Zhi-Xiong Xiao

Role of p63 in Development, Tumorigenesis and Cancer Progression

The p53-related protein p63 has pleiotropic functions, including cell proliferation, survival, apoptosis, differentiation, senescence, and aging. The p63 gene is expressed as multiple isoforms that either contain an N-terminal p53-homologous transactivation domain (TAp63) or that lack this domain (ΔNp63). Multiple studies have demonstrated that p63 plays a crucial role in stratified epithelial development, and have shown the importance of p63 for maintaining proliferation potential, inducing differentiation, and preventing senescence. Additionally, much research focuses on the role of p63 in cancer progression. Clinical evidence suggests that p63 may play a role in inhibiting metastasis. Similarly, genetic mice models together with cell culture data strongly indicate that p63 deficiency may be a causative factor for metastatic spread. Moreover, the role of p63 in cancer metastasis has been shown to be greatly related to the ability of mutant p53 to promote cancer malignancy. However, there is still much confusion as to what the role of each specific isoform is. In this review, we highlight some of the major findings in the current literature regarding the role of specific p63 isoforms in development, tumorigenesis, and particularly in cancer metastasis.

Deubiquitylase OTUD3 regulates PTEN stability and suppresses tumorigenesis

PTEN is one of the most frequently mutated tumour suppressors and reduction in PTEN protein stability also plays a role in tumorigenesis. Although several ubiquitin ligases for PTEN have been identified, the deubiquitylase for de-polyubiquitylation and stabilization of PTEN is less defined. Here, we report OTUD3 as a deubiquitylase of PTEN. OTUD3 interacts with, de-polyubiquitylates and stabilizes PTEN. Depletion of OTUD3 leads to the activation of Akt signalling, induction of cellular transformation and cancer metastasis. OTUD3 transgenic mice exhibit higher levels of the PTEN protein and are less prone to tumorigenesis. Reduction of OTUD3 expression, concomitant with decreased PTEN abundance, correlates with human breast cancer progression. Furthermore, we identified loss-of-function OTUD3 mutations in human cancers, which either abolish OTUD3 catalytic activity or attenuate the interaction with PTEN. These findings demonstrate that OTUD3 is an essential regulator of PTEN and that the OTUD3–PTEN signalling axis plays a critical role in tumour suppression.

Histone methyltransferase SETDB1 regulates liver cancer cell growth through methylation of p53

SETDB1 is a histone H3K9 methyltransferase that has a critical role in early development. It is located within a melanoma susceptibility locus and facilitates melanoma formation. However, the mechanism by which SETDB1 regulates tumorigenesis remains unknown. Here we report the molecular interplay between SETDB1 and the well-known hotspot gain-of-function (GOF) TP53 R249S mutation. We show that in hepatocellular carcinoma (HCC) SETDB1 is overexpressed with moderate copy number gain, and GOF TP53 mutations including R249S associate with this overexpression. Inactivation of SETDB1 in HCC cell lines bearing the R249S mutation suppresses cell growth. The TP53 mutation status renders cancer cells dependent on SETDB1. Moreover, SETDB1 forms a complex with p53 and catalyses p53K370 di-methylation. SETDB1 attenuation reduces the p53K370me2 level, which subsequently leads to increased recognition and degradation of p53 by MDM2. Together, we provide both genetic and biochemical evidence for a mechanism by which SETDB1 regulates cancer cell growth via methylation of p53.

Pin1 modulates p63α protein stability in regulation of cell survival, proliferation and tumor formation

The homolog of p53 gene, p63, encodes multiple p63 protein isoforms. TAp63 proteins contain an N-terminal transactivation domain similar to that of p53 and function as tumor suppressors; whereas ΔNp63 isoforms, which lack the intact N-terminal transactivation domain, are associated with human tumorigenesis. Accumulating evidence demonstrating the important roles of p63 in development and cancer development, the regulation of p63 proteins, however, is not fully understood. In this study, we show that peptidyl-prolyl isomerase Pin1 directly binds to and stabilizes TAp63α and ΔNp63α via inhibiting the proteasomal degradation mediated by E3 ligase WWP1. We further show that Pin1 specifically interacts with T538P which is adjacent to the P550PxY543 motif, and disrupts p63α–WWP1 interaction. In addition, while Pin1 enhances TAp63α-mediated apoptosis, it promotes ΔNp63α-induced cell proliferation. Furthermore, knockdown of Pin1 in FaDu cells inhibits tumor formation in nude mice, which is rescued by simultaneous knockdown of WWP1 or ectopic expression of ΔNp63α. Moreover, overexpression of Pin1 correlates with increased expression of ΔNp63α in human oral squamous cell carcinoma samples. Together, these results suggest that Pin1-mediated modulation of ΔNp63α may have a causative role in tumorigenesis.

ATM and LKB1 dependent activation of AMPK sensitizes cancer cells to etoposide-induced apoptosis.

