Anti-tumor effects of the eIF4A inhibitor didesmethylrocaglamide and its derivatives in human and canine osteosarcomas.

Inhibition of translation initiation using eIF4A inhibitors like (-)-didesmethylrocaglamide [(-)-DDR] and (-)-rocaglamide [(-)-Roc] is a potential cancer treatment strategy as they simultaneously diminish multiple oncogenic drivers. We showed that human and dog osteosarcoma cells expressed higher levels of eIF4A1/2 compared with mesenchymal stem cells. Genetic depletion of eIF4A1 and/or 2 slowed osteosarcoma cell growth. To advance preclinical development of eIF4A inhibitors, we demonstrated the importance of (-)-chirality in DDR for growth-inhibitory activity. Bromination of DDR at carbon-5 abolished growth-inhibitory activity, while acetylating DDR at carbon-1 was tolerated. Like (-)-DDR, (±)-DDR, and (-)-Roc, (±)-DDR-acetate increased γH2A.X levels and induced G2/M arrest and apoptosis. Consistent with translation inhibition, these rocaglates decreased the levels of several mitogenic kinases, the STAT3 transcription factor, and the stress-activated protein kinase p38. However, phosphorylated p38 was greatly enhanced in treated cells, suggesting activation of stress response pathways. RNA sequencing identified RHOB as a top upregulated gene in both (-)-DDR- and (-)-Roc-treated osteosarcoma cells, but the Rho inhibitor Rhosin did not enhance the growth-inhibitory activity of (-)-DDR or (-)-Roc. Nonetheless, these rocaglates potently suppressed tumor growth in a canine osteosarcoma patient-derived xenograft model. These results suggest that these eIF4A inhibitors can be leveraged to treat both human and dog osteosarcomas.

Anti-tumor Effects of the eIF4A Inhibitor Didesmethylrocaglamide and Its Derivatives in Human and Canine Osteosarcomas.

Inhibition of translation initiation using eIF4A inhibitors like (-)-didesmethylrocaglamide [(-)-DDR] and (-)-rocaglamide [(-)-Roc] is a potential cancer treatment strategy as they simultaneously diminish multiple oncogenic drivers. We showed that human and dog osteosarcoma cells expressed high levels of eIF4A1/2, particularly eIF4A2. Genetic depletion of eIF4A1 and/or 2 slowed osteosarcoma cell growth. To advance preclinical development of eIF4A inhibitors, we demonstrated the importance of (-)-chirality in DDR for growth-inhibitory activity. Bromination of DDR at carbon-5 abolished growth-inhibitory activity, while acetylating DDR at carbon-1 was tolerated. Like DDR and Roc, DDR-acetate increased the γH2A.X levels and induced G2/M arrest and apoptosis. Consistent with translation inhibition, these rocaglates decreased the levels of several mitogenic kinases, the STAT3 transcription factor, and the stress-activated protein kinase p38. However, phosphorylated p38 was greatly enhanced in treated cells, suggesting activation of stress response pathways. RNA sequencing identified RHOB as a top upregulated gene in both DDR- and Roc-treated osteosarcoma cells, but the Rho inhibitor Rhosin did not enhance the growth-inhibitory activity of (-)-DDR or (-)-Roc. Nonetheless, these rocaglates potently suppressed tumor growth in a canine osteosarcoma patient-derived xenograft model. These results suggest that these eIF4A inhibitors can be leveraged to treat both human and dog osteosarcomas.

Pharmacokinetic Exposures Associated With Oral Administration of Sorafenib in Dogs With Spontaneous Tumors.

