2 lmol/L, H1781: from 5 nmol/L to 0.4 lmol/L) over a period of 6 months (stepwise escalation method), or use of a high concentration of afatinib from the start of culture (Calu3 and H2170: 2 lmol/L, H1781: 0.4 lmol/L) until 6 months (high concentration method). Finally, afatinib-resistant sublines designated as H2170-ARS, Calu3-ARS and H1781-ARS were established by the stepwise escalation method, and those designated as H2170-ARH, Calu3-ARH and H1781-ARH were established by the high concentration method (Table 1).

| Reagents and cell cultures
Acquired resistance was defined as an IC50 value of over a 100-fold higher than that for the parental cell line. Afatinib and docetaxel were purchased from Synkinase (San Diego, CA, USA) and Wako Pure Chemical Industries (Osaka, Japan), respectively. Crizotinib and elacridar were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Details of the phospho-receptor tyrosine kinase array and the cell proliferation assay are included in Document S1.

| DNA and RNA extraction
Genomic DNA were extracted from the cell lines using the DNeasy Blood and Tissue Kit (Qiagen, Venlo, the Netherlands), and total RNA were extracted using the RNeasy Mini Kit (Qiagen). Complementary DNA (cDNA) was synthesized from total RNA using a

| Direct sequencing
We determined the mutational status of the tyrosine kinase domain of HER2 by direct sequencing; the PCR conditions employed are shown in Table S1B.

| Western blot analysis
The total cell lysate was extracted with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich) and Complete Mini (Roche, Basel, Switzerland). Western blot analysis was carried out using the conventional method using the following  Table S1C.

| Cell growth inhibition assay
Cells were cultured with or without afatinib for 72 hours and the sensitivity to the drug was determined by a modified MTS assay using CellTiter 96 Aqueous One Solution Reagent (Promega, Fitchburg, WI, USA), as previously described. 28 The antiproliferative effect of each drug was expressed in terms of the IC50 value, which refers to the concentration of the drug required to inhibit cell proliferation by 50%.

| Statistical analysis
All data were analyzed using the JMP v 9.0.0 software (SAS Insti- HER2-amplified cell lines (Calu3 and H2170) and 1 HER2-mutant cell line (H1781). To investigate the genotypic changes in the cells with acquired resistance to afatinib, we examined the copy number of HER2, EGFR and MET, gain of which is well known as a mechanism underlying the resistance to HER inhibitors. 29 Significant copy number gains of MET were detected in the Calu3-ARS cells ( Figure 1A).
In addition, the HER2 copy number was almost lost in the H2170-ARS and H2170-ARH cells. Copy number analysis also revealed progressive decrease of the HER2 copy number of H2170-ARS cells with increasing number of passages, eventually leading to the disappearance of the amplified HER2 alleles ( Figure 1B). To investigate the mechanism underlying the progressive decrease of the copy number of HER2, we performed the FISH assay and found that all of H2170 parental cells harbored amplified HER2 alleles ( Figure 1C).  Figure 2A); furthermore, the resistant cells had acquired a spindle-like shape in contrast to the morphology of the corresponding parental Calu3 cells ( Figure 2B). These findings suggest the occurrence of epithelial-to-mesenchymal transition (EMT) in the Calu3-ARH, which was considered as being the mechanism underlying the resistance to afatinib.
Next, we investigated the expression levels of ALDH1A1, ABCB1, CD44, Oct-4 and CD133, which are putative CSC markers in NSCLC ( Figure 2C and Figure S2A). ALDH1A1 was found to be overexpressed in the H1781-ARS and H1781-ARH cells as compared to the parental H1781 cells. Western blot analysis also revealed overexpression of ALDH1A1 in the H1781-ARS and H1781-ARH cells ( Figure 2A). These cell lines with acquired resistance of afatinib were non-adhesive and capable of forming sphere-like clusters ( Figure 2B).
We conducted a sphere formation assay to examine the cellular functional features of CSC-like properties, 28 and found that H1781-ARS and H1781-ARH cells acquired higher ability to form spheres in suspension culture compared with parental H1781 cells ( Figure S2B).
To examine the CSC-like properties in vivo, we conducted tumor transplantation experiments. 28,30,31 We subcutaneously implanted H1781, H1781-ARS and H1781-ARH cells to examine the tumorforming capability in NOD/SCID mice. We found that H1781-ARS and H1781-ARH cells exhibited higher tumorigenicity than parental H1781 cells. The H1781-ARS and H1781-ARH cells established larger tumors with shorter latencies than the parental H1781 cells ( Figure S2C). These findings suggest that the H1781-ARS and H1781-ARH cells had acquired CSC-like properties, which could be considered as the mechanism underlying the resistance to afatinib.
ABCB1 was overexpressed in the H2170-ARS and H2170-ARH cells, but not in either the H1781-ARS or H1781-ARH cells ( Figure 2C).
CD44, Oct-4 and CD133 overexpression was not detected in any of the resistant cell lines.

