HER2 G776S Mutation Promotes Oncogenic Potential in Colorectal Cancer Cells when Accompanied by Loss of APC Function

doi:10.21203/rs.3.rs-594302/v1

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HER2 G776S Mutation Promotes Oncogenic Potential in Colorectal Cancer Cells when Accompanied by Loss of APC Function

https://doi.org/10.21203/rs.3.rs-594302/v1

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Background

Clinical cancer genome sequencing detects oncogenic variants that are potential targets for cancer treatment, but it also detects variants of unknown significance that may interact and affect the pathophysiology of the tumor; however, these interactions are not fully understood. In this study, we examined the interactions of a minor HER2 mutation (G776S) and APC mutations, which were detected by cancer genome sequencing of samples from a patient with colorectal cancer.

Methods

We transfected HER2-G776S mutant- or HER2 wild type- expressing vectors into several cell lines, HeLa, FHC, CACO-2 and COLO-320, to evaluate their effects on HER2 phosphorylation and kinase activity, HER2 downstream signaling (phosphorylation of AKT and MAPK), and anchorage-independent growth ability. APC- knockout cells and APC overexpressing cells were established to investigate the effect of APC function on the HER2 signaling pathway. We also evaluated the efficacy of a HER2 tyrosine kinase inhibitor on xenograft tumors derived from HER2-G776S transfected cells.

Results

HER2 G776S mutation increased the kinase activity and phosphorylation of HER2 protein, but these effects were weaker than those of the other HER2 driver mutation. HER2 G776S did not activate HER2-downstream signal pathways, such as ERK and AKT phosphorylation, in cells with wild-type APC (HeLa and FHC cells). By contrast, HER2 G776S increased the activation of HER2 downstream signaling, especially ERK phosphorylation, and anchorage-independent cell growth in cells with an APC mutation (CACO-2 and COLO-320) and APC-knockout HeLa cells. Wild-type APC overexpression in HER2 G776S-transfected COLO-320 cells neutralized ERK phosphorylation. Loss of APC function increased Wnt pathway activity but also increased RAS–GTP, which increased ERK phosphorylation triggered by HER2 G776S transfection. Afatinib, a pan-HER tyrosine kinase inhibitor, inhibited tumor growth of HER2 G776S-transfected COLO-320 xenografts

Conclusions

HER2 G776S mutation acts as a weak oncogenic driver, but it also increases HER2–ERK signaling activity by increasing RAS–GTP production when APC function is simultaneously impaired. These results suggest that even weakly active mutations may be therapeutic targets, and the use of this strategy may contribute to the development of HER2-targeted therapy for colorectal cancer.

Cancer Biology

Oncology

afatinib

APC

colorectal cancer

HER2

RAS

VUS (variants of uncertain significance)

In recent years, the development of next-generation sequencing (NGS) has enabled the detailed analysis of the cancer genome in individual cancer patients, and targeting of oncogenic driver alterations has been shown to be effective in several types of cancers [1–4]. One of the possible targets among oncogenic driver alterations is the human epidermal growth factor receptor 2 (HER2) [5, 6], also known as ERBB2, which is a member of the epidermal growth factor receptor (EGFR) family of homologous transmembrane receptor tyrosine kinases [7]. HER2-targeted therapies have improved outcomes for cancer patients with HER2 gene amplification and overexpression [8, 9]. Somatic point mutations in HER2 can also be detected by NGS in a number of cancers [10, 11]. Several major HER2 mutations, such as V777L in breast cancer [12] and HER2 exon 20 insertion mutations in lung cancer [13], have been shown to be driver mutations that promote oncogenicity through constant phosphorylation of HER2 and are expected to be therapeutic targets.

In colorectal cancer (CRC), somatic point mutations in HER2 are detected in 4–6% of cases [6, 14], most of which are diverse single nucleotide variants [6, 10]. HER2 V777L and HER2 V842I are the well-known mutations in CRC [5], and these mutations have been shown to be associated with phosphorylation of extracellular signal-regulated kinase (ERK) and increased anchorage-independent cell growth activity [15]. These previous reports indicate the significance of HER2 mutations in CRC; however, numerous other HER2 mutations remain variants of unknown significance. Moreover, clinical NGS often detects multiple gene variants in a single patient [3, 16], and these variants may interact with each other and affect cancer cell tumorigenesis. However, the interactions between gene variants are not fully understood.

