Sensitivity of hematopoietic stem cells to mitochondrial dysfunction by SdhD gene deletion.

It is established that hematopoietic stem cells (HSC) in the hypoxic bone marrow have adapted their metabolism to oxygen-limiting conditions. This adaptation includes suppression of mitochondrial activity, induction of anerobic glycolysis, and activation of hypoxia-inducible transcription factor 1α (Hif1α)-dependent gene expression. During progression of hematopoiesis, a metabolic switch towards mitochondrial oxidative phosphorylation is observed, making this organelle essential for determining cell fate choice in bone marrow. However, given that HSC metabolism is essentially oxygen-independent, it is still unclear whether functional mitochondria are absolutely required for their survival. To assess the actual dependency of these undifferentiated cells on mitochondrial function, we have performed an analysis of the hematopoiesis in a mouse mutant, named SDHD-ESR, with inducible deletion of the mitochondrial protein-encoding SdhD gene. This gene encodes one of the subunits of the mitochondrial complex II (MCII). In this study, we demonstrate that, in contrast to what has been previously established, survival of HSC, and also myeloid and B-lymphoid progenitors, depends on proper mitochondrial activity. In addition, gene expression analysis of these hematopoietic lineages in SDHD-ESR mutants calls into question the proposed activation of Hif1α in response to MCII dysfunction.

E-cadherin downregulation sensitizes PTEN-mutant tumors to PI3Kß silencing.

Alterations in phosphatidylinositol 3-kinase (PI3K) and in PTEN (phosphatase and tensin homolog), the negative regulator of the PI3K pathway, are found in nearly half of human tumors. As PI3Kß, the main isoform activated in PTEN-mutant tumors, has kinase-dependent and -independent activities, we compared the effects of depleting vs. drug-inhibiting PI3Kß kinase activity in a collection of diverse tumor types and in a set of bladder carcinoma cell lines grown as xenografts in mice. PI3Kß depletion (by intratumor injection of PIK3CB siRNA) induced apoptosis and triggered regression of PTEN-mutant tumors more efficiently than PI3Kß inhibition. A small proportion of these tumors was resistant to PI3Kß downregulation; we analyzed what determined resistance in these cases. Using add-back experiments, we show that both PTEN mutation and low E-cadherin expression are necessary for PI3Kß dependence. In bladder carcinoma, loss of E-cadherin expression coincides with N-cadherin upregulation. We found that PI3Kß associated with N-cadherin and that PIK3CB depletion selectively disrupted N-cadherin cell adhesions in PTEN-mutant bladder carcinoma. These results support the use of PIK3CB interfering RNA as a therapeutic approach for high-risk bladder cancers that show E-cadherin loss and express mutant PTEN.

Myelomatous plasma cells display an aberrant gene expression pattern similar to that observed in normal memory B cells.

Memory B cells (MBCs) remain in a quiescent state for years, expressing pro-survival and anti-apoptotic factors while repressing cell proliferation and activation genes. During their differentiation into plasma cells (PCs), their expression pattern is reversed, with a higher expression of genes related to cell proliferation and activation, and a lower expression of pro-survival genes. To determine whether myelomatous PCs (mPCs) share characteristics with normal PCs and MBCs and to identify genes involved in the pathophysiology of multiple myeloma (MM), we compared gene expression patterns in these three cell sub-types. We observed that mPCs had features intermediate between those of MBCs and normal PCs, and identified 3455 genes differentially expressed in mPCs relative to normal PCs but with a similar expression pattern to that in MBCs. Most of these genes are involved in cell death and survival, cell growth and proliferation and protein synthesis. According to our findings, mPCs have a gene expression pattern closer to a MBC than a PC with a high expression of genes involved in cell survival. These genes should be physiologically inactivated in the transit from MBC to PC, but remain overexpressed in mPCs and thus may play a role in the pathophysiology of the disease.

The CD27+ memory B cells display changes in the gene expression pattern in elderly individuals.

