DNA polymerase eta (POLH), a target of p53 tumor suppressor, plays a key role in translesion DNA synthesis (TLS). Loss of POLH is responsible for human cancer prone syndrome, Xeroderma Pigmentosum Variant (XPV). Due to its critical role in DNA repair and genome stability, POLH expression and activity are regulated by multiple pathways. In this study, we found that the levels of both POLH transcript and protein were decreased upon knockdown of the transcript encoding poly(rC)-binding protein 1 (PCBP1). We also found that the half-life of POLH mRNA was markedly decreased upon knockdown of PCBP1. Moreover, we found that PCBP1 directly bound to POLH 3'UTR and the PCBP1-binding site in POLH mRNA is an atypical AU-rich element. Finally, we showed that the AU-rich element in POLH 3'UTR was responsive to PCBP1 and sufficient for PCBP1 to regulate POLH expression. Altogether, we uncovered a novel mechanism by which POLH expression is controlled by PCBP1 via mRNA stability.
The p53 gene is mutated in more than 50% of human tumors. Mutant p53 exerts an oncogenic function and is often highly expressed in cancer cells due to evasion of proteasome-dependent degradation. Thus, reactivating proteasome-dependent degradation of mutant p53 protein is an attractive strategy for cancer management. Previously, we found that arsenic trioxide (ATO), a drug for acute promyelocytic leukemia, degrades mutant p53 protein through a proteasome pathway. However, it remains unclear what is the E3 ligase that targets mutant p53 for degradation. In current study, we sought to identify an E3 ligase necessary for ATO-mediated degradation of mutant p53. We found that ATO induces expression of Pirh2 E3 ligase at the transcriptional level. We also found that knockdown of Pirh2 inhibits, whereas ectopic expression of Pirh2 enhances, ATO-induced degradation of mutant p53 protein. Furthermore, we found that Pirh2 E3 ligase physically interacts with and targets mutant p53 for polyubiquitination and subsequently proteasomal degradation. Interestingly, we found that ATO cooperates with HSP90 or HDAC inhibitor to promote mutant p53 degradation and growth suppression in tumor cells. Together, these data suggest that ATO promotes mutant p53 degradation in part via induction of the Pirh2-dependent proteasome pathway.
P73, a member of the p53 family, plays a critical role in neural development and tumorigenesis. Due to the usage of two different promoters, p73 is expressed as two major isoforms, TAp73 and ?Np73, often with opposing functions. Here, we reported that transcriptional factor DEC1, a target of the p53 family, exerts a distinct control of TAp73 and ?Np73 expression. In particular, we showed that DEC1 was able to increase TAp73 expression via transcriptional activation of the TAp73 promoter. By contrast, Np73 transcription was inhibited by DEC1 via transcriptional repression of the ?Np73 promoter. To further explore the underlying mechanism, we showed that DEC1 was unable to increase TAp73 expression in the absence of HDAC8, suggesting that HDAC8 is required for DEC1 to enhance TAp73 expression. Furthermore, we found that DEC1 was able to interact with HDAC8 and recruit HDAC8 to the TAp73, but not the ?Np73, promoter. Together, our data provide evidence that DEC1 and HDAC8 in differentially regulate TAp73 and ?Np73 expression, suggesting that this regulation may lay a foundation for a therapeutic strategy to enhance the chemosensitivity of tumor cells.
P63, a p53 family member, plays pivotal roles in epidermal development, aging, and tumorigenesis. Thus, understanding how p63 expression is controlled has biological and clinical importance. RBM24 is an RNA-binding protein and shares a high sequence similarity with RBM38, a critical regulator of p63. In the current study, we investigated whether RBM24 is capable of regulating p63 expression. Indeed, we found that ectopic expression of RBM24 decreased, whereas knockdown of RBM24 increased, the levels of p63 transcript and protein. To explore the underlying mechanism, we found that RBM24 was able to bind to multiple regions in the p63 3 untranslated region and subsequently, destabilize p63 transcript. Furthermore, we showed that the 3 untranslated region in p63 transcript and the RNA-binding domain in RBM24 were required for RBM24 to bind p63 transcript and consequently, inhibit p63 expression. Taken together, our data provide evidence that RBM24 is a novel regulator of p63 via mRNA stability. Implications: Our study suggests that p63 is regulated by RBM24 via mRNA stability, which gives an insight into understanding how posttranscriptional regulatory mechanisms contribute to p63 expression.
