Dual HER2 Targeting Impedes Growth of HER2 Gene Amplified Uterine Serous Carcinoma Xenografts

Abstract

Purpose

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that commonly harbors HER2 gene amplification. We investigated the effectiveness of HER2 inhibition using lapatinib and trastuzumab in vitro and in xenografts derived from USC cell lines and USC patient derived xenografts (PDXs).

Experimental Design

Immunohistochemistry and fluorescence in situ hybridization were performed to assess HER2 expression in 42 primary USC specimens. ARK1, ARK2 and SPEC2 cell lines were treated with trastuzumab or lapatinib. Cohorts of mice harboring xenografts derived from ARK2 and SPEC2 cell lines and EnCa1 and EnCa2 primary human USC samples were treated with either vehicle, trastuzumab, lapatinib or the combination of trastuzumab and lapatinib. Acute and chronic post treatment tumor samples were assessed for downstream signaling alterations and examined for apoptosis and proliferation.

Results

HER2 gene amplification (24%) correlated significantly with HER2 protein over-expression (55%). All models were impervious to single agent trastuzumab treatment. Lapatinib decreased in vitro proliferation of all cell lines and in vivo growth of HER2 amplified xenografts (ARK2, EnCa1). In addition, dual therapy with trastuzumab and lapatinib resulted in significant anti-tumor activity only in ARK2 and EnCa1 tumors. Dual HER2 therapy induced on target alteration of downstream MAPK and PI3K pathway mediators only in HER2 amplified models, and was associated with increased apoptosis and decreased proliferation.

Conclusions

While trastuzumab alone did not impact USC growth, dual anti-HER2 therapy with lapatinib led to improved inhibition of tumor growth in HER2 amplified USC and may be a promising avenue for future investigation.

Introduction

Endometrial cancer (EnCa) is the most common gynecologic malignancy affecting nearly 50,000 women in the United States annually (1). Approximately 80-85% of women who undergo comprehensive surgical staging with or without post-operative radiation therapy will be cured of their disease with a low recurrence risk (2, 3). In contrast, the distinct subset of women who present with high-grade carcinomas of various histologic types including high-grade endometrioid, uterine serous carcinoma (USC) and carcinosarcoma are at increased risk for recurrence of aggressive disease and chemotherapy resistance (4, 5). These cases account for the majority of the 8,000 annual deaths from EnCa (5-8). The limitations of conventional cytotoxic and radiation therapies to treat women with these aggressive tumors highlight the dire need for scientific investigation to understand molecular signatures that may confer sensitivity to targeted therapy.

Human epidermal growth factor receptor 2 (HER2), also called HER2/neu or c-erbB2, is a well-characterized member of the human epidermal growth factor receptor superfamily that consists of three other tyrosine kinase receptors (HER1/EGFR, HER3 and HER4)(9). The HER2 gene encodes a 185-kDa transmembrane tyrosine kinase receptor and is located on chromosome 17q21. When activated, HER2 can dimerize and induce signal transduction through the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways (10). This downstream activation leads to induction of genes that can promote oncogenic transformation via cell survival, proliferation, angiogenesis and metastasis. Unlike the other epidermal growth factor receptors, HER2 has no known ligand, highlighting the fact that it may be constitutively activated and could act independently to drive an invasive phenotype (9). Amplification of the HER2 (ERBB2) gene and over-expression of the HER2 protein have been described in many human malignancies including breast, colon, gastric, esophageal, ovarian and endometrial. For some of these cancers, anti-HER2 therapies have become a mainstay of treatment (11, 12).

HER2 protein over-expression or gene amplification has been utilized most successfully in breast cancer as a potent biomarker to select those women most likely to respond to anti-HER2 therapies, such as trastuzumab, a monoclonal antibody, or lapatinib, a small molecule tyrosine kinase inhibitor. In breast cancer, nearly 30% of tumors have been found to harbor HER2 expression via gene amplification or protein over-expression, and are thus designated as HER2 “positive”. While HER2 over-expression was initially associated with the most guarded prognosis in breast cancer, the advent of targeted anti-HER2 therapy has resulted in women with HER2 positive tumors having one of the most favorable prognoses (12, 13). Currently, trastuzumab, pertuzumab (both humanized monoclonal antibodies to the HER2 extracellular domain), trastuzumab emantisine (antibody conjugate to cytotoxic mertansine) as well as lapatinib (a dual HER1/HER2 small molecular tyrosine kinase inhibitor) are FDA approved agents for women with HER2 positive local and metastatic breast cancer to be used in concert with conventional cytotoxic chemotherapy (14-17).

