Rodolfo M. Alarcon

A role of miR‐27 in the regulation of adipogenesis

MicroRNAs (miRNAs) are involved in a plethora of important biological processes, from embryonic development to homeostasis in adult tissues. Recently, miRNAs have emerged as a class of epigenetic regulators of metabolism and energy homeostasis. We have investigated the role of miRNAs in the regulation of adipogenic differentiation. In this article, we demonstrate that the miR‐27 gene family is downregulated during adipogenic differentiation. Overexpression of miR‐27 specifically inhibited adipocyte formation, without affecting myogenic differentiation. We also found that expression of miR‐27 resulted in blockade of expression of PPARγ and C/EBPα, the two master regulators of adipogenesis. Importantly, expression of miR‐27 was increased in fat tissue of obese mice and was regulated by hypoxia, an important extracellular stress associated with obesity. Our data strongly suggest that miR‐27 represents a new class of adipogenic inhibitors and may play a role in the pathological development of obesity.

Regulation of p53 by Hypoxia: Dissociation of Transcriptional Repression and Apoptosis from p53-Dependent Transactivation

ABSTRACT Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.

In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection

Hypoxia is a key determinant of tumor aggressiveness, yet little is known regarding hypoxic global gene regulation in vivo. We used the hypoxia marker EF5 coupled with laser-capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. Through microarray analysis, we identified several mRNAs (including the HIF targets Vegf, Glut-1, and Hsp27) with increased levels under hypoxia compared with normoxia both in vitro and in vivo. However, we also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. Intriguingly, the mRNA levels of a substantial number of immunomodulatory and DNA repair proteins including CXCL9, CD3D, and RAD51 were found to be downregulated in hypoxic areas in vivo, consistent with a protumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic versus normoxic tumor regions. Moreover, CD8(+) T cells, which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. This is the first study to analyze the influence of hypoxia on mRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors.

Oxygen and Cell Fate Decisions.

Molecular oxygen has been known to play a critical role in a wide range of biological processes including glycolysis, mitochondrial respiration, angiogenesis, pulmonary functions, and cardiovascular activities. An emerging theme has developed in recent years that oxygen has significant impact on embryonic development, maintenance of stem cells, and cellular differentiation or cell fate decisions. Among the notable observations, early embryonic development takes place in a hypoxic microenvironment. Hematopoietic stem cells appear to be located in hypoxic regions within the bone marrow. Majority of the current observations have shown that hypoxia seems to prevent cellular differentiation and to maintain pluripotency of stem/progenitor cells. Genetic studies have demonstrated a critical role of hypoxia-inducible factors 1α and 2α in embryonic development. These intriguing observations demonstrate an important role of molecular oxygen in such fundamental biological processes as stem cell maintenance and regulation of cell fate decisions. Herein, we describe some of the latest advances in the biology of molecular oxygen and provide our perspectives on the potential impact of these interesting findings.

p53 mediates apoptosis induced by c-Myc activation in hypoxic or gamma irradiated fibroblasts.

Deregulated c-Myc expression leads to a cellular state where proliferation and apoptosis are equally favored depending on the cellular microenvironment. Since the apoptotic sensitivity of many cells is influenced by the status of the p53 tumor suppressor gene, we investigated whether the induction of apoptosis by DNA damage or non-genotoxic stress are also influenced by the p53 status of cells with altered c-Myc activity. Rat-1 fibroblasts expressing a conditional c-Myc allele (c-MycER), were transfected to express an antisense RNA complimentary to p53 mRNA. Expression of antisense p53 RNA decreased p53 protein levels and delayed p53 accumulation following c-Myc activation. Under hypoxic or low serum conditions, cells expressing antisense p53 were substantially more resistant to c-Myc-induced apoptosis than were control cells. c-Myc activation also sensitized Rat-1 cells to radiation-induced apoptosis. Rat-1 cells expressing antisense p53 RNA were more resistant to apoptosis induced by the combined effects of c-Myc activation and gamma irradiation. In a similar manner, apoptosis induced by c-Myc in serum starved, hypoxic or gamma irradiated fibroblasts was also inhibited by Bcl-2. These data indicate that p53 is involved in c-Myc-mediated apoptosis under a variety of stresses which may influence tumor growth, evolution and response to therapy.

Abstract 5004: In vivo profiling of hypoxic mRNA and miRNA expression in gliomas and head and neck tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Hypoxia plays a key role in tumor aggressiveness and radiation resistance, yet little is known regarding hypoxic global gene regulation in vivo. Although the regulation of mRNA and miRNA expression by hypoxia has been investigated in isolation in vitro, it has not yet been analyzed directly in tumor samples. We have used the hypoxia marker EF5 coupled with laser capture microdissection (LCM) to isolate RNA from viable hypoxic and normoxic regions of 9L experimental rat gliomas and from human head and neck (H&N) tumor samples. This was followed by microarray analysis of mRNA expression and comparison of this signature with that obtained from treating 9L cells with hypoxia in vitro. Through this, we have identified several mRNAs (including the HIF targets Vegf, Glut-1 and Hsp27) with increased levels under hypoxia compared to normoxia both in vitro and in vivo. We also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. An intriguing finding was the hypoxic-downregulation of a number of immunomodulatory and DNA repair proteins including CXCL9, CD3D and RAD51 in vivo, consistent with a pro-tumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic vs. normoxic tumor regions. Moreover, CD8+ T cells which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. Global microRNA expression changes have been reported to occur in response to hypoxia in vitro. Hence, our second objective was to identify the cluster of miRNAs differentially expressed in hypoxic vs. normoxic regions of the human tumors using TaqMan® Array MicroRNA Cards. We employed the same technique used with the 9L tumors to analyze miRNA expression patterns in hypoxic vs. normoxic samples from H&N cancer patients who have been administered EF5 prior to surgical removal of the tumor. Consistent with published in vitro studies, miR-210 emerged as a major miRNA robustly induced in the hypoxic regions. We also confirmed miR-210 induction by an individual real time-PCR assay and found it to be upregulated > 2-fold in all hypoxic RNA samples. In addition, we have also found several miRNAs whose levels were upregulated and downregulated in hypoxic vs. normoxic areas. This is the first study to analyze the influence of hypoxia on mRNA and miRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5004. doi:10.1158/1538-7445.AM2011-5004