Chen-Hsuan Liu

Molecular Identification of Canine Podocalyxin-Like Protein 1 as a Renal Tubulogenic Regulator

GP135 is an apical membrane protein expressed in polarized MDCK epithelial cells. When cultured in three-dimensional collagen gel, MDCK cells form branching tubules in response to hepatocyte growth factor stimulation in a manner that simulates the embryonic renal development. During this process, GP135 displays transient loss of membranous localization but reappears at the cell surface when nascent lumen emerges from the developing tubules. Despite being used for decades as the canonical hallmark of apical surface, the molecular identity and the significance of the dynamic expression of GP135 during the tubulogenic process remain elusive. For exploring the function of GP135, the full-length cDNA encoding GP135 was obtained. Sequence alignments and features analysis confirm GP135 as a canine homolog of podocalyxin, confirming the finding of an earlier independent study. Immunohistochemical assays on canine kidney sections identified both glomerular and tubular distribution of GP135 along the nephron. Mutant MDCK cells expressing siRNA targeted at two regions of GP135 show defects in hepatocyte growth factor-induced tubulogenesis. Re-expression of full-length and an O-linked glycosylation abbreviated construct of GP135 could recapitulate the tubulogenesis process lacking in siRNA knockdown cells; however, a deletion construct devoid of the cytoplasmic domain failed to rescue the phenotype. In summary, the data identify the MDCK apical domain marker GP135 as a tubular form of podocalyxin and provide evidence for its importance in renal tubulogenesis.

KIT gene exon 11 mutations in canine malignant melanoma

The proto-oncogene KIT encodes a receptor tyrosine kinase which has been shown to be upregulated in canine melanomas. KIT mutations lead to constitutive phosphorylation and activation of KIT in the absence of ligand binding. The presence of KIT mutations and KIT protein expression was examined in a cohort of 49 dogs with canine malignant melanoma. An exon 11 synonymous nucleotide 1743C→T mutation was identified in five cases in which one also harbored a L579P mutation. Tumors that harbored the KIT exon 11 mutation(s) correlated significantly with disease recurrence (P = 0.05). All 36 melanomas available for immunohistochemical analysis showed either weak (16 cases, 44.4%) or strong (20 cases, 55.6%) expression of the KIT protein. The five KIT mutation carriers were all strongly positive for KIT by immunohistochemical staining. These findings suggest that a subset of canine malignant melanomas harbors a KIT exon 11 mutation.

Chicken HSP70 DNA vaccine inhibits tumor growth in a canine cancer model.

Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4(+) subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true clinical potential.

Overexpression of α-enolase correlates with poor survival in canine mammary carcinoma.

Backgroundα-Enolase (ENO1) is a key glycolytic enzyme implicated in the development of many human cancers including breast cancer. Increased expression of ENO1 has recently been reported in estrogen (ER)-positive human breast cancer patients. The present study examined the expression of ENO1 and assessed its significance in canine mammary carcinoma.ResultsImmunohistochemical staining was employed to investigate the expression of ENO1 in 82 cases of canine mammary tumor (32 benign tumors and 50 carcinomas). Quantification of immunohistochemistry was carried out using Quick score and the results showed cytoplasmic ENO1 overexpression in 9 of the 50 carcinomas (18%). Overexpression of ENO1 correlated significantly with shorter cause-specific survival (P = 0.019), but was not associated with ER positivity in canine mammary carcinoma.ConclusionsOur findings suggest that overexpression of ENO1 may be used as a prognostic marker for poor outcome in canine mammary carcinoma.