| Drug sensitivities of the afatinib-resistant cells showing MET amplification
The sensitivities of the cells to various drugs are shown in Table 2.
The cell growth inhibition assay showed that the Calu3-ARS cells showing MET amplification were insensitive to monotherapy with crizotinib, which is a MET inhibitor, but sensitive to combined treatment with afatinib plus crizotinib ( Figure 3A)

| Sensitivity of the afatinib-resistant cells to chemotherapeutic agents
The H2170-ARS and H2170-ARH cells, which had lost HER2 amplification, also acquired resistance to docetaxel (Table 2). To investigate whether their resistance mechanism was associated with overexpression of ATP-binding Cassette sub-family B Member 1 (ABCB1), which is well known to induce resistance to chemotherapy, we inhibitor elacridar ( Figure S3A,B). We found that elacridar recovered the sensitivity to docetaxel, but not to afatinib in H2170-ARS and H2170-ARH cells. These findings were similar to those in our previous report using docetaxel-resistant NSCLC cells with EGFR-mutations. 32 In contrast, the other afatinib-resistant cell lines, including Calu3-ARH, H1781-ARS and H1781-ARH, which showed EMT or had acquired CSC-like features, showed maintained sensitivity to docetaxel, because ABCB1 was not upregulated in these acquired resistance cell lines.

| DISCUSSION
In this study, we established multiple cell lines that exhibited acquired resistance to the irreversible pan-HER inhibitor afatinib, previous study using EGFR-mutant NSCLC cells, we reported that the method of exposure of cell cultures to drugs influences the mechanisms of acquired resistance to HER inhibitors (gefitinib and afatinib). 28,33 Consistent with that report, in this study also, conducted using HER2-altered NSCLC cells, the resistance mechanisms were affected by the manner of drug exposure. The resistance mechanism in the Calu3-ARS cells established by the stepwise escalation method appeared to be MET amplification, whereas that in the Calu3-ARH cells established by the high drug concentration method was considered to be possibly attributable to the acquisition of EMT features.
MET amplification is one of the major mechanisms underlying the acquisition of resistance to HER inhibitors in EGFR-mutant NSCLC. 28,29,33,34 MET amplification has also been reported to contribute to trastuzumab resistance in HER2-positive breast cancer cells. 35 In this study, we identified MET amplification as one of the In afatinib-resistant cell lines established from H2170 cells showing HER2 amplification, the copy number of HER2 decreased gradually during the course of exposure to afatinib. We previously reported loss of the EGFR-mutant allele and progressive decrease of the EGFR copy number in an EGFR-mutant NSCLC cell line during gefitinib exposure. 28 However, to the best of our knowledge, loss of HER2 amplification during afatinib treatment has never been reported before in in vitro examinations. However, loss of HER2 amplification has sometimes been reported after trastuzumab treatment in clinical HER2-positive breast or gastric cancer patients. [36][37][38][39] Our finding seems to be consistent with these latter clinical reports.
Loss of HER2 amplification in the residual disease following neoadjuvant trastuzumab-based therapy has been reported to be associated with a poorer relapse-free survival when compared with maintained HER2 expression. 40 The biological basis of these genetic alterations has not been clarified yet, and might include tumor heterogeneity, genetic drift toward a more aggressive tumor phenotype, or selective pressure of adjuvant therapy. 38