Memory B cells (MBCs) have a very long life-span as compared to naïve B cells (NBCs), remaining viable for years. It could predispose them to suffer misbalances in the gene expression pattern at the long term, which might be involved in the development of age-related B-cell disorders. In order to identify genes whose expression might change during life, we analyzed the gene expression patterns of CD27- NBCs versus CD27+ MBCs in young and old subjects. Using microarray assays we observed that the expression pattern of CD27- NBCs versus CD27+ MBCs is significantly different. Furthermore, in order to evaluate the age effect, we compared the gene expression pattern of young versus aged subjects in both cell populations. Interestingly, we did not find significant differences in the CD27- NBC population between young and aged individuals, whereas we found 925 genes differentially expressed in CD27+ MBCs. Among these genes, 193 were also differentially expressed in CD27+ MBCs as compared to CD27- NBCs, most of them involved in cell survival, cell growth and proliferation, cellular development and gene expression. We conclude that gene expression profiles of CD27- NBCs and CD27+ MBCs are different. Moreover, whereas the gene expression pattern of CD27+ MBCs varies with age, the same does not happen in CD27- NBCs. This suggests that MBCs undergo time-dependent changes which could underlie a higher susceptibility to dysfunction with age. This article is protected by copyright. All rights reserved.

Genetically modeled mice with mutations in mitochondrial metabolic enzymes for the study of cancer.

Mitochondrial dysfunction has long been implicated in progression of cancer. As a paradigm, the "Warburg effect," which by means of a switch toward anaerobic metabolism enables cancer cells to proliferate in oxygen limiting conditions, is well established. Besides this metabolic transformation of tumors, it has been discovered that mutations in genes encoding mitochondrial proteins are the etiological factors in different types of cancer. This confers to mitochondrial dysfunction a causative role, rather than resultant, in tumor genesis beyond its role in tumor progression and development. Mitochondrial proteins encoded by tumor-suppressor genes are part of the succinate-dehydrogenase, the fumarate-hydratase, and the mitochondrial isocitrate-dehydrogenase enzymes, all of them participating in the Krebs cycle. The spectrum of tumors associated with mutations in these genes is becoming larger and varies between each enzyme. Several mechanisms of tumorigenesis have been proposed for the different enzymatic defects, most of them based on studies using cellular and animal models. Regarding the molecular pathways implicated in the oncogenic transformation, one of the first accepted theories was based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as "pseudo-hypoxic drive." This mechanism has been linked to the three types of mutations, thus suggesting a central role in cancer. However, other alternative molecular processes, such as oxidative stress or altered chromatin remodeling, have been also proposed to play an onco-pathogenic role. In the recent years, the role of oncometabolites, a new concept emerged from biochemical studies upon these tumors, has acquired relevance as responsible for tumor formation. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. In this review, we summarize the results obtained from mouse strains genetically modified in the three different enzymes.

A conditional mouse mutant in the tumor suppressor SdhD gene unveils a link between p21(WAF1/Cip1) induction and mitochondrial dysfunction.

Mutations in mitochondrial complex II (MCII; succinate dehydrogenase, Sdh) genes cause familiar pheochromocytoma/paraganglioma tumors. Several mechanisms have been proposed to account for Sdh-mutation-induced tumorigenesis, the most accepted of which is based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as "pseudo-hypoxic drive". Other molecular processes, such as oxidative stress, apoptosis, or chromatin remodeling have been also proposed to play a causative role. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. Moreover, the biological factors that determine the tissue-specificity of these tumors have not been identified. In this work, we made use of the inducible SDHD-ESR mouse, a conditional mutant in the SdhD gene, which encodes the small subunit of MCII, and that acts as a tumor suppressor gene in humans. The analysis of the Hif1α pathway in SDHD-ESR tissues and in two newly derived cell lines after complete SdhD loss -a requirement for hereditary paraganglioma type-1 tumor formation in humans- partially recapitulated the "pseudo-hypoxic" response and rendered inconsistent results. Therefore, we performed microarray analysis of adrenal medulla and kidney in order to identify other early gene expression changes elicited by SdhD deletion. Our results revealed that each mutant tissue displayed different variations in their gene expression profiles affecting to different biological processes. However, we found that the Cdkn1a gene was up-regulated in both tissues. This gene encodes the cyclin-dependent kinase inhibitor p21(WAF1/Cip1), a factor implicated in cell cycle, senescence, and cancer. The two SDHD-ESR cell lines also showed accumulation of this protein. This new and unprecedented evidence for a link between SdhD dysfunction and p21(WAF1/Cip1) will open new avenues for the study of the mechanisms that cause tumors in Sdh mutants. Finally, we discuss the actual role of Hif1α in tumorigenesis.