The ETS family member GABPα modulates androgen receptor signalling and mediates an aggressive phenotype in prostate cancer

In prostate cancer (PC), the androgen receptor (AR) is a key transcription factor at all disease stages, including the advanced stage of castrate-resistant prostate cancer (CRPC). In the present study, we show that GABPα, an ETS factor that is up-regulated in PC, is an AR-interacting transcription factor. Expression of GABPα enables PC cell lines to acquire some of the molecular and cellular characteristics of CRPC tissues as well as more aggressive growth phenotypes. GABPα has a transcriptional role that dissects the overlapping cistromes of the two most common ETS gene fusions in PC: overlapping significantly with ETV1 but not with ERG target genes. GABPα bound predominantly to gene promoters, regulated the expression of one-third of AR target genes and modulated sensitivity to AR antagonists in hormone responsive and castrate resistant PC models. This study supports a critical role for GABPα in CRPC and reveals potential targets for therapeutic intervention.

Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke

Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury.

The CXCR2 antagonist, SCH-527123, shows antitumor activity and sensitizes cells to oxaliplatin in preclinical colon cancer models

Colorectal cancer is the second most common cause of cancer-related death in the United States. Recent studies showed that interleukin-8 (IL-8) and its receptors (CXCR1 and CXCR2) are significantly upregulated in both the tumor and its microenvironment, and act as key regulators of proliferation, angiogenesis, and metastasis. Our previous study showed that IL-8 overexpression in colorectal cancer cells triggers the upregulation of the CXCR2-mediated proliferative pathway. The aim of this study was to investigate whether the CXCR2 antagonist, SCH-527123, inhibits colorectal cancer proliferation and if it can sensitize colorectal cancer cells to oxaliplatin both in vitro and in vivo. SCH-527123 showed concentration-dependent antiproliferative effects in HCT116, Caco2, and their respective IL-8-overexpressing variants colorectal cancer cell lines. Moreover, SCH-527123 was able to suppress CXCR2-mediated signal transduction as shown through decreased phosphorylation of the NF-κB/mitogen-activated protein kinase (MAPK)/AKT pathway. These findings corresponded with decreased cell migration and invasion, while increased apoptosis in colorectal cancer cell lines. In vivo results verified that SCH-527123 treatment decreased tumor growth and microvessel density when compared with vehicle-treated tumors. Importantly, these preclinical studies showed that the combination of SCH-527123 and oxaliplatin resulted in a greater decrease in cell proliferation, tumor growth, apoptosis, and angiogenesis that was superior to single-agent treatment. Taken together, these findings suggest that targeting CXCR2 may block tumor proliferation, migration, invasion, and angiogenesis. In addition, CXCR2 blockade may further sensitize colorectal cancer to oxaliplatin treatment.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

The dual EGFR/HER-2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN-38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER-2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN-38, the active metabolite of irinotecan (CPT-11), in colon and gastric cancer cell lines. Concentration-dependent antiproliferative effects of both lapatinib and SN-38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08-11.7 muM; SN-38 range 3.6-256 nM). Lapatinib potently inhibited the growth of a HER-2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER-2 expression. The combination of lapatinib and SN-38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN-38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN-38 when compared to SN-38 treatment alone. Finally, the combination of lapatinib and CPT-11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT-11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas.

Serum Amyloid P-Component Prevents Cardiac Remodeling in Hypertensive Heart Disease

The potential for serum amyloid P-component (SAP) to prevent cardiac remodeling and identify worsening diastolic dysfunction (DD) was investigated. The anti-fibrotic potential of SAP was tested in an animal model of hypertensive heart disease (spontaneously hypertensive rats treated with SAP [SHR - SAP] × 12 weeks). Biomarker analysis included a prospective study of 60 patients with asymptomatic progressive DD. Compared with vehicle-treated Wistar-Kyoto rats (WKY-V), the vehicle-treated SHRs (SHR-V) exhibited significant increases in left ventricular mass, perivascular collagen, cardiomyocyte size, and macrophage infiltration. SAP administration was associated with significantly lower left ventricular mass (p < 0.01), perivascular collagen (p < 0.01), and cardiomyocyte size (p < 0.01). Macrophage infiltration was significantly attenuated in the SHR-SAP group. Biomarker analysis showed significant decreases in SAP concentration over time in patients with progressive DD (p < 0.05). Our results indicate that SAP prevents cardiac remodeling by inhibiting recruitment of pro-fibrotic macrophages and that depleted SAP levels identify patients with advancing DD suggesting a role for SAP therapy.

Cardiomyopathy in Offspring of Pregestational Diabetic Mouse Pregnancy

PURPOSE: To investigate cardiomyopathy in offspring in a mouse model of pregestational type 1 diabetic pregnancy.