p21, a cyclin-dependent kinase inhibitor, is necessary for proper control of the cell cycle and premature senescence. Thus, p21 expression needs to be tightly controlled. In this study, we found that Rbm24, an RNA-binding protein and a target gene of the p53 protein, can regulate p21 expression via mRNA stability. Specifically, we showed that Rbm24 is induced by DNA damage and Mdm2 inhibitor Nutlin-3. We also found that p53 protein binds to and activates the promoter of the Rbm24 gene. Moreover, we found that overexpression of Rbm24 increases, whereas knockdown of Rbm24 decreases, p21 mRNA and protein expression. In addition, we demonstrated that overexpression of Rbm24 enhances the half-life of p21 transcript. Consistent with this, we provided evidence that Rbm24 binds to the 3 untranslated region (3 UTR) of p21 transcript and an AU/U-rich element in the p21 3 UTR is necessary for Rbm24 to increase p21 expression. Finally, we showed that the RNA recognition motif in Rbm24 is required for binding to p21 transcript and subsequently for inducing p21 expression. Altogether, we uncover that Rbm24 is a novel player in the p53 pathway, which may be explored to restore proper cell cycle control in p53-deficient tumors via p21.
The RNPC1 RNA-binding protein, also called Rbm38, is a target of p53 and a repressor of p53 mRNA translation. Thus, the p53-RNPC1 loop is critical for modulating p53 tumor suppression, but it is not clear how the loop is regulated. Here, we showed that RNPC1 is phosphorylated at Ser195 by glycogen synthase kinase 3 (GSK3). We also showed that GSK3 promotes p53 mRNA translation through phosphorylation of RNPC1. Interestingly, we found that the phosphor-mimetic mutant S195D and the deletion mutant ?189-204, which lacks the GSK3 phosphorylation site, are unable to repress p53 mRNA translation due to loss of interaction with eukaryotic translation factor eIF4E on p53 mRNA. Additionally, we found that phosphorylated RNPC1, RNPC1-S195D, and RNPC1(?189-204) promote p53 mRNA translation through interaction with eukaryotic translation factor eIF4G, which then facilitates the assembly of the eIF4F complex on p53 mRNA. Furthermore, we showed that upon inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, GSK3 is activated, leading to increased RNPC1 phosphorylation and increased p53 expression in a RNPC1-dependent manner. Together, we postulate that the p53-RNPC1 loop can be explored to increase or decrease p53 activity for cancer therapy.
Macrophage inhibitory cytokine-1 (MIC-1), a secreted cytokine, is a direct target of p53 and known to play a role in cell proliferation, apoptosis, cell metastasis, and angiogenesis through autocrine and paracrine signaling. Previous studies have shown that serum levels of MIC-1 closely parallel cancer progression and are being explored as a diagnostic tool. MIC-1 has also shown potential as a therapeutic agent as it has exhibited several anti-carcinogenic activities. Thus, MIC-1 displays two opposing effects: tumor suppression versus promotion. However, it remains unclear whether MIC-1 is regulated by a mechanism other than transcription and how MIC-1 exerts its tumor suppression. In this study, we show that overexpression of RNA-binding protein RNPC1 can increase, whereas knockdown or knock-out of RNPC1 decreases, MIC-1 transcript and protein levels. Additionally, we demonstrate that RNPC1 can bind to MIC-1 mRNA via an AU-rich element within MIC-1 3-UTR and then enhances MIC-1 mRNA stability. Finally, to explore the functional significance of MIC-1, we showed that knockdown of MIC-1 can decrease RNPC1-induced cell growth suppression. Altogether, we uncover a novel mechanism by which MIC-1 can be regulated through RNPC1 via mRNA stability.
The tumor suppressor protein p53 plays a crucial role in coordinating cellular processes, such as cell cycle arrest, apoptosis, and senescence. The nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a homophilic adhesion molecule and involved in nerve regeneration. Interestingly, Ninj1 is found to be overexpressed in human cancer, but its role in tumorigenesis is not clear. Here, we found that Ninj1 is transcriptionally regulated by p53 and can be induced by DNA damage in a p53-dependent manner. We also found that knockout or knockdown of Ninj1 increases p53 expression potentially through enhanced p53 mRNA translation. In addition, we found that Ninj1 deficiency suppresses cell proliferation but enhances apoptosis and premature senescence in a p53-dependent manner. Consistent with this, we found that mice heterozygous in ninj1 are hypersensitive to ionizing radiation-induced lethality, along with increased expression of p53 in thymus. Taken together, we provided evidence that Ninj1 is a p53 target and modulates p53 mRNA translation and p53-dependent premature senescence, cell proliferation, apoptosis, and radiation-induced mortality in vitro and in vivo. Thus, we postulate that as a membrane adhesion molecule, Ninj1 is an ideal target to regulate p53 activity via the p53-Ninj1 loop.
