Yingjuan Qian

Examination of the expanding pathways for the regulation of p21 expression and activity.

p21(Waf1/Cip1/Sdi1) was originally identified as an inhibitor of cyclin-dependent kinases, a mediator of p53 in growth suppression and a marker of cellular senescence. p21 is required for proper cell cycle progression and plays a role in cell death, DNA repair, senescence and aging, and induced pluripotent stem cell reprogramming. Although transcriptional regulation is considered to be the initial control point for p21 expression, there is growing evidence that post-transcriptional and post-translational regulations play a critical role in p21 expression and activity. This review will briefly discuss the activity of p21 and focus on current knowledge of the determinants that control p21 transcription, mRNA stability and translation, and protein stability and activity.

p53, a Target of Estrogen Receptor (ER) α, Modulates DNA Damage-induced Growth Suppression in ER-positive Breast Cancer Cells

Background: ERα and p53 are transcription factors that play important roles in breast cancer. Results: ERα transcriptionally regulates p53, which then modulates DNA damage-induced growth suppression. Conclusion: p53 is a target of ERα and is responsible for the sensitivity of ERα-positive breast cancer cells to DNA damage. Significance: Loss of ERα, causing a decrease in p53 expression, could lead to tumors resistant to both antiestrogen and chemotherapy. In response to genotoxic stress, the p53 tumor suppressor induces target genes for cell cycle arrest, apoptosis, and DNA repair. Although p53 is the most commonly mutated gene in all human cancers, it is only mutated in about 20% of breast cancers. 70% of all breast cancer cases are estrogen receptor (ER)-positive and express ERα. ER-positive breast cancer generally indicates good patient prognosis and treatment responsiveness with antiestrogens, such as tamoxifen. However, ER-positive breast cancer patients can experience loss or a reduction in ERα, which is associated with aggressive tumor growth, increased invasiveness, poor prognosis, and loss of p53 function. Consistent with this, we found that p53 is a target gene of ERα. Specifically, we found that knockdown of ERα decreases expression of p53 and its downstream targets, MDM2 and p21. In addition, we found that ERα activates p53 transcription via binding to estrogen response element half-sites within the p53 promoter. Moreover, we found that loss of ERα desensitizes, whereas ectopic expression of ERα sensitizes, breast cancer cells to DNA damage-induced growth suppression in a p53-dependent manner. Altogether, this study provides an insight into a feedback loop between ERα and p53 and a biological role of p53 in the DNA damage response in ER-positive breast cancers.

DNA polymerase eta is targeted by Mdm2 for polyubiquitination and proteasomal degradation in response to ultraviolet irradiation

DNA polymerase eta (PolH), the product of the xeroderma pigmentosum variant (XPV) gene and a Y-family DNA polymerase, plays a pivotal role in translesion DNA synthesis. Loss of PolH leads to early onset of malignant skin cancer in XPV patients and increases UV-induced carcinogenesis. Thus, the pathways by which PolH expression and activity are controlled may be explored as a strategy to prevent UV-induced cancer. In this study, we found that Mdm2, a RING finger E3 ligase, promotes PolH degradation. Specifically, we showed that knockdown of Mdm2 increases PolH expression in both p53-proficient and -deficient cells. In addition, we showed that UV-induced PolH degradation is attenuated by Mdm2 knockdown. In contrast, ectopically expression of Mdm2 decreases PolH expression, which can be abrogated by the proteasome inhibitor MG132. Moreover, we showed that Mdm2 physically associates with PolH and promotes PolH polyubiquitination in vivo and in vitro. Finally, we showed that knockdown of Mdm2 increases the formation of PolH replication foci and decreases the sensitivity of cells to UV-induced lesions in a PolH-dependent manner. Taken together, we uncovered that Mdm2 serves as an E3 ligase for PolH polyubiquitination and proteasomal degradation in cells under the basal condition and in response to UV irradiation.