Like breast cancer, USC has been shown to harbor a 10-30% rate of HER2 gene amplification, with up to 70% of tumors exhibiting HER2 protein over-expression (18-20). HER2 over-expressing USC has been associated with decreased overall survival (19). Preclinical in vitro data has suggested that cells derived from HER2 gene amplified USC tumors are more responsive to anti-HER2 therapies compared to cells derived from non-amplified tumors (21). Despite promising preclinical data, the two published phase II trials of anti-HER2 therapy in recurrent EnCa manifested poor responses. One trial evaluated the efficacy of lapatinib in patients with persistent or recurrent EnCa regardless of histology and HER2 status, and found a 3% partial response rate (22, 23). Another recent phase II trial pre-selected patients with HER2 positive recurrent endometrial tumors and administered the HER2 monoclonal antibody trastuzumab (24). Unlike an extensive body of breast and gastric cancer literature suggesting HER2 over-expression to be a biomarker for response to anti-HER2 therapy (25, 26), trastuzumab treatment revealed no responses in this trial with HER2 positive EnCa patients (24). While there is disagreement regarding why lapatinib and trastuzumab as single agents failed to demonstrate any significant durable clinical benefit in EnCa, these trials suggest that single agent anti-HER2 therapies have limited effect, possibly due to innate or drug-induced resistance pathways (27).

In breast cancer, investigators are propounding the concept of dual anti-HER2 therapy, where biologic therapeutics targeting different aspects of the HER2 protein may someday obviate the need for conventional chemotherapy for women with HER2 positive breast cancer (28). Dual anti-HER2 therapy remains untested in HER2 positive EnCa and given the emerging breast cancer experiences, we explored HER2 expression in USC, and used in vitro and in vivo models to test single and dual anti-HER2 therapy in USC cell lines and patient derived xenografts (PDXs) with and without HER2 gene amplification.

Methods

Tissue Samples

Using an institutional review board (IRB) approved protocol, a retrospective cohort of 42 patients with USC, who were surgically treated at our institution between 2000 and 2012, was established. Formalin fixed, paraffin embedded primary USC specimens of all patients were obtained from the pathology department.

Immunohistochemistry

Paraffin embedded USC tissue sections of 5 μm thickness were subjected to immunohistochemistry (IHC) for HER2 using the HercepTest™ (Dako, Carpinteria, CA), following the manufacturer's recommendations. The intensity and pattern of the HER2 membrane immunostaining were evaluated, and all samples were scored by a pathologist on a 0 - 3+ scale with 0 representing no staining, 1+ representing weak staining in >10% of invasive tumor cells, 2+ representing moderate intensity in >10% of invasive carcinoma cells or intense staining in < 10%, and 3+ staining defined as intense circumferential membranous staining in > 10% of the invasive carcinoma.

USC xenograft sections (5 μm) were examined for Ki-67 expression levels by IHC. Antigen retrieval was performed using a 10 mM citrate buffer, and slides were treated with 3% hydrogen peroxide. The blocking reagent and antibody diluent of a M.O.M. kit (Vector Laboratories, Burlingame, CA) were used following the manufacturer's instructions. Sections were incubated with a primary antibody against Ki-67 (clone MIB-1, Dako), followed by incubation with an anti-mouse secondary antibody (M.O.M. kit, Vector Laboratories). Slides were then treated with Vectastain ABC reagents (Vector Laboratories) and further visualized using 3,3'-diaminobenzidine chromogen (DAB, Dako). The percentage of Ki-67 stained nuclei was determined by counting four different fields of each tumor sample. The number of cells counted was 400 cells ± 100 per field. Sections with no primary antibody were used as negative controls.

Fluorescence in situ hybridization

To determine the HER2 gene copy number of primary USC samples and USC cell line derived xenografts, fluorescence in situ hybridization (FISH) was performed on 5 μm thick tissue sections. A PathVysion HER2 DNA Probe Kit (Abbott Laboratories, Abbott Park, IL) was used, consisting of a LSI HER2 probe with SpectrumOrange label and a CEP 17 control probe with a SpectrumGreen tag directed against the centromere region of chromosome 17. Counterstaining was carried out using Vectashield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). All samples were visualized and scored using the CytoVision® platform (Leica Biosystems, Buffalo Grove, IL). For each specimen, the HER2 to CEP 17 ratio was determined by counting the red (HER2) and green (CEP 17) signals in a minimum of 50 nuclei. Samples with a HER2 to CEP 17 ratio greater than 2.0 were considered amplified.

Cell culture

Three established human, non-immortalized USC cell lines (ARK1, ARK2 and SPEC2) were generously provided in 2011 by Dr. I. Fidler (MD Anderson, Houston, TX) and Dr. A. Santin (Yale University, New Haven, CT) and have been characterized in previous reports.(30, 31). Each cell line was derived from a patient with USC. We authenticated these cell lines by confirming the HER2 protein and gene status, as well as serous histology via pathologic review. ARK1 and ARK2 cell lines were cultured in RPMI 1640 medium (Corning) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin (Life Technologies). SPEC2 cells were cultured in MEM medium containing Earle's salts and L-glutamine, supplemented with 10% FBS, 1 mM sodium pyruvate, 2% MEM Vitamin Solution and 1% MEM Non-Essential Amino Acids (Life Technologies, Grand Island, NY). BT-474 breast cancer cells were cultured in DMEM/F12 medium with L-glutamine, supplemented with 10% FBS. All cell lines were maintained in an atmosphere containing in 5% CO₂ at 37 °C.