Histone deacetylases (HDACs) play important roles in fundamental cellular processes, and HDAC inhibitors are emerging as promising cancer therapeutics. p73, a member of the p53 family, plays a critical role in tumor suppression and neural development. Interestingly, p73 produces two classes of proteins with opposing functions: the full-length TAp73 and the N-terminally truncated ?Np73. In the current study, we sought to characterize the potential regulation of p73 by HDACs and found that histone deacetylase 1 (HDAC1) is a key regulator of TAp73 protein stability. Specifically, we showed that HDAC1 inhibition by HDAC inhibitors or by siRNA shortened the half-life of TAp73 protein and subsequently decreased TAp73 expression under normal and DNA damage-induced conditions. Mechanistically, we found that HDAC1 knockdown resulted in hyperacetylation and inactivation of heat shock protein 90, which disrupted the interaction between heat shock protein 90 and TAp73 and thus promoted the proteasomal degradation of TAp73. Functionally, we found that down-regulation of TAp73 was required for the enhanced cell migration mediated by HDAC1 knockdown. Together, we uncover a novel regulation of TAp73 protein stability by HDAC1-heat shock protein 90 chaperone complex, and our data suggest that TAp73 is a critical downstream mediator of HDAC1-regulated cell migration.
p53, a guardian of the genome, exerts its tumor suppression activity by regulating a large number of downstream targets involved in cell cycle arrest, DNA repair, apoptosis, and cellular senescence. Although p53-mediated apoptosis is able to kill cancer cells, a role for cellular senescence in p53-dependent tumor suppression is becoming clear. Mouse studies showed that activation of p53-induced premature senescence promotes tumor regression in vivo. However, p53-mediated cellular senescence also leads to aging-related phenotypes, such as tissue atrophy, stem cell depletion, and impaired wound healing. In addition, several p53 isoforms and two p53 homologs, p63 and p73, have been shown to play a role in cellular senescence and/or aging. Importantly, p53, p63, and p73 are necessary for the maintenance of adult stem cells. Therefore, understanding the dual role the p53 protein family in cancer and aging is critical to solve cancer and longevity in the future. In this chapter, we provide an overview on how p53, p63, p73, and their isoforms regulate cellular senescence and aging.
Mutation of the p53 gene is the most common genetic alteration in human malignances and associated clinically with tumor progression and metastasis. To determine the effect of mutant p53 on epithelial differentiation, we developed three-dimensional culture (3-D) of Madin-Darby canine kidney (MDCK) cells. We found that parental MDCK cells undergo a series of morphological changes and form polarized and growth-arrested cysts with hollow lumen, which resembles branching tubules in vitro. We also found that upon knockdown of endogenous wild-type p53 (p53-KD), MDCK cells still form normal cysts in 3-D culture, indicating that p53-KD alone is not sufficient to disrupt cysts formation. However, we found that ectopic expression of mutant R163H (human equivalent R175H) or R261H (human equivalent R273H) in MDCK cells leads to disruption of cyst polarity and formation of invasive aggregates, which is further compounded by knockdown of endogenous wild-type p53. Consistently, we found that expression of E-cadherin, ?-catenin, and epithelial-to-mesenchymal transition (EMT) transcription factors (Snail-1, Slug and Twist) is altered by mutant p53, which is also compounded by knockdown of wild-type p53. Moreover, the expression level of c-Met, the hepatocyte growth factor receptor and a key regulator of kidney cell tubulogenesis, is enhanced by combined knockdown of endogenous wild-type p53 and ectopic expression of mutant R163H or R261H but not by each individually. Together, our data suggest that upon inactivating mutation of the p53 gene, mutant p53 acquires its gain of function by altering morphogenesis and promoting cell migration and invasion in part by upregulating EMT and c-Met.