METHODS: Pregestational diabetes was induced with STZ administration in female C57BL6/J mice that were subsequently mated with healthy C57BL6/J males. Offspring were sacrificed at embryonic day 18.5 and 6-week adolescent and 12-week adult stages. The size and number of cardiomyocyte nuclei and also the extent of collagen deposition within the hearts of diabetic and control offspring were assessed following cardiac tissue staining with either haematoxylin and eosin or Picrosirius red and subsequently quantified using automated digital image analysis.

RESULTS: Offspring from diabetic mice at embryonic day 18.5 had a significantly higher number of cardiomyocyte nuclei present compared to controls. These nuclei were also significantly smaller than controls. Collagen deposition was shown to be significantly increased in the hearts of diabetic offspring at the same age. No significant differences were found between the groups at 6 and 12 weeks.

CONCLUSIONS: Our results from offspring of type 1 diabetic mice show increased myocardial collagen deposition in late gestation and have increased myocardial nuclear counts (hyperplasia) as opposed to increased myocardial nuclear size (hypertrophy) in late gestation. These changes normalize postpartum after removal from the maternal intrauterine environment.

Btk regulates macrophage polarization in response to lipopolysaccharide

Bacterial Lipopolysaccharide (LPS) is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (-\-)) mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(-/-) macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(-/-) macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(-/-) macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (-/-) mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation.

Circulating miRNAs: Potential Novel Biomarkers for Hepatopathology Progression and Diagnosis of Schistosomiasis Japonica in Two Murine Models

BACKGROUND: Schistosomiasis remains a major public health issue, with an estimated 230 million people infected worldwide. Novel tools for early diagnosis and surveillance of schistosomiasis are currently needed. Elevated levels of circulating microRNAs (miRNAs) are commonly associated with the initiation and progression of human disease pathology. Hence, serum miRNAs are emerging as promising biomarkers for the diagnosis of a variety of human diseases. This study investigated circulating host miRNAs commonly associated with liver diseases and schistosome parasite-derived miRNAs during the progression of hepatic schistosomiasis japonica in two murine models.

METHODOLOGY/PRINCIPAL FINDINGS: Two mouse strains (C57BL/6 and BALB/c) were infected with a low dosage of Schistosoma japonicum cercariae. The dynamic patterns of hepatopathology, the serum levels of liver injury-related enzymes and the serum circulating miRNAs (both host and parasite-derived) levels were then assessed in the progression of schistosomiasis japonica. For the first time, an inverse correlation between the severity of hepatocyte necrosis and the level of liver fibrosis was revealed during S. japonicum infection in BALB/c, but not in C57BL/6 mice. The inconsistent levels of the host circulating miRNAs, miR-122, miR-21 and miR-34a in serum were confirmed in the two murine models during infection, which limits their potential value as individual diagnostic biomarkers for schistosomiasis. However, their serum levels in combination may serve as a novel biomarker to mirror the hepatic immune responses induced in the mammalian host during schistosome infection and the degree of hepatopathology. Further, two circulating parasite-specific miRNAs, sja-miR-277 and sja-miR-3479-3p, were shown to have potential as diagnostic markers for schistosomiasis japonica.

CONCLUSIONS/SIGNIFICANCE: We provide the first evidence for the potential of utilizing circulating host miRNAs to indicate different immune responses and the severity of hepatopathology outcomes induced in two murine strains infected with S. japonicum. This study also establishes a basis for the early and cell-free diagnosis of schistosomiasis by targeting circulating schistosome parasite-derived miRNAs.

Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential

To further investigate the importance of insulin signaling in the growth, development, sexual maturation and egg production of adult schistosomes, we have focused attention on the insulin receptors (SjIRs) of Schistosoma japonicum, which we have previously cloned and partially characterised. We now show, by Biolayer Interferometry, that human insulin can bind the L1 subdomain (insulin binding domain) of recombinant (r)SjIR1 and rSjIR2 (designated SjLD1 and SjLD2) produced using the Drosophila S2 protein expression system. We have then used RNA interference (RNAi) to knock down the expression of the SjIRs in adult S. japonicum in vitro and show that, in addition to their reduced transcription, the transcript levels of other important downstream genes within the insulin pathway, associated with glucose metabolism and schistosome fecundity, were also impacted substantially. Further, a significant decrease in glucose uptake was observed in the SjIR-knockdown worms compared with luciferase controls. In vaccine/challenge experiments, we found that rSjLD1 and rSjLD2 depressed female growth, intestinal granuloma density and faecal egg production in S. japonicum in mice presented with a low dose challenge infection. These data re-emphasize the potential of the SjIRs as veterinary transmission blocking vaccine candidates against zoonotic schistosomiasis japonica in China and the Philippines.

IL-1α and inflammasome-independent IL-1β promote neutrophil infiltration following alum vaccination

Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to alum-induced innate and adaptive immune responses is controversial and the role of IL-1α following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1α and IL-1β are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1β at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1β secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL-1α and IL-1β are both necessary for alum-induced neutrophil influx in vivo.