Drugs

Lapatinib was purchased from LC Laboratories (Woburn, MA). Trastuzumab was obtained from our institution's clinical pharmacy.

USC model genotyping

An adapted version of the Applied Biosystems (ABI) Prism SNaPshot multiplex system was used to genotype 3 mm core samples from the EnCa1 and EnCa2 primary tumors, as well as extracted genomic DNA from the three non-immortalized human cell lines ARK1, ARK2 and SPEC2 as previously described.(29) This clinical mutational profiling platform screens for 130 well-characterized mutations that are distributed across 15 cancer genes including AKT1, APC, BRAF, CTNNB1, EGFR, ERBB2, IDH1, KIT, KRAS, MAP2K1, NOTCH1, NRAS, PIK3CA, PTEN, and TP53.(29)

In vitro treatment of USC cell lines

ARK1, ARK2 and SPEC2 were seeded on 6-well plates and serum starved using growth medium containing 1% FBS. The following day, cells were treated in duplicate with increasing concentrations of lapatinib or trastuzumab in growth medium with 1% FBS. Treated cells were incubated for 2 days (lapatinib) or 5 days (trastuzumab), then collected and counted. With trastuzumab dose response experiments, the HER2 over-expressing breast cancer cell line BT-474 was co-treated to serve as positive control for drug response. After having determined the IC₅₀ dose of lapatinib for each cell line, cells treated with this dose for 48 hours were used for western blotting analyses of members of the PI3K and MAPK signaling pathways.

Generation and propagation of USC xenografts

All mouse studies were carried out in compliance with the Institutional Animal Care and Use Committee guidelines. Xenografts derived from ARK2 or SPEC2 cells were established by subcutaneous (s.c.) injection of cultured cells into 6 - 8 week old female NOD/SCID mice (Jackson Laboratory, Bar Harbor, ME), in a 1:1 suspension of PBS and Matrigel (BD Biosciences, San Jose, CA). Patient derived USC xenografts (PDXs) were established as previously described . Briefly, primary human USC specimens were obtained under an IRB approved tissue collection protocol. They were enzymatically processed, followed by depletion of endothelial and hematopoietic cells. The remaining purified USC cells were suspended in PBS with Matrigel (1:1) and injected s.c. into 6 - 8 week old female NOD/SCID mice. Xenograft formation was monitored regularly, and mice were euthanized by CO₂ inhalation when tumors had reached a diameter of 15 - 20 mm. Tumors were then excised and enzymatically processed, followed by depletion of H-2K^d positive mouse cells. The resulting single tumor cells were re-suspended in PBS/Matrigel (1:1) and re-injected s.c. into female NOD/SCID mice. Serial transplantation of USC xenografts resulted in the generation of cohorts of mice that each harbored a tumor derived from the same primary USC. A total of 217 mice were euthanized in order to conduct the described in vivo experiments.

Treatment of mice harboring USC xenografts

Mice bearing xenografts derived from ARK2 or SPEC2 cells were randomized into two groups of 5 (ARK2) or 6 (SPEC2) mice with equivalent average tumor volumes. The formula [length in mm x width in mm x height in mm] / 2 derived from caliper measurements was used to calculate tumor volumes as has previously been described (32). One arm was treated with lapatinib (150 mg/kg) and the control arm received its vehicle (0.5% hydroxypropyl-methylcellulose, 0.1% Tween 80), administered by oral gavage once daily for 6 days per week. Because the efficacy of trastuzumab in an immunocompromised NOD/SCID animal model could be impaired, we administered single agent trastuzumab (10 mg/kg) or vehicle to a cohort of 7 NOD/SCID mice bearing BT-474 cell line derived xenografts with matched tumor growth. Subsequently, cohorts of mice harboring ARK2 or SPEC2 cell line derived xenografts as well as EnCa1 or EnCa2 PDXs were randomly divided into four groups of 6 (ARK2, EnCa2) or 7 (SPEC2, EnCa1) mice each. The different treatment regimens were as follows: 1) vehicle control: 0.5% hydroxypropyl-methylcellulose, 0.1% Tween 80 by oral gavage (lapatinib vehicle) and sterile water by intra-peritoneal (i.p.) injection (trastuzumab vehicle); 2) i.p. injection of trastuzumab (10 mg/kg) and oral gavage of the lapatinib vehicle; 3) lapatinib (150 mg/kg) by oral gavage and administration of the trastuzumab vehicle by i.p. injection; and 4) lapatinib by oral gavage and trastuzumab by i.p. injection. Trastuzumab and its vehicle were administered twice weekly, while lapatinib and its vehicle were administered once daily for 6 days per week as has been previously described. Tumors were measured every 3 - 4 days with calipers and mice were weighed weekly. Treatment periods spanned 14 - 21 days. At the end of each treatment study, mice were euthanized and xenografts were harvested. Portions of each xenograft were snap frozen as well as formaldehyde fixed and paraffin embedded for further analyses.

