Albert Taiching Liao

Characterization of STAT3 activation and expression in canine and human osteosarcoma

BackgroundDysregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a key participant in tumor cell survival, proliferation, and metastasis and is often correlated with a more malignant tumor phenotype. STAT3 phosphorylation has been demonstrated in a subset of human osteosarcoma (OSA) tissues and cell lines. OSA in the canine population is known to exhibit a similar clinical behavior and molecular biology when compared to its human counterpart, and is often used as a model for preclinical testing of novel therapeutics. The purpose of this study was to investigate the potential role of STAT3 in canine and human OSA, and to evaluate the biologic activity of a novel small molecule STAT3 inhibitor.MethodsTo examine STAT3 and Src expression in OSA, we performed Western blotting and RT-PCR. OSA cells were treated with either STAT3 siRNA or small molecule Src (SU6656) or STAT3 (LLL3) inhibitors and cell proliferation (CyQUANT), caspase 3/7 activity (ELISA), apoptosis (Western blotting for PARP cleavage) and/or viability (Wst-1) were determined. Additionally, STAT3 DNA binding after treatment was determined using EMSA. Expression of STAT3 targets after treatment was demonstrated with Western blotting, RT-PCR, or gel zymography.ResultsOur data demonstrate that constitutive activation of STAT3 is present in a subset of canine OSA tumors and human and canine cell lines, but not normal canine osteoblasts. In both canine and human OSA cell lines, downregulation of STAT3 activity through inhibition of upstream Src family kinases using SU6656, inhibition of STAT3 DNA binding and transcriptional activities using LLL3, or modulation of STAT3 expression using siRNA, all resulted in decreased cell proliferation and viability, ultimately inducing caspase-3/7 mediated apoptosis in treated cells. Furthermore, inhibition of either Src or STAT3 activity downregulated the expression of survivin, VEGF, and MMP2, all known transcriptional targets of STAT3.ConclusionThese data suggest that STAT3 activation contributes to the survival and proliferation of human and canine OSA cells, thereby providing a potentially promising target for therapeutic intervention. Future investigational trials of LLL3 in dogs with spontaneous OSA will help to more accurately define the role of STAT3 in the clinical setting.

A novel small molecule Met inhibitor, PF2362376, exhibits biological activity against osteosarcoma.

The receptor tyrosine kinase Met is dysregulated in several human cancers including osteosarcoma (OSA) in which overexpression is a negative prognostic indicator and enforced Met expression in normal osteoblasts leads to genomic instability and malignant transformation. Met is also known to be inappropriately expressed in canine OSA tumour samples and cell lines. The purpose of this study was to evaluate the potential utility of an orally bioavailable small molecule Met inhibitor, PF2362376, against canine OSA cell lines as a prelude to future clinical work. PF2362376 inhibited phosphorylation of Met, Gab-1, Erk and Akt, but not of Src or STAT3. Furthermore, PF2362376 inhibited proliferation of canine OSA cell lines and induced cell death at biologically achievable concentrations. Last, activities associated with Met signalling including migration, invasion, branching morphogenesis and colony formation in soft agar were blocked by PF2362376. These studies support the notion that Met is a relevant target for therapeutic intervention in OSA.

Characterization, expression and function of c-Met in canine spontaneous cancers

Aberrant expression of the proto-oncogene c-Met has been noted in a variety of human cancers. To better define the potential role of Met dysregulation in canine cancer, the canine Met, hepatocyte growth factor (HGF) and HGF activator were cloned. Inappropriate expression of Met was present in canine tumour cell lines derived from a wide variety of cancers. Furthermore, both HGF and HGF activator were also expressed in several of these cell lines, providing evidence of a possible autocrine loop of Met activation. Stimulation of tumour cell lines with recombinant human HGF induced Met autophosphorylation, as well as activation of the downstream signalling elements Gab-1, Akt and Erk1/2. Scattering of tumour cells and migration across a defect occurred in response to HGF stimulation. The Met inhibitor PHA665752 blocked both HGF-induced phosphorylation of canine Met and HGF-mediated cell cycling, scattering and migration. These studies provide evidence that Met dysregulation may play a role in the biology of canine cancer and lay the groundwork for future studies employing Met inhibitors.