p63, a transcription factor and p53 family protein, plays a crucial role in tumor suppression and development of various epithelial tissues. While p63 expression is controlled mostly by post-translational modifications, recent studies indicate that transcriptional and posttranscriptional regulations are essential for proper p63 expression. Here, we investigated the regulation of p63 expression by poly (C)-binding protein 1 (PCBP1, also known as hnRNP-E1 and ?CP1). We found that knockdown of PCBP1 decreases the level of p63 transcript and protein. We also found that PCBP1 regulates the stability of p63 mRNA via binding to p63 3UTR. Additionally, we found that a CU-rich element (CUE) in p63 3UTR is bound by and responsive to PCBP1. Together, we conclude that PCBP1 regulates p63 expression via mRNA stability.
Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multiple MCF10A cell lines in which PUMA, p21, or both were stably knocked down. We found that morphogenesis of MCF10A cells was altered modestly by knockdown of either PUMA or p21 alone but markedly by knockdown of both PUMA and p21. Moreover, we found that knockdown of PUMA and p21 leads to loss of E-cadherin expression along with increased expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, we found that knockdown of ?Np73, which antagonizes the ability of wide-type p53 and TA isoform of p73 to regulate PUMA and p21, mitigates the abnormal morphogenesis and EMT induced by knockdown of PUMA or p21. Together, our data suggest that PUMA cooperates with p21 to regulate normal acinus formation and EMT.
Castration-resistant prostate cancer continues to rely on androgen receptor (AR) expression. AR plays a central role in the development of prostate cancer and progression to castration resistance during and after androgen deprivation therapy. Here, we identified miR-let-7c as a key regulator of expression of AR. miR-let-7c suppresses AR expression and activity in human prostate cancer cells by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation of human prostate cancer cells. Down-regulation of Let-7c in prostate cancer specimens is inversely correlated with AR expression, whereas the expression of Lin28 (a repressor of let-7) is correlated positively with AR expression. Our study demonstrates that the miRNA let-7c plays an important role in the regulation of androgen signaling in prostate cancer by down-regulating AR expression. These results suggest that reconstitution of miR-let-7c may aid in targeting enhanced and hypersensitive AR in advanced prostate cancer.
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 p73 gene, a homologue of the p53 tumor suppressor, is expressed as TA and ?N isoforms. TAp73 has similar activity as p53 and functions as a tumor suppressor whereas ?Np73 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.
Canine and human lymphoma share similar characteristics in disease development and response to therapy. Translational research can be furthered using tools such as canine cell lines to model therapeutic compounds and strategies. We developed 5 B-cell lymphoma cell lines from dogs with confirmed large B-cell lymphoma. These cell lines were CD3, CD18, CD20, and CD90 positive with variable CD79a, CD1c and CD34 expression. All cell lines were tumorigenic in Nu/nu mice and were wild type for p53. Canine lymphoma cell lines serve as an important resource for translational lymphoma research.
Polymerase eta (PolH) is necessary for translesion DNA synthesis, and PolH deficiency predisposes xeroderma pigmentosum variant (XPV) patients to cancer. Due to the critical role of PolH in translesion DNA synthesis, the activity of PolH is tightly controlled and subjected to multiple regulations, especially posttranslational modifications. Here, we show that PolH-dependent lesion bypass and intracellular translocation are regulated by Pirh2 E3 ubiquitin ligase through monoubiquitination. Specifically, we show that Pirh2, a target of the p53 tumor suppressor, monoubiquitinates PolH at one of multiple lysine residues. We also show that monoubiquitination of PolH inhibits the ability of PolH to interact with PCNA and to bypass UV-induced lesions, leading to decreased viability of UV-damaged cells. Moreover, we show that monoubiquitination of PolH alters the ability of PolH to translocate to replication foci for translesion DNA synthesis of UV-induced DNA lesions. Considering that Pirh2 is known to be overexpressed in various cancers, we postulate that in addition to mutation of PolH in XPV patients, inactivation of PolH by Pirh2 via monoubiquitination is one of the mechanisms by which PolH function is controlled, which might be responsible for the development and progression of some spontaneous tumors wherein PolH is not found to be mutated.