To study the effects of acute treatment on downstream targets of HER2, mice bearing ARK2, SPEC2, EnCa1 or EnCa2 xenografts received a single dose of either vehicle, trastuzumab, lapatinib or trastuzumab and lapatinib. Mice were euthanized and xenografts were harvested 6 hours after treatment. Tumor portions were snap frozen and other pieces formaldehyde fixed and paraffin embedded.

Western Blotting

Frozen xenograft samples or pelleted cells were lysed using a buffer of Mammalian Protein Extraction Reagent (Thermo Scientific, Tewksbury, MA) supplemented with kinase, protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Protein lysates were resolved on 10% Bis-Tris gels (NuPAGE Novex, Life Technologies) and transferred to PVDF membranes (Millipore, Billerica, MA). After blocking, membranes were incubated overnight with the primary antibody at 4°C. Primary antibodies directed against phospho-Akt (p-Akt, Thr308), Akt, phospho-Erk1/2 (p-Erk1/2, Thr202/Tyr204), Erk1/2, phospho-PRAS40 (p-PRAS40, Thr246), PRAS40, phospho-p70S6K (p-p70S6K, Thr389) and p70S6K (all Cell Signaling, Danvers, MA) were used. The dilution for all primary antibodies was 1:1000. The blots were then probed with a horseradish peroxidase conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX) and developed using a chemiluminescent detection reagent (ECL Prime, GE Healthcare Life Sciences, Pittsburgh, PA). Each blot was stripped with 1 M glycine when the same membrane was used to re-probe with another primary antibody. Finally, all membranes were stripped and re-probed with either a mouse anti-Pan-actin antibody (1:10000, NeoMarkers, Fremont, CA) or a rabbit anti-GAPDH antibody (1:1000, Cell Signaling) to verify equal protein loading.

TUNEL assay

Apoptosis was assessed in paraffin embedded xenograft sections of 5 μm thickness, collected at the end of each four-arm treatment experiment. The DeadEnd™ Fluorometric TUNEL System (Promega, Madison , WI) was used, according to the manufacturer's instructions with minor modifications. A negative control without addition of rTdT enzyme was included. Detection of fragmented DNA was conducted on a fluorescence microscope (Nikon Eclipse, Tokyo, Japan). For each sample, the number of DAPI-stained nuclei and the number of TUNEL-positive nuclei were quantified in four randomly selected fields using Fiji software (ImageJ, Bethesda, MD). The TUNEL index was calculated as the number of TUNEL-positive cells ×100/Total number of nuclei.

Statistical Analysis

Fisher exact testing was utilized for comparison of proportions. Survival analysis was done using the Kaplan-Meier method along with a Cox proportional hazards model incorporating age and stage of disease as continuous variables. Two-way ANOVA analysis was used to determine the statistical significance of the effects of lapatinib treatment on the different cell lines in vitro. Statistical significance of the observed differences in xenograft growth and mouse weights between the different treatment arms was assessed with non-parametric Wilcoxon rank sum tests. One-way ANOVA analyses were performed to determine significance of the observed differences in Ki-67 and TUNEL positive cell counts. Stata software version 11.1, (StataCorp, LP, College Station, TX) and GraphPad Prism software version 6 (GraphPad Software, Inc., La Jolla, CA) was used, and a p-value of < 0.05 was considered statistically significant.

Results

Prevalence of HER2 protein over-expression and HER2 gene amplification in USC

HER2 protein expression as well as HER2 gene copy number were analyzed in a cohort of 42 primary USC tissue blocks. Supplemental Table 1 summarizes the clinical characteristics of the corresponding patients. The mean age of the cohort was 68.7 years, and all stages of disease were represented with an average overall survival rate of 2.4 years from diagnosis. IHC for HER2 revealed 2+ or 3+ protein expression, in 55% of the cohort (Figure 1A and 1B). A lower rate of HER2 gene amplification (HER2 to CEP 17 ratio > 2.0) was observed by FISH, with 10 of 42 USC samples (24%) found to be amplified (Figure 1A and 1B). HER2 gene amplification significantly associated with HER2 protein over-expression in our cohort (p < 0.02, Figure 1B). When comparing clinical outcome of HER2 amplified USCs with non-amplified tumors, a worse overall survival rate was observed in patients harboring HER2 amplification when controlling for age and stage (p = 0.015, Figure 1C).

Figure 1.

HER2 gene amplification was observed in 24% of USC specimens, which was significantly associated with HER2 protein over-expression and worse overall survival. A) Representative images of FISH (upper), 100X and IHC, 10X (lower) for HER2: upper left HER2 non-amplified; upper right HER2 amplification; lower left HER2 immunostaining score 1+; lower right HER2 immunostaining score 3+. IHC Scale bars: 100 μm. B) Overview of FISH and IHC findings. A significant association between HER2 gene amplification and 2+ or 3+ HER2 protein expression was found (p < 0.02). Low protein expression was defined as a score of 0 or 1+, and high HER2 protein expression as a score of 2+ or 3+. C) Kaplan-Meier curve showing the overall survival differences between patients with tumors harboring HER2 gene amplification and patients with tumors with normal HER2 gene status. In a Cox proportional hazards model incorporating age and stage, HER2 amplification independently correlated with overall survival, with a median of 1.2 years for patients with HER2 amplification as opposed to 2.9 years for those with normal HER2 gene status (p = 0.02).