In this study, we focused on a patient with CRC treated in our hospital whose HER2 G776S mutation (c.2326G > A, p.Gly776Ser) and an APC nonsense mutation were detected by clinical NGS. HER2 G776S is located in the tyrosine kinase domain in HER2 (amino acid 721–975). This domain has been found to have more frequent mutations compared with other domains [6, 17]. Several mutations such as A775_G776insYVMA and G776 > VC have been annotated as activating variants in non-small-cell lung carcinoma[18]. However, this G776S mutation is rare, and its significance is not fully understood. Here, we examined the oncogenic function and treatment sensitivity of the HER2 G776S mutation and assessed the interaction between the HER2 G776S mutation and loss of APC function.

Clinical sequencing using an NGS-based multiplex gene assay

The formalin-fixed paraffin-embedded tumor tissue of the patient was submitted to EA Genomics (Morrisville, NC) to obtain the profile using an NGS-based multiplex gene assay (OncoPrime (3); Mitsui Knowledge Industry, Tokyo, Japan) covering 215 cancer-related genes (Supplementary Table 1).

Cell lines and investigation of their genetic features

HeLa cells were purchased from JCRB Cell Bank (Ibaraki, Japan), COLO-320 and Ba/F3 cells were purchased from Riken BioResource Center (Ibaraki, Japan), and FHC cells were purchased from ATCC (Manassas, VA). NIH/3T3 and CACO-2 cells were kindly provided by the Gastroenterology Department, Kyoto University, in April 2016. CACO-2 cells were validated using Short Tandem Repeat analysis by the Cell Line Authentication Service of Promega (Madison, WI) in March 2021. The composition of the medium for each cell is shown in Supplementary Table 2. All cells were cultured at 37°C in a 5% CO2 incubator.

The genetic features of the human cell lines, HeLa (cervical cancer cells), CACO-2 (colorectal cancer cells), and COLO-320 (colorectal cancer cells) were examined using the database Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle). Because FHC (fetal human colon epithelial cells) are not available in the database, we examined their genetic features by targeted NGS using the Ion AmpliSeq Comprehensive Cancer Panel (Thermo Fisher Scientific, Waltham, MA) in accordance with the manufacturer’s instructions. Variants of genes associated with the HER2 signaling pathway and Wnt/β-catenin pathway in the cell lines are shown in Supplementary Table 3. HER2 copy number changes were analyzed by quantitative polymerase chain reaction (PCR) using the qBiomarker Copy Number PCR Assay Kit (Qiagen, Hilden, Germany). HER2 primers (VPH117-0412044A) and reference primer set (VPH000-0000000A) were purchased from Qiagen.

Design and construction of the expression plasmid vectors

pDONR223-ERBB2[19], which is a plasmid vector harboring human HER2 cDNA, was a gift from William Hahn and David Root (Addgene plasmid #23888; http://n2t.net/addgene:23888; RRID:Addgene_23888). pCMV-Neo-Bam APC was a gift from Bert Vogelstein (Addgene plasmid # 16507; http://n2t.net/addgene:16507; RRID:Addgene_16507) [20].

We established plasmid expression vectors containing human cDNA encoding full-length HER2 wild-type (WT) and mutant HER2 G776S. In addition, an expression vector for mutant HER2 G776 > VC, a known driver mutation in lung cancer [21], was established as a positive control. To construct WT HER2 expression vectors, the coding sequence of HER2 cDNA was amplified by PCR using the pDONR223-ERBB2 as a template, and the PCR fragment was subcloned into the pcDNA3.1 vector (Thermo Fisher Scientific) using an In-Fusion HD Cloning Kit (Takara, Otsu, Japan). The resultant vectors are referred to as pcDNA_HER2-WT. To construct mutant HER2 expression vectors, HER2 G776S and HER2 G776 > VC were directly induced into the pcDNA_HER2 WT vector by site-directed mutagenesis using a PrimeSTAR Mutagenesis Basal Kit (Takara) with the mutagenic primers. The resultant vectors are referred to as pcDNA_HER2 G776S and pcDNA_HER2 G776 > VC. The sequences of oligo DNAs used for vector construction (PCR primers and mutagenic primers) are shown in Supplementary Table 4.