The p53 pathway is critical for tumor suppression, as the majority of human cancer has a faulty p53. Here, we identified RNPC1, a p53 target and a RNA-binding protein, as a critical regulator of p53 translation. We showed that ectopic expression of RNPC1 inhibited, whereas knockdown of RNPC1 increased, p53 translation under normal and stress conditions. We also showed that RNPC1 prevented cap-binding protein eIF4E from binding p53 mRNA via its C-terminal domain for physical interaction with eIF4E, and its N-terminal domain for binding p53 mRNA. Consistent with this, we found that RNPC1 directly binds to p53 5 and 3untranslated regions (UTRs). Importantly, we showed that RNPC1 inhibits ectopic expression of p53 in a dose-dependent manner via p53 5 or 3 UTR. Moreover, we showed that loss of RNPC1 in mouse embryonic fibroblasts increased the level of p53 protein, leading to enhanced premature senescence in a p53-dependent manner. Finally, to explore the clinical relevance of our finding, we showed that RNPC1 was frequently overexpressed in dog lymphomas, most of which were accompanied by decreased expression of wild-type p53. Together, we identified a novel p53-RNPC1 autoregulatory loop, and our findings suggest that RNPC1 plays a role in tumorigenesis by repressing p53 translation.
Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2(-/-) mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.
p53 is frequently mutated in tumor cells, and mutant p53 is often highly expressed due to its increased half-life. Thus, targeting mutant p53 for degradation might be explored as a therapeutic strategy to manage tumors that are addicted to mutant p53 for survival. Arsenic trioxide, a drug for patients with acute promyelocytic leukemia, is found to target and degrade a class of proteins with high levels of cysteine residues and vicinal thiol groups, such as promyelocytic leukemia protein (PML) and PML-retinoic acid receptor ? fusion protein. Interestingly, wild type p53 is accumulated in cells treated with arsenic compounds, presumably due to arsenic-induced oxidative stresses. In this study, we found that wild type p53 is induced by arsenic trioxide in tumor cells, consistent with published studies. In contrast, we found that arsenic compounds degrade both endogenous and ectopically expressed mutant p53 in time- and dose-dependent manners. We also found that arsenic trioxide decreases the stability of mutant p53 protein through a proteasomal pathway, and blockage of mutant p53 nuclear export can alleviate the arsenic-induced mutant p53 degradation. Furthermore, we found that knockdown of endogenous mutant p53 sensitizes, whereas ectopic expression of mutant p53 desensitizes, tumor cells to arsenic treatment. Taken together, we found that mutant p53 is a target of arsenic compounds, which provides an insight into exploring arsenic compound-based therapy for tumors harboring a mutant p53.
Mutant p53 is not only deficient in tumor suppression but also acquires additional activity, called gain of function. Mutant p53 gain of function is recapitulated in knock-in mice that carry one null allele and one mutant allele of the p53 gene. These knock-in mice develop aggressive tumors compared with p53-null mice. Recently, we and others showed that tumor cells carrying a mutant p53 are addicted to the mutant for cell survival and resistance to DNA damage. To further define mutant p53 gain of function, we used the MCF-10A three-dimensional model of mammary morphogenesis. MCF-10A cells in three-dimensional culture undergo a series of morphological changes and form polarized and growth-arrested spheroids with hollow lumen, which resembles normal glandular architectures in vivo. Here, we found that endogenous wild-type p53 in MCF-10A cells was not required for acinus formation, but knockdown of endogenous wild-type p53 (p53-KD) led to partial clearance of cells in the lumen due to decreased apoptosis. Consistent with this, p53-KD altered expression patterns of the cell adhesion molecule E-cadherin, the cytoskeletal marker ?-catenin, and the extracellular matrix protein laminin V. We also found that ectopic expression of the mutant G245S led to a phenotype similar to p53-KD, whereas a combination of ectopic expression of siRNA-resistant G245S with p53-KD led to a less cleared lumen. In contrast, ectopic expression of mutant R248W, R175H, and R273H disrupted normal acinus architectures with filled lumen and led to formation of irregular and multiacinus structures regardless of p53-KD. In addition, these mutants altered normal expression patterns and/or levels of E-cadherin, ?-catenin, laminin V, and tight junction marker ZO-1. Furthermore, epithelial-to-mesenchymal transitions (EMT) markers, Snail, Slug, and Twist, were highly induced by mutant p53 and/or p53-KD. Together, we postulate that EMT represents a mutant p53 gain of function and mutant p53 alters cell polarity via EMT.
