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Major changes in maternal physiology during pregnancy and lactation can have a large impact on the immune and neuroendocrine systems. One of the most significant changes, observed in rats and mice, is hyporesponsiveness of the hypothalamic pituitary adrenal axis (HPAA) in response to inflammation, restraint, and other psychological stressors during late pregnancy and lactation. This attenuation, however, has not been well characterized in ruminant animals and may be relevant to their susceptibility to inflammatory diseases during these periods. Thus, the intent of this study was to characterize responsiveness of the ovine HPAA to inflammatory challenge during pregnancy and lactation. Ewes from early (33 d), middle (55 d), and late (138 d) pregnancy, as well as early lactation (10 d), were challenged i.v. with a bolus dose of 400 ng of Escherichia coli lipopolysaccharide (LPS)/kg of BW or saline. A corresponding group of nonpregnant ewes was also challenged with LPS to serve as positive control animals for each pregnancy and lactation study. Responsiveness of the HPAA was assessed by measuring the 4-h change in serum cortisol concentration after LPS challenge. The cortisol increase after LPS challenge was elevated (P < 0.01) in pregnant ewes during late pregnancy over that of nonpregnant animals. In contrast, the characteristic temperature response associated with systemic LPS challenge was decreased (P < 0.01) during early pregnancy and lactation compared with nonpregnant or nonlactating animals. Serum IL-6 concentrations were measured to assess whether changes in HPAA responsiveness during pregnancy or lactation were attributed to changes in proinflammatory signaling to the HPAA. Interestingly, enhanced cortisol responsiveness during late pregnancy was correlated with increased (P < 0.01) serum IL-6 concentrations, indicating that IL-6 may contribute to enhanced HPAA responsiveness during this period. Serum IL-6 concentrations during early and midpregnancy did not increase in response to LPS challenge, indicating that HPAA activation during periods of pregnancy may be independent of IL-6 production. Key words: hypothalamic-pituitary-adrenal axis, lactation, lipopolysaccharide, ovine, pregnancy

Abstract Background During systemic gram-negative bacterial infections, lipopolysaccharide (LPS) ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg) using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR). Results Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg) and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep. Conclusion We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4 wt overexpression as murine myoblasts expressing FGFR4 V550E undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4 V550E were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4 V550E tumor-derived cell and human RMS cell viability. FGFR4 V550E -overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC 50 values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.

Background: Leiomyosarcoma (LMS) is a common type of soft tissue sarcoma that responds poorly to standard chemotherapy. Thus the goal of this study was to identify novel selective therapies that may be effective in leiomyosarcoma by screening cell lines with a small molecule library comprised of 480 kinase inhibitors to functionally determine which signalling pathways may be critical for LMS growth., Methods: LMS cell lines were screened with the OICR kinase library and a cell viability assay was used to identify potentially effective compounds. The top 10 % of hits underwent secondary validation to determine their EC50 and immunoblots were performed to confirm selective drug action. The efficacy of combination drug therapy with doxorubicin (Dox) in vitro was analyzed using the Calcusyn program after treatment with one of three dosing schedules: concurrent treatment, initial treatment with a selective compound followed by Dox, or initial treatment with Dox followed by the selective compound. Single and combination drug therapy were then validated in vivo using LMS xenografts., Results: Compounds that targeted PI3K/AKT/mTOR pathways (52 %) were most effective. EC50s were determined to validate these initial hits, and of the 11 confirmed hits, 10 targeted PI3K and/or mTOR pathways with EC50 values <1 μM. We therefore examined if BEZ235 and BKM120, two selective compounds in these pathways, would inhibit leiomyosarcoma growth in vitro. Immunoblots confirmed on-target effects of these compounds in the PI3K and/or mTOR pathways. We next investigated if there was synergy with these agents and first line chemotherapy doxorubicin (Dox), which would allow for earlier introduction into patient care. Only combined treatment of BEZ235 and Dox was synergistic in vitro. To validate these findings in pre-clinical models, leiomyosarcoma xenografts were treated with single agent and combination therapy. BEZ235 treated xenografts (n = 8) demonstrated a decrease in tumor volume of 42 % whereas combining BEZ235 with Dox (n = 8) decreased tumor volume 68 % compared to vehicle alone., Conclusions: In summary, this study supports further investigation into the use of PI3K and mTOR inhibitors alone and in combination with standard treatment in leiomyosarcoma patients.

While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53-/- myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53-/- myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum.

Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA) system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues) would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins KrasG12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively). Disrupting the G1 checkpoint CDKN2 (p16/p19-/-) resulted in sarcoma in 30% of p16/p19-/- xN-tva mice with a median latency of 23 weeks (range 8-40 weeks). A similar incidence occurred in p16/p19-/- xBKE mice (32%), however, a shorter median latency (10.4 weeks) was observed. p16/p19-/- xAKE mice also developed sarcomas (24% incidence; median 9 weeks) yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. KrasG12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research.