Assessment of Hypoxia Inducible Factor Levels in Cancer Cell Lines upon Hypoxic Induction Using a Novel Reporter Construct

Hypoxia Inducible Factor (HIF) signaling pathway is important for tumor cells with limited oxygen supplies, as it is shown to be involved in the process of proliferation and angiogenesis. Given its pivotal role in cancer biology, robust assays for tracking changes in HIF expression are necessary for understanding its regulation in cancer as well as developing therapies that target HIF signaling. Here we report a novel HIF reporter construct containing tandem repeats of minimum HIF binding sites upstream of eYFP coding sequence. We show that the reporter construct has an excellent signal to background ratio and the reporter activity is HIF dependent and directly correlates with HIF protein levels. By utilizing this new construct, we assayed HIF activity levels in different cancer cell lines cultured in various degrees of hypoxia. This analysis reveals a surprising cancer cell line specific variation of HIF activity in the same level of hypoxia. We further show that in two cervical cancer cell lines, ME180 and HeLa, the different HIF activity levels observed correlate with the levels of hsp90, a cofactor that protects HIF against VHL-independent degradation. This novel HIF reporter construct serves as a tool to rapidly define HIF activity levels and therefore the therapeutic capacity of potential HIF repressors in individual cancers

Wnt signaling exerts an antiproliferative effect on adult cardiac progenitor cells through IGFBP3.

RATIONALE: Recent work in animal models and humans has demonstrated the presence of organ-specific progenitor cells required for the regenerative capacity of the adult heart. In response to tissue injury, progenitor cells differentiate into specialized cells, while their numbers are maintained through mechanisms of self-renewal. The molecular cues that dictate the self-renewal of adult progenitor cells in the heart, however, remain unclear. OBJECTIVE: We investigate the role of canonical Wnt signaling on adult cardiac side population (CSP) cells under physiological and disease conditions. METHODS AND RESULTS: CSP cells isolated from C57BL/6J mice were used to study the effects of canonical Wnt signaling on their proliferative capacity. The proliferative capacity of CSP cells was also tested after injection of recombinant Wnt3a protein (r-Wnt3a) in the left ventricular free wall. Wnt signaling was found to decrease the proliferation of adult CSP cells, both in vitro and in vivo, through suppression of cell cycle progression. Wnt stimulation exerted its antiproliferative effects through a previously unappreciated activation of insulin-like growth factor binding protein 3 (IGFBP3), which requires intact IGF binding site for its action. Moreover, injection of r-Wnt3a after myocardial infarction in mice showed that Wnt signaling limits CSP cell renewal, blocks endogenous cardiac regeneration and impairs cardiac performance, highlighting the importance of progenitor cells in maintaining tissue function after injury. CONCLUSIONS: Our study identifies canonical Wnt signaling and the novel downstream mediator, IGFBP3, as key regulators of adult cardiac progenitor self-renewal in physiological and pathological states

Disrupted in Schizophrenia 1 Regulates Neuronal Progenitor Proliferation via Modulation of GSK3β/β-Catenin Signaling

The Disrupted in Schizophrenia 1 (DISC1) gene is disrupted by a balanced chromosomal translocation (1; 11) (q42; q14.3) in a Scottish family with a high incidence of major depression, schizophrenia, and bipolar disorder. Subsequent studies provided indications that DISC1 plays a role in brain development. Here, we demonstrate that suppression of DISC1 expression reduces neural progenitor proliferation, leading to premature cell cycle exit and differentiation. Several lines of evidence suggest that DISC1 mediates this function by regulating GSK3β. First, DISC1 inhibits GSK3β activity through direct physical interaction, which reduces β-catenin phosphorylation and stabilizes β-catenin. Importantly, expression of stabilized β-catenin overrides the impairment of progenitor proliferation caused by DISC1 loss of function. Furthermore, GSK3 inhibitors normalize progenitor proliferation and behavioral defects caused by DISC1 loss of function. Together, these results implicate DISC1 in GSK3β/β-catenin signaling pathways and provide a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders.National Alliance for Research on Schizophrenia and Depression (U.S.) (Young Investigator Award)Natural Sciences and Engineering Research Council of Canada (Postdoctoral Award)Human Frontier Science Program (Strasbourg, France) (Fellowship)Singleton FellowshipNational Institutes of Health (U.S.) (Grant NS37007

Simvastatin Promotes Adult Hippocampal Neurogenesis by Enhancing Wnt/β-Catenin Signaling