The p63 gene, a member of the p53 family, is expressed as TA and ?N isoforms. ?Np63 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 ?Np63 is regulated by the transcription factor DEC1, a p53 family target. We also showed that the ability of DEC1 to regulate ?Np63 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 ?Np63 promoter. Interestingly, we found that growth suppression induced by HDAC inhibitors is attenuated by ectopic expression of DEC1 in a ?Np63-dependent manner. In addition, we showed that ectopic expression of DEC1 inhibits, whereas knockdown of DEC1 promotes, keratinocyte differentiation via modulating ?Np63 expression. Finally, we showed that DEC1 cooperates with HDAC inhibitors to further decrease keratinocyte differentiation. Together, we conclude that ?Np63 is a novel target of DEC1 and HDAC2 and modulates the efficacy of HDAC inhibitors in growth suppression and keratinocyte differentiation.
RNA-binding proteins (RBPs) play a major role in many post-transcriptional processes, including mRNA stability, alternative splicing and translation. PCBP4, also called MCG10, is an RBP belonging to the poly(C)-binding protein family and a target of p53 tumor suppressor. Ectopic expression of PCBP4 induces cell-cycle arrest in G? and apoptosis. To identify RNA targets regulated by PCBP4 and further decipher its function, we generated multiple cell lines in which PCBP4 is either inducibly over-expressed or knocked down. We found that PCBP4 expression decreases cyclin-dependent kinase inhibitor p21 induction in response to DNA damage. We also provided evidence that PCBP4 regulates p21 expression independently of p53. In addition, we showed that a deficiency in PCBP4 enhances p21 induction upon DNA damage. To validate PCBP4 regulation of p21, we made PCBP4-deficient mice and showed that p21 expression is markedly increased in PCBP4-deficient primary mouse embryo fibroblasts compared to that in wild-type counterparts. Finally, we uncovered that PCBP4 binds to the 3-UTR of p21 transcript in vitro and in vivo to regulate p21 mRNA stability. Taken together, we revealed that PCBP4 regulates both basal and stress-induced p21 expression through binding p21 3-UTR and modulating p21 mRNA stability.
G protein-coupled receptors (GPRs) constitute one of the largest families of membrane proteins encoded by the human genome. Upon binding to various ligands, these seven-transmembrane receptors play an essential role in many physiological processes, including neurotransmission, immunity, inflammation, regulation of mood and behavior. In view of their important functions, aberrant expression and activity of GPRs have been implicated in a wide spectrum of diseases, including tumorigenesis. GPR87, a cell surface GPR related to the LPA receptor family, is overexpressed in diverse carcinomas and plays an essential role in tumor cell survival. In our recent work, we uncovered that GPR87 expression is regulated by the tumor suppressor p53 and by DNA damage in a p53-dependent manner. Moreover, we found that a lack of GPR87 triggers an increase in p53, concomitant with a decrease in Akt, which results in the sensitization of tumor cells to DNA damage-induced apoptosis and growth suppression. Altogether, we uncovered an essential function for GPR87 in p53-dependent cell survival in response to stress signals. Due to their unique structure, localization and ligand binding ability, GPRs have been extensively used for drug development and are the most common targets of commercial drugs. Although studies are required to determine GPR87 natural ligand(s) and signaling pathways, GPR87 is undoubtedly a very promising novel target for cancer prevention and treatment.
The tumor suppressor p53 is known to be able to trigger apoptosis in response to DNA damage, oncogene activation, and certain chemotherapeutic drugs. In addition to its transcriptional activation, a fraction of p53 translocates to mitochondria at the very early stage of apoptosis, which eventually contributes to the loss of mitochondrial membrane potential, generation of reactive oxygen species (ROS), cytochrome c release, and caspase activation. However, the mitochondrial events that affect p53 translocation are still unclear. Since mitochondrial uncoupling has been suggested to contribute to cancer development, herein, we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by mitochondrial uncoupling using chemical protonophores, and further verified the results using a siRNA approach in murine skin epidermal JB6 cells. Our results showed that mitochondrial uncoupling blocked p53 mitochondrial translocation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter to induce p53-mediated apoptosis in skin carcinogenesis. This blocking effect, in turn, led to preservation of mitochondrial functions, and eventually suppression of caspase activity and apoptosis. Moreover, uncoupling protein 2 (UCP2), a potential suppressor of ROS in mitochondria, is important for TPA-induced cell transformation in JB6 cells. UCP2 knock down cells showed enhanced p53 mitochondrial translocation, and were less prone to form colonies in soft agar after TPA treatment. Altogether, our data suggest that mitochondrial uncoupling may serve as an important regulator of p53 mitochondrial translocation and p53-mediated apoptosis during early tumor promotion. Therefore, targeting mitochondrial uncoupling may be considered as a novel treatment strategy for cancer.