Transfection and establishment of stably transfected cells

The plasmids were transfected into cells using Lipofectamine LTX and Plus Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Control cells were mock transfected with the same amount of the pcDNA or pCMV empty vector. Experiments using the transiently transfected cells were performed 48 hours after transfection. To establish stably transfected cells with these plasmids, the transfected cells were cultured in 400 µg/mL hygromycin B (Nacalai Tesque, Kyoto, Japan) medium for 14–20 days. After drug selection, we aligned HER2 expression to the same level using cell sorting on a FACSAria III flow cytometer (BD Biosciences, Franklin Lakes, NJ) using allophycocyanin-conjugated anti-human HER2 antibody (Biolegend, San Diego, CA). Seven days after cell sorting, the experiments using stably transfected cells were performed.

Cell-based transfection assays using murine cell lines, NIH/3T3 and Ba/F3 cells

Focus formation assays were performed using NIH/3T3 cells stably transfected with pcDNA-mock, HER2- WT, G776S, or G776 > VC expression plasmid vectors. The cells (3×10⁵) were plated into 6 cm-diameter dishes and incubated in DMEM supplemented with 5% calf serum. Cells were fixed 12–14 days later and stained with a solution containing 30% methanol and 0.4% crystal violet. Foci larger than 1 mm in diameter were scored.

Ba/F3 transformation assay were performed using IL-3-dependent Ba/F3 cells stably transfected with the above plasmids. The cells (1×10⁵) were washed three times in PBS and seeded in complete medium without interleukin 3, and the number of viable cells was counted 5 days after seeding. Viability was determined by trypan blue exclusion.

Establishment of APC-knockout HeLa cells using the CRISPR-Cas9 system

APC knockout (KO) cells were generated using LentiCRISPR v2, which was a gift from Feng Zhang (Addgene plasmid #52961; http://n2t.net/addgene:52961; RRID:Addgene_52961) [22], following the manufacturer’s instructions. The sequences of single guide RNA (sgRNA) and oligos are listed in Supplementary Table 4. The sgRNA for APC KO was designed using GPP sgRNA Designer (https://portals.broadinstitute.org/gpp/public/). Cloning of the lentiCRISPR targeting APC was performed in the backbone of the all-in-one lentiCRISPR v2 vector, which contains the inserts Cas9 and the puromycin resistance marker. Forward and reverse oligos were mixed, annealed, and ligated with the BsmBI-digested lentiCRISPR backbone and transformed into Stbl3 chemically competent Escherichia coli (Invitrogen, Carlsbad, CA). Single bacterial colonies were selected and transferred to culture for the minipreparation of plasmids (QIAprep Spin Miniprep Kit; Qiagen). The established plasmids were verified by Sanger sequencing.

The resultant plasmids were transfected into HEK293T cells to produce viral particles using Trans-Lentiviral Packaging Kits (Horizon, Cambridge, UK) following the manufacturer’s instructions. HeLa cells (3 ⋅ 10⁵) were incubated in DMEM containing these viral particles and 10 µg/mL Polybrene. The plate was centrifuged at 1800 rpm at 32°C for 1 h, and 2 days after the initial infection, the cells were refreshed with medium containing puromycin (1.0 µg/mL). After drug selection, single-cell colonies were isolated using cloning rings. Successful APC KO in clones was confirmed by Western blotting and TCF/LEF reporter assays.

Kinase activity assay of WT and mutant HER2 proteins

HeLa cells were transiently transfected with pcDNA-mock, HER2 WT, G776S, or G776 > VC as mentioned above and lysed with RIPA buffer (Nacalai Tesque) 48 hours after transfection. Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

The purified proteins were eluted into elution buffer under nondenaturing conditions and incubated with Poly-(4:1 Glu, Tyr) substrate peptide (Cosmo Bio, Tokyo, Japan) along with the kinase reaction mix from the ADPsensor Universal Kinase Activity Assay Kit (BioVision, Milpitas, CA) following the manufacturers’ instructions. The fluorescent signal was measured using an Infinite F200 PRO microplate reader (Tecan Group, Ltd., Männedorf, Switzerland).

Western blotting

The total cell lysate was extracted with lysis buffer, which is a mixture of RIPA buffer (Nacalai Tesque) and PhosSTOP phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO). Western blot analysis was performed as described previously [23]. The antibodies and enhanced chemiluminescence solution are described in Supplementary Table 5. Full-length APC was detected with APC antibody (#2504) from Cell Signaling Technology, and truncated APC was detected with APC antibody (sc-9998) from Santa Cruz Biotechnology (Dallas, TX) raised against the N-terminus of APC. β-Actin was used as the loading control.

