MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP.

Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through miR-101, which targets proteasome biogenesis to control overall protein turnover and tumor cell proliferation.

The fibronectin leucine-rich repeat transmembrane protein Flrt2 is required in the epicardium to promote heart morphogenesis.

The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium.

Targeting the heat shock protein 90: a rational way to inhibit macrophage migration inhibitory factor function in cancer.

PURPOSE OF REVIEW: Macrophage migration inhibitory factor (MIF), originally identified as a proinflammatory cytokine, is highly elevated in many human cancer types, independent of their histological origin. MIF's tumour promoting activities correlate with tumour aggressiveness and poor clinical prognosis. Genetic depletion of MIF in mouse cancer models results in significant inhibition of cell proliferation and induction of apoptosis, making it an attractive target for anticancer therapies. Here, we summarize the current possibilities to inhibit MIF function in cancer. RECENT FINDINGS: All known small molecule MIF inhibitors antagonize MIF's enzymatic function. However, a recent knockin mouse model suggested that protein interactions play a bigger biological role in tumour cell growth regulation than MIF's enzymatic activity. Thus, alternative strategies are important for targeting MIF. Recently, we identified that MIF in cancer cells is highly stabilized through the heat shock protein 90 machinery (HSP90). Thus, MIF is an HSP90 client. Pharmacological inhibition of the Hsp90 ATPase activity results in MIF degradation in several types of cancer cells. This provides a new way to inhibit MIF function independent of its enzymatic activity. SUMMARY: Targeting the HSP90 machinery is a promising way to inhibit MIF function in cancer. Along with MIF and dependent on the molecular make-up of the tumour, a large number of other critical tumourigenic proteins are also destabilized by HSP90 inhibition, overall resulting in a profound block of tumour growth.

Involvement of Fractin in TGF-beta-induced apoptosis in oligodendroglial progenitor cells.

Transforming growth factor-beta (TGF-beta) induces apoptotic cell death during the development of the nervous system. We recently identified that TGF-beta induced apoptosis in oligodendroglial progenitor cells (primary cells as well as oligodendroglial cell line OLI-neu) is characterized by down-regulation of Bcl-xl. In this report, we now focused on mechanisms that mediate TGF-beta dependent Bcl-xl down-regulation in oligodendroglial cells. We showed that the caspase-specific cleavage product Fractin is produced in oligodendroglial cells during TGF-beta-mediated apoptosis, which represents an early event of the cascade. Cleavage of actin into Fractin was dependent on functional actin and caspases, and occurred simultaneously with a Fractin-Bcl-xl-interaction. This Fractin-Bcl-xl interaction indicated a connection between Bcl-xl down-regulation and Fractin appearance, since Bcl-xl regulation was also dependent on caspases and functional actin, and an overexpression of Fractin induced a Bcl-xl protein down-regulation. Further analysis of Fractin-Bcl-xl interaction in other culture systems confirmed these data. In conclusion, we show that Fractin is not only an apoptotic marker, but has indeed a functional role in apoptotic signaling in oligodendrocytes.

TGF-beta superfamily members, ActivinA and TGF-beta1, induce apoptosis in oligodendrocytes by different pathways.

Activins and transforming growth factor (TGF)-betas, members of the TGF-beta superfamily, affect numerous physiological processes, including apoptosis, in a variety of organs and tissues. Apoptotic functions of TGF-betas, in contrast to those of the activins, are well documented in the developing and adult nervous system. TGF-betas operate in a context-dependent manner and cooperate with other cytokines in the regulation of apoptosis. In this study, we show, for the first time, an apoptotic function of ActivinA in the nervous system, i.e. in oligodendroglial progenitor cells. Using the oligodendroglial cell line OLI-neu, we show that ActivinA acts autonomously, without cooperating with TGF-beta. In contrast to the mechanism of TGF-beta-mediated apoptosis involving Bcl-xl down-regulation, Bcl-xl in ActivinA-induced apoptosis is classically sequestered by the BH3-only protein Puma. Puma expression is controlled by the transcription factor p53 as demonstrated by experiments with the p53 inhibitor Pifithrin-alpha. Furthermore, in the apoptotic TGF-beta pathway, caspase-3 is activated, whereas in the apoptotic ActivinA pathway, apoptosis-inducing factor is released to trigger DNA fragmentation. These data suggest that TGF-beta and ActivinA induce apoptosis in oligodendrocytes by different apoptotic pathways.

HSP90 inhibitor antagonizing MIF: The specifics of pleiotropic cancer drug candidates.

Constitutively stabilized HSP90 client proteins are crucial for cancer cell survival and proliferation. Thus, despite-or perhaps because of-their pleiotropic effects on variety of critical oncoproteins, HSP90 inhibitors represent a promising new class of anticancer drugs. We identified MIF as an essential HSP90 client protein in a murine model of Her2-overexpressing breast cancer.

Inhibiting the HSP90 chaperone destabilizes macrophage migration inhibitory factor and thereby inhibits breast tumor progression.