Hydatidosis in a Chapman's zebra (Equus burchelli antiquorum)

A 14-year-old male Chapmans zebra had been treated for a cervical granulomatous cellulitis for 3 months prior to sudden death associated with myocardial fibrosis. Incidental findings at necropsy included multiple 3–8-cm unilocular cysts in the liver and lungs. Cysts contained either a clear fluid with or without hydatid sand or a gelatinous substance with foci of mineralization. Light and scanning electron microscopic examination of the hydatid sand revealed free protoscolices as well as intact and ruptured brood capsules with protoscolices attached to the germinal membrane. The protoscolices had 2 rows of 36–38 rostellar hooks with a length of 25–30 μm. The cyst wall consisted of inner germinal, intermediate laminated, and outer adventitial layers. Hydatidosis caused by Echinococcus granulosus was diagnosed based on the unilocular cysts, multiple protoscolices formed in a brood capsule, typical trilayered cyst wall, and herbivorous intermediate host. This is the first reported case of animal hydatidosis in Taiwan. The infection is thought to have been established 12 years ago in South Africa prior to importation.

Downregulation of the KLF4 transcription factor inhibits the proliferation and migration of canine mammary tumor cells

Canine mammary tumor (CMT) is the most common neoplasm in female dogs, and over 50% of CMTs are diagnosed as malignant. Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and is associated with cell proliferation, differentiation, migration, and apoptosis. Although the role of KLF4 is still controversial in various human cancers, KLF4 has been identified as an oncogene in human breast cancer. Moreover, high KLF4 expression is correlated with an aggressive phenotype in CMT. Therefore, investigating the function of KLF4 may help better understand the pathogenesis of CMT. In this study, partial sequences of canine KLF4 and KLF4 expression were identified in various normal canine tissues, as well as CMT cells and Madin-Darby canine kidney (MDCK) cells. Kenpaullone, a small molecule inhibitor of KLF4, downregulated KLF4 expression in CMT cells and reduced CMT cell proliferation, migration, and colony formation in soft agar. Kenpaullone treatment induced S and G2/M phase arrest in CMT and MDCK cells, and induced death in CMT cells, but not in MDCK cells. It was concluded that KLF4 is expressed in various normal canine tissues, and downregulation of KLF4 inhibited CMT cell proliferation and migration, and induced cell death. The results of this study suggest that KLF4 may represent a suitable therapeutic target for CMT therapy.

Scirrhous gastric carcinoma with mediastinal invasion in a dog

An 8-year-old male Rottweiler was presented for recurrent episodes of dysphagia and vomiting with chronic weight loss. Radiography revealed a mediastinal mass in the heart base region. Necropsy revealed a firm, white mediastinal mass extending along the distal esophagus, through the diaphragm, to the gastric cardia, leftward to the convex visceral aspect of the fundus, and rightward along the lesser curvature of the stomach to the pyloric antrum. The gastric lymph node was enlarged and the omentum contained several nodules. Histologically, deep fundic mucosa contained pleomorphic, vacuolated cells with intracytoplasmic mucin, which was hyaluronidase resistant. Neoplastic cells were cytokeratin positive and vimentin negative. Transmural invasion was evidenced by the presence of cytokeratin-positive cells between smooth muscle bundles of the gastric wall. The mediastinal mass was composed of clusters of neoplastic cells in a stroma of dense and loose connective tissue. Neoplastic cells were also within blood and lymphatic vessels, tracheobronchial and gastric lymph nodes, and around peripheral nerves. This carcinoma most likely arose from the gastric fundus and extended to the cardia, from where it advanced proximally to the mediastinum as well as further rightward along the lesser curvature, demonstrating an anatomic continuity suggestive of a direct invasion. Metastasis, evidenced by the presence of lymphatic, blood, and perineural tumor emboli, also occurred.

Expression of autophagy-related protein beclin-1 in malignant canine mammary tumors

BackgroundAutophagy is a self-catabolic mechanism that degrades unnecessary cellular components through lysosomal enzymes. Beclin-1, an autophagy-related protein, establishes the first connection between autophagy and tumorigenesis. The purpose of this study is to assess the Beclin-1 expression pattern and to determine its prognostic significance in patients with malignant canine mammary tumor (CMT).ResultsWe examined Beclin-1 expression in 70 cases of malignant CMTs by immunohistochemistry. Cytoplasmic Beclin-1 expression was significantly weaker in cancer cells than in nearby normal mammary glands (p < 0.001). Low cytoplasmic expression (57.14%) was associated with older age, lower degree of tubular formation, increased mitotic activity, higher histologic grade, and extensive necrosis. Low nuclear expression (40%) was connected with older age, lower degree of tubular formation, extensive necrosis, and negative for Her2/neu overexpression. Univariate survival analysis showed that Beclin-1 cytoplasmic expression was a poor prognostic factor for overall survival rate (p < 0.001). Multivariate survival analysis demonstrated that Beclin-1 cytoplasmic expression is an independent prognostic factor (p = 0.016).ConclusionsLoss of Beclin-1 is associated with aggressive clinicopathologic features and poor overall survival. The results suggest that Beclin-1 plays an important role in tumor progression of malignant CMTs.

Elevated Krüppel-like factor 4 transcription factor in canine mammary carcinoma

BackgroundKrüppel-like factors (KLFs) are critical regulators of biological and physiological systems and have been extensively studied for their roles in cell proliferation, differentiation and survival in the context of cancer. Among the KLFs, KLF4 is highly expressed in human breast cancers and plays an oncogenic role. The present study examined the expression of KLF4 and assessed its significance in canine mammary carcinoma.ResultsImmunohistochemistry was employed to investigate the expression of KLF4 in 142 cases of canine mammary tumor. 75 of the 142 (52.8%) cases were histologically confirmed as mammary carcinoma. Quantification of immunohistochemistry was carried out using Quick score which multiply the staining intensity by the percentage of positive cells. High KLF4 expression was identified in 44 of the 75 (59%) dogs with mammary carcinoma and none in the benign cases. High KLF4 expression occurred only in the tumor cells and not the adjacent normal cells in mammary carcinoma (P < 0.001). Moreover, the high expression level of KLF4 expression was statistically associated with poor grade, late stage, histological subtypes of simple and complex carcinoma, and shorter 24-month survival. The Kaplan-Meier survival analysis also indicated that dogs with high nuclear KLF4 expression had a significantly shorter survival than those with low/moderate KLF4 expression (P = 0.011).ConclusionsKLF4 is highly and frequently expressed in canine mammary carcinoma and correlates with a more aggressive phenotype.

Functional assessment of expiratory flow pattern in feline lower airway disease

Feline lower airway disease (FLAD) is a chronic respiratory disease of which there is a lack of information on functional assessment in current veterinary medicine. The purposes of this study were to investigate expiratory pattern and evaluate the diagnostic utility of functional parameters in cats with FLAD. Thirty-three client-owned cats (23 FLAD cats and 10 control cats) were studied. Under quiet tidal breathing, pseudo-tidal breathing flow-volume loop (pTBFVL) was obtained from a barometric whole body plethysmography (BWBP) device. There were significant differences in the shapes of expiratory, but not inspiratory, curves between FLAD and control cats. The incidence of the presence of concave expiratory curve indicating lower airway obstruction was 74% in FLAD cats. To assess the diagnostic utility of pTBFVL indices in cats with FLAD, area under the receiver–operator curve was 0.86 for PEF/EF50 (peak expiratory flow divided by expiratory flow at end expiratory volume plus 50% tidal volume); a cuff-off value of PEF/EF50 >1.51 distinguished normal from FLAD (73.9% sensitivity, 100% specificity). There were no significant differences in traditionally measured BWBP parameters (ie, enhanced pause) between cats with and without FLAD in the present study. In conclusion, underlying change on expiratory flow pattern during natural tidal breathing existed in FLAD cats, and selected pTBFVL indices were useful in discriminating FLAD from normal cats. Tidal breathing pattern depicted by pseudoflow–pseudovolume loops from a BWBP system could be a non-invasive tool for functional assessment in client-owned cats.