The present study aims to determine the effect of AMPK on etoposide-induced apoptosis of cancer cells. Our results revealed that etoposide induced AMPK activation in prostate C4-2 cancer cells, an event that was attenuated by ATM siRNA. In A549 cells that lack LKB1, AMPK was unable to be activated by etoposide, which was restored by introduction of LKB1. Likewise, silencing LKB1 in C4-2 cells impaired AMPK activation. Finally, etoposide displayed a potent pro-apoptotic effect in cancer cells with functional LKB1 and AMPK. Thus, our results establish a linear relationship of ATM, LKB1 and AMPK in response to the DNA damage drug.

ΔNp63α activates CD82 metastasis suppressor to inhibit cancer cell invasion.

P63 is a p53 family member involved in multiple facets of biology, including embryonic development, cell proliferation, differentiation, survival, apoptosis, senescence and aging. The p63 gene encodes multiple protein isoforms either with (TAp63) or without (ΔNp63) the N-terminal transactivation domain. Amounting evidence suggests that p63 can function as a tumor suppressor, yet the precise molecular mechanisms, and particularly the specific roles of TAp63 and ΔNp63 in cancer progression, are still largely unclear. Here, we demonstrated that ΔNp63α, the predominant isoform expressed in epithelial cells and squamous cell carcinomas, inhibits cell invasion. Affymetrix gene expression profiling, combined with gain- and loss-of-function analyses and chromatin immunoprecipitation, indicated that cluster of differentiation 82 (CD82), a documented metastasis suppressor, is a direct transcriptional target of ΔNp63α. Expression of ΔNp63α inhibited outgrowth in Matrigel and cancer cell invasion, which was largely reversed by specific ablation of CD82. Conversely, ΔNp63α knockdown led to increased cell invasion, which was reversed by ectopic expression of CD82. Moreover, inhibition of glycogen synthase kinase-3β (GSK3β) by either pharmacological inhibitors or by RNA interference resulted in the downregulation of ΔNp63α and CD82 expression, concomitant with increased cell invasion, independently of β-catenin. Furthermore, decreased expression of p63 and CD82 is correlated with cancer progression. Taken together, this study reveals that ΔNp63α upregulates CD82 to inhibit cell invasion, and suggests that GSK3β can regulate cell invasion by modulating the ΔNp63α–CD82 axis.

Regulation of p63 Protein Stability via Ubiquitin-Proteasome Pathway

The p53-related p63 gene encodes multiple protein isoforms, which are involved in a variety of biological activities. p63 protein stability is mainly regulated by the ubiquitin-dependent proteasomal degradation pathway. Several ubiquitin E3 ligases have been identified and some protein kinases as well as other kinds of proteins are involved in regulation of p63 protein stability. These regulators are responsive to diverse extracellular signaling, resulting in changes of the p63 protein levels and impacting different biological processes.

MUC1 activates JNK1 and inhibits apoptosis under genotoxic stress.

The MUC1 transmembrane glycoprotein is aberrantly overexpressed in diverse human carcinomas and has been shown to inhibit apoptosis induced by genotoxic agents. In the present work, we report that MUC1 binds to and activates JNK1, an important member of the mitogen-activated protein kinases (MAPK) superfamily. The physical interaction between MUC1 cytoplasmic domain (MUC1-CD) and JNK1 was established by GST-pull-down assay in vitro and co-immunoprecipitation assay in vivo. We show that MUC1 activates JNK1 and inhibits cisplatin-induced apoptosis in human colon cancer HCT116 cells. Pharmacological inhibition of JNK or knockdown of JNK significantly reduces the ability of MUC1 to inhibit cisplatin-induced apoptosis. Together, our data indicate that MUC1 can inhibit apoptosis via activating JNK1 pathway in response to genotoxic anticancer agents.

p53 Degradation by a Coronavirus Papain-like Protease Suppresses Type I Interferon Signaling

Background: The molecular mechanism of coronavirus PLPs suppressing the innate immune response remains unclear. Results: PLP2 induces the degradation of p53 through stabilizing MDM2, and IRF7 is a novel target gene of p53. Conclusion: PLP2 inhibits the p53-mediated production of type I IFN and apoptosis to ensure viral growth. Significance: We identify the mechanism with which coronavirus induces the low dosage IFN production. Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling.

MDMX exerts its oncogenic activity via suppression of retinoblastoma protein

Inactivation of the retinoblastoma protein (RB) has a major role in the development of human malignancies. We have previously shown that MDM2, an ubiquitin E3 ligase and major negative regulator of p53, binds to and promotes proteasome-mediated degradation of RB. MDMX, a homolog of MDM2, also binds to and inhibits p53 transactivation activity, yet it does not possess intrinsic ubiquitin ligase activity. Here, we show that MDMX binds to and promotes RB degradation in an MDM2-dependent manner. Specifically, the MDMX C-terminal ring domain binds to the RB C-pocket and enhances MDM2–RB interaction. Silencing MDMX induces RB accumulation, cell cycle arrest and senescence-like phenotypes, which are reverted by simultaneous RB knockdown. Furthermore, MDMX ablation leads to significant retardation of xenograft tumor growth, concomitant with RB accumulation. These results demonstrate that MDMX exerts oncogenic activity via suppression of RB, and suggest that both MDM2 and MDMX could be chemotherapeutic targets.