Intracellular macrophage migration inhibitory factor (MIF) often becomes stabilized in human cancer cells. MIF can promote tumor cell survival, and elevated MIF protein correlates with tumor aggressiveness and poor prognosis. However, the molecular mechanism facilitating MIF stabilization in tumors is not understood. We show that the tumor-activated HSP90 chaperone complex protects MIF from degradation. Pharmacological inhibition of HSP90 activity, or siRNA-mediated knockdown of HSP90 or HDAC6, destabilizes MIF in a variety of human cancer cells. The HSP90-associated E3 ubiquitin ligase CHIP mediates the ensuing proteasome-dependent MIF degradation. Cancer cells contain constitutive endogenous MIF-HSP90 complexes. siRNA-mediated MIF knockdown inhibits proliferation and triggers apoptosis of cultured human cancer cells, whereas HSP90 inhibitor-induced apoptosis is overridden by ectopic MIF expression. In the ErbB2 transgenic model of human HER2-positive breast cancer, genetic ablation of MIF delays tumor progression and prolongs overall survival of mice. Systemic treatment with the HSP90 inhibitor 17AAG reduces MIF expression and blocks growth of MIF-expressing, but not MIF-deficient, tumors. Together, these findings identify MIF as a novel HSP90 client and suggest that HSP90 inhibitors inhibit ErbB2-driven breast tumor growth at least in part by destabilizing MIF.

While p73 is essential, p63 is completely dispensable for the development of the central nervous system.

The ancient p53 paralogs p63 and p73 regulate specific tissue formation, cell survival and cell death via their TA and ΔN isoforms. Targeted disruption of the p73 locus leads to severe defects in the development of the central nervous system (CNS), and p73 has recently been shown to be an essential regulator of neural stem cell maintenance and differentiation in both embryonal and adult neurogenesis. In contrast, global p63-/- mice lack skin and limbs. Moreover, p63 is detectable in embryonic cortex. It has previously been proposed to also play critical pro-death and pro-survival roles in neural precursors of the developing sympathetic and central nervous system, respectively, based on experimental overexpression and siRNA-mediated knockdown of p63. Here we perform an extensive analysis of the developing central nervous system in global p63-/- mice and their wildtype littermates. Brain and spinal cord of embryos and newborn mice were assessed in vivo for neuroanatomy, histology, apoptosis, proliferation, stemness and differentiation, and in vitro for self-renewal and maturation in neurosphere assays. None of these analyses revealed a detectable phenotype in p63-/- mice. Hence, despite the profound impact of p63 on the development of stratified epithelia and limbs, p63 is completely dispensable for proper development of the central nervous system. Thus, despite their strong homology, the non-overlapping tissue specificity of p63 and p73 functions appears more pronounced than previously anticipated.

Functional inactivation of endogenous MDM2 and CHIP by HSP90 causes aberrant stabilization of mutant p53 in human cancer cells.

The tight control of wild-type p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53, which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wild-type p53 tumors. Importantly, mutant p53 hyperstabilization is critical for oncogenic gain of function of mutant p53 in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutant p53 knock-in mice established that normal mutant p53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutant p53. We find that in human cancer cells, endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wild-type p53, the many mutant p53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wild-type p53 cancer cells, we show that in mutant p53 cancer cells, this HSP90 interaction blocks the endogenous MDM2 and CHIP (carboxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with HSP90 either by RNA interference against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacologic inhibition of Hsp90 activity with 17AAG (17-allylamino-17-demethoxygeldanamycin) destroys the complex, liberates mutant p53, and reactivates endogenous MDM2 and CHIP to degrade mutant p53. Of note, 17AAG induces a stronger viability loss in mutant p53 than in wild-type p53 cancer cells. Our data support the rationale that suppression of mutant p53 levels in vivo in established cancers might achieve clinically significant effects.

The yeast CPC2/ASC1 gene is regulated by the transcription factors Fhl1p and Ifh1p.

CPC2/ASC1 is one of the most abundantly transcribed genes in Saccharomyces cerevisiae. It encodes a ribosome-associated Gbeta-like WD protein, which is highly conserved from yeast to man. Here, we show that CPC2 transcription depends on the carbon source and is induced during utilization of the sugar glucose. CPC2 promoter deletion and insertion analyses identified two upstream activation sequence elements for CPC2, which are required for basal expression and regulation. One of these upstream activation sequence elements has an ATGTACGGATGT motif, which has previously been described as a putative binding site for the forkhead-like transcription factor Fhl1p. Deletion of FHL1 reduces CPC2 transcription significantly in presence of glucose, but has no effect when the non-fermentable carbon source ethanol is provided. Increased amounts of the Fhl1p co-regulator Ifh1p induce CPC2 transcription even when ethanol is utilized. These data suggest that the interaction between Fhl1p and Ifh1p is critical for the regulation of CPC2 transcription during utilization of different carbon sources.