Sorafenib is a multi-kinase small molecule inhibitor that targets serine/threonine and tyrosine kinases including the RAF kinase family, VEGFR-2, and PDGFR. The aim of this study was to evaluate the systemic pharmacokinetics of a previously defined tolerable oral dose of sorafenib in tumor-bearing dogs. Six client-owned dogs with a cytologic or histologic diagnosis of cancer were enrolled in this open-label, tolerability study. Dogs were administered sorafenib at an intended dose of 3 mg/kg and serum samples were obtained for analysis of sorafenib serum concentrations at 0, 1, 2, 6, 12, 24, 48, 72, 96, and 168 h post-drug administration. Median time to peak serum sorafenib concentration occurred at 4 h (range 2-12 h) resulting in an average serum concentration of 54.9 ± 33.5 ng/mL (118.2 ± 72.1 nM). Mean sorafenib levels declined by over 70% relative to peak serum concentrations by 24 h in all dogs, suggesting the value of at least twice daily administration. Doses of 3 mg/kg were well-tolerated and no patients in the study experienced adverse events that were attributable to sorafenib. Future trials in dogs with cancer are recommended at this dosing schedule to assess the effect of sorafenib administration on anti-tumor efficacy signals and relevant pharmacodynamic target modulation in vivo.

Characterizing the metabolic role of STAT3 in canine osteosarcoma.

Signal transducer and activator of transcription 3 (STAT3) dysregulation has been characterized in canine OS, with previous data suggesting that constitutive STAT3 activation contributes to survival and proliferation in OS cell lines in vitro. Recently, the contribution of STAT3 to tumour metabolism has been described across several tumour histologies, and understanding the metabolic implications of STAT3 loss may elucidate novel therapeutic approaches with synergistic activity. The objective of this work was to characterize metabolic benchmarks associated with STAT3 loss in canine OS. STAT3 expression and activation was evaluated using western blotting in canine OS cell lines OSCA8 and Abrams. STAT3 was deleted from these OS cell lines using CRISPR-Cas9, and the effects on proliferation, invasion and metabolism (respirometry, intracellular lactate) were determined. Loss of STAT3 was associated with decreased basal and compensatory glycolysis in canine OS cell lines, without modulation of cellular proliferation. Loss of STAT3 also resulted in diminished invasive capacity in vitro. Interestingly, the absence of STAT3 did not impact sensitivity to doxorubicin in vitro. Our data demonstrate that loss of STAT3 modulates features of aerobic glycolysis in canine OS impacting capacities for cellular invasions, suggesting a role for this transcription factor in metastasis.

Serum IL-6 and MCP-1 concentrations in dogs with lymphoma before and after doxorubicin treatment as a potential marker of cellular senescence.

BACKGROUND: Chemotherapy can induce cellular senescence and a secretory phenotype characterized by an increased expression of inflammatory cytokines, such as IL-6 and MCP-1. Increased IL-6 and MCP-1 serum concentrations have been documented in dogs with lymphoma, but no studies have evaluated the effects of chemotherapy on cytokine concentrations. OBJECTIVES: To measure IL-6 and MCP-1 in 16 client-owned dogs with lymphoma, at baseline and before and after doxorubicin, as a potential marker for senescence and correlate cytokine concentrations with treatment response and toxicities. METHODS: Serum IL-6 and MCP-1 concentrations at baseline, 0-h, 3-h, 6-h, 24-h and 1 week post doxorubicin were measured using a canine ELISA. We hypothesized that IL-6 and MCP-1 concentrations would increase following doxorubicin as a result of induction of cellular senescence. RESULTS: IL-6 concentrations were unchanged from baseline to 0-h but significantly decreased 1 week post doxorubicin (p = 0.001) compared to 0-6 h (p = 0.045) and 24-h (p = 0.001) time points. MCP-1 concentrations significantly decreased from baseline to 0-h (p = 0.003). Compared to 0-6 h, MCP-1 concentrations transiently increased at 24-h (p = 0.001) and decreased at 1 week (p = 0.014) post doxorubicin. Changes in IL-6 and MCP-1 concentrations did not correlate with leukocyte count, response to treatment or chemotherapy toxicities. CONCLUSIONS: Changes in IL-6 and MCP-1 concentrations did not support doxorubicin-induced cellular senescence or correlate with leukocyte count, response to treatment or chemotherapy toxicity. However, our results suggest that remission status and doxorubicin treatment may influence cytokine concentrations and future studies are warranted to investigate the role of these cytokines as biomarkers.