The G Protein–Coupled Receptor 87 Is Necessary for p53-Dependent Cell Survival in Response to Genotoxic Stress

p53 regulates an array of target genes, which mediates p53 tumor suppression by inducing cell cycle arrest, apoptosis, and cell survival. G protein-coupled receptors belong to a superfamily of cell surface molecules and are known to regulate cell proliferation, migration, and survival. Here, we found that G protein-coupled receptor 87 (GPR87) was up-regulated by p53 and by DNA damage in a p53-dependent manner. We also found that p53 directly regulated GPR87 potentially via a p53-responsive element in the GPR87 gene. To investigate the role of GPR87 in the p53 pathway, we generated multiple RKO and MCF7 cell lines in that GPR87 can be inducibly overexpressed or knocked down by a tetracycline-inducible system. We found that overexpression of GPR87 had little effect on cell growth. However, GPR87 knockdown sensitized cancer cells to DNA damage-induced growth suppression via enhanced p53 stabilization and activation. Importantly, the prosurvival activity of GPR87 can be reversed by knockdown of p53. Together, our results suggested that GPR87 is essential for p53-dependent cell survival in response to DNA damage. Thus, due to its expression on the cell surface and its role in cell survival, GPR87 may be explored as a novel therapeutic target for cancer treatment and prevention.

The p53-Estrogen Receptor Loop in Cancer

Tumor suppressor p53 maintains genome stability by regulating diverse cellular functions including cell cycle arrest, apoptosis, senescence and metabolic homeostasis. Mutations in the p53 gene occur in almost all human cancers with a frequency of up to 80%. However, it is only 20% in breast cancers, 18% in endometrial cancers and 1.5% in cervical cancers. Estrogen receptor alpha (ERα) plays a pivotal role in hormone-dependent cancer development and the status of ERα is used for designing treatment strategy and for prognosis. A closer look at the cross-talk between p53 and ERα has revealed that their activities are mutually regulated. This review will summarize the current body of knowledge on p53, ERα and ERβ in cancer. Clinical correlations between estrogen receptors and p53 status have also been reported. Thus, this review will discuss the relationship between p53 and ERs at both the molecular and clinical levels.

Pirh2 RING‐finger E3 ubiquitin ligase: Its role in tumorigenesis and cancer therapy

The ubiquitin‐dependent proteasome system plays a critical role in many cellular processes and pathogenesis of various human diseases, including cancer. Although there are a large number of E3 ubiquitin ligases, the majority are RING‐finger type E3s. Pirh2, a target of p53 transcription factor, contains a highly conserved C3H2C3 type RING domain. Importantly, Pirh2 was found to regulate a group of key factors dedicated to the DNA damage response, such as p53, p73, PolH, and c‐Myc. Interestingly, Pirh2 was upregulated or downregulated in different types of cancers. These suggest that Pirh2 is implicated in either promoting or suppressing tumor progression in a tissue‐dependent manner. This review will focus on the major findings in these studies and discuss the potential to explore Pirh2 as a cancer therapeutic target.

Pirh2 E3 Ubiquitin Ligase Modulates Keratinocyte Differentiation through p63

p63, a homologue of the tumor suppressor p53, is essential for the development of epidermis and limb. p63 is highly expressed in epithelial cell layer and acts as a molecular switch that initiates epithelial stratification. However, the mechanisms controlling p63 protein level is still far from fully understood. Here, we demonstrate a regulatory protein for the p63 activity. We found that Pirh2 E3 ubiquitin ligase physically interacts with p63 and targets p63 for polyubiquitination and subsequently proteasomal degradation. We also found that ectopic expression of Pirh2 in HaCaT cells suppresses cell proliferation. Consistent with this, we found that along with altered expression of ΔNp63 protein, ectopic expression of Pirh2 promotes, whereas knockdown of Pirh2 inhibits, keratinocyte differentiation. Collectively, our data suggest that Pirh2 plays a physiologically relevant role in keratinocyte differentiation through posttranslational modification of p63 protein.

The p73 Tumor Suppressor Is Targeted by Pirh2 RING Finger E3 Ubiquitin Ligase for the Proteasome-dependent Degradation

Background: The status of p73 expression is linked to the sensitivity of tumor cells to therapy, but how p73 expression is regulated remains uncertain. Results: Pirh2 E3 ubiquitin ligase promotes the proteasomal turnover of TAp73. Conclusion: Pirh2 is a novel E3 ligase of p73. Significance: Targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy. The p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and ΔN isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas ΔNp73 has both pro- and anti-survival functions. While p73 is rarely mutated in spontaneous tumors, the expression status of p73 is linked to the sensitivity of tumor cells to chemotherapy and prognosis for many types of human cancer. Thus, uncovering its regulators in tumors is of great interest. Here, we found that Pirh2, a RING finger E3 ubiquitin ligase, promotes the proteasome-dependent degradation of p73. Specifically, we showed that knockdown of Pirh2 up-regulates, whereas ectopic expression of Pirh2 down-regulates, expression of endogenous and exogenous p73. In addition, Pirh2 physically associates with and promotes TAp73 polyubiquitination both in vivo and in vitro. Moreover, we found that p73 can be degraded by both 20 S and 26 S proteasomes. Finally, we showed that Pirh2 knockdown leads to growth suppression in a TAp73-dependent manner. Taken together, our findings indicate that Pirh2 promotes the proteasomal turnover of TAp73, and thus targeting Pirh2 to restore TAp73-mediated growth suppression in p53-deficient tumors may be developed as a novel anti-cancer strategy.

Differentiated embryo-chondrocyte expressed gene 1 regulates p53-dependent cell survival versus cell death through macrophage inhibitory cytokine-1

Activation of p53 upon DNA damage induces an array of target genes, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which the cell fate is controlled by p53 remains to be clarified. Previously, we showed that DEC1, a basic helix–loop–helix transcription factor and a target of p53, is capable of inducing cell cycle arrest and mediating DNA damage-induced premature senescence. Here, we found that ectopic expression of DEC1 inhibits, whereas knockdown of DEC1 enhances, DNA damage-induced cell death. Surprisingly, we showed that the anti–cell-death activity of DEC1 is p53 dependent, but DEC1 does not directly modulate p53 expression. Instead, we showed that DEC1 inhibits the ability of p53 to induce macrophage inhibitory cytokine-1 (MIC-1), but not other prosurvival/proapoptotic targets, including p21 and Puma. Importantly, we showed that upon binding to their respective response elements on the MIC-1 promoter, DEC1 and p53 physically interact on the MIC-1 promoter via the basic helix–loop–helix domain in DEC1 and the tetramerization domain in p53, which likely weakens the DNA-binding activity of p53 to the MIC-1 promoter. Finally, we found that depletion of MIC-1 abrogates the ability of DEC1 to attenuate DNA damage-induced cell death. Together, we hypothesize that DEC1 controls the response of p53-dependent cell survival vs. cell death to a stress signal through MIC-1.

ΔNp63, a Target of DEC1 and Histone Deacetylase 2, Modulates the Efficacy of Histone Deacetylase Inhibitors in Growth Suppression and Keratinocyte Differentiation

The p63 gene, a member of the p53 family, is expressed as TA and ΔN isoforms. ΔNp63 is the predominant isoform expressed in cells of epithelial origin and frequently overexpressed in cancers. However, what regulates p63 expression is uncertain. Here, we showed that ΔNp63 is regulated by the transcription factor DEC1, a p53 family target. We also showed that the ability of DEC1 to regulate ΔNp63 is enhanced by histone deacetylase (HDAC) inhibitors or knockdown of histone deacetylase 2 (HDAC2). Consistent with this, we found that DEC1 and HDAC2 physically interact and knockdown of HDAC2 leads to increased binding of DEC1 to the ΔNp63 promoter. Interestingly, we found that growth suppression induced by HDAC inhibitors is attenuated by ectopic expression of DEC1 in a ΔNp63-dependent manner. In addition, we showed that ectopic expression of DEC1 inhibits, whereas knockdown of DEC1 promotes, keratinocyte differentiation via modulating ΔNp63 expression. Finally, we showed that DEC1 cooperates with HDAC inhibitors to further decrease keratinocyte differentiation. Together, we conclude that ΔNp63 is a novel target of DEC1 and HDAC2 and modulates the efficacy of HDAC inhibitors in growth suppression and keratinocyte differentiation.