Potential therapeutic efficiency of pan-ERBB inhibitors for canine glioma

Canine glioma is one of the most common brain tumors with poor prognosis, making effective chemotherapy highly desirable. Previous studies have suggested that ERBB4, a signaling molecule involving one of the epidermal growth factor receptors (EGFR), may be a promising therapeutic target. In this study, the anti-tumor effects of pan-ERBB inhibitors, which can inhibit the phosphorylation of ERBB4, were evaluated both in vitro and in vivo using a canine glioblastoma cell line. The results demonstrated that both afatinib and dacomitinib effectively reduced the expression of phosphorylated ERBB4, and significantly decreased the number of viable cells, ultimately prolonging the survival time of orthotopically xenografted mice. Further downstream of ERBB4, afatinib was found to suppress the expression of phosphorylated Akt and phosphorylated Extracellular signal-related kinases1 and 2 (ERK1/2) and induced apoptotic cell death. Thus, pan-ERBB inhibition is a promising therapeutic strategy for the treatment of canine gliomas.


Introduction
Canine glioma is a most common brain tumors that is standardly treated by surgical excision and/or radiation therapy (Rossmeisl and Pancotto 2020).Lomustine and temozolomide are used empirically as chemotherapy because they can penetrate the blood-brain barrier (Rossmeisl et al. 2020).However, the use of monotherapy with these drugs in the presence of gross disease or as an adjuvant therapy after surgery or radiation therapy may not provide clinical benefit (Rossmeisl et al. 2020;Hidalgo et al. 2022).Therefore, effective chemotherapies are needed to improve the prognosis of canine glioma.
ERBB4 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases.EGFR

Reagents and antibodies
Afatinib (Tokyo Chemical Industry, Tokyo, Japan), dissolved in dimethyl sulfoxide (DMSO), was added to the cell culture media to assess its impact on cell growth and protein expression.Dacomitinib (Tokyo Chemical Industry) was studied similarly.Z-VAD-FMK (Medical and biological laboratories, Tokyo, Japan) was utilized to block pancaspase activity at a concentration of 25 µM.DMSO was used as a negative control.

Western blotting
Total protein was extracted from whole cells as previously described (Noguchi et al. 2011).The protein content was measured with a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA).Lysate proteins (10 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for western blotting.The proteins were electroblotted onto a polyvinylidene fluoride membrane (PerkinElmer Life Sciences, Boston, MA, USA).Details of the method used after blotting have been described previously (Noguchi et al. 2011).The antibodies were diluted in Tris buffered saline with Tween 20 (TBS-T) containing 2% bovine serum albumin and 0.01% sodium azide.The loading control was prepared by re-incubating the same membrane with an anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA).The bands were visualized by a chemiluminescent agent (ECL Pro, PerkinElmer), and the densitometric value of each band was calculated using Multi Gauge software (Fuji film, Tokyo Japan) and normalized to the value of β-actin.

Flowcytometric analysis
The cells were treated with afatinib or dacomitinib, with or without Z-VAD-FMK, for 24 h.Then, 1.0 × 10 6 cells were detached by trypsinization and stained using an Annexin V-FITC Apoptosis detection kit (NACALAI TESQUE, INC., Kyoto, Japan) according to the manufacturer's protocol.The number of apoptotic cells was quantified using CytoFLEX (Beckman Coulter, Brea, CA, USA).

Orthotopic mice xenograft model
2.5 × 10 5 J3T-1 cells were cultured in 2 µl DMEM for stereotactic injection into 5-week-old nude mice (Japan SLC, Hamamatsu, Japan).Following a previous study (Torrini et al. 2022), a Hamilton syringe was inserted into a burr hole located 2.5 mm lateral and 1 mm anterior of bregma and 3.0 mm deep into the striatum using a stereotactic device (Narishige, Tokyo, Japan).Tumor cells were stereotactically injected at a rate of 1 µL/10 s using a microinjector (Narishige).From the 7th day after the transplantation, each drug was orally administered five consecutive days per week for five weeks using a cannula.The drugs were dissolved in 0.5% methylcellulose solution (FUJIFILM Wako Pure Chemical, Osaka, Japan), which was used as a control.Afatinib and dacomitinib were administered at doses of 20 mg/kg and 15 mg/kg, respectively, which were determined based on the FDA interview form and a previous study (Yoshioka et al. 2019).If any neurologic abnormalities or a > 20% reduction in body weight was observed, the mice were euthanized.The brains of all mice were cut into four sections along the transverse plane as shown in Fig. 1c, fixed with 10% neutral-buffered formaldehyde, and embedded in paraffin.Paraffin-embedded samples were sectioned at a thickness of 5 μm and histopathologically evaluated after staining with hematoxylin and eosin (H&E).Animal experiment protocol was approved by the Committee for Animal Research and Welfare of Osaka Prefecture University.