Soft agar colony-formation assay

Anchorage-independent cell growth activity was assessed using a soft agar colony-formation assay. Base layers of growth medium containing 1.0% low melting point agarose (Lonza, Basel, Switzerland) were poured into six-well plates and allowed to solidify. Cells (2.5 ⋅ 10⁴ per well) were plated in triplicate in the top layer comprising growth medium containing 0.67% agarose. In the treatment group, gefitinib (1.0 µM), afatinib (0.1 µM), or dimethyl sulfoxide (DMSO) vehicle (0.5%) was added to the cultures. Ten days after seeding, the plates were stained with crystal violet (Sigma-Aldrich) for 1 h. Colonies measuring > 50 µm were counted using a Biorevo BZ-9000 inverted microscope (Keyence, Osaka, Japan).

TCF/LEF activity assay

Wnt/β-catenin signaling activity was monitored using the TCF/LEF Reporter Kit (BPS Bioscience, San Diego, CA) following the manufacturer’s instructions. In brief, cells (1 ⋅ 10⁴/per well) were seeded into a 96-well plate. The next day, the TCF/LEF luciferase reporter vectors were transfected into cells, and the cells were incubated for 48 h. Cells were harvested, lysed, and analyzed using the Dual-Luciferase Reporter Assay System (Promega) to measure luciferase activity.

RAS–GTP assay

To examine RAS activity, we measured the amount of RAS–GTP from cell lysates using an RAS Activation G-LISA Kit (Cytoskeleton, Denver, CO) according to the manufacturer’s instruction. The signal was measured using an Infinite 200 PRO multiplate reader (Tecan Group Ltd.) at a wavelength of 490 nm. The results are expressed as the fold increase in activity compared with the control group.

Cell proliferation assay

Cell proliferation was quantified using a WST-1 assay (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. Cells (1 ⋅ 10⁴ cells/well) were seeded into 96-well plates. All data were obtained using an Infinite 200 PRO multiplate reader (Tecan Group Ltd.) at a wavelength of 450 nm.

Xenograft models and in vivo afatinib treatment

Six-week-old BALB/cAJcl-nu/nu mice (CLEA Japan, Tokyo, Japan) were used for in vivo assays. To establish xenograft tumors derived from COLO-320 cells stably transfected with pcDNA-mock, HER2-WT, or HER2-G776S, each cell line (2 ⋅ 10⁶ cells) was suspended in 50% Matrigel (BD Biosciences) and injected subcutaneously into the flanks of the mice. Tumor volume was measured with calipers every 3 or 4 days and was calculated using the empirical formula: tumor volume = ½ ⋅ [(the shortest diameter)² ⋅ (the longest diameter)]. When the tumor volume was about 100 mm³, mice were randomly allocated to two groups (n = 6 in each group) that received either vehicle or 25 mg⁄kg afatinib orally by gavage once daily for 6 days per week. After 3 weeks of treatment, the mice were sacrificed, and the tumor pathology was evaluated. Ki-67-positive cells were scored by counting at least 1000 cells per low-power field under a Biorevo BZ-9000 light microscope (Keyence).

All animal experiments conformed to the relevant regulatory standards and were approved by the Institutional Animal Care and Use Committee of Kyoto University (Med Kyo 17253) and the Ethics Committee of Kyoto University (G1182-1).

Statistics

All statistical analyses were performed using JMP 15 (SAS Institute Inc., Cary, NC). Two-tailed Student’s t tests were used to compare data between two groups, and One-way ANOVA and post-hoc Tukey Honest Significant Difference (HSD) tests were used to analyze three or more groups, unless otherwise indicated. The interaction between two factors was assessed using two-way ANOVA. Error bars represent ± SD. P < 0.05 was considered to be significant.

NGS-based multiplex gene assay for molecular profiling analysis and variant annotation in a CRC patient

The patient, a 57-year-old man, was diagnosed with colorectal adenocarcinoma with liver metastasis (Fig. 1A). After resection of the primary lesion, eight courses of chemotherapy with mFOLFOX6 plus cetuximab (anti-EGFR antibody) were performed. Subsequently, a right hepatic lobectomy was performed, but recurrence occurred in the liver, lung and brain, and the patient was referred to Kyoto University Hospital for clinical NGS examination .

