A long-standing but poorly understood observation in stem cell biology is that the quiescence of the adult stem cells associates with their longterm functions ( 1 -6 ). The molecular pathway that keeps them in quiescence is largely obscure, although recent studies have implicated stem cell niches ( 4 ) and cell-intrinsic functions of p21 ( 5 ) and Pten ( 6 ) in this process. The signifi cance of quiescence in stem cell function is bolstered as genetic disruption of its quiescence almost invariably inactivates the hematopoietic stem cell (HSC) function ( 4 -6 ). Nevertheless, it is largely unclear how an active metabolism is incompatible with a normal HSC function.In addition to cell-intrinsic factors, accumulating data demonstrated that residence of adult HSCs in the BM niches is essential for their quiescence and long-term functions ( 7 -9 ). Because exposure to high levels of oxygen damages the functions of HSCs ( 10 -15 ), it has been proposed that hypoxia is important for HSC functions. However, the underlying molecular mechanism of how hypoxia maintains the stemness is unknown.In Drosophila and in vitro -cultured mammalian cells, hypoxia activates tuberous sclerosis complex (TSC), which can inhibit the target of rapamycin (TOR), through REDD1 and AMP-activated protein kinase ( 16 -20 ). Whether this pathway operates in the HSCs has yet to be tested.The mammalian TOR (mTOR) pathway has emerged as a key regulator for cellular metabolism. Accumulating data have demonstrated that mTOR regulates several important cellular functions, including protein synthesis, autophagy, endocytosis and nutrient uptake ( 21 ). An increased mTOR activity results in increased cellular growth and nonmalignant growth of cells in solid organs ( 22,23 The tuberous sclerosis complex (TSC) -mammalian target of rapamycin (mTOR) pathway is a key regulator of cellular metabolism. We used conditional deletion of Tsc1 to address how quiescence is associated with the function of hematopoietic stem cells (HSCs). We demonstrate that Tsc1 deletion in the HSCs drives them from quiescence into rapid cycling, with increased mitochondrial biogenesis and elevated levels of reactive oxygen species (ROS). Importantly, this deletion dramatically reduced both hematopoiesis and self-renewal of HSCs, as revealed by serial and competitive bone marrow transplantation. In vivo treatment with an ROS antagonist restored HSC numbers and functions. These data demonstrated that the TSC -mTOR pathway maintains the quiescence and function of HSCs by repressing ROS production. The detrimental effect of up-regulated ROS in metabolically active HSCs may explain the well-documented association between quiescence and the " stemness " of HSCs.

Despite clear epidemiological and genetic evidence for X-linked prostate cancer risk, all prostate cancer genes identified are autosomal. Here we report somatic inactivating mutations and deletion of the X-linked FOXP3 gene residing at Xp11.23 in human prostate cancer. Lineage-specific ablation of FoxP3 in the mouse prostate epithelial cells leads to prostate hyperplasia and prostate intraepithelial neoplasia. In both normal and malignant prostate tissues, FOXP3 is both necessary and sufficient to transcriptionally repress cMYC, the most commonly over-expressed oncogene in prostate cancer as well as among the aggregates of other cancers. FOXP3 is an X-linked prostate tumor suppressor in the male. Since the male has only one X chromosome, our data represents a paradigm of “single-genetic-hit” inactivation-mediated carcinogenesis.

S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex.Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2. IntroductionCancer pathogenesis involves both the inactivation of tumor suppressor genes and the activation of oncogenes (1, 2). One of the most fascinating aspects of cancer biology is the interaction between cancer suppressor genes and oncogenes. Most of these interactions are at posttranslational levels. For instance, proteins encoded by tumor suppressor genes can inactivate oncogenes. One of the most clearly studied cases is tumor suppressor Rb, which inhibits the E2F family members of oncogenes (1,2). Conversely, oncogenes can overcome the tumor suppressor proteins. For example, S-phase kinase-associated protein 2 (SKP2) causes the degradation of tumor suppressor FOXO (3) as well as CDK inhibitors, such as p27 (4, 5). It is less clear whether such antagonism exists at the transcriptional level. However, a recent study suggests that FOXO may repress the expression of cyclin D (6), a well-known oncogene. Likewise, we have recently demonstrated that forkhead box P3 (FOXP3), an X-linked tumor suppressor, represses transcription of ERBB2/HER2 oncogene (7).The high-level expression of SKP2 was reported in a significant proportion of cancers (5). SKP2 is a component of the E3 ubiquitin ligase SCF with specificity for CDK inhibitor p27. However, under physiological conditions, Skp2 expression is maximal at the S and G2 phases and appears to primarily mediate p27 degradation at the G2 (8, 9) but not G0 and G1 phases (10). As such, Skp2 was found to be essential for progression into mitosis in cell cycles (4, 9, 11). Targeted mutation of Skp2 causes delayed animal growth and cellular polyplo...

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Abstractp21 loss has been implicated in conferring oncogenic activity to known tumor suppressor gene KLF4 and cancer drug tamoxifen. Regulators of p21, therefore, play critical roles in tumorigenesis. Here, we report that X-linked tumor suppressor FOXP3 is essential for p21 expression in normal epithelia and that lack of FOXP3 is associated with p21 down-regulation in breast cancer samples. A specific FOXP3 binding site in the intron 1 is essential for p21 induction by FOXP3. FOXP3 specifically inhibited binding of histone deacetylase 2 (HDAC2) and HDAC4 to the site and increased local histone H3 acetylation. Short hairpin RNA silencing of either HDAC2 or HDAC4 is sufficient to induce p21 expression. Our data provides a novel mechanism for transcription activation by FOXP3 and a genetic mechanism for lack of p21 in a large proportion of breast cancer. [Cancer Res 2009;69(6):2252-9]