The development of intratumoral hypoxia is a universal hallmark of rapidly growing solid tumors. Adaptation to the hypoxic environment, which is critical for tumor cell survival and growth, is mediated primarily through a hypoxia-inducible factor (HIF)-dependent transcriptional program. HIF activates genes that facilitate crucial adaptive mechanisms including increased glucose uptake and glycolysis and tumor angiogenesis, making it an important therapeutic target. However, the HIF-dependent transcriptional mechanism remains incompletely understood, and targeting HIF is a difficult endeavor. Here, we show that the orphan nuclear receptor estrogen-related receptors (ERRs) physically interact with HIF and stimulate HIF-induced transcription. Importantly, ERRs appear to be essential for HIF's function. Transcriptional activation of hypoxic genes in cells cultured under hypoxia is largely blocked by suppression of ERRs through expression of a dominant negative form of ERR or treatment with a pharmacological ERR inhibitor, diethylstilbestrol. Systematic administration of diethylstilbestrol severely diminished growth and angiogenesis of tumor xenografts in vivo. Because nuclear receptors are outstanding targets for drug discovery, the findings not only may offer mechanistic insights into HIF-mediated transcription but also may open new avenues for targeting the HIF pathway for cancer therapy.angiogenesis ͉ energy metabolism ͉ nuclear hormone receptor R apidly growing solid tumors tend to outstrip the supply of oxygen and nutrients provided by blood vessels, resulting in regions of low oxygen levels (hypoxia). Cancer cells undergo adaptation to persist in the hostile hypoxic environment. The adaptive response to hypoxia is controlled primarily by the hypoxia-inducible factor (HIF), a master regulator of hypoxic gene expression and oxygen homeostasis (1-3). HIF is a heterodimeric transcription factor comprising an oxygen-regulated ␣-subunit (HIF1␣ or -2␣) and a constitutively expressed and stable ␤-subunit (HIF␤). Under normal oxygen tension, the HIF␣ subunit is subjected to prolyl hydroxylation catalyzed by a set of oxygen-and ferrous ion-dependent prolyl hydroxylases. Hydroxylated HIF␣ is recognized by the tumor-suppressor protein von Hippel-Lindau (VHL), a component of an E3 ubiquitin ligase complex. Subsequently, HIF␣ becomes polyubiquitinated and is targeted for rapid degradation by the proteasomal system. Oxygen deprivation, or administration of iron chelators or cobaltous ion (a classic hypoxia mimetic, which competes with ferrous ion), suppresses hydroxylase activity, allowing HIF␣ to escape the VHL-mediated destruction and to accumulate and dimerize with the constitutively present HIF␤. The binding of the HIF␣-HIF␤ heterodimer along with the transcriptional coactivator p300/CBP to the cognate hypoxia-responsive element (HRE) augments the expression of a plethora of hypoxic genes that carry such elements within their promoters or enhancers (4). The HIF-orchestrated transcriptional program is directly responsible ...

SUMMARY Canonical Wnt signaling pathway, mediated by the transcription factor β-catenin, plays critical roles in embryonic development, and represents an important therapeutic target. In a zebrafish-based in vivo screen for small molecules that specifically perturb embryonic dorsoventral patterning, we discovered a novel compound, named windorphen, which selectively blocks the Wnt signal required for ventral development. Windorphen exhibits remarkable specificity toward β-catenin-1 function, indicating that the two β-catenin isoforms found in zebrafish are not functionally redundant. We show that windorphen is a selective inhibitor of p300 histone acetyl transferase, a co-activator that associates with β-catenin. Lastly, windorphen robustly and selectively kills cancer cells that harbor Wnt-activating mutations, supporting the therapeutic potential of this novel Wnt inhibitor class.

The possibility of using cell-based therapeutics to treat cardiac failure has generated significant interest since the initial introduction of stem cell-based technologies. However, the methods to quickly and robustly direct stem cell differentiation towards cardiac cell types have been limited by a reliance on recombinant growth factors to provide necessary biological cues. We report here the use of dorsomorphin homologue 1 (DMH1), a second-generation small molecule BMP inhibitor based on dorsomorphin, to efficiently induce beating cardiomyocyte formation in mouse embryonic stem cells (ESCs) and to specifically upregulate canonical transcriptional markers associated with cardiac development. DMH1 differs significantly from its predecessor by its ability to enrich for pro-cardiac progenitor cells that respond to late-stage Wnt inhibition using XAV939 and produce secondary beating cardiomyocytes. Our study demonstrates the utility of small molecules to complement existing in vitro cardiac differentiation protocols and highlights the role of transient BMP inhibition in cardiomyogenesis.

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.