As shown in Fig. 1B, three somatic mutations, HER2 G776S mutation, APC nonsense mutation, and TP53 missense mutation, were detected from his liver metastasis specimen by the NGS-based multiplex gene assay. Other mutations related to the HER2 signaling pathway (KRAS, NRAS, BRAF, and PIK3CA) were not detected. We also confirmed the HER2 G776S mutation in this specimen using Sanger sequencing (Fig. 1C). HER2 copy number amplification was not detected by real-time quantitative PCR (Supplementary Fig. 1).

According to the COSMIC database (v91; http://cancer.sanger.ac.uk/cosmic/), the amino acid position G776 is in the kinase domain of HER2, and several mutations in this region, such as G776 > VC, have been registered as oncogenic genes (Fig. 1D). G776S is a rare mutation that has been detected in three cases: two cases of gastric cancer and one case of urothelial tract cancer. However, its significance has not been elucidated.

Effect of HER2 G776S mutation on kinase activity and phosphorylation and its evaluation in classical cell-based transfection assays

We established plasmid expression vectors containing human cDNA encoding full-length HER2 WT (Supplementary Fig. 2A), mutant HER2 G776S, and G776 > VC as a positive control (Supplementary Fig. 2B). To evaluate the effect of HER2 G776S on kinase activity, we transiently transfected the HER2 WT or HER2 mutant expression vectors into HeLa cells, which do not have oncogenic variants in RTK-RAS-ERK pathway or Wnt/β-actin pathway. (Supplementary Table 3). We confirmed by immunoprecipitation that HER2 proteins were successfully generated in the transfected cells (Fig. 2A).

We measured their kinase activity toward peptide substrates. The kinase activity was significantly higher for HER2 G776S protein than WT protein but was less than that of HER2 G776 > VC (Fig. 2B). We assessed the phosphorylation of HER2 and EGFR, which is one of the dimerization partners of HER2, in WT-, G776S- and G776 > VC-transfected HeLa cells. As shown in Fig. 2C, G776S and G776 > VC transfection increased the phosphorylation of both HER2 and EGFR. However, similar to the results for kinase activity, the degree of HER2 and EGFR phosphorylation was weaker for G776S transfection than for G776 > VC transfection.

Next, we evaluated the oncogenic potential of HER2 G776S using classical cell-based transfection assay using murine cell lines, NIH/3T3 focus formation assay [24], and Ba/F3 transformation assay [4, 25]. NIH/3T3 cells acquired the focus-forming ability by transfection with G776 > VC but not with G776S (Fig. 2D). Similarly, Ba/F3 cells exhibited IL3-independent proliferation upon transfection with G776 > VC but not G776S (Fig. 2E). These findings suggest that HER2 G776S had weaker phosphorylation capacity and kinase activity than the known HER2 mutation and did not promote the transformed phenotype in the transfection assays using murine cells.

Differences in the effects of HER2 G776S mutation on activation of the HER2 signaling pathway between APC-intact and APC-mutant cells

To assess the effect of the HER2 G776S mutation on the HER2 downstream signaling pathway in human cancer cells, we transfected HER2 WT or HER2 G776S expression vectors into several human cell lines: HeLa, FHC, CACO-2 and COLO-320 cells. As described in the Materials and methods section, these cell lines have no HER2 mutations or amplifications and no mutation in the HER2 downstream signals genes, KRAS, NRAS, BRAF or PIK3CA (Supplementary Table 3). HeLa and FHC cells are APC WT, and CACO-2 and COLO-320 harbor APC mutations. Using Western blotting, we consistently detected full-length APC in HeLa and FHC cells, and truncated APC in CACO-2 and COLO-320 cells (Fig. 3A). Using the TCF/LEF reporter assay, we confirmed that the Wnt activity was relatively higher in CACO-2 and COLO-320 cells than in the other two cell lines (HeLa and FHC cells) (Fig. 3B).

As shown in Fig. 3C, G776S transfection increased the amount of phosphorylated HER2 compared with WT transfection in all cell lines. Of note, G776S transfection increased the phosphorylation of the HER2 downstream signaling proteins, AKT and ERK1/2, in CACO-2 and COLO-320 cells (with mutant APC) but not in HeLa and FHC cells (with WT APC). Next, we established stably transfected FHC and COLO-320 cells and examined the effects of HER2 G776S transfection on cell proliferation and anchorage-independent cell growth activity. Transfection with HER2 WT or G776S did not affect cell proliferation in FHC or COLO-320 cells (Supplementary Fig. 3). By contrast, G776S transfection significantly increased anchorage-independent cell growth activity in COLO-320 cells (mutant APC) but not in FHC cells (WT APC). WT transfection did not alter anchorage-independent growth in either cell line (Fig. 3D).

To evaluate the effects of loss of APC function on the HER2 signaling pathway, we used CRISPR-Cas9 to establish APC-KO HeLa cells. As described in Supplementary Table 3, we used HeLa cells in this experiment because they have no oncogenic variant in RTK-RAS-ERK pathway or Wnt/β-actin pathway. As shown in Fig. 4A, we confirmed the loss of full-length APC protein in APC-KO cells. We used the TCF/LEF reporter assay to confirm that the Wnt/β-catenin signaling pathway was significantly activated in APC-KO cells compared with nontargeting control (NTC) cells (Fig. 4B).

To examine the effects of APC deficiency on the HER2 signaling pathway, we assessed the amount and phosphorylation of HER2 and downstream signal proteins, including detection of RAS–GTP (activated RAS), in APC-KO cells and NTC cells. APC KO significantly increased the amount of RAS–GTP (Fig. 4C) and slightly increased phosphorylated ERK 1/2 (Fig. 4A), whereas there was no apparent change in the amount or phosphorylation of other proteins of the HER2 signaling pathway (HER2, CRAF, MEK and AKT) (Fig. 4A and Supplementary Fig. 4A). These results suggest that APC KO increases RAS–GTP as well as the activity of the Wnt pathway without affecting phosphorylation of HER2 and other downstream signals.

To assess the effects of activation of the HER2 signaling pathway in G776S-transfected APC-KO cells, we next transiently transfected the HER2 WT or HER2 G776S expression plasmid vector into APC-KO and NTC cells. As shown in Fig. 3D, HER2 G776S transfection massively increased the phosphorylation of downstream proteins in the HER2 pathway, especially ERK1/2, in APC-KO cells. By contrast, G776S transfection did not affect AKT or ERK1/2 phosphorylation in NTC cells (Fig. 4D). G776S transfection and KO of APC synergistically increased RAS-GTP. (Interaction P < 0.01, two-way ANOVA) (Fig. 4E). Moreover, G776S transfection in APC-KO cells significantly increased anchorage-independent growth, whereas growth was not affected by mock or HER2 WT transfection in APC-KO cells (Fig. 4F).

Next, we cotransfected the APC WT expression vector with the HER2 WT or HER2 G776S expression vector into COLO-320 cells. HER2 G776S transfection increased the phosphorylation of AKT and ERK1/2 in cells without APC transfection, whereas their phosphorylation was not increased in cells with APC transfection (Fig. 5D). In COLO-320 cells, the amount of RAS–GTP was significantly increased by HER2 G776S transfection, but was not increased when APC was cotransfected (Fig. 5E).

To elucidate further the effect of Wnt/β-catenin pathway activity on RAS–GTP, we cultured COLO-320 cells with ICG-001, a Wnt pathway inhibitor that antagonizes β-catenin/TCF-mediated transcription [26]. As shown in Fig. 5F, ICG-001 dose-dependently suppressed Wnt pathway activity in COLO-320 (APC mutant) cells but did not change the amount of RAS–GTP (Fig. 5G). These results indicate that inhibition of transcription downstream of the Wnt pathway dose not suppress the amount of RAS–GTP.

Inhibition by afatinib of the proliferation and tumor growth of colon cancer cells harboring HER2 G776S and APC mutations

To investigate the efficacy of HER2-targeted therapy in colon cancer cells harboring HER2 G776S, we examined the cytotoxic effect of afatinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI), on COLO-320 cells (mutant APC) stably transfected with HER2 WT or HER2 G776S. Cells were also treated with gefitinib (EGFR TKI) for comparison. As shown in Fig. 6A, HER2 WT or G776S transfection increased EGFR and HER2 phosphorylation in COLO-320 cells, and afatinib inhibited both EGFR and HER2 phosphorylation. By contrast, gefitinib did not inhibit phosphorylation of either protein. Afatinib strongly suppressed AKT and ERK1/2 phosphorylation in HER2 G776S-transfected cells. Similarly, afatinib inhibited the cell viability of HER2 G776S-transfected COLO-320 cells compared with HER2 WT-transfected cells, but gefitinib did not alter their viability (Fig. 6B and Supplementary Fig. 5). In addition, as shown in Fig. 6C, afatinib significantly inhibited the anchorage-independent growth acquired by HER2 G776S transfection, but gefitinib did not. By contrast, gefitinib and afatinib did not alter the anchorage-independent growth of HER2 WT-transfected cells.

Next, we investigated the antitumor effects of afatinib using COLO-320 cells stably transfected with HER2 WT or HER2 G776S in a xenograft mouse model. Afatinib did not inhibit the growth of xenograft tumors in WT-transfected cells, but it significantly suppressed the growth of HER2 G776S-transfected cells compared with the vehicle (DMSO) control group (Fig. 6D). Evaluation of the pathology revealed that HER2 G776S-transfected tumors treated with afatinib had a sparse cell density compared with those in the vehicle control group (Fig. 6E). We confirmed that Ki-67 expression was significantly suppressed by afatinib treatment in HER2 G776S-transfected xenograft tumors but not in HER2 WT-transfected tumors (Fig. 6F).

In this study, we report on the function of the HER2 G776S mutation, which was detected in a patient with CRC who harbored an APC nonsense mutation. HER2 G776S transfection slightly increased the phosphorylation and kinase activity of HER2 but did not increase HER2 downstream signaling activity in cells with APC WT. We found that HER2 G776S increased the downstream signaling activity in cells with loss of APC function through an increase in the amount of RAS–GTP. We also found that afatinib, a pan-HER TKI, inhibited tumor growth of HER2 G776S-transfected COLO-320 cells with mutant APC.

Although most HER2 kinase domain mutations are categorized as activating [10, 17], the kinase activity of HER2 G776S was weaker than that of G776 > VC and insufficient to activate downstream signaling. However, when accompanied by APC mutations, HER2 G776S activated downstream signaling and functioned as an oncogenic driver mutation. These results are consistent with previous reports showing that the activities of mutant HER2 are diverse and that some of these do not result in apparent oncogenic changes [12, 27]. The significance of these less active mutations is not clear, but they may acquire oncogenic potential through the coexistence of other genetic variants, such as APC mutations. APC mutations occur in more than 80% of CRCs [28], and may have a significant impact on the role and distribution of mutant HER2 in CRC.

Although APC loss of function activates the Wnt pathway through the accumulation of β-catenin [20], our results show that loss of APC function increases the amount of activated RAS (RAS–GTP) as well as the activity of the Wnt pathway. This suggests the possibility of cross talk between the HER2–RAS–ERK pathway and the Wnt/β-catenin pathway. Our data are consistent with those of previous reports showing that APC and RAS mutations affect each other through interactions between these two pathways [29, 30]. In more recent studies, loss of APC function has been shown to stabilize the activated RAS, which results in increased function of the mutant RAS and leads to an early stage of colorectal carcinogenesis [31, 32]. Although little has been reported on the interaction between HER2 mutations and APC mutations, our findings suggest that the HER2 G776S mutation induces significant activation of the ERK pathway via the increase in activated RAS due to loss of APC function (Fig. 7). In our study, ICG-001, a β-catenin/TCF inhibitor, reduced Wnt activity but did not affect the amount of activated RAS. This result suggests that the cross talk with RAS occurs upstream of β-catenin in the Wnt pathway and that suppression downstream of the Wnt pathway alone does not inhibit ERK signaling.

We evaluated whether this HER2 mutation may be a therapeutic target in the treatment of CRC. We found that afatinib was effective in suppressing the growth of xenograft tumors derived from HER2 G776S-transfected COLO-320 cells with mutant APC. This result suggests that HER2 G776S acted as a driver mutation in the COLO-320 cells and that the tumors were strongly dependent on the mutated HER2. This patient was not treated with HER2-targeted therapy because the HER2 G776S mutation was not known to be oncogenic in advance. However, we believe that this study will help to provide a rationale for targeted therapy in future cases.

Our study has some limitations. First, cells were analyzed when expressing increased levels of the mutant HER2 and not endogenous levels. Analysis of overexpression is complicated because overexpression of HER2 itself causes some degree of transforming activity [7]. Therefore, we compared the transforming potential between HER2 mutants and HER2 WT whose expression levels were maintained at the same level using cell sorting and flow cytometry. Second, this study was conducted on a minor HER2 mutation observed in a single clinical case and not on other minor HER2 mutations. However, it is difficult to analyze several HER2 mutations observed in multiple cases because minor mutations are innumerable and the coexisting other gene mutations vary between cases.