Cell line genotyping

Multiplex tumor analysis of both EnCa1 and EnCa2 revealed no gain of function mutations, although the testing confirmed the heightened HER2 gene dosage that was observed on FISH analysis. No gain of function mutation was detected in the ARK2 cell line, though a PIK3CA mutation (1624G to A, E542K) was confirmed in ARK1 as has been described (33), and SPEC2 was found to harbor an NRAS mutation (34G to T, G12C) of uncertain clinical significance.

HER2 inhibition using lapatinib and trastuzumab in USC cell lines

HER2 protein over-expression (3+ staining) and gene amplification were demonstrated in ARK1 and ARK2 derived xenografts by IHC and FISH, while SPEC2 derived tumors were shown to harbor low protein expression and normal HER2 gene status (Supplementary Figure 1). In all three USC cell lines, treatment with trastuzumab showed no effect on cell proliferation (data not shown). However, a dose dependent reduction in cell number was found in ARK1 and ARK2 cells after lapatinib treatment as compared to vehicle controls. ARK2 cells showed the strongest response to lapatinib treatment in vitro, with observed IC₅₀ values of 0.05 μM for ARK2 and 0.5 μM for ARK1 (p < 0.0001, Figure 2A). ARK1 was sensitive at higher doses compared to ARK2 likely as a result of the PIK3CA gene mutation that uncouples the HER2 inhibition.

Figure 2.

Lapatinib treatment of USC cell lines decreased cell proliferation as well as phosphorylated downstream signaling proteins. A) ARK1, ARK2 and SPEC2 cells were incubated with increasing concentrations of lapatinib in duplicate for 48 hours, followed by quantification of viable cells. A dose dependent reduction in cell count was observed in HER2 amplified ARK1 and ARK2 cells, with the highest sensitivity to lapatinib observed in ARK2 cells (p < 0.0001). The non-HER2 amplified SPEC2 cells were found to be relatively resistant to lapatinib treatment, in which a dose as high as 2 μM was needed to decrease cell numbers with approximately 50%. Average relative changes in cell numbers as compared to untreated controls are shown. Error bars represent the standard error of the mean. B) Cells of each line were treated with 0.5 μM (ARK1), 0.05 μM (ARK2) or 2 μM (SPEC2) lapatinib or medium only and collected 48 hours after treatment. Cells were used for western blotting analyses of total and phosphorylated Akt and Erk. Lapatinib treatment led to reduced levels of p-Akt and p-Erk in ARK2 cells, while a decrease in p-Akt but not p-Erk was found in lapatinib treated ARK1 cells and a decrease in p-Erk but not p-Akt was seen in lapatinib treated SPEC2 cells. Expression of Pan-actin or GAPDH was used as loading control.

We next studied the effect of lapatinib treatment on signaling molecules downstream of HER2, by assessing protein expression and phosphorylation of members of the PI3K and MAPK pathways. Western blotting analyses showed decreased levels of phosphorylated Akt (Thr308) in ARK1 and ARK2 cells and reduced expression of phosphorylated Erk in ARK2 and SPEC2 cells after treatment with lapatinib, compared with untreated controls (Figure 2B). The levels of total Akt and Erk proteins were not affected by treatment with lapatinib.

HER2 inhibition with lapatinib and trastuzumab in USC xenografts

HER2 protein over-expression and HER2 gene amplification were observed in EnCa1 xenografts, while low levels of the HER2 protein and gene were found in EnCa2 xenografts (Supplementary Figure 1). The single agent lapatinib experiments employed two cohorts of mice bearing xenografts derived from either ARK2 or SPEC2. A reduction of tumor growth was observed in ARK2 xenografts (p = 0.02), but not in SPEC2 xenografts, following treatment with lapatinib as compared to vehicle treated tumors (Supplementary Figure 2).

To further explore the effects of anti-HER2 therapy, four mouse cohorts harboring tumors derived from ARK2 and SPEC2 as well as EnCa1 and EnCa2 were used to study the anti-tumor efficacy of trastuzumab alone, lapatinib alone and the combination of trastuzumab and lapatinib (Figure 3). Single agent trastuzumab treatment induced significant regression of BT-474 xenografts (p < 0.03), confirming effective targeting of HER2 using trastuzumab in immunocompromised mice (data not shown). In all four USC xenograft cohorts, no significant inhibition of tumor growth was observed following treatment with trastuzumab alone as compared to vehicle treated controls. However, significantly superior anti-tumor activity was observed with the combination of trastuzumab and lapatinib in ARK2 tumors, compared with lapatinib alone and trastuzumab alone (p < 0.01). Similarly, the strongest reduction of EnCa1 tumor growth was found in the dual therapy arm and while statistically different from vehicle, there was no statistical improvement over single agent lapatinib treatment which also significantly inhibited EnCa1 tumor growth (p < 0.03). In both tumors lacking HER2 gene amplification (EnCa2 and SPEC2), no therapy induced anti-tumor activity. Importantly, mouse weights revealed no statistically significant alterations during the treatment period of all xenograft cohorts (Supplementary Figure 3).

