Xiaohua Su

TAp63 Prevents Premature Aging by Promoting Adult Stem Cell Maintenance

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 (TAp63(fl/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.

P63 steps into the limelight: Crucial roles in the suppression of tumorigenesis and metastasis

The role of p63 in cancer has been an area of intense debate and controversy. Is TP63 (which encodes p63) a tumour suppressor gene or an oncogene? This debate is partly due to the complexity of the gene. There are several p63 isoforms — some with tumour suppressive functions and others with oncogenic functions. In this Opinion article, we focus on the recent advances in understanding p63 biology and its roles in cancer. In this regard, we discuss the role of p63 in multiple stem cell compartments, ageing, in the response to DNA damage and in DNA repair. Finally, we highlight the importance of understanding the interactions between all three p53 family members and the potential impact of this knowledge on cancer therapy and regenerative medicine.

TAp63 is a master transcriptional regulator of lipid and glucose metabolism.

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.

Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf

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.

IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo.

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the β cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.

Induced multipotency in adult keratinocytes through down-regulation of Δnp63 or DGCR8

Significance The p53 family member deltaNp63 (ΔNp63) is required for transcriptional activation of the microprocessor complex subunit DGCR8 in epidermal cells, leading to terminal differentiation of tissues such as the epidermis. We show here that loss of ΔNp63 leads to the generation of cells with self-renewing but limited differentiation capacity. When DGCR8 is reexpressed in cells deficient for ΔNp63, these cells can terminally differentiate into all three germ layers. We dubbed these cells induced multipotent stem cells because of their remarkable plasticity and ability to differentiate into multiple cell lineages. Based on our results using human keratinocytes, we predict that epidermal cells can be extracted from patient skin biopsies and reprogrammed into multipotent stem cells by knockdown of ΔNp63 or DGCR8. The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63−/− epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63−/− epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.

Inhibiting glutaminase in acute myeloid leukemia: metabolic dependency of selected AML subtypes

Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.

IκB Kinase β (IKKβ) Inhibits p63 Isoform γ (TAp63γ) Transcriptional Activity

Background: The functional regulation of p63 Isoform γ (TAp63γ) by IκB Kinase β (IKKβ) remains unknown, although they were previously shown to bind to each other. Results: IKKβ, but not its kinase dead mutant, suppressed the transcriptional activity of TAp63γ by disrupting its interaction with p300. Conclusion: IKKβ negatively regulates the transcriptional activity of TAp63γ. Significance: IKKβ may favor cell proliferation by inhibiting TAp63γ activity. Previously, we reported that IκB kinase-β(IKKβ) phosphorylates and stabilizes TAp63γ. However, the effect of this phosphorylation on TAp63γ transcriptional activity remains unclear. In this study, we showed that overexpression of IKKβ, but not its kinase dead mutant and IKKα, can surprisingly inhibit TAp63γ transcriptional activity as measured by luciferase assays and real-time PCR analyses of p63 target genes. This inhibition was impaired by ACHP, an IKKβ inhibitor, and enhanced by TNFα that activates IKKβ. Consistently, IKKβ inhibited the binding between TAp63γ and p300, a co-activator of TAp63γ, and consequently counteracted the positive effect of p300 on TAp63γ transcriptional activity. Through phosphorylation site prediction and mass spectrometry, we identified that Ser-4 and Ser-12 of p63 are IKKβ-targeting residues. As expected, IKKβ fails to suppress the transcriptional activity of the S4A/S12A double mutant p63. These results indicate that IKKβ can suppress TAp63γ activity by interfering with the interaction between TAp63γ and p300.

Ccdc3: A new P63 target involved in regulation of liver lipid metabolism

TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, upregulates CCDC3 expression by directly binding to its enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity, but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but conversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance and steatosis formation in transgenic CCDC3 mice on high-fat diet (HFD) by reducing the expression of hepatic PPARγ and its target gene CIDEA as well as other genes involved in de novo lipogenesis. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on HFD. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.

Abstract 2331: Deletion of ΔNp63 and ΔNp73 in p53 deficient mice results in TAp63 and TAp73 compensation of p53 tumor suppression in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC p53 tumor suppressor undergoes mutational loss in majority of cancers contributing to tumor formation. Therapeutic strategies are aimed towards p53 overexpression in tumors or to identify targets that compensate for p53-functional loss. p63 & p73, share structural similarities to p53, making them excellent candidates for therapeutic compensation of p53. Unlike p53, p63 and p73 do not undergo mutational loss and their role in tumorigenesis is being delineated. p63 and p73 have two major isoforms, the transactivation (TA), with activities similar to p53 and the delta (Δ)N- isoform with oncogenic functions. Inhibition of TAp63 and TAp73 is observed in cancers as a consequence of overexpression of ΔN isoforms of p63 and p73. In disparity, recent studies report, tumor suppressive properties of ΔNp63 and ΔNp73 in activating genes involved in DNA repair and apoptosis. To define the functional roles of ΔNp63 and ΔNp73 in cancer, mouse models targeting the ΔN isoforms were generated. We observed that, ΔNp63+/- and ΔNp73−/− mice on a p53−/− background had lower thymic lymphoma incidence compared to the p53−/− mice. I found TAp63 and TAp73 up regulated in the double mutant mice that correspond with an increase in p53-downstream apoptotic (PUMA, Noxa, BAX) and cell cycle targets (p21, p16, PML). This suggests that ablation of ΔN isoforms mediate TAp63 and TAp73 up regulation inducing apoptosis or cell cycle arrest by activation of p53-downstream targets. To further demonstrate this, I ablated ΔNp63 and ΔNp73 in vivo in p53−/- mice thymic lymphoma by administering adenoviral-CRE specifically to the thymus. The CRE-treated mice had a significant thymic lymphoma regression within 3 weeks as imaged by MRI in comparison to the mock-treated mouse cohorts. Additionally, RNA-Seq analysis from CRE-treated versus untreated mice, has identified novel metabolic genes with apoptotic or cell-cycle functions. We further report, ΔNp63 and ΔNp73 to bind to promoter site of TAp63 and TAp73 by chromatin immunoprecipitation (ChIP). This supports the notion that ablation of ΔN isoforms of p63 and p73 restores the function of TAp63 and TAp73 thus compensating for p53-tumor suppressive function in vivo. To test, if ablation of ΔN isoforms reduces tumorigenesis in human cancers, ΔNp63 and ΔNp73 were knocked down in human cancer cell lines were p53 expression was ablated or mutated. TAp63 and TAp73 were upregulated in ΔNp63/ΔNp73 knock down human cancer cell lines. However, induction of apoptosis or cell-cycle arrest was observed in p53-deleted cancer cell lines in comparison to the p53-mutated cell lines. This highlights the co-repressive effect of mutant p53, preventing activation of TAp63/TAp73 downstream targets. Current work is aimed towards overcoming mutant p53 effect in these cancer cell lines. Thus, targeting the ΔNp63/ΔNp73 compensates for p53-functional loss mediating tumor suppression. Citation Format: Avinashnarayan Venkatanarayan, Deepavali Chakravarti, Xiaohua Su, Santosh Sandur, Lingzhi Liu, Eliot Fletcher Sananikone, Payal Raulji, Cristian Coarfa, William Norton, Preethi Gunaratne, Elsa Renee Flores. Deletion of ΔNp63 and ΔNp73 in p53 deficient mice results in TAp63 and TAp73 compensation of p53 tumor suppression in vivo . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2331. doi:10.1158/1538-7445.AM2013-2331