Expression and prognostic value of TRPM7 in canine mammary tumours

Abstract Canine mammary gland tumour (CMTs) are one of the most commonly found tumours in intact female dogs. A previous study on canine mammary glands demonstrated the presence of the transient receptor potential melastatin 7 (TRPM7) ion channels in healthy canine mammary tissues. However, the significance of TRPM7 in CMT is not yet known. TRPM7 is a Ca2+ and Mg2+ permeable cation channel that contains a protein kinase domain. The aim of this study was to determine TRPM7 expression in 57 benign and malignant CMT tissues of dogs using immunohistochemistry (IHC) and evaluate its correlation with clinicopathological features and explore the potential prognostic value of TRPM7 in a prospective survival study. IHC analysis shows that TRPM7 was expressed in the cytoplasm of neoplastic epithelial cells. Moreover, TRPM7 expression was significantly associated with tumour malignancy (P = .027), Ki‐67 index (P < .0001) and metastasis (P < .0001). Survival curve analysis indicates that high TRPM7 expression was significantly associated with poor disease‐free (P = .035) and overall survival (P = .011) in malignant CMTs. Our results demonstrate that TRPM7 is expressed in CMTs and that its expression is positively correlated with clinicopathological parameters. Thus, TRPM7 was assumed to be a potential prognostic factor for CMTs.


| INTRODUCTION
Canine mammary gland tumour (CMTs) account for 70% of all tumours in intact female dogs 1,2 and can be benign or malignant in nature. CMTs are hormone-dependent and might recur after surgical removal or metastasize to other organs, in particular, the lymph node and lungs. 3 The prognostic factors of cancer include histological type, tumour grade, mode of tumour growth, lymph node status and tumour size. 4 These clinicopathological factors are important for evaluating and determining a prognosis as well as predicting the molecular cancer behaviour. In humans and animals, ion channels have been newly identified as prognostic factors that could potentially lead to the identification of new therapeutic targets. Deregulation of Ca 2+ homeostasis has been implicated in mammary gland disease. [5][6][7][8] Moreover, Ca 2+ and Mg 2+ ion channels play an important role in cell proliferation, differentiation, apoptosis and oncogenesis. 9,10 Transient receptor potential (TRP) is a plasma membrane ion channel that regulates the permeability of Ca 2+ and Mg 2+ across the plasma membrane of animal cells. 7 TRP channel activity is important for essential hallmarks of tumorigenesis. Therefore, TRP channels Seulji Lee and Sungin Lee contributed equally to this work.
have not only been suggested as clinical markers but also as promising anticancer targets in recent years. TRP channels were first discovered in a TRP-mutant strain of the fruit fly Drosophila, and are categorized into six subfamilies based on their amino acid sequences: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin) and TRPP (polycystin) channels. 11 Dhennin-Duthille et al 10 reported that increased expression of TRP channels is a useful biomarker for the diagnosis, prognosis and/or treatment of human breast ductal adenocarcinoma. Accumulating evidence has indicated that increased expression of TRP channels can be used as a biomarker for several human malignancies. [12][13][14] TRPC, TRPM and TRPV expression levels were shown to be correlated with malignant growth and cancer progression. 15 Transient receptor potential melastatin 7 (TRPM7) channel is widely expressed in various organs, including the heart, lung, liver, brain and spleen. 16 It is overexpressed in various types of cancers, such as ovarian carcinoma, retinoblastoma, neck and head carcinoma, prostate cancer, lung cancer and pancreatic adenocarcinoma. [17][18][19][20] Furthermore, an increased expression of TRPM7 channel is correlated with breast cancer progression and metastasis. 21 Another study showed that siRNA-mediated knockdown of TRPM7 expression in MCF-7 cells impairs biological functions, highlighting the importance of TRPM7 expression in human breast cancer epithelial cells. 9 Aberrant TRPM7 expression in human breast and pancreatic cancers is closely correlated with clinicopathological parameters, such as tumour grade, Ki-67 proliferation index and patient survival time. 22 A previous study had proven that TRPM7 is necessary for pancreatic cancer cell invasion. 23 Despite abundant knowledge on TRPM7-related carcinogenic pathways in human breast cancer, its role in CMT pathogenesis remains poorly understood. In addition, a previous study described the presence and distribution of TRPM7 channels in canine normal mammary gland tissue using RT-PCR, immunohistochemistry (IHC) and western blotting. 24 Since TRPM7 has not been studied in the context of clinical value, its prognostic value in CMTs is still unknown.
In this study, we aimed to investigate the presence of TRPM7 in CMTs by IHC and to evaluate the correlation between TRPM7 and clinicopathological features of dogs to explore the potential prognostic value of TRPM7 in survival study.
Antigen retrieval was carried out using a 2100-retriever pressure cooker Ltd, Paisley, England). The antibody used against TRPM7 was previously validated for canine tissues. 24 The sections were subsequently incubated with secondary antibodies HRP anti-goat IgG (ImmPACTTM, Vector, California) and HRP anti-rabbit IgG (ImmPACTTM, Vector) for 1 hour. The slides were incubated in 3,3 0 -diaminobenzidine tetrahydrochloride (ImmPACTTM) diaminobenzidine (DAB) peroxidase substrate kit, Vector) for 90 seconds and the reaction was stopped by immersion in distilled water. In a previous study, normal C57BL/6J mouse brain tissues were used as positive control. 24,27 Negative control samples were incubated in the absence of the primary antibodies to rule out non-specific binding by the secondary antibodies. The tissue sections were counterstained with Mayer's haematoxylin, dehydrated in graded alcohol and cleared in xylene.
The slides were washed with PBS between each procedure. Immunostained slides were scanned using an Olympus BX51 microscope (Olympus, Japan) with appropriate light filters (Tucsen, Fuzhou, China).

| Quantitation of IHC staining
IHC results were analysed using Aperio ImageScope version 12.3.0.5056 (Aperio, Vista, California). IHC slide images were analysed using the Aperio program. These algorithms used colour de-convolution to separate DAB from the haematoxylin counterstain. The algorithm to determine the intensity of cytoplasmic and nuclear staining for each slide was used to calculate the staining intensity and percent of target labelled by digitally analysing the colour intensity. The output staining intensities ranged from 0 (negative) to +3 (strong positive) and were correlated with conventional manual scoring methods. 28 Aperio Cytoplasm V2 algorithm was used to analyse cytoplasmic positivity of TRPM7 expression, based on criteria for TRPM7 in human breast cancer. 29,30 The positive expression of TRPM7 in cytoplasm was quantitatively assessed, for each sample in 10 randomly selected high-power fields (400×), as follows: 0 (negative), +1 (weakly positive), +2 (moderately positive) and + 3 (strongly positive). Staining intensities for TRPM7 expression ranged from 0 to 3 and were correlated with conventional manual scoring methods.
Aperio Nuclear V9 algorithm was used to analyse nuclear positivity of Ki-67. An algorithm for analysing nuclear immunoreactivity was used to measure the percentage of immunoreactive cells. 31

| Follow-up data
All dogs with CMTs were evaluated before surgery, 3 weeks after surgery, and every 3 months for at least 2 years. Owners were instructed to contact the hospital at any time, even if it was not related with CMT, whenever they discovered abnormalities. Assessment of metastasis and recurrence of tumours was carried out by physical examination, thoracic radiograph (three views), ultrasound (abdomen), FNA, biopsy, autopsy and/or CT scan (if required) at the Seoul National University hospital and/or referred to another animal hospital.
Whenever new mammary gland lesions were discovered, lymph nodes were considered clinically abnormal or lesions were detected in any other organ, additional examinations (eg, FNA, excisional biopsy and CT scan) were performed to rule out other neoplasms and/or to identify local recurrences or metastatic disease.

| Statistical analysis
Correlation between TRPM7 and clinicopathological parameters was analysed using Fisher exact test, the chi-square and the linear-bylinear association test. Kaplan-Meier survival curves were plotted and compared using the log-rank test. All the statistical analyses were performed using SPSS software (SPSS, Chicago, Illinois).
Disease-free survival (DFS) was defined as the interval (months) from primary surgical treatment to the date of detection of the first local recurrence or development of metastases. Overall survival (OS) was calculated from the date of primary surgical treatment to the time of death from the cancer. In the OS study, the dogs were censored if and when they died from causes unrelated to mammary tumours, were lost to follow-up, or were alive 2 years after surgery. In the DFS study, the dogs were censored if and when they were lost to follow-up, died from causes unrelated to mammary tumours before developing signs of metastatic disease, or were free of metastases 24 months postsurgery. Each tick mark represents the time at which a patient was censored. A P < .05 was considered to be statistically significant.

| Dogs
A total of 57 dogs diagnosed with CMTs were included in this study.
The signalment data are presented in Table 1. The median age of dogs with benign CMTs was 11.00 years (range: 6-16 years) and was similar to that of dogs with malignant CMTs (11.94 years; range: 6-15 years).

| Immunolocalization
IHC staining showed TRPM7 expression in the cytoplasm of neoplastic epithelial cells. Cytoplasmic immunoreactivity in benign tumours was weak compared to that in malignant tumours. In addition, no immunoreactivity was observed in myoepithelial cells of complex adenomas and mesenchymal areas of benign mixed tumours. A cell population was observed in the mesenchymal area, and TRPM7 expression in these cells was higher than that in the adjacent non-cancerous cells ( Figure 1A-D).

| Correlation between TRPM7 overexpression and clinical outcome
The

| DISCUSSION
TRPM7 is a ubiquitously expressed protein and plays a prominent role in early embryogenesis and organogenesis. 34,35 It is involved in cell cycle progression, adhesion, survival and migration of cancer cells. 36 Although cytoplasm at elevated levels in higher grade CMTs. This pattern was also observed in the pancreas wherein TRPM7 was expressed in apical plasma membrane of pancreatic ductal epithelia and in the cytoplasm of pancreatic adenocarcinoma cells. 37 We investigated whether other TRP channels show differential immunoreactivity (plasma membrane or cytoplasm) in normal cell and tumour state. TRPM8 protein was localized to the plasma membrane of cells in the normal prostate tissue, whereas its channel showed severely internalized pattern of TRPM8 in tumour tissues. 38,39 Taken together, these data suggest that IHC staining results of pancreatic Previous results and the current study have provided basic data for future studies to evaluate TRPM7 expression and OS in canine mammary gland tumour. These findings suggest that TRPM7 might have prognostic value and, ultimately, we need to discuss the potential function of TRPM7 channel-kinase as a biomarker and therapeutic target for veterinary oncology. 45

| CONCLUSION
In this study, we showed that TRPM7 is expressed in the cytoplasm of benign and malignant CMTs. We observed higher TRPM7 expression in malignant CMTs than in benign CMTs, indicating that TRPM7 is involved in cancer progression. Furthermore, we demonstrated that TRPM7 overexpression is positively associated with prognostic factors such as tumour grade, Ki-67 index and metastasis. In addition, our findings demonstrate that high-TRPM7 expression is significantly associated with DFS and OS. However, further studies are required to understand the mechanism by which TRPM7 overexpression promotes the development of CMTs.

CONFLICT OF INTEREST
The authors have declared that no competing interests exist. The funders had no role in the design of the research; in the collection, analyses or interpretation of data; in the writing of the article, or in the decision to publish the results.

ETHICS APPROVAL
This study is a retrospective investigation carried out on archived tissue samples of CMT. The protocol was reviewed and approved by the Institutional Animal Care and Used Committee of Seoul National University (SNU-190314-6-1). Informed consent was obtained from the owners for the collection and use of tissue samples.

DATA AVAILABILITY STATEMENT
The data that support the findings of this research are available from the corresponding author upon reasonable request.