Statins improve recovery from traumatic brain injury and show promise in preventing Alzheimer disease. However, the mechanisms by which statins may be therapeutic for neurological conditions are not fully understood. In this study, we present the initial evidence that oral administration of simvastatin in mice enhances Wnt signaling in vivo. Concomitantly, simvastatin enhances neurogenesis in cultured adult neural progenitor cells as well as in the dentate gyrus of adult mice. Finally, we find that statins enhance Wnt signaling through regulation of isoprenoid synthesis and not through cholesterol. These findings provide direct evidence that Wnt signaling is enhanced in vivo by simvastatin and that this elevation of Wnt signaling is required for the neurogenic effects of simvastatin. Collectively, these data add to the growing body of evidence that statins may have therapeutic value for treating certain neurological disorders

WIKI4, a novel inhibitor of tankyrase and Wnt/ß-catenin signaling.

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells. Furthermore, we demonstrate that WIKI4 mediates its effects on Wnt/ß-catenin signaling by inhibiting the enzymatic activity of TNKS2, a regulator of AXIN ubiquitylation and degradation. While TNKS has previously been shown to be the target of small molecule inhibitors of Wnt/ß-catenin signaling, WIKI4 is structurally distinct from previously identified TNKS inhibitors

Targeted BRAF Inhibition Impacts Survival in Melanoma Patients with High Levels of Wnt/β-Catenin Signaling

<div><p>Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of β-catenin immunohistochemical expression in pretreatment <i>BRAF</i>-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.</p></div

Lower levels of nuclear β-catenin significantly correlate with improved survival on BRAFi in post-hoc analysis.

<p>(A) In exploratory post-hoc analysis, patients with nuclear β-catenin scores in the lowest quartile exhibited a statistically-significant improvement in overall survival (p = 0.037) compared to the remainder of the cohort. (B) A similar trend towards improvement with patients in the lowest quartile was seen with progression-free survival, but did not reach statistical significance (p = 0.099).</p

Activated ERK1 and ERK2 inhibit Wnt/β-catenin signaling in melanoma cells.

<p>(A) Levels of ERK1/2 phosphorylation (ppERK1/2) were compared by immunoblot in BRAFi-naïve A375 melanoma cells and A375 cells treated long-term for 4–6 weeks with BRAFi (2 µM vemurafenib) or WNT3A + BRAFi. Long-term treatment with BRAFi results in re-activation of ERK1/2 phosphorylation. No significant changes were seen in levels of total ERK1/2 or β-tubulin. (B) Human A375 melanoma cells were transfected with control siRNA or siRNA targeting ERK1 and ERK2, either individually or in combination (ERK1/2) at a concentration of 20nM. After 48 hours, transfected cells were then cultured overnight in the absence or presence of WNT3A conditioned media. For comparison, cells transfected with control siRNA were also treated with BRAFi (1 µM PLX4720). Apoptosis was measured by the cleavage of PARP1. Specific knockdown of ERK1 and ERK2 were visualized by loss of the appropriate bands detected using antibodies targeting phosphorylated ERK1/2 (ppERK1/2) or total ERK1/2. AXIN1 abundance decreased with knockdown of ERK1 and ERK2, either individually or in combination, paralleling the observed decrease in abundance seen with PLX4720.</p

Long-term treatment with BRAFi downregulates cellular responses to WNT3A.

<p>Melanoma cell lines were cultured long-term (4–6 weeks) in 2 µM vemurafenib (BRAFi) in the absence or presence of continually-replenished WNT3A conditioned media (WNT3A and WNT3A + BRAFi). Cells were then treated with acutely with BRAF in the absence and presence of WNT3A conditioned media, and responses were compared to BRAFi-naïve cells (column sets 1–4). Transcriptional activation of Wnt/β-catenin signaling was assessed by quantitative-PCR-based measurements of the target gene <i>AXIN2</i>, normalized to <i>GAPDH</i> (upper bar sets). Cellular apoptosis was assessed through the detection of cleaved PARP by flow-cytometry (lower bar sets). Note that long-term treatment with BRAFi, WNT3A or WNT3A + BRAFi significantly attenuates activation of <i>AXIN2</i> transcription and enhancement of apoptosis by WNT3A. For each cell line, one-way ANOVA with Bonferroni's post-test was performed to determine statistical significance (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094748#pone.0094748.s001" target="_blank">Table S1</a>).</p