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.

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.

Gene-expression profiling to identify genes related to spontaneous tumor regression in a canine cancer model.

Microarray transcriptome study in cancer has been commonly used to investigate tumorigenic mechanisms. The unique growth pattern of spontaneous regression (SR) after progressive (P) growth in canine transmissible venereal tumor (CTVT) provides a valuable cancer model to study the genome-wide differences in samples between the two stages of growth. In this study, Affymetrix analysis was performed based on the canine genome to compare the gene expression profiles of CTVT P- and SR-phase tumors. A total of 459 (278 up-regulated and 181 down-regulated) genes were identified as being differentially-expressed during the SR phase by the 2-fold method. Further analysis of these genes revealed that the expression of three genes associated with IL-6 production -TIMD-4, GPNMB and PLTP - was significantly higher in SR-phase tumors than in P-phase tumors; these results were also confirmed by real time RT-PCR in tumor tissues of beagles. In addition, we found that Th17-related genes were over-expressed in the SR phase, suggesting autoimmune responses involvement in tumor regression. Although the interaction between CTVT and host immunity were partially investigated in previous studies, our results enable us to gain new insight into the genes and possible mechanisms involved in tumor regression and reveal potentially useful targets for cancer therapy.

Chemotherapy-induced neutropenia is associated with prolonged remission duration and survival time in canine lymphoma.

Myelosuppression is one of the most common side effects of chemotherapy. The aim of this study was to determine whether chemotherapy-induced neutropenia is a positive prognostic indicator for remission and survival time in dogs with lymphoma. Fifty dogs with multicentric lymphoma received CHOP-based (C-cyclophosphamide; H-hydroxydaunorubicin; O-vincristine; P-prednisolone) chemotherapy using conventional dosages. Complete blood counts were recorded to determine the presence or absence of neutropenia after treatment. Toxicity, remission, and survival times were recorded and analysed. Thirteen dogs had chemotherapy-induced neutropenia and 37 had no neutropenia during the study period. No statistical difference was found between the groups for signalment or the presence of historical negative prognostic factors, except for bodyweight (P = 0.02). The median first remission times in the neutropenia and no neutropenia groups were 812 and 219 days, respectively (P <0.01). The median survival times of dogs in the neutropenia and no neutropenia groups were 952 and 282 days, respectively (P <0.01). Dogs with lymphoma that had chemotherapy-induced neutropenia exhibited significantly increased remission and survival times compared with dogs without neutropenia. Chemotherapeutic dosages may be adjusted individually to induce neutropenia without severe adverse effects in order to achieve longer remission and survival times.

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.

Overexpression of chemokine ligand 7 is associated with the progression of canine transmissible venereal tumor

BackgroundChemokines play multiple roles in the development and progression in a variety of tumors. Chemokine (C-X-C motif) ligand 7 (CXCL7) has been found associated with pro-inflammatory responses, but its role in cancer growth remains unclear. Our previous study showed that R phase tumor infiltrating lymphocytes (TILs) produced large amounts of interleukin (IL)-6 which antagonized transforming growth factor (TGF)-β derived from CTVT to diminish the immune-suppressive microenvironment. Now we intend to determine the expression pattern of CXCL7 and the role of IL-6/TGF-β in CXCL7 induction during spontaneous progressive (P) and regressive (R) phases in canine transmissible venereal tumor (CTVT).ResultsWe have demonstrated that CXCL7 expressed at high level in P phase and down-regulated in R phase by western blot and real-time PCR. This suggested that CXCL7 expression was negatively correlated with the tumor growth. Co-culturing TILs with CTVT cells was found to reduce CXCL7 expression, while adding IL-6 blocking antibody reversed it. Moreover, in P phase CTVT, while IL-1β and TGF-β had no obvious effect on CXCL7 expression, IL-6 was found significantly to reduce CXCL7 expression in a dose-dependent manner. The mRNA expression results of CXCL7 receptor, CXCR2, further confirmed the effects of IL-6 concentration on the CXCL7 expression.ConclusionCXCL7 overexpression might be associated with the progressive growth of CTVT. The results shown here also suggest the role of CXCL7 in cancer development and the potential as the anti-cancer therapeutic target.

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.