Antioxidant 1 copper chaperone gene expression and copper levels in dog osteosarcoma patients

Abstract

Twenty-four dogs with OS underwent limb amputation. Serum, OS tumour, and normal bone were harvested at time of surgery. RNA was extracted and gene expression was performed using quantitative polymerase chain reaction (qPCR). Tissue and blood copper concentrations were also determined with spectrophotometry. Compared to bone, tumour samples had significantly higher expressions of antioxidant 1 copper chaperone (ATOX1, p = .0003). OS tumour copper levels were significantly higher than that of serum (p < .010) and bone (p = .038). Similar to our previous observations in mouse and human OS, dog OS demonstrates overexpression of genes that regulate copper metabolism (ATOX1), and subsequent copper levels. Dogs with OS may provide a robust comparative oncology platform for the further study of these factors, as well as potential pharmacologic interventions.

1 |. INTRODUCTION

Osteosarcoma (OS) is an aggressive primary tumour of bone with high metastatic potential. It is the most common primary bone malignancy in both humans and dogs.^1,2 OS is rare in humans, mainly affecting children and adolescents with an incidence of approximately 1 per million.² Conversely, OS is 27 times more common in dogs.^3,4 Dog OS closely resembles human OS: both species share similarities in biologic behaviour, disease progression, and treatments, making dog OS a useful model for the study of this disease.^5–7 The aetiology for OS has not yet been elucidated.

Animal models are important to enhance the understanding of the pathogenesis of OS and the development of therapeutic strategies. Similar to human OS, the primary tumour in dogs most commonly originates from the medullary cavity of metaphyseal bone and rapidly metastasizes to the lungs.⁸ Treatment with local control alone does not provide favourable prognosis as the most dogs succumb to metastatic disease or owners elect euthanasia within 6 months.^9,10 In both species, surgery plus chemotherapy remains the treatment paradigm of choice.^11,12 Most chemotherapeutic protocols include platinum-based compounds with or without doxorubicin.^13,14 In both species, the addition of adjuvant chemotherapy has been associated with increased survival times over surgery alone.^15,16 Humans with only the primary tumour at time of diagnosis have 5-year event-free survival of approximately 70% compared with 27.4% in patients with evidence of metastatic disease.^2,8 In dogs with only primary tumour at time of diagnosis, the 1-year survival rate after treatment is approximately 45%, compared to 76 days median survival in dogs with metastatic disease.^17–20 Novel therapeutic treatments are needed given the failure to improve outcomes of both human and dogs with OS for over 30 years.^9,21–25

Our group has demonstrated that the antioxidant enzyme and cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) is highly upregulated in metastatic murine and human OS tumours.^26,27 ALDH is responsible for metabolizing aldehydes into carboxylic acids which may permit cancer cells to resist the oxidative stress imparted by chemotherapeutic agents.^28–31 We also observed that metastatic human OS tumours have reduced copper concentrations compared with non-metastatic OS tumours.³² Conversely, OS patients with metastases displayed increased levels of blood serum copper levels compared with patients who did not develop metastases.³²

Copper is an essential trace nutrient metal involved in multiple cellular processes. Ingested copper is absorbed in the stomach and small intestine³³ and transported to the liver via portal circulation followed by redistribution to extrahepatic tissues.³⁴ The main route of copper removal is via biliary excretion into the faeces, although a small amount is excreted by the kidneys.³⁵ Copper enters cells by the plasma membrane transport proteins copper transporter 1 (CTR1).³⁶ Chaperone proteins that facilitate copper delivery to multiple destinations include copper chaperone for superoxide dismutase (CCS) and its target zinc-dependent superoxide dismutase (SOD1).³⁷ SOD1 protects oxidative cell damage from reactive oxygen species.³⁸ Antioxidant 1 copper chaperone (ATOX1), shuttles copper to the copper-transporting ATPases: ATP7A or ATP7B.³⁹ These proteins transport newly synthesized cuproenzymes through the secretory pathway.⁴⁰ Breiter et al evaluated copper measurements in human OS patients and observed that serum copper levels were significantly elevated (p < .0001) in all 18 patients with untreated tumours compared to controls.⁴¹ To our knowledge, no previous studies report copper levels of serum, normal bone, and OS-affected bone in canine OS patients. The reported reference interval for normal healthy canine serum copper concentrations is 132–712 ng/mL.⁴²

Disulfiram (Dis) is an FDA approved anti-alcoholism drug which (1) acts as an irreversible inhibitor of ALDH activity and (2) potently complexes with copper which has been suggested to enhance Dis’s antineoplastic effects.^43–49 It has been demonstrated that human OS cells are sensitive to ALDH inhibition with Dis, which raises the possibility of using ALDH inhibition as a therapeutic strategy in OS.²⁷

The objectives of our study were to evaluate the gene expressions of ALDH and enzymes involved with copper metabolism between dog primary OS tumours and normal bone. We also compared copper levels in OS tumours, normal bone, and peripheral blood. We hypothesized that the OS tumours would demonstrate higher copper levels and ALDH expression than normal bone samples. We further hypothesized that copper metabolism genes would be differentially expressed between OS tumours and normal bone.

