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)).publishedVersio

The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.</p> <p>Methods</p> <p>Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT^®), apoptosis (SensoLyte^®Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.</p> <p>Results</p> <p>Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.</p> <p>Conclusions</p> <p>These data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound <it>in vivo</it>.</p

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/βcatenin, TGFβ, 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

Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis

Background: While microRNA (miRNA) expression is known to be altered in a variety of human malignancies contributing to cancer development and progression, the potential role of miRNA dysregulation in malignant mast cell disease has not been previously explored. The purpose of this study was to investigate the potential contribution of miRNA dysregulation to the biology of canine mast cell tumors (MCTs), a well-established spontaneous model of malignant mast cell disease.Methods: We evaluated the miRNA expression profiles from biologically low-grade and biologically high-grade primary canine MCTs using real-time PCR-based TaqMan Low Density miRNA Arrays and performed real-time PCR to evaluate miR-9 expression in primary canine MCTs, malignant mast cell lines, and normal bone marrow-derived mast cells (BMMCs). Mouse mast cell lines and BMMCs were transduced with empty or pre-miR-9 expressing lentiviral constructs and cell proliferation, caspase 3/7 activity, and invasion were assessed. Transcriptional profiling of cells overexpressing miR-9 was performed using Affymetrix GeneChip Mouse Gene 2.0 ST arrays and real-time PCR was performed to validate changes in mRNA expression.Results: Our data demonstrate that unique miRNA expression profiles correlate with the biological behavior of primary canine MCTs and that miR-9 expression is increased in biologically high grade canine MCTs and malignant cell lines compared to biologically low grade tumors and normal canine BMMCs. In transformed mouse malignant mast cell lines expressing either wild-type (C57) or activating (P815) KIT mutations and mouse BMMCs, miR-9 overexpression significantly enhanced invasion but had no effect on cell proliferation or apoptosis. Transcriptional profiling of normal mouse BMMCs and P815 cells possessing enforced miR-9 expression demonstrated dysregulation of several genes, including upregulation of CMA1, a protease involved in activation of matrix metalloproteases and extracellular matrix remodeling.Conclusions: Our findings demonstrate that unique miRNA expression profiles correlate with the biological behavior of canine MCTs. Furthermore, dysregulation of miR-9 is associated with MCT metastasis potentially through the induction of an invasive phenotype, identifying a potentially novel pathway for therapeutic intervention. © 2014 Fenger et al.; licensee BioMed Central Ltd

MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines

<div><p>Background</p><p>Osteosarcoma (OSA) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a fundamental role in cancer. The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous model of the human disease.</p><p>Methodology and principal findings</p><p>RT-qPCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. In canine OSA cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of down-regulated miR-34a target genes showed enrichment of several biological processes related to cell invasion and motility. Lastly, we validated changes in miR-34a putative target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells overexpressing miR-34a and identified KLF4 and VEGFA as direct target genes of miR-34a. Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes.</p><p>Conclusions</p><p>These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA.</p></div

Overexpression of miR-34a in canine OSA8 cells significantly alters gene expression.

<p>Total RNA was isolated from canine OSA8 cells expressing control or pre-miR-34a lentiviral constructs from three separate transduction experiments and next-generation sequencing was performed to identify differences in gene transcript expression. Supervised hierarchical cluster analysis of 111 genes differentially expressed in OSA8 cells expressing either empty vector (EV) or miR-34a (miR34a) as determined by one-way ANOVA comparison test (<i>p</i> ≤ 0.0001). Color areas indicate relative expression of each gene after log2 transformation with respect to the gene median expression (red and green colors denote high and low expression, respectively).</p

Identification of putative miR-34a target genes dysregulated by miR-34a overexpression in canine OSA cell lines.

<p>Transcriptional profiling of canine OSA8 cells expressing pre-miR-34a (miR-34a) or empty vector (EV) control was performed by next-generation sequencing to identify genes showing differential expression with miR-34a overexpression. RT-qPCR was performed to independently validate changes in gene expression for putative miR-34a targets (A) KLF4, (B) SEM3AE, and (C) VEGFA altered by miR-34a overexpression in both canine OSA8 and Abrams cell lines. Data presented show the mean fold change (relative to GAPDH control) ± standard deviation (Student’s <i>t</i>-test, *<i>p</i> < 0.01).</p

KLF4 and VEGFA are direct targets of miR-34a.

<p>(A) Predicted miR-34a binding sites in the 3’UTR of canine KLF4 and VEGFA. Reporter constructs harboring wild-type (WT) or seed sequence-mutated (MUT) miR-34a binding sites for canine KLF4 and VEGFA were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector. (B, C) Canine OSA8 cells were co-transfected with control or miR-34a mimics and pmirGLO vectors, possessing either the WT or MUT 3’UTR sequence for KLF4 or VEGFA. Cells were harvested after 24 h and assayed for dual luciferase activity. Luciferase activity was normalized to <i>Renilla</i> control reporter activity. Data shown are means ± standard deviation from a representative of three independent experiments (one-way ANOVA followed by Tukey-Kramer <i>post-hoc</i> test, *<i>p</i> < 0.05).</p

Ectopic expression of miR-34a in canine OSA cells does not affect cell proliferation or cell cycle distribution.

<p>(A) Canine OSA8 and Abrams cells were transduced with pre-miR-34a lentiviral constructs or empty control vector and sorted to greater than 95% purity based on GFP expression 72 hours following infection. Total RNA was isolated and RT-qPCR was performed as described above immediately prior to plating cells to confirm transduction efficiency miR-34a levels in wild-type (WT), empty vector (EV), and miR-34a expressing cells (*<i>p</i> < 0.05). Multiple group comparisons were analyzed by one-way ANOVA followed by Tukey-Kramer <i>post-hoc</i> comparison. (B) OSA8 and Abrams cells transduced with either empty control or pre-miR-34a lentiviral constructs were plated in complete media and the cell proliferation was assessed at 24, 48, and 72 hours using the BrdU incorporation assay. Cell proliferation was measured at 490 nm. Values of optical density (OD) are expressed as means +/- SD of 3 independent experiments. (C) OSA8 and Abrams cells expressing empty vector or pre-miR-34a lentivirus vector were incubated in complete media for 24 hours. Cells were then evaluated for effects on cell cycle using propidium iodide (PI) staining and flow cytometry. Three independent experiments were performed, and 1 representative result is presented. Linear mixed effects models were applied to OSA8 and Abrams cell line miR-34a expression, proliferation and cell cycling data to take account of the correlation among observations from the same replicates. No statistically significant difference in cell proliferation or cell cycle distribution was detected in OSA8 or Abrams cells expressing empty vector or pre-miR-34a vector for any of the tested time points (Student’s <i>t</i>-test).</p

Evaluation of putative miR-34a target expression in primary canine OSA tumors.

<p>(A) RT-qPCR evaluating mature miR-34a expression in primary canine OSA tumors (N = 9) and normal canine osteoblasts demonstrated that the mean expression of miR-34a was significantly reduced in OSA tumors compared to osteoblasts (Bars: SD. Statistical analysis: one-way ANOVA followed by Tukey-Kramer <i>post-hoc</i> comparison, *<i>p</i> < 0.05). All reactions were performed in triplicate and results were normalized to snU6. Transcript levels of putative miR-34a target genes (B) KLF4, (C) SEM3AE, and (D) VEGFA were assessed in primary canine OSA tumors and normal canine osteoblasts using RT-qPCR. All reactions were performed in triplicate and results were normalized to GAPDH. Raw data were log transformed to reduce variance and skewness. Linear mixed effects models were applied to expression data to account for the correlation of the observations from the same batch.</p