Myosin VI is an unconventional motor protein and functions in a variety of intracellular processes such as cell migration, vesicular trafficking, and homeostasis of the Golgi complex. Previously, we found that myosin VI is up-regulated in RKO, LS174T, and H1299 cells by DNA damage in a p53-dependent manner and mediates the pro-survival function of p53. Here, we showed that the levels of myosin VI protein were markedly inhibited in MCF7 and LNCaP cells by topoisomerase I-II inhibitors. However, the levels of myosin VI transcript were decreased only by topoisomerase I inhibitors. We also found that the levels of myosin VI protein were markedly inhibited in MCF7 cells by wild-type p53 but not tumor-derived mutant p53. Surprisingly, we found that the level of myosin VI transcript was slightly increased instead of decreased in MCF7 cells by p53, suggesting that a mechanism other than transcriptional repression is involved. Additionally, we found that on the myosin VI promoter, the level of acetylated histone H3 was markedly decreased, whereas that of p53 and acetylated histone H4 was slightly increased in MCF7 cells upon treatment with topoisomerase I-II inhibitors. Finally, we showed that overexpression of myosin VI enhances, whereas knockdown of myosin VI decreases, DNA damage-induced stabilization of p53, and consequently, knockdown of myosin VI de-sensitizes MCF7 cells to DNA damage-induced apoptosis. Taken together, as a mediator of the p53 pro-survival pathway and a marker of malignancy in some tumors, differential regulation of myosin VI in various tumor cells by topoisomerase inhibitors dictates whether knockdown of myosin VI inhibits, rather than enhances, the susceptibility of tumor cells to some therapeutic agents, which might be explored for designing a proper therapeutic strategy.
P63, a p53 family tumor suppressor, is involved in many cellular processes, including growth suppression and differentiation. Thus, p63 activity needs to be tightly controlled. Here, we found that RNPC1, a RNA-binding protein and a target of the p53 family, regulates p63 mRNA stability and consequently p63 activity. Specifically, we showed that overexpression of RNPC1 decreases, whereas knockdown of RNPC1 increases, the half-life of p63 transcript, which leads to altered p63 expression. Consistent with this, we showed that RNPC1 binds the AU-/U-rich elements in p63 3 UTR in vitro and in vivo and the RRM domain in RNPC1 is required for binding, and regulating the stability of, p63 transcript. Furthermore, we showed that RNPC1 promotes keratinocyte differentiation by repressing p63 expression. Together, we uncovered a previously undetected mechanism by which p63 expression is regulated via mRNA stability and a novel regulatory feedback loop between RNPC1 and p63.
Tumor cells, including SW480 carcinoma cells that carry a mutant p53, are addicted to the mutant for their survival and resistance to growth suppression by chemotherapeutic agents. Here, we investigated whether various classes of p53 mutants share a common property and functional domains necessary for mutant p53 gain of function. To test this, we generated SW480 cell lines in which endogenous mutant R273H/P309S can be inducibly or stably knocked down, whereas a small interfering RNA-resistant mutant p53 along with a mutated functional domain can be inducibly or stably expressed. We found that both contact-site (R248W and R273H) and conformation (G245S and R249S) mutants are able to maintain the transformed phenotypes of SW480 cells conferred by endogenous mutant p53. We also found that activation domains 1-2 and the proline-rich domain are required for mutant p53 gain of function. Interestingly, we showed that the C-terminal basic domain, which is required for wild-type p53 activity, is an inhibitory domain for mutant p53. Furthermore, we showed that deletion of the basic domain enhances, whereas a mutation in activation domains 1-2 and deletion of the proline-rich domain abolish mutant p53 to regulate Gro1 and Id2, both of which are regulated by and mediate endogenous mutant p53 gain of function. These results indicate that both conformation and contact-site mutants share a property for cell transformation, and the domains critical for wild-type p53 tumor suppression are also required for mutant p53 tumor promotion. Thus, the inhibitory basic domain and the common property for p53 mutants can be explored for targeting tumors with mutant p53.