2 |. METHODS

2.1 |. Sample collection

This was a prospective study of dogs with non-metastatic OS who underwent limb amputation surgery by a board-certified veterinary surgeon. This study was approved by the BluePearl Science IRB (IRB# PAY.05.03.2019). After informed consent from dog owners was obtained, whole blood, OS tumour, and normal bone samples were obtained at the time of surgery. Dogs were staged for pulmonary metastases via three-view chest radiographs that were determined to be macroscopically negative by a veterinary radiologist. Normal bone was taken from the opposite end of the tumour-bearing limb. Radiographic confirmation was performed to ensure that this portion of the bone was disease-free. Whole blood was collected in Streck tubes, then processed into serum and buffy coat samples and stored in −80°C. Tumour and normal bone were placed in two separate tubes containing unsupplemented DMEM for transportation. Bone samples were removed from DMEM and preserved by immersion in liquid nitrogen for 60 s and then stored in −80°C. All samples were processed in a tissue culture hood using sterile technique.

2.2 |. Bone samples

Following harvest and storage, tumour and normal bone samples were pulverized into a fine powder with a Cryomill (Spex Freezer Mill 8670, Metuchen, NJ, USA). Cryomilling was performed through one 5-min cooling cycle and one 3-min milling cycle. Powdered sample was then transferred into plastic centrifuge tubes and stored at −80°C.

2.3 |. RNA expression analysis

Hundred grams of powdered bone and tumour samples were digested in 500 μL trizol (Invitrogen) using the Bullet Blender (Next Advance) for 5 min at 4°C. D418 and D17 dog OS cells were plated into 6-well plates at a concentration of 1.25 million cells/well in 2 mL media. RNA extraction from tumour and normal bone was performed using the RNeasy Lipid Tissue Mini Kit (Qiagen). RNA quantification was then confirmed using the Nanodrop 2000 1-position Spectrophotometer (Thermo Scientific). cDNA was obtained from 2 μg of total RNA using the Reverse Transcriptase Kit (Applied Biosystems). qPCR was performed using 100 ng of cDNA, SYBR Green Supermix (Bio-Rad) and shelf ready Primer sets (BioRad) (Supplemental Table 1). The qPCR was performed according to manufactures recommendations. Briefly, 40 cycle setup was as follows: Activation 95°C for 2 min, Denaturation 95°C for 5 s, Annealing/extension 60°C for 30 s, Melt curve 65–95°C for 5 s/step. Technical replicates x3 were performed. Delta CT values were obtained from the average Ct of this house-keeping gene. RNA expression comparisons between bone and tumour samples were performed for the Genes seen in Table 1.

TABLE 1.

Genes of interest evaluated.

GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
ALDH1A1	Aldehyde dehydrogenase 1A1
SLC31A1 (CTR1)	Solute carrier family 31 member 1 (copper uptake protein 1)
MDR1	Multidrug resistant gene 1
AR	Androgen receptor
ABCG2	ATP-binding cassette subfamily G member 2
ATOX1	Antioxidant protein 1
BMP4	Bone morphogenetic protein 4
VEGFA	Vascular endothelial growth factor A
ATP7B	ATPase copper transporting beta
ATP7A	ATPase copper transporting alpha
SOD1	Superoxide dismutase 1

2.4 |. Copper analysis

Dog tumour, bone, and serum samples were processed and stored as described above. Copper concentrations were determined using a PerkinElmer Analyst 600 atomic absorption spectrophotometer adjusted to detect copper (324.8 nm) as previously described.^32,51 The final copper concentrations were calculated as total μg/ml. Intracellular bone and tumour copper concentrations were calculated as (final sample concentration of copper [ng/mL]/(total sample protein concentration [mg/ml])) and recorded as ng copper/mg protein.

3 |. STATISTICAL ANALYSIS

Statistical analysis of gene expression and copper levels were conducted through Mann–Whitney U tests, and ANOVA, respectively. A p < .05 was considered statistically significant. All statistical analyses were performed using the Prism 9.5 software (GraphPad, La Jolla, CA).