This study shows that the HER2 G776S mutation triggers HER2 downstream signaling activation when accompanied by APC loss of function and that this may be a potential therapeutic target in CRC. Our results suggest that the oncogenicity and potential targeting of HER2 mutations in CRC should be considered in the context of APC function. Our findings provide novel insights into HER2 functions and strategies to target them in CRC.

NGS: next-generation sequencing, HER2: human epidermal growth factor receptor 2, EGFR: epidermal growth factor receptor, CRC: colorectal cancer, ERK: extracellular signal-regulated kinase, sgRNA: single guide RNA, NTC: nontargeting control, TKI: tyrosine kinase inhibitor

Ethics approval and consent to participate

This study has been approved by the research ethics committee of Kyoto University (G1182-1). Written informed consent was obtained from the patient for participation in this study.

Consent for publication

Written informed consent was obtained from the patient for publication in the appropriate journal.

Availability of data and materials

The datasets generated and analyzed during this study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that there are no competing interests.

Funding

This work was financially supported by the Japan Society for the Promotion of Science KAKENHI (18H02692).

Authors’ contributions

YM, SO and MM wrote the manuscript and formatted figures. YM, OK, YN, TI and YY were involved in conducting experiments and data analysis. AM, TS, SK, JM, AY, MK and SM participated in the design of the review and draft the manuscript. The authors read and approved the final manuscript.

Acknowledgments

The authors are grateful for technical assistance from the Center for Anatomical, Pathological and Forensic Medical Researches and the Medical Research Support Center, Kyoto University Graduate School of Medicine.

Conflict of Interest Statement: The authors declare no competing interests.

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SupplementalyFIgure1.docx
Supplementary Figures 1 (. docx): Copy number analysis of HER2 using qPCR. genomic DNA extracted from FFPE was analyzed. Lovo and Colo-320 cells are listed for comparison.
SupplementalyFIgure2.docx
Supplementary Figures 2 (. docx): Structure of HER2 WT and mutant HER2 expression plasmid vectors. (A) Genetic map and organization of pcDNA_HER2-WT. The circular image was generated with SnapGene Viewer (v. 5.1.; GSL Biotech LLC, San Diego, CA). (B) DNA Sequence of HER2 WT, G776S, and G776>VC. The bases generated by mutation and insertion are surrounded by squares.
SupplementalyFIgure3.docx
Supplementary Figures 3 (. docx): Cell proliferative potential of HER2 G776S transfected colon cells. FHC and COLO-320 cells stably transfected with mock or the HER2 expression vectors were seeded in 96 wells (1×104 cells per well) with complete medium. Their cell proliferation was evaluated by WST-1 assay on days 1, 3 and 5, respectively (n=6).
SupplementalyFIgure4.docx
Supplementary Figures 4 (. docx): The effect of APC-KO or APC-overexpression on HER2-ERK pathway. Phosphorylation and expression of the HER2 downstream signaling in APC-KO HeLa cells (A) or APC-expressing COLO-320 cells (B).
SupplementalyFIgure5.docx
Supplementary Figure 5 (. docx): Efficacy and IC50 of afatinib for colon cells transfected with HER2 G776S. COLO 320 cells (mutant APC) transfected with an empty vector, HER2 WT, or HER2 G776S were treated with afatinib or gefitinib for 24 h and the IC50 values were determined using a WST-1 cell proliferation assay (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions.
SupplementalyTable1.docx
Supplementary Table 1 (. docx). Target genes of the comprehensive genomic analysis.
SupplementalyTable2.docx
Supplementary Table 2 (. docx): The medium and supplements used in this study.
SupplementalyTable3.docx
Supplementary Table 3 (. docx). Genetic characteristics of colon cell lines and HeLa cells.
SupplementalyTable4.docx
Supplementary Table 4 (. docx): Sequence of primers and oligos.
SupplementalyTable5.docx
Supplementary Table 5 (. docx): Antibodies, reagents and equipment used in Western blotting.

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HER2 G776S Mutation Promotes Oncogenic Potential in Colorectal Cancer Cells when Accompanied by Loss of APC Function

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