Characterization of mesothelin gene expression in dogs and overexpression in canine mesotheliomas

Abstract

Introduction: Canine mesotheliomas are uncommon malignant tumors typically detected late. Minimally invasive diagnostic biomarkers would facilitate diagnosis at earlier stages, thereby improving clinical outcomes. We hypothesized that mesothelin could be used as a reliable diagnostic biomarker for canine mesotheliomas since it has been used as a cancer biomarker for human mesothelioma. We aimed to explore and characterize mesothelin gene expression in dogs and assess its use as a diagnostic biomarker for canine mesotheliomas.

Materials and methods: We quantified expressed canine mesothelin transcripts via reverse transcription polymerase chain reaction (RT-PCR) and sequenced them using ribonucleic acid (RNA) extracted from a canine mesothelioma cell line. After confirming mesothelin expression, we assessed its levels in major organ tissues and compared them with those in the mesothelioma tissues using quantitative PCR (qPCR). Mesothelin overexpression in mesotheliomas was detected, and we further compared its levels using qPCR between mesotheliomas and non-mesotheliomas using tumor tissues and clinical sample effusions, confirming its significance as a diagnostic biomarker for canine mesothelioma.

Results: Mesothelin complementary deoxyribonucleic acid (cDNA) was amplified via RT-PCR, yielding a single band of expected upon DNA electrophoresis. Sequence analyses confirmed it as a predicted canine mesothelin transcript from the genome sequence database. Comparative sequence analysis of the deduced amino acid sequence of the expressed canine mesothelin demonstrated molecular signature similarities with the human mesothelin. However, the pre-sequence of canine mesothelin lacks the mature megakaryocyte potentiating factor (MPF) portion, which is typically cleaved post-translationally with furin. Mesothelin expression was quantified via qPCR revealing low levels in the mesothelial and lung tissues, with negligible expression in the other major organs. Canine mesothelin exhibited significantly higher expression in the canine mesotheliomas than in the noncancerous tissues. Moreover, analysis of clinical samples using qPCR demonstrated markedly elevated mesothelin expression in canine mesotheliomas compared to non-mesothelioma cases.

Discussion and conclusion: Canine mesothelin exhibits molecular and biological characteristics akin to human mesothelin. It could serve as a vital biomarker for diagnosing canine mesotheliomas, applicable to both tissue- and effusion-based samples.

Keywords: biomarker; cancer; diagnosis; effusion; immunotherapy; megakaryocyte potentiating factor; sequence analysis; tissue.

Figure 1

Canine mesothelin variants and primer designs. A database search showed nine canine mesothelin splicing variants. Canine mesothelin splicing variants are likely a product of alternative selection of the 3′ or 5′ splice sites. The target region for reverse transcriptase-polymerase chain reaction (RT-PCR) was exons 5–8, with the reverse primer designed to span the exon boundary. The amplicon sequence shared 100% identity with the canine mesothelin variant 2 (XM_038669678). Predesigned TaqMan probes and primer sets for mesothelin, designed to cover all variants, were used for quantitative PCR (qPCR); therefore, allowing evaluation of the changes in the overall mesothelin gene expression, irrespective of the variant that was considered biologically crucial.

Figure 2

Comparing the amino acid sequences of the canine and human mesothelin. In the region where mature mesothelin is coded (red), canine mesothelin isoform 2 (XP_038525606), encoded by its variant 2, demonstrated substantial (70.34%) sequence identity with the human mesothelin isoform 1 (NP_001170826), the predominant isoform of mesothelin in humans. Comparison of canine and human mesothelin amino acids sequence revealed canine mesothelin could have a potential signal peptide (residues 37–69, corresponding to residues 1–37 of human mesothelin depicted in purple line), furin protease cleavage site (a predicted cleavage site at Arginine¹⁰⁹, corresponding to Arginine²⁹⁵ of human mesothelin), disulfide bond (Cysteine¹³⁹ and Cysteine¹⁶³, corresponding to Cysteine³⁰² and Cysteine³²⁶ of human mesothelin depicted in orange thick arrow), glycosylation residues (N-linked glycan at Asparagine²²⁵ and Asparagine³⁵², corresponding to Asparagine³⁸⁸ and Asparagine⁵¹⁵ of human mesothelin depicted in blue arrowhead), and GPI-anchorage attachment site (a predicted cleavage site at Serine⁴³⁵, corresponding to Serine⁵⁹⁸ of human mesothelin). Moreover, functionally important amino acids in human mesothelin (starts; tyrosine³¹⁸, tryptophan³²¹, glutamic acid³²⁴) were all conserved in canine mesothelin (tyrosine¹⁵⁵, tryptophan¹⁵⁸, glutamic acid¹⁶¹).

Figure 3

Mesothelin expression distribution in dogs and overexpression in canine mesotheliomas. The mesothelin expression distribution in dogs was analyzed using quantitative polymerase chain reaction (qPCR). Mesothelin expression was low in the mesothelial and lung tissues, whereas only negligible expression was observed in the other major organ tissues. Mesothelin was overexpressed (p < 0.001) in the mesothelioma cells.

Figure 4

Analysis of mesothelin expression in various tumors in dogs. Mesothelin expression in various canine tumors was examined using quantitative polymerase chain reaction (qPCR). (A) Significantly higher mesothelin levels were detected in the mesotheliomas than in non-mesothelioma (non-MS) tumors (p < 0.001). (B) Among the non-MS tumors, epithelial tumors expressed significantly higher mesothelin levels than mesenchymal tumors (p = 0.045). (C) Malignant tumors exhibited mesothelin levels similar to those in the benign tumors (p = 0.656). (D) When comparing malignant tumors, epithelial malignancies showed relatively higher expression levels than soft tissue sarcomas and other malignant tumors. Mesotheliomas exhibited greater overexpression compared to the non-MS malignancies.

Figure 5

Analysis of mesothelin expression in the effusions in dogs. Mesothelin expression in the clinical effusion samples was analyzed using quantitative polymerase chain reaction (qPCR). Mesothelin was overexpressed in the mesothelioma (MS) effusions compared to the non-mesothelioma (non-MS) effusions (p < 0.001), underscoring the diagnostic utility of mesothelin expression analysis in canine mesothelioma effusions.