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Lysyl oxidase (LOX) is an extracellular metalloenzyme which mediates crosslinking of collagen and elastin. It has been reported to play a pivotal role in cancer metastasis especially in women suffering from breast cancer. The present study is the first to evaluate the gene expression levels of LOX by Real time-polymerase chain reaction (Real time-PCR) in dogs with mammary tumor besides molecular cloning and expression of canine lysyl oxidase gene (lox). Real time-PCR studies showed a significant upregulation (threefold higher) of lox in mammary tumor cases as compared to healthy dogs indicating its possible diagnostic and prognostic role in canine mammary tumors (CMTs). Cloning and sequencing of lox gene revealed 1230 bp CDS which is mostly conserved in C-terminal region. Sequence analysis of canine lox showed that it shares 99% homology with the predicted sequence available on NCBI and had greatest identity with the lox gene from cat. Protein structure predicted with homology modelling was validated by Ramachandran plot analysis which revealed most (approximately 95%) of the amino acids in favoured region. Additionally, recombinant lysyl oxidase expressed as His-tagged fusion protein in prokaryotic expression vector (pPROExHTa) was used in an ELISA for detection of circulating protein LOX in serum of CMT subjects. Receiver operating characteristics analysis of the ELISA revealed high sensitivity (90%) and specificity (85%) with histopathology as reference standard. Taken together, we propose LOX as a diagnostic biomarker and a putative prognostic candidate in CMT cases.

Background: Mammaglobin, a member of secretoglobin family has been recognized as a breast cancer associated protein. Though the exact function of the protein is not fully known, its expression has been reported to be upregulated in human breast cancer.We focused on studying the expression of mammaglobin-B gene and protein in canine mammary tumor (CMT) tissue. Expression of mammaglobin-B mRNA and protein were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively., Results: High levels of mammaglobin-B mRNA expression (6.663 ± 0.841times) was observed in CMT as compared to age and breed matched healthy controls. Further, expression of mammaglobin-B protein was detected in paraffin-embedded mammary tumor tissues from the same subjects by IHC. Mammaglobin-B protein was overexpressed only in 6.67% of healthy mammary glands while, a high level of its expression was scored in 76.7% of the CMT subjects. Moreover, no significant differences in terms of IHC score and qRT-PCR score with respect to CMT histotypes or tumor grades were observed, indicating that mammaglobin-B over-expression occurred irrespective of CMT types or grades., Conclusion: Overall, significantly increased expression of mammaglobin-B protein was found in CMTs with respect to healthy mammary glands, which positively correlates to its transcript. These findings suggest that overexpression of mammaglobin-B is associated with tumors of canine mammary glands.

Heat shock proteins (Hsp) are molecular chaperones that are responsible for protein folding and maintenance of cellular homeostasis. Hsp90, an important member of HSP family, has an important role in breast cancer. Glucose-regulated protein 94 (Grp94) is the endoplasmic reticulum paralog of Hsp90 encoded by Hsp90B1 gene. To test if this protein is overexpressed in dogs with mammary tumor, we estimated and compared its serum levels in healthy dogs and that of dogs with mammary tumors. Hsp90B1 mRNA expression was measured in tumorous and healthy mammary tissues (from age- and breed-matched dogs) by real-time PCR. The gene was found to be overexpressed in mammary tumors (3.586 ± 0.067 times). Further, it was heterologously expressed in a prokaryotic system as 90 kDa protein. A recombinant Grp94-based sandwich ELISA was developed to quantify serum Grp94 in dogs with mammary tumors. Based on receiver-operating characteristics' analysis, the assay was found to be 90.62% sensitive and 93.75% specific for a cutoff value of 0.35 with respect to histopathological staining in diagnosing the disease. The t test showed that serum Grp94 levels were significantly elevated (92.97 ± 3.62 ng/ml) in dogs with mammary tumors compared with healthy controls (10.30 ± 0.79 ng/ml) (p < 0.0001). These findings suggest that Grp94 might act as a potential biomarker for prognosis of canine mammary tumors and monitoring its therapy.