Figure 3.

Anti-HER2 therapies of USC xenografts stratified by HER2 gene amplification. Significant anti-tumor activity of lapatinib and a synergistic effect of lapatinib with trastuzumab were observed in HER2 amplified USC xenografts (ARK2, EnCa1), while no effect was seen in non-amplified xenografts (SPEC2, EnCa2). Mice bearing xenografts derived from ARK2 or SPEC2 cell lines or EnCa1 or EnCa2 primary USCs were treated with either vehicle, lapatinib alone, trastuzumab alone or the combination of lapatinib and trastuzumab. Tumors were measured twice weekly, and the percent change in tumor volume compared to baseline volume (100%) is shown. Error bars represent the standard error of the mean. While no significant anti-tumor effect of trastuzumab, dual therapy with lapatinib and trastuzumab impeded the growth of HER2 amplified ARK2 xenografts when compared to all other arms (p < 0.01). Dual therapy with lapatinib and trastuzumab delayed tumor growth in the EnCa1 xenografts compared to vehicle controls (p < 0.02), though the latter was not statistically different from single agent trastuzumab or lapatinib. In both non-HER2 amplified models (SPEC2 and EnCa2), lapatinib and trastuzumab did not affect xenograft growth. * p < 0.02, ** p < 0.01.

Effects of HER2 inhibition in vivo on downstream signaling proteins

Activity of the PI3K and MAPK pathways was assessed by studying the phosphorylated and total protein levels of Akt, PRAS40, p70S6K and Erk1/2 in xenografts harvested 6 hours after administration of a single dose of trastuzumab, lapatinib, both agents or vehicles only (Figure 4). Reduced levels of p-Akt, p-PRAS40 and p-p70S6K as well as p-Erk1/2 were observed in ARK2 and EnCa1 xenografts following dual therapy with trastuzumab and lapatinib, as compared to vehicle controls. While lapatinib alone led to decreased protein expression of p-Akt and p-Erk, trastuzumab alone did not affect expression of the analyzed proteins. Unlike ARK2 and EnCa1 tumors, HER2 therapies failed to alter phosphorylated protein levels of the analyzed PI3K and MAPK pathway members in the non-HER2 gene amplified SPEC2 and EnCa2 xenografts.

Figure 4.

Dual treatment with lapatinib and trastuzumab led to decreased levels of downstream phosphorylated signaling proteins only in HER2 amplified USC xenografts. Mice harboring ARK2, SPEC2, EnCa1 or EnCa2 tumors were treated with a single dose of vehicle (V), trastuzumab (T), lapatinib (L) or both drugs (T+L) and euthanized 6 hours later. Xenografts were collected and utilized to assess protein levels of total and phosphorylated Akt, Erk1/2, PRAS40 and p70S6K. Western blotting analyses revealed reduced levels of p-Akt, p-Erk, p-PRAS40 and p-p70S6K in ARK2 and EnCa1 tumors after combination treatment with trastuzumab and lapatinib, while no effect of treatment was seen in non-HER2 amplified SPEC2 and EnCa2 xenografts. Levels of the analyzed total proteins were not affected by any treatment regimen. GAPDH was used as loading control.

Effects of HER2 inhibition on cell proliferation and apoptosis

To analyze the effect of treatment with trastuzumab, lapatinib and dual therapy on proliferation and apoptosis, xenograft samples harvested at the end of each treatment experiment were subjected to Ki-67 IHC as well as TUNEL analysis. Significantly decreased expression of Ki-67 was observed in ARK2 and EnCa1 samples treated with lapatinib alone or the combination of trastuzumab and lapatinib, compared with vehicle treated tumors (Figure 5). Treatment with trastuzumab alone led to a smaller though significant decrease in ARK2 Ki-67 levels, while no significant difference was seen in EnCa1 tumors. Increased TUNEL staining was found in ARK2 and EnCa1 xenografts treated with lapatinib with and without trastuzumab, compared with vehicle controls (p < 0.05, Figure 6). These differences in Ki-67 and TUNEL staining were not seen in SPEC2 and EnCa2 treated samples (Supplementary Figures 4 and 5).

Figure 5.

Lapatinib with and without trastuzumab significantly decreased proliferation of HER2 amplified ARK2 and EnCa1 xenografts. Tumors harvested at the completion of each in vivo treatment study were used to assess cell proliferation by immunohistochemistry for Ki-67. The percentage of Ki-67 positive nuclei was significantly lower in ARK2 tumors (A) treated with trastuzumab, lapatinib or both agents, compared with vehicle treatment (C). Lapatinib, alone and in combination with trastuzumab, led to a significantly stronger reduction in Ki-67 stained cells as compared to treatment with trastuzumab alone. In EnCa1 xenografts (B), a significant decrease in the percentage of Ki-67 stained nuclei was found following treatment with lapatinib or the combination of lapatinib and trastuzumab (D). Magnification 20x. Scale bars: 100 μm. Error bars represent the standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 6.

Increased apoptosis was observed in ARK2 and EnCa1 tumors following treatment with lapatinib or the combination of lapatinib and trastuzumab. Xenografts collected at the end of the in vivo treatment experiments were examined for apoptosis using TUNEL analysis. Representative images of TUNEL stained (green) sections with DAPI counterstain (blue) of the differently treated ARK2 (A) and EnCa1 (B) tumors are shown. The percentage TUNEL positive cells was significantly higher in ARK2 (C) and EnCa1 (D) xenografts treated with lapatinib with or without trastuzumab, compared with vehicle controls. Trastuzumab alone had no effect on tumor cell apoptosis. Scale bars: 50 μm. Error bars represent the standard error of the mean. * p < 0.05, ** p < 0.01.

Discussion

HER2 gene amplification has been shown to be a prevalent and prognostic signature that in other disease sites has been effectively targeted for significant clinical benefit (34). Echoing clinical trial experience, the data presented here support that USCs present with innate trastuzumab resistance that may be mitigated by administering concurrent lapatinib, an anti-HER2 therapy that targets the intracellular domain of the receptor. While in vitro models demonstrated a reduction in cell viability regardless of HER2 gene amplification status when treated with lapatinib, only HER2 gene amplified xenografts (ARK2, EnCa1) responded to lapatinib in vivo, with the most robust responses observed with the use of combination lapatinib and trastuzumab. This is the first report to our knowledge that has utilized cell line derived and patient derived USC xenografts to suggest that HER2 gene amplification can be a biomarker associated with response to anti-HER2 therapies in USC. Despite unsuccessful efforts in the clinic to utilize HER2 inhibition in high-grade EnCa, these data support a role for the use of dual anti-HER2 therapy in gene amplified USC as has been propounded for the treatment of trastuzumab-resistant breast carcinoma (35).

This preclinical investigation confirms and extends the findings of numerous investigations that have sought to characterize HER2 expression in USC. Early reports of HER2 protein over-expression and gene amplification demonstrated this to be a prevalent signature associated with aggressive disease and a worse prognosis (19, 36, 37). The rates reported were in the 17-30% range for HER2 gene amplification, not dissimilar from that observed in breast and gastric cancers where trastuzumab was found to offer significant benefit (38). Santin and colleagues later refined this experience, showing that HER2 expression was over-represented in African American women compared to Caucasian women (39). This finding might lend insight into the increased mortality observed in this population despite similar medical access and care. Most investigators suggested that HER2 gene amplification could be a potent biomarker for the selection of women most likely to respond to trastuzumab prompting the Gynecologic Oncology Group (GOG) to initiate a trial in recurrent EnCa. Despite accruing over 30 women, all with HER2 over-expressing recurrent endometrial tumors, no responses to trastuzumab were observed(24). While many authors suggested the trial was flawed as it lacked a high proportion of EnCas known to harbor the highest HER2 expression (African American subjects and serous histology), the complete lack of signal may indicate that HER2 over-expressing EnCa presents with innate trastuzumab resistance.

In this investigation, we tested trastuzumab and found that in both the USC cell line and patient derived xenografts studied, trastuzumab failed to induce any alterations in vitro or in vivo. We believe this effect was not due to failure of antibody therapies in our models, as trastuzumab induced significant anti-tumor activity in the HER2 over-expressing BT-474 cells. Interestingly, when lapatinib was utilized, all cell lines regardless of gene amplification status demonstrated a decrease in cell viability with higher doses needed for this response in the non-amplified SPEC2 cells. The most robust response to lapatinib was observed in ARK2, likely because SPEC2 lacks HER2 gene amplification, and ARK1 was found to harbor a PIK3CA gene mutation both of which would act to render the tumor cells less sensitive to anti-HER2 therapies.

Unlike the in vitro experiments, however, the in vivo studies demonstrated that lapatinib as single agent and in combination with trastuzumab induced significant tumorstatic effects only in those tumors harboring HER2 gene amplification (ARK2, EnCa1). In the non-amplified SPEC2 and EnCa2 xenografts, a complete lack of response to any administered therapy was seen. These models strongly support the hypothesis that HER2 gene amplification is a biomarker for response to HER2 inhibition in USC as has been shown in breast and gastric carcinomas (11, 34, 40).