Characterization of receptor tyrosine kinase activation and biological activity of toceranib phosphate in canine urothelial carcinoma cell lines.

BACKGROUND: Urothelial carcinoma (UC) accounts for > 90% of canine tumors occurring in the urinary bladder. Toceranib phosphate (TOC) is a multi-target receptor tyrosine kinase (RTK) inhibitor that exhibits activity against members of the split kinase family of RTKs. The purpose of this study was to evaluate primary UC tumors and UC cell lines for the expression and activation of VEGFR2, PDGFRα, PDGFRß, and KIT to assess whether dysregulation of these RTKs may contribute to the observed biological activity of TOC. RESULTS: Transcript for VEGFR2, PDGFRα, PDGFRß, and KIT was detected in all UC tissue samples and UC cell lines. The Proteome Profiler™ Human Phospho-RTK Array Kit (R & D Systems) provided a platform to assess phosphorylation of 42 different RTKs in primary UC tumors and UC cell lines. Evidence of PDGFRα and PDGFRß phosphorylation was present in only 11% or 33% of UC tumors, respectively, and 25% of UC cell lines. Treatment of UC cell lines with TOC had no significant impact on cell proliferation, including UC cell lines with evidence of PDGFRß phosphorylation. CONCLUSIONS: Phosphorylation of several key RTKs targeted by TOC is present in a small subset of primary UC tumors and UC cell lines, suggesting that these RTKs do not exist in a state of continuous activation. These data suggest that activation of RTKs targeted by TOC is present in a small subset of UC tumors and UC cell lines and that treatment with TOC at physiologically relevant concentrations has no direct anti-proliferative effect on UC cells.

Development of an exosomal gene signature to detect residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning.

Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups ("healthy", "osteosarcoma", "other bone tumor", or "non-neoplastic disease"). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as "osteosarcoma detected" or "osteosarcoma-NOT detected". Dogs in a validation set whose post-treatment samples were classified as "osteosarcoma-NOT detected" had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.

The selective inhibitor of nuclear export (SINE) verdinexor exhibits biologic activity against canine osteosarcoma cell lines.

Verdinexor (KPT-335) is a novel orally bioavailable selective inhibitor of nuclear export (SINE) compound that inhibits the function of the nuclear export protein Exportin 1 (XPO1/CRM1). In the present study, we sought to characterize the expression of XPO1 in primary canine osteosarcoma (OS) tumour samples, OS cell lines and normal osteoblasts and evaluate the in vitro activity of verdinexor alone or in combination with doxorubicin. Canine OS cell lines and a subset of primary OS tumours showed increased XPO1 transcript and protein expression as compared with normal canine osteoblast cells. All canine OS cell lines exhibited dose-dependent growth inhibition and increased caspase 3,7 activity in response to low nanomolar concentrations of verdinexor (IC50 concentrations ranging from 21 to 74 nM). Notably, growth inhibition of normal canine osteoblast cell lines treated with verdinexor was observed at high micromolar concentrations (IC50  = 21 µM). The combination of verdinexor and doxorubicin resulted in potent inhibition of cell viability and demonstrated synergetic activity in three canine OS cell lines. Concordantly, OS cell lines showed increased γH2A.X foci following treatment with doxorubicin and recovery in verdinexor compared with cells treated with doxorubicin and recovered in normal media for 24 hours. These findings demonstrate that verdinexor has biologic activity against canine OS cell lines at physiologically relevant doses and suggest that XPO1 inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in canine OS.

Plasma Cytokeratin 18 and fecal Alpha-1 Antitrypsin concentrations in dogs with osteosarcoma receiving carboplatin chemotherapy.