Matrix metalloproteinase-3 is invariably upregulated in cancerous condition. So we aimed to determine serum level of MMP-3 in canine mammary tumors. The gene was expressed in E. coli system as ~43kDa recombinant protein, which was refolded, purified, and confirmed. Hyperimmune serum was raised against the expressed protein in rabbits and mice to standardize sandwich ELISA. ROC analysis revealed largest area under the curve of 0.998 with sensitivity (100%) and specificity (95%) for a cut-off value of 0.363 with respect to histopathological staining. The finding of the present study indicates that MMP-3 can act as a potential molecular marker for serodiagnosis of canine mammary carcinomas.

All four members of the tissue inhibitors of metalloproteinase (TIMP) family have been reported to be over-expressed in breast cancer cells in vitro. Dysregulation of TIMP-4 expression predicts poor prognosis in cancers. The present study evaluated the association of the expression levels of TIMP-4 with mammary tumor development in dogs, measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mammary tissue samples were collected from healthy canine mammary gland and from tumor subjects. TIMP-4 expression was found to be upregulated (5.856 times) in complex canine mammary carcinomas. Also, TIMP-4 mature peptide was expressed heterologously in E. coli. The recombinant protein was purified by Ni- NTA affinity chromatography and further confirmed by western blotting. The rTIMP-4 was found to be functionally active and could inhibit matrix metalloproteinase 11(MMP-11) activity. Immunization of mice with rTIMP-4 resulted in increased antigen specific serum antibody titer, and this serum could be suitably used to detect and quantify the protein in sera of dogs with mammary tumors. TIMP-4 could act as a marker of canine mammary tumors. To the best of our knowledge, this is the first report of heterologous expression of TIMP-4 from complex canine mammary carcinomas.

Mammaglobin is a glycoprotein exhibiting homology to uteroglobin gene family. Although the biological function of the protein is not yet known it has been reported to act as marker for breast cancer in women. This study reports the expression of mammaglobin gene in canine mammary tumor condition. The gene was cloned, sequenced and heterologously expressed in Escherichia coli host system as 12 kDa (approx.) recombinant fusion protein. The expressed protein was further purified to homogeneity and confirmed by western blotting. Hyperimmune sera were raised against the expressed protein in rabbits and mice to standardize sandwich ELISA for relative quantification of circulating protein in the sera of dogs with mammary tumors. Based on receiver-operating characteristics analysis, the test was found to be 90% sensitive and 95% specific for a cut-off value of 0.177 with respect to histopathological staining in diagnosing canine mammary tumors and the protein level was not elevated in other diseased conditions. These findings indicate that it can act as a novel molecular marker for detecting mammary tumors in canines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinases (MMPs) are reported to be involved in tumor growth, apoptosis, angiogenesis, invasion, and development of metastases. These are zinc containing metalloproteases, known for their role in extracellular matrix degradation. MMP-11 (stromelysin3) is reported to be highly expressed in breast cancer, therefore it may act as marker enzyme for breast cancer progression. The present work was carried out to produce recombinant canine (Canis lupus familiaris) MMP-11 lacking the signal and propeptide in E. coli by optimizing its expression and purification in biologically active form and to functionally characterize it. A bacterial protein expression vector pPROEX HTc was used. The MMP-11 mature peptide encoding gene was successfully cloned and expressed in E. coli and the purified recombinant enzyme was found to be functionally active. The recombinant enzyme exhibited caseinolytic activity and could be activated by Trypsin and 4-Amino phenyl mercuric acetate (APMA). However Ethylene diamine tertra acetate (EDTA) inhibited the enzyme's caseinolytic activity. The recombinant enzyme degraded extracellular matrix constituents and facilitated migration of MDCK (Madin-Darby canine kidney) cells through BD Biocoat Matrigel invasion chambers. These results suggest that in vivo MMP-11 could play a significant role in the turnover of extracellular matrix constituents.