Aberrant expression of microRNAs (miRNAs) and the enzymes that control their processing have been reported in multiple biological processes including primary and metastatic tumours1–6, but the mechanisms governing this are not clearly understood. Here we show that TAp63, a p53 family member, suppresses tumorigenesis and metastasis, and coordinately regulates Dicer and miR-130b to suppress metastasis. Metastatic mouse and human tumours deficient in TAp63 express Dicer at very low levels, and we found that modulation of expression of Dicer and miR-130b markedly affected the metastatic potential of cells lacking TAp63. TAp63 binds to and transactivates the Dicer promoter, demonstrating direct transcriptional regulation of Dicer by TAp63. These data provide a novel understanding of the roles of TAp63 in tumour and metastasis suppression through the coordinate transcriptional regulation of Dicer and miR-130b and may have implications for the many processes regulated by miRNAs.

SUMMARY The cellular mechanisms that regulate the maintenance of adult tissue stem cells are still largely unknown. We show here that the p53 family member, TAp63, is essential for maintenance of epidermal and dermal precursors and that, in its absence, these precursors senesce and skin ages prematurely. Specifically, we have developed a TAp63 conditional knockout mouse and used it to ablate TAp63 in the germline (TAp63−/−) or in K14-expressing cells in the basal layer of the epidermis (TAp63fl/fl;K14cre+). TAp63−/− mice age prematurely and develop blisters, skin ulcerations, senescence of hair follicle-associated dermal and epidermal cells, and decreased hair morphogenesis. These phenotypes are likely due to loss of TAp63 in dermal and epidermal precursors since both cell types show defective proliferation, early senescence, and genomic instability. These data indicate that TAp63 serves to maintain adult skin stem cells by regulating cellular senescence and genomic stability, thereby preventing premature tissue aging.

The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis.

SUMMARY TAp63 prevents premature aging suggesting a link to genes that regulate longevity. Further characterization of TAp63−/− mice revealed that these mice develop obesity, insulin resistance, and glucose intolerance, similar to those seen in mice lacking two key metabolic regulators, Silent information regulator T1 (Sirt1) and AMPK. While the roles of Sirt1 and AMPK in metabolism have been well studied, their upstream regulators are not well understood. We found that TAp63 is important in regulating energy metabolism by accumulating in response to metabolic stress and transcriptionally activating Sirt1, AMPKα2, and LKB1 resulting in increased fatty acid synthesis and decreased fatty acid oxidation. Moreover, we found that TAp63 lowers blood glucose levels in response to metformin. Restoration of Sirt1, AMPKα2, and LKB1 in TAp63−/− mice rescued some of the metabolic defects of the TAp63−/− mice. Our study defines a role for TAp63 in metabolism and weight control.

Mice lacking p63 cannot form skin, exhibit craniofacial and skeletal defects, and die soon after birth. The p63 gene regulates a complex network of target genes, and disruption of p63 has been shown to affect the maintenance of epithelial stem cells, the differentiation of keratinocytes, and the preservation of the adhesive properties of stratified epithelium. Here, we show that inactivation of p63 in mice is accompanied by aberrantly increased expression of the Ink4a and Arf tumour suppressor genes. In turn, anomalies of the p63-null mouse affecting the skin and skeleton are partially ameliorated in mice lacking either Ink4a or Arf. Rescue of epithelialization is accompanied by restoration of keratinocyte proliferative capacity both in vivo and in vitro and by expression of markers of squamous differentiation. Thus, in the absence of p63, abnormal upregulation of Ink4a and Arf is incompatible with skin development.

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of β2-spectrin (β2SP, encoded by SPTBN1), a SMAD adaptor for TGF-β signaling, is causally associated with BWS; however, a role of TGF-β deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-β signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-β-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-β inducible and facilitates TGF-β-mediated repression of TERT transcription via interactions with β2SP and SMAD3. This regulation was abrogated in TGF-β-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-β pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Mechanisms regulating the transition of mammary epithelial cells (MECs) to mammary stem cells (MaSCs) and to tumor-initiating cells (TICs) have not been entirely elucidated. The p53 family member, p63, is critical for mammary gland development and contains transactivation domain isoforms, which have tumor-suppressive activities, and the ΔN isoforms, which act as oncogenes. In the clinic, p63 is often used as a diagnostic marker, and further analysis of the function of TAp63 in the mammary gland is critical for improved diagnosis and patient care. Loss of TAp63 in mice leads to the formation of aggressive metastatic mammary adenocarcinoma at 9–16 months of age. Here we show that TAp63 is crucial for the transition of mammary cancer cells to TICs. When TAp63 is lost, MECs express embryonic and MaSC signatures and activate the Hippo pathway. These data indicate a crucial role for TAp63 in mammary TICs and provide a mechanism for its role as a tumor- and metastasis-suppressor in breast cancer.

Background: The incidence of cardiovascular disease (CVD) is higher in HIV + patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for CVD. However, the molecular mechanisms that link cART and CVD are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species (ROS)-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. Methods: Peripheral whole blood from cART-treated HIV + individuals and non-treated HIV − individuals was treated with H 2 O 2 (200 μM) for 4 minutes, and p90RSK activity in CD14 + monocytes was measured. Plaque formation in the carotids were also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK specific inhibitor (FMK-MEA) or dominant negative p90RSK (DN-p90RSK), and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild type and DN-p90RSK transgenic mice. Results: Monocytes from HIV + patients exhibited higher levels of p90RSK activity and were also more sensitive to ROS than monocytes from HIV − individuals. A multiple linear regression analysis involving cART, Reynolds CV risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the two significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H 2 O 2-induced over-activation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Lastly, the data obtained from our gain-or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. Conclusions: cART increased monocyte/macrophage sensitivity to ROS in HIV + individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and pro-inflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing CVD, especially in HIV + patients treated with cART.