Statistics
Each examination was performed in triplicate.The calculated data was compared using unpaired 2-tailed Student's t-test or one-way ANOVA with Tukey's method using Microsoft Excel add-in software Statcel3 (OMS publication, Tokyo, Japan).The survival time of the mice was assessed by the log-rank test using GraphPad Prism8.Statistical p-value of < 0.05 was considered statistically significant.

Pan-ERBB inhibitors exhibited anti-tumor effects on J3T-1 cells
First, we examined the effects of afatinib and dacomitinib on viable cell number and protein expressions.Both afatinib and dacomitinib significantly reduced the number of J3T-1 cells in a dose-dependent manner, as assessed using one-way ANOVA (Fig. 2a).The effect of afatinib was significantly stronger than that of dacomitinib (5 µM).IC50 values for these reagents were 3.3 µM (afatinib) and 5.1 µM (dacomitinib), respectively.Both afatinib and dacomitinib successfully decreased the expression of p-ERBB4 (Fig. 2b).Furthermore, afatinib, but not dacomitinib, suppressed the expression of the downstream molecules of ERBB4, p-Akt, and p-ERK1/2.Additionally, both afatinib and dacomitinib

Pan-ERBB inhibitor prolonged the survival time of orthotopic xenografted mice
Finally, we validated the effect of pan-ERBB inhibitors on survival time of the mice xenograft model (Fig. 3a).Since these inhibitors exhibited anti-tumor effects in vitro, we evaluated their effect in the mice model by orally administering afatinib and dacomitinib to mice transplanted with J3T-1 cells.The results showed that the survival time was significantly prolonged in the mice treated with either afatinib or dacomitinib compared to those treated with 0.5% methylcellulose (Fig. 3b).The median survival time for each group was as follows: control, 20 days; afatinib, 38 days; and dacomitinib, 34 days.However, there was no significant difference between mice treated with afatinib and dacomitinib.Although tumor tissue was observed in all mice increased the level of cleaved caspase-3, with the effect of afatinib being greater than that of dacomitinib.

A pan-caspase inhibitor partly suppressed apoptosis induced by afatinib
We then evaluated the induction of apoptosis by afatinib as it increased the level of cleaved caspase-3.The co-treatment with Z-VAD-FMK and afatinib resulted in a decrease in the expression of cleaved caspase-3 in comparison to the treatment with afatinib alone (Fig. 2a).Additionally, the treatment with Z-VAD-FMK restored the expression level of p-ERK1/2 in the cells and significantly decreased the number of Annexin V-positive cells in cells treated with afatinib (Fig. 2b).Moreover, Z-VAD-FMK attenuated the decreased number f viable cells by afatinib treatment (Fig. 2c).compounds exerted a significant effect on J3T-1 cells in vitro and in vivo.However, afatinib showed greater effects, such as cell growth suppression, signaling inhibition, and induction of cell death, than dacomitinib.These findings suggest that pan-ERBB inhibitors have potential as therapeutic drugs for canine glioma.
However, the comprehensive antitumor mechanisms of pan-ERBB inhibitors remain unclear, while those of afatinib include inhibition of PI3K/Akt and MAPK signaling, known downstream signaling pathways of ERBB4, and induction of apoptosis.In the current study, afatinib inhibited MAPK signaling, whereas in our previous study (Noguchi et al. 2021), it suppressed PI3K/Akt signaling.Protein expression levels of downstream molecules were evaluated 12 h after treatment with afatinib or dacomitinib at a dose of 5 µM and 24 h after treatment with afatinib at a dose of euthanized before the end of study (Day 42; Fig. 3c), histologic evaluation revealed that no tumor tissue was observed in the brains of mice alive at the end of the study (Fig. 3d; afatinib goup n = 2; dacomitinib group n = 1).It was not possible to compare tumor size among groups accurately, as the tumor tissues did not form nodule.

Discussion
Previously, we reported that, compared to gefitinib (EGFR inhibitor) and lapatinib (EGFR and ERBB2 inhibitor), afatinib demonstrated an antitumor effect on canine glioma cells (Noguchi et al. 2021).In this study, we conducted a comparison of the antitumor effects of afatinib and dacomitinib, both in vitro and in vivo.The results showed that both 10 µM.Therefore, afatinib may inhibit MAPK signaling in the initial phase and subsequently inhibit PI3K/Akt signaling.Although Z-VAD-FMK did not completely restore the decreased viable cell number caused by afatinib treatment, the expression level of cleaved caspase-3 was restored by the pan-caspase inhibitor.Additionally, as previously reported (Wu et al. 2011), Z-VAD-FMK increased the level of p-ERK1/2.The antitumor mechanisms of p-ERBB inhibitors may include STAT3 signaling, which is also a downstream molecule of ERBB4 (Longo et al. 2019).
In this study, we did not evaluate whether the drugs could reduce tumor size in vivo, as the experimental design focused on survival time.Nevertheless, we considered that the pan-ERBB inhibitors assessed in the current study potentially could delay tumor growth, as no tumor growth was observed in some mice alive at the end of the experiment.On the other hand, the mortality rate of mice administered each drug was 60% (afatinib) and 80% (dacomitinib), indicating that the pan-ERBB inhibitors could not achieve the complete tumor growth suppression.Thus, the dose of the drugs might be insufficient.Further studies to validate the effect of the drugs on tumor growth need to be conducted using an in vivo imaging system.In addition, the tumor suppressive effect of the pan-ERBB inhibitors combined with other drugs such as temozolomide needs to be evaluated.
This study had numerous limitations.Firstly, we examined the effects only on the J3T-1 cell line.To validate the effects of the pan-ERBB inhibitors, it is necessary to evaluate their antitumor effects using other cell lines.Furthermore, the cell line was not genetically evaluated to determine whether it originated from a canine in the current study, although the cell line has been used as a canine astrocytoma cell line in the previous several studies (Inoue et al. 2012;Onishi et al. 2013;Lee et al. 2021).Secondly, the superiority of one of the two drugs was not concluded and should be decided based on their toxicity, as well as antitumor effects.
In conclusion, pan-ERBB inhibitors show promise as drugs for treating canine glioma.Further studies, including the clinical trials, could lead to the development of new strategies for the treatment of canine glioma.

Fig. 1
Fig. 1 In vitro antitumor effects of pan-ERBB inhibitors.a. Reduction in the viable cell number by the treatment with afatinib or dacomitinib at indicated doses.Cell number was counted 48 h after the treatment.Data are expressed as the mean + SD (n = 3).b.Protein expression in J3T-1 cells treated with afatinib or dacomitinib at a dose of 5 µM, assessed by western blotting.Protein extraction was performed at 12 h after the treatment.Densitometric values were indicated below each band.*p < 0.05, for the difference between the cells treated with afatinib and dacomitinib, assessed by the unpaired 2-tailed Student t-test

Fig. 2
Fig. 2 Anti-apoptotic effect of Z-VAD-FMK on afatinib-inducing cell death.a.Protein expression levels assessed by western blotting.Protein extraction was performed at 12 h after the treatment.Densitometric values were indicated below each band.b.Flowcytometric analysis in the cells stained Annexin V (AV) and propidium iodide (PI).Assay was performed 24 h after the treatment.c.Number of viable cells at 48 h after the treatment.Afatinib and dacomitinib were administered at a dose of 10 µM and Z-VAD-FMK at 25 µM.Afatinib or dacomitinib and Z-VAD-FMK were concurrently treated.*p < 0.05, for the difference between the cells treated with afatinib alone and afatinib and Z-VAD-FMK assessed by one-way ANOVA following Tukey's method.Calculated data are expressed as the mean + SD (n = 3)

Fig. 3
Fig. 3 Antitumor effect of pan-ERBB inhibitors on orthotopic xenografted mice.a. Location of transplantation of J3T-1 cells and the administration schedule.b.Kaplan Meier survival curves of mice.*p < 0.05, for the difference between the bracketed groups assessed by log-rank test.c.The sites of tissue section indicated by dash lines (right image; star, an injection site) and H&E-stained representative histological images of FFPE samples of forebrain at the site of middle dash line from orthotopic xenografted mice at 20 days (Control), 31 days (afatinib), and 34 days (dacomitinib) after transplantation (left panels; high power field).d.H&E-stained histological images of FFPE samples of forebrain without any observable tumor growth at 42 days (end of study) after transplantation.Bar, 1 mm (high power field, 50 μm)