Gastrointestinal (GI) toxicosis is a common side effect of cytotoxic chemotherapy treatment in humans and dogs. Measurement of cytokeratin 18 (CK18), an intracellular structural protein released during epithelial apoptosis, and Alpha1-Antitrypsin (A1AT) in faeces provides a mechanism for evaluating damage to the intestinal mucosa secondary to cytotoxic chemotherapy. Our goal was to evaluate the clinical utility of plasma CK18 and faecal A1-AT levels as non-invasive biomarkers of cytotoxic chemotherapy induced GI toxicity. We conducted a prospective cohort study in dogs (N = 10) with osteosarcoma undergoing amputation followed by carboplatin chemotherapy. We hypothesized that plasma CK18 and faecal A1-AT levels would increase following carboplatin administration due to drug-induced GI epithelial damage/apoptosis, and that plasma CK18 and faecal A1-AT levels would correlate with severity of GI toxicity. Mean baseline plasma CK18 concentration was variable amongst patients; however, CK18 concentration prior to carboplatin chemotherapy treatment was not significantly different from CK18 levels after treatment. There was significant intra and inter-patient variability in mean faecal A1-AT levels at baseline. Mean A1-AT concentration did not change significantly from day 0 to day 21. Gastrointestinal toxicity was minimal; therefore, we were unable to determine the association of plasma CK18 and faecal A1-AT concentrations with development of GI toxicosis. In this study population, plasma CK18 and faecal A1-AT concentration were not clinically useful biomarkers for the detection of GI toxicosis secondary to carboplatin administration. Further prospective evaluation of CK18 and A1-AT as biomarkers of drug-induced GI toxicity is warranted in a larger cohort of dogs receiving cytotoxic chemotherapy. AVMA clinical trial registration number: AAHSD004827.

Characterization of WWOX expression and function in canine mast cell tumors and malignant mast cell lines.

BACKGROUND: The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior. METHODS/RESULTS: WWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs. CONCLUSIONS: These data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.

Evaluation of microwave ablation for local treatment of dogs with distal radial osteosarcoma: A pilot study.

OBJECTIVE: To evaluate the feasibility and safety of microwave ablation (MWA) as a modality to induce tumor necrosis within distal radial osteosarcoma (OSA). STUDY DESIGN: Pilot study. ANIMALS: Six client-owned dogs with distal radius OSA confirmed by cytological examination. METHODS: Dogs underwent computed tomography for surgical planning before general anesthesia for fluoroscopy-guided ablation. Computed tomography was repeated 48 hours after MWA, before amputation. The ablated tumor was evaluated with histopathology. RESULTS: Six dogs underwent MWA of distal radius OSA. A lower power setting (30 W) was selected for the first two dogs to avoid collateral soft tissue damage. The power was increased to 75 W for the last four dogs. The temperature was maintained between 45°C and 55°C (113 °F-131 °F) at the bone/soft tissue interface. Tumor necrosis varied between 30% and 90% (median, 55%) according to histopathology. No intraoperative or periprocedural complications were observed. CONCLUSION: Microwave ablation induced variable tumor necrosis and did not induce immediate postablation complications in these six dogs with distal radius OSA. CLINICAL SIGNIFICANCE: These results justify further evaluation of MWA as a potential modality to treat primary bone lesions in dogs.

S100A4 mRNA-protein relationship uncovered by measurement noise reduction.

Intrinsic biological fluctuation and/or measurement error can obscure the association of gene expression patterns between RNA and protein levels. Appropriate normalization of reverse-transcription quantitative PCR (RT-qPCR) data can reduce technical noise in transcript measurement, thus uncovering such relationships. The accuracy of gene expression measurement is often challenged in the context of cancer due to the genetic instability and "splicing weakness" involved. Here, we sequenced the poly(A) cancer transcriptome of canine osteosarcoma using mRNA-Seq. Expressed sequences were resolved at the level of two consecutive exons to enable the design of exon-border spanning RT-qPCR assays and ranked for stability based on the coefficient of variation (CV). Using the same template type for RT-qPCR validation, i.e. poly(A) RNA, avoided skewing of stability assessment by circular RNAs (circRNAs) and/or rRNA deregulation. The strength of the relationship between mRNA expression of the tumour marker S100A4 and its proportion score of quantitative immunohistochemistry (qIHC) was introduced as an experimental readout to fine-tune the normalization choice. Together with the essential logit transformation of qIHC scores, this approach reduced the noise of measurement as demonstrated by uncovering a highly significant, strong association between mRNA and protein expressions of S100A4 (Spearman's coefficient ρ = 0.72 (p = 0.006)). KEY MESSAGES: • RNA-seq identifies stable pairs of consecutive exons in a heterogeneous tumour. • Poly(A) RNA templates for RT-qPCR avoid bias from circRNA and rRNA deregulation. • HNRNPL is stably expressed across various cancer tissues and osteosarcoma. • Logit transformed qIHC score better associates with mRNA amount. • Quantification of minor S100A4 mRNA species requires poly(A) RNA templates and dPCR.

Radiation-induced sarcoma in a cat following hypofractionated, palliative intent radiation therapy for large-cell lymphoma.

CASE SUMMARY: A 5-year-old female spayed domestic shorthair cat was presented with a 4.5 × 3 cm ulcerated cutaneous mass on the nasal bridge with extension into the nasal cavity. Tissue biopsy was obtained and a diagnosis of large-cell lymphoma was confirmed on histopathology. The cat was started on prednisolone and injectable chemotherapy; however, only a partial response was observed. A CT scan revealed a highly infiltrative mass with extensive subcutaneous involvement, extending into the nasal cavity, resulting in lysis of numerous nasal and facial bones. The cat received hypofractionated, palliative intent radiation therapy (four fractions of 8 Gray) and a complete clinical response was achieved. Nine months after radiation therapy, minimal residual intranasal disease was observed on advanced imaging. Sixty-nine months after the completion of radiotherapy, a mass was observed dorsal to the right eye within the previous radiation field. CT scan revealed a mass associated with the right frontal sinus with extension throughout the nasal cavity and facial bones. Histopathology was consistent with a moderately differentiated sarcoma. Seventy-one months post-radiation therapy, the cat developed neurologic clinical signs and was humanely euthanized. Radiation-induced sarcoma was suspected based on human criteria, which included history of irradiation and tumor development within the irradiated field, a latent period after irradiation prior to the development of the second tumor and histopathologic confirmation of a different malignant neoplasm at the irradiated site. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first report of a malignant radiation-induced sarcoma in a cat. Based on this case, radiation-induced sarcomas should be considered as a late-term side effect associated with radiation therapy in cats.

MicroRNA expression in the cerebrospinal fluid of dogs with and without cervical spondylomyelopathy.

BACKGROUND: Osseous-associated cervical spondylomyelopathy (OA-CSM) is a common condition of the cervical vertebral column that affects giant dog breeds. MicroRNAs (miRNAs) are small RNAs that regulate gene expression, and recent data suggest that circulating miRNAs present in biological fluids may serve as potential biomarkers for disease. The miRNA profiles of cerebrospinal fluid (CSF) from healthy dogs and dogs clinically affected by OA-CSM have not been described. OBJECTIVE: To characterize the expression levels of miRNAs present in the CSF of normal Great Danes and identify differentially expressed miRNAs in the CSF of Great Danes clinically affected with OA-CSM. ANIMALS: Client-owned dogs: 12 control, 12 OA-CSM affected. METHODS: Cerebrospinal fluid samples were collected prospectively. MicroRNA expression was evaluated using the NanoString nCounter platform and quantitative real-time PCR. RESULTS: We identified 8 miRNAs with significant differential expression. MiR-299-5p and miR-765 had increased expression levels in the CSF of OA-CSM-affected dogs, whereas miR-494, miR-612, miR-302-d, miR-4531, miR-4455, and miR-6721-5p had decreased expression levels in OA-CSM affected dogs compared to clinically normal dogs. Quantitative real-time PCR was performed to validate the expression levels of 2 miRNAs (miR-494 and miR-612), and we found a 1.5-fold increase in miR-494 expression and a 1.2-fold decrease in miR-612 in the CSF of the OA-CSM affected group (P = .41 and .89, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Data generated from our study represent an initial characterization of the miRNA profile of normal canine CSF and suggest that a distinct CSF miRNA expression profile is associated with OA-CSM.

Canine osteosarcoma genome sequencing identifies recurrent mutations in DMD and the histone methyltransferase gene SETD2.

Osteosarcoma (OS) is a rare, metastatic, human adolescent cancer that also occurs in pet dogs. To define the genomic underpinnings of canine OS, we performed multi-platform analysis of OS tumors from 59 dogs, including whole genome sequencing (n = 24) and whole exome sequencing (WES; n = 13) of primary tumors and matched normal tissue, WES (n = 10) of matched primary/metastatic/normal samples and RNA sequencing (n = 54) of primary tumors. We found that canine OS recapitulates features of human OS including low point mutation burden (median 1.98 per Mb) with a trend towards higher burden in metastases, high structural complexity, frequent TP53 (71%), PI3K pathway (37%), and MAPK pathway mutations (17%), and low expression of immune-associated genes. We also identified novel features of canine OS including putatively inactivating somatic SETD2 (42%) and DMD (50%) aberrations. These findings set the stage for understanding OS development in dogs and humans, and establish genomic contexts for future comparative analyses.

Emerging roles of and therapeutic strategies targeting BRD4 in cancer.

The Bromodomain and Extra-terminal (BET) family of proteins were first recognized as important epigenetic regulators in inflammatory processes; however, there is increasing evidence to support the notion that BET proteins also play a critical role in 'reading' chromatin and recruiting chromatin-regulating enzymes to control gene expression in a number of pathologic processes, including cancer. To this end, the mechanisms by which BET proteins regulate chromatin remodeling and promote tumor-associated inflammation have been heavily studied over the past decade. This article to review the biology of BET protein dysfunction in promoting tumor-associated inflammation and cancer progression and the application of small molecule inhibitors that target specific BET proteins, alone or in combination with immunomodulatory agents as a novel therapeutic strategy for cancer patients.

Plasma cytokeratin-18 concentrations as noninvasive biomarker of early gastrointestinal toxicosis in dogs receiving toceranib.

BACKGROUND: No biomarkers for the early detection of gastrointestinal (GI) toxicosis secondary to antineoplastic treatment are recognized in veterinary medicine. Toceranib causes GI toxicosis in dogs. HYPOTHESIS/OBJECTIVE: To assess if changes in plasma cytokeratin 18 (CK18) concentration, measured in dogs being treated with toceranib phosphate, can predict the onset of GI toxicosis. We hypothesize that an increase in CK18 concentrations will be detected before the development of GI toxicosis in dogs treated with toceranib phosphate. ANIMALS: Twenty healthy client-owned dogs and 25 client-owned dogs with surgically excised mast cell tumor (MCT). METHODS: Prospective cohort study. Dogs were treated with toceranib (2.75 mg/kg PO q48h). Plasma was collected weekly for 4 weeks. Plasma CK18 concentration was measured on days 0, 7, 14, 21, and 28. vascular endothelial growth factor was measured on days 0 and 28. RESULTS: Mean plasma CK18 concentration on day 0 in dogs with MCT was not significantly different than healthy controls (313.5 ± 592.8 pg/mL, 119.7 ± 76.9 pg/mL, mean ± SD P = 0.27). Mean plasma CK18 concentration decreased by 98.69 pg/mL from day 0 to day 28 (P < 0.001). Plasma CK18 concentration was not a significant predictor of the development of signs of GI toxicosis. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma CK18 concentration was not a clinically useful biomarker for the early detection of GI toxicosis secondary to toceranib administration in dogs with MCTs.

miR-133a function in the pathogenesis of dedifferentiated liposarcoma.

BACKGROUND: Sarcomas are malignant heterogeneous tumors of mesenchymal derivation. Dedifferentiated liposarcoma (DDLPS) is aggressive with recurrence in 80% and metastasis in 20% of patients. We previously found that miR-133a was significantly underexpressed in liposarcoma tissues. As this miRNA has recently been shown to be a tumor suppressor in many cancers, the objective of this study was to characterize the biological and molecular consequences of miR-133a underexpression in DDLPS. METHODS: Real-time PCR was used to evaluate expression levels of miR-133a in human DDLPS tissue, normal fat tissue, and human DDLPS cell lines. DDLPS cells were stably transduced with miR-133a vector to assess the effects in vitro on proliferation, cell cycle, cell death, migration, and metabolism. A Seahorse Bioanalyzer system was also used to assess metabolism in vivo by measuring glycolysis and oxidative phosphorylation (OXPHOS) in subcutaneous xenograft tumors from immunocompromised mice. RESULTS: miR-133a expression was significantly decreased in human DDLPS tissue and cell lines. Enforced expression of miR-133a decreased cell proliferation, impacted cell cycle progression kinetics, decreased glycolysis, and increased OXPHOS. There was no significant effect on cell death or migration. Using an in vivo xenograft mouse study, we showed that tumors with increased miR-133a expression had no difference in tumor growth compared to control, but did exhibit an increase in OXPHOS metabolic respiration. CONCLUSIONS: Based on our collective findings, we propose that in DDPLS, loss of miR-133a induces a metabolic shift due to a reduction in oxidative metabolism favoring a Warburg effect in DDLPS tumors, but this regulation on metabolism was not sufficient to affect DDPLS.

Classical NF-κB Metabolically Reprograms Sarcoma Cells Through Regulation of Hexokinase 2.

BACKGROUND: Metabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB) play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS) and osteosarcoma (OS), has not been characterized. METHODS: Classical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XFe24 was used to analyze oxygen consumption rate as a measure of aerobic respiration. RESULTS: Inhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK) 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells. CONCLUSION: These findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.

Osteosarcoma: Accelerating Progress Makes for a Hopeful Future.

Patients who develop osteosarcoma in 2017 receive treatment that remains essentially unchanged since the 1970s. Outcomes likewise remain largely unimproved. Large, collaborative, multinational efforts to improve therapy have evaluated strategies leveraging both cytotoxic intensification and immunomodulatory agents. While these have confirmed our capacity to conduct such trials, results have proved largely disappointing. This has motivated efforts to focus on the basic biology of osteosarcoma, where understanding remains poor but has improved significantly. Recent advances have identified characteristic genetic features of osteosarcoma, including profound chromosomal disruption, marked patient-patient heterogeneity, and a paucity of recurrent mutations. Analyses suggest genesis in early catastrophic genetic events, although the nature of the inciting events remains unclear. While p53 and Rb inactivation occurs in most osteosarcomas, the landscape of associated driver mutations has proved extensive. Few mutations recur with high frequency, though patterns continue to emerge that suggest recurrent alterations within specific pathways. Biological pathways implicated in osteosarcoma biology through genetic and other preclinical studies include PI3K/mTOR, WNT/ßcatenin, TGFß, RANKL/NF-κB, and IGF. Unfortunately, clinical studies evaluating targeted agents have to date yielded disappointing results, as have studies examining modern immunotherapeutics. It remains unclear whether this pattern of clinical failures exposes inadequacies of our preclinical models, unrealistic expectations for single-agent responses in heavily pretreated patients, or biology less relevant than suggested. Nearly all patients who succumb to osteosarcoma develop lung metastases, which exhibit marked chemoresistance. Much scientific effort has recently sought to enhance our mechanistic understanding of metastasis biology. This research has potential to reveal novel targets for preventing and treating metastasis and for uncovering key vulnerabilities of osteosarcoma cells. Efforts to implement drug development strategies that leverage clinical studies in veterinary patients have potential to accelerate the translation of novel experimental regimens toward human studies. These could reduce costs and development timelines, prioritize agents, and refine regimens prior to human clinical trials. The rise of philanthropic groups focused on osteosarcoma has enhanced cross-disciplinary and cross-institutional focus and provided much needed resources. Transformative new therapies will likely arise from collaborative, interdisciplinary efforts that extend our understanding of osteosarcoma's most basic inner workings.