4 |. RESULTS

4.1 |. Demographics

There were 24 dogs in the final analysis. All dogs had OS diagnosed by histopathology between October 2019 and August 2021. Breeds varied, with a predominance of Rottweilers (5) and Greyhounds (4). Metaphyseal tumour location varied, with a predominance of distal radius (right: 5; left: 4), proximal humerus (right: 3; left: 3), distal tibia (left: 4; right: 2) (Table 2).

TABLE 2.

Patient demographics.

Demographic	Values
Age (mean)	8.0 ± 2.8 years
Body weight (mean)	38.9 ± 11.6 kg
Female (spayed)	6
Males (neutered)	13
Males (intact)	5
Location of tumour
Distal radius	9
Proximal humerus	6
Distal tibia	6
Proximal tibia	1
Distal femur	1
Proximal femur	1
Breed
Rottweiler	5
Greyhound	4
Golden retriever	3
American pit bull terrier	2
German shorthaired pointer	2
Other	8

Note: Values expressed in n (%) or mean ± SD.

4.2 |. Gene expression

Quantitative PCR (qPCR) was performed on genes of listed in Table 1. Tumour samples had higher expression levels of ALDH1A1 (p = 0.4286) but this was not statistically significant. Tumour samples had a significantly greater expression of ATOX1 compared with normal bone samples (p = .0003). As expected, expression of CTR1 was greater in tumour than normal bone, but this was not statistically significant (p = 0.7900). Expression of the copper efflux pump ATP7B was lower in tumour samples compared with bone samples as expected, but also not significantly significant (p = 0.1851) (Figure 1).

FIGURE 1.

Fold change of genes ALDH1A1, CRT1, ATOX1, and ATP7B between normal bone and tumour samples. Our data demonstrate gene expression differences between normal bone (blue bars) and OS (red bars). While several factors trended to greater expression in tumours compared with normal bone, only ATOX1 reached statistical significance (p = .0003). Values are depicted as mean and standard deviation. These findings are commensurate with our previously published observations in murine and human OS cells and tissues.^{31,50,53–55}

4.3 |. Copper levels

Copper levels in canine OS cells were increased compared with serum and normal bone. Average copper levels of serum (505 ng/mL, SD = 124.62) and bone (576 ng/mg, SD = 268.72) were not statistically significant from each other (p = 0.724). However, average copper levels found in OS tumour samples was 809 ng/mg (SD = 452.98), significantly higher than that of both serum (p < .010) and bone (p = .038) (Figure 2).

FIGURE 2.

Copper levels: Copper levels in canine OS cells are increased compared to serum and normal bone. Average copper levels of serum (505 ng/mL, SD = 124.62) and bone (576 ng/mg, SD = 268.72) were not statistically significant from each other (p = 0.724). However, average copper levels found in OS tumour samples was 809 ng/mg (SD = 452.98), significantly higher than that of both serum (p < .010) and bone (p = .038).

5 |. DISCUSSION

OS is a devastating bone malignancy that is rare in humans but much more common in dogs. Dog OS may thus serve as a model to study OS pathophysiology. Our study sought to evaluate gene expressions and copper concentrations in dog OS tumours, normal bone, and serum. We observed that ATOX1 expression was significantly greater in OS tumours compared with normal bone. Tumour copper levels were significantly greater than either normal bone or serum. Genes including ALDH1A1, CTR1, and ATP7B trended commensurately with our previous observations in mouse and human OS, but did not reach statistical significance.

ATOX1 is an intracellular copper transporter that plays a key role in copper regulation. Previous studies have also shown that its inhibition sensitizes OS cells to carboplatin chemotherapy.⁵² Dysregulation of copper metabolism was supported by our observation that ATOX1 is significantly overexpressed in OS tumours compared with normal bone, and further substantiated by our copper concentration analyses. These demonstrated significantly greater copper concentrations in OS tumours than normal bone and serum.

There are a number of limitations to our study. One is limited sample size (n = 24). There was also a limited ability to obtain nucleic acid from bone samples. We were unable to obtain metastatic samples.

In conclusion, we have demonstrated significant overexpression of ATOX1 in dog OS tumours compared with normal bone, which corroborates the observation of significantly greater copper concentrations in OS tumours compared with normal bone and serum from the same animals. The expression of ALDH1A1 and other copper transport genes did not reach statistical significance. These data, in combination with our group’s previous mouse and human data, suggest that copper transport mechanisms may have importance in OS biology. Future studies will comprehensively explore copper transport in primary and metastatic OS and may provide clues to novel systemic treatments for both dogs and humans.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.