Prognostic significance of circulating microRNA-214 and -126 in dogs with appendicular osteosarcoma receiving amputation and chemotherapy

Abstract Background Dogs with appendicular osteosarcoma (OSA) receiving standard amputation and adjuvant chemotherapy demonstrate variable outcome with treatment; however, additional biomarkers would be helpful for predicting their outcome. In the present study, we assessed the potential of circulating microRNA-214 (miR-214) and − 126 (miR-126) to predict time to metastasis and death in dogs with OSA treated with amputation and chemotherapy. Results Seventy-six dogs that fully met inclusion criteria were included in the analysis. The criteria included (1) a diagnosis of appendicular OSA without metastases at diagnosis, (2) treatment by amputation and chemotherapy using carboplatin, doxorubicin, cisplatin, or a combination of these agents. Circulating miR-214 and -126 levels at the time before treatment were measured by using RT-qPCR. High circulating miR-214 and serum alkaline phosphatase (ALP) significantly predicted short disease-free survival (DFS) and overall survival (OS). Conversely, high circulating miR-126 significantly predicted prolonged DFS and OS. An integrated approach using circulating miR-214, − 126, and serum ALP showed better accuracy in the prediction of DFS and OS and identification of long-term survivors than prediction using only ALP. Other variables (age, weight, sex, monocyte counts, and primary tumor site) were associated with neither DFS nor OS. miRNA levels did not strongly correlate with histopathological indices. Conclusions Circulating miR-214, − 126, and an integrated prognostic score have strong potential to predict the outcome of canine appendicular OSA patients receiving amputation and chemotherapy

In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma

<div><p>While extremely rare in humans, hemangiosarcoma (HSA) accounts for nearly 2% of canine neoplasia, and is characterized by both aggressive local growth/invasion and a high rate of metastasis. Both canine and human HSA exhibit sustained aberrant PI3K/Akt/mTOR pathway signaling. The purpose of this study was to examine the <i>in vitro</i> effects of a novel dual PI3K/mTOR inhibitor, VDC-597, in three canine HSA cell lines (DEN-, CIN-, and SB-HSA). VDC-597 suppressed activation of both Akt and 4eBP1 in canine HSA cells in a dose-dependent fashion, with an IC50 of approximately 0.3 uM, a concentration predicted to be clinically achievable based on preliminary early-phase canine and human studies. VDC-597 dose-dependently reduced proliferation, migration, and vascular endothelial growth factor production in HSA cells, while promoting tumor cell apoptosis. VDC-597 demonstrated additive antiproliferative effects when combined with doxorubicin. These results suggest that inhibitors of the PI3K/mTOR pathway may act against multiple components of the neoplastic process, including proliferation/apoptosis, chemosensitivity, migration, and angiogenesis, and justify the evaluation of PI3K/mTOR inhibitors in canine, and potentially human, HSA.</p></div

Chemical structure of VDC-597.

<p>Chemical structure of VDC-597.</p

In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma - Fig 3

<p>(A)50% Inhibitory concentrations following a 72-hour exposure to VDC-597 in canine tumor cell lines. Canine tumor cells were incubated in C/10 EMEM or C/10 EMEM with various concentrations of VDC-597 for 72 hours, followed by determination of relative viable cell number using a bioreductive fluorometric assay. IC50s were calculated using nonlinear regression, and presented values indicate means from 3 separate experiments. (B). VDC-597 Dose-Dependently Inhibits the Growth of Canine Hemangiosarcoma Cells. Canine HSA cells were exposed to varying concentrations of VDC-597 followed by determination of relative viable cell number as above. Calculated IC50s were 0.23, 0.69 and 0.71 uM for CIN-, SB-, and DEN-HSA respectively. Results shown indicate means of 3 separate experiments. Error bars indicate standard error measurements.</p

The dual PI3K/mTOR Inhibitor VDC-597 reduces downstream phosphorylation of Akt and 4eBP1 in canine hemangiosarcoma cells.

<p>DEN, CIN, and SB-HSA were incubated for 24 hours in standard conditions with either C/10 EMEM or C/10 EMEM with VDC-597 (range of concentrations for DEN-HSA, 1 uM for CIN- and SB-HSA), followed by protein extraction and western analysis. The addition of VDC-597 reduced downstream phosphorylation of both Akt and 4EBP1, as assessed by comparing the ratio of phospho-protein to total protein in each condition.</p

VDC-597 in combination with doxorubicin provides additive inhibitory effects on canine hemangiosarcoma cell growth.

<p>DEN, CIN, and SB-HSA cells were incubated in C/10 EMEM with varying concentrations of doxorubicin, or co-treated in C/10 EMEM with VDC-597 and doxorubicin for 72 hours followed by determination of relative viable cell number as above. Error bars indicate standard error measurements.</p

VDC-597 inhibits proliferation and induces apoptosis of canine hemangiosarcoma cells.

<p>NucLight Red-expressing SB-HSA cells were incubated for 72 hours with C/10 or C/10 with various concentrations of VDC-597 with the addition of YoYo-1 iodide reagent to label apoptotic cells. The number of viable (<b>A</b>) and number of apoptotic (<b>B</b>) cells per well were determined using an IncuCyte real-time videomicroscopy system. Area under the curve for each condition was calculated. Asterisks indicate <i>P</i> values less than 0.05. Error bars indicate standard error measurements.</p

VDC-597 inhibits VEGF production in canine hemangiosarcoma cells.

<p>DEN, CIN, and SB-HSA cells were incubated in C/10 EMEM with various concentrations of VDC-597. 24 hours later, supernatants were harvested and utilized in a canine VEGF-specific ELISA. Relative viable cell number was concurrently assessed and VEGF concentration normalized to cell number. Error bars represent standard error measurements. Asterisks indicate <i>P</i> values less than 0.05.</p

Antibodies utilized for western analysis.

<p>Antibodies utilized for western analysis.</p

WNT7B in fibroblastic foci of idiopathic pulmonary fibrosis

<p>Abstract</p> <p>Background</p> <p>Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial pneumonia causing a loss of respiratory surface area due to a proliferative fibrotic response involving hyperplastic, hypertrophic, and metaplastic epithelium, cystic honeycomb change, septal expansion, and variable inflammation. Wnt (wingless) signaling glycoproteins are known to be involved in lung development and tissue repair, and are up-regulated in patients with IPF. Based on previous qRT-PCR data showing increased Wnt7B in lungs of IPF patients, a systematic, quantitative examination of its tissue site distribution was undertaken.</p> <p>Methods</p> <p>Tissue samples from the Lung Tissue Research Consortium (LTRC) of 39 patients diagnosed with mild to severe IPF/usual interstitial pneumonia (UIP) and 19 normal patients were examined for the immunolocalization of Wnt7B.</p> <p>Results</p> <p>In normal lung, moderate Wnt7B reactivity was confined to airway epithelium, smooth muscle of airways and vasculature, and macrophages. IPF lung showed strong Wnt7B reactivity in fibroblastic foci, dysplastic airway and alveolar epithelium, and in highly discrete subepithelial, basement membrane-associated regions. All reactive sites were sized and counted relative to specific microscopic regions. Those in the subepithelial sites were found in significantly greater numbers and larger relative area compared with the others. No reactive sites were present in normal patient controls.</p> <p>Conclusions</p> <p>The results demonstrate Wnt7B to be expressed at high concentrations in regions of active hyperplasia, metaplasia, and fibrotic change in IPF patients. In this context and its previously established biologic activities, Wnt7B would be expected to be of potential importance in the pathogenesis of IPF.</p