The anti-tumor effects following HER2 blockade in ARK2 and EnCa1 xenografts were associated with increased apoptosis and decreased proliferation as has been shown in lung and head and neck carcinomas (41, 42), as well as reduced levels of signaling proteins of the PI3K and MAPK pathways. The most potent acute abrogation of p-Erk, p-Akt, p-PRAS40 and p-p70S6K was seen following dual treatment with lapatinib and trastuzumab, supporting a rationale for the synergistic inhibition of tumor growth observed. Unlike breast carcinoma, where HER2 inhibition has been primarily associated with blockades in the PI3K signaling pathway, these findings in USC suggest down regulation of both MAPK and PI3K signaling with dual therapy (43). In the two different xenograft examples lacking HER2 gene amplification (SPEC2, EnCa2), no downstream alterations could be discerned. This suggests that without the HER2 signature, both trastuzumab and lapatinib fail to produce meaningful changes in the target pathways.

It is unclear why HER2 gene amplified USC is impervious to trastuzumab though this phenomenon has been noted in many other non-breast adenocarcinomas found to harbor HER2 positive tumors (34). In breast carcinoma, much clinical and preclinical effort has been focused on trastuzumab resistant tumor cells. Pohlman et al. (44) and others (45) have reviewed this topic extensively, generally in the setting of breast cancer where trastuzumab is a mainstay of therapy for HER2 over-expressing tumors. Many factors are likely to contribute to this observed pre-clinical and clinical resistance in endometrial cancer including a high prevalence of PI3K pathway activation via PIK3CA mutation and PTEN loss of function. One potential mechanism of resistance implicates a truncated variant of HER2, p95HER2 that lacks the extracellular domain (ECD) where trastuzumab binds, thus decreasing therapeutic efficacy (46). High expression of this variant has been associated with trastuzumab resistance and lapatinib sensitivity in breast cancer, and prospective trials are underway to validate p95HER2 expression as a biomarker associated with response to specific HER2 therapies (47, 48). Elevated ECD levels have been observed in USC (49), though the expression of p95HER2 in HER2 over-expressing USC has yet to be defined and would require prospective validation as a biomarker associated with resistance to trastuzumab.

Investigators have suggested that trastuzumab resistance may be overcome through the use of dual anti-HER2 therapies that interact with the receptor in different ways, either by prohibiting dimerization or tyrosine kinase function. Scaltriti et al. described how breast cancer cell line therapy with lapatinib led to a potent upregulation of HER2 protein expression which subsequently sensitized previously resistant cells to trastuzumab (50). In the USC xenografts used in this study, concurrent trastuzumab and lapatinib treatment led to the statistically most robust anti-tumor activity when compared to vehicle only in the HER2 gene amplified tumors.

Interestingly, the ARK2 cell line derived xenografts exhibited uniform HER2 gene amplification with a ratio > 15.0 in all nuclei counted, whereas the patient derived EnCa1 xenografts demonstrated more heterogeneity regarding HER2 gene amplification. In the latter, a gene copy ratio of 9.0 was found though not all tumor cells demonstrated increased HER2 gene copy numbers. Intra-tumor heterogeneity of HER2 gene amplification was also observed in the retrospective cohort, and has been noted recently in the literature (20). In the present study, lower anti-tumor activity of dual HER2 inhibition was seen in the EnCa1 xenografts with lower prevalence of HER2 amplified cells when compared with the ARK2 model in which tumor regression was observed. Extrapolating from these pilot data, merging dual anti-HER2 therapy with conventional cytotoxics or an additional biologic could be a rational method to target the non-amplified tumor cells that commonly co-exist in HER2 amplified tumors.

Currently, there are no clinical trials registered in the United States examining dual anti-HER2 therapies in HER2 over-expressing EnCa. Two trials that are utilizing HER2 over-expression as a biomarker are a phase II trial of trastuzumab in concert with paclitaxel and carboplatin for upfront therapy of USC (NCT01367002) and a phase I trial testing lapatinib with ixabepilone for recurrent uterine carcinoma and carcinosarcoma. The preclinical data described in this study underscore the potential for USC to harbor innate trastuzumab resistance and suggest that combination therapy with lapatinib induces significant cell death and in vivo anti-tumor activity. Further investigation of dual HER2 inhibition in HER2 gene amplified USC may be warranted in a clinical trial setting.

Translational Relevance.

Biologic therapies targeting HER2 (ERBB2) have become a mainstay of effective therapy for breast tumors found to harbor HER2 gene amplification. While a subset of uterine serous carcinoma (USC) exhibit HER2 gene amplification, single agent anti-HER2 directed therapy has failed to produce responses in clinical trials, even those that selected for tumors over-expressing HER2. This investigation utilized non-immortalized USC cell lines and USC patient derived xenografts (PDXs) to test the efficacy of lapatinib and trastuzumab, as single agent and combination therapy. We have demonstrated that all HER2 amplified models exhibited trastuzumab resistance that was overcome with the addition of lapatinib. Anti-tumor responses were associated with significantly increased apoptosis and decreased proliferation as well as decreased Akt and Erk signaling. These data support a potential role for dual anti-HER2 therapy in women with HER2 gene amplified USC.