Cellular senescence is a permanent cell cycle arrest and a potent tumor suppression mechanism. The p53 tumor suppressor is a sequence-specific transcription factor and acts as a central hub sensing various stress signals and activating an array of target genes to induce cell cycle arrest, apoptosis, and senescence. Recent reports showed that restoration of p53 induces premature senescence and tumor regression in mice with hepatocarcinomas or sarcomas. Thus, p53-mediated senescence is capable of eliminating cancer cells in vivo. p63 and p73, two homologues of p53, have similar function in cell cycle arrest and apoptosis. However, the role of p63 and p73 in cellular senescence is elusive. In this review, we will discuss how p53 regulates senescence and future studies about p53 family members in senescence.
P21, a cyclin-dependent kinase inhibitor, plays a pivotal role in the cell-cycle regulation in response to stress stimuli. P21 expression is highly regulated through transcriptional, post-transcriptional and post-translational mechanisms. Previously, we and others showed that p21 expression is regulated through p21 mRNA stability by RNPC1, a target of the p53 family and HuR, a member of the ELAV family RNA-binding proteins. HuR carries three highly conserved RNA recognition motifs (RRMs) whereas RNPC1 carries one. Here we found that the ability of RNPC1 to regulate p21 mRNA stability is dependent on HuR. We also found that RNPC1 and HuR physically interact, and the RRM domain in RNPC1 and RRM3 in HuR are necessary for their interaction. Interestingly, we found that RNPC1 and HuR, both of which can bind AU-rich elements (AREs) in p21 3-UTR, preferentially bind the upstream and downstream AREs, respectively. Finally, we showed that the RNA-binding activity of HuR to p21 transcript was enhanced by RNPC1 in vitro and in vivo. Together, we hypothesize that RNPC1 modulates the RNA-binding activity of, and cooperates with, HuR to regulate p21 mRNA stability.
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.
DNA polymerase eta (PolH), a Y family translesion polymerase, is required for repairing UV-induced DNA damage, and loss of PolH is responsible for early onset of malignant skin cancers in patients with xeroderma pigmentosum variant (XPV), an autosomal recessive disorder. Here, we show that PolH, a target of the p53 tumor suppressor, is a short-half-life protein. We found that PolH is degraded by proteasome, which is enhanced upon UV irradiation. We also found that PolH interacts with Pirh2 E3 ligase, another target of the p53 tumor suppressor, via the polymerase-associated domain in PolH and the RING finger domain in Pirh2. In addition, we show that overexpression of Pirh2 decreases PolH protein stability, whereas knockdown of Pirh2 increases it. Interestingly, we found that PolH is recruited by Pirh2 and degraded by 20S proteasome in a ubiquitin-independent manner. Finally, we observed that Pirh2 knockdown leads to accumulation of PolH and, subsequently, enhances the survival of UV-irradiated cells. We postulate that UV irradiation promotes cancer formation in part by destabilizing PolH via Pirh2-mediated 20S proteasomal degradation.
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.
Mutant p53 gain of function contributes to cancer progression, increased invasion and metastasis potentials, and resistance to anticancer therapy. The ability of mutant p53 to acquire its gain of function is shown to correlate with increased expression of progrowth genes, such as c-MYC, MDR1, and NF-kappaB2. However, most of the published studies to identify mutant p53 target genes were performed in a cell system that artificially overexpresses mutant p53. Thus, it remains unclear whether such mutant p53 targets can be regulated by endogenous physiological levels of mutant p53. Here, we utilized SW480 and MIA-PaCa-2 cells, in which endogenous mutant p53 can be inducibly knocked down, to identify mutant p53 target genes that potentially mediate mutant p53 gain of function. We found that knockdown of mutant p53 inhibits GRO1 expression, whereas ectopic expression of mutant R175H in p53-null HCT116 cells increases GRO1 expression. In addition, we found that endogenous mutant p53 is capable of binding to and activating the GRO1 promoter. Interestingly, ectopic expression of GRO1 can rescue the proliferative defect in SW480 and MIA-PaCa-2 cells induced by knockdown of mutant p53. Conversely, knockdown of endogenous GRO1 inhibits cell proliferation and thus abrogates mutant p53 gain of function in SW480 cells. Taken together, our findings define a novel mechanism by which mutant p53 acquires its gain of function via transactivating the GRO1 gene in cancer cells. Thus, targeting GRO1 for cancer therapy would be applicable to a large portion of human tumors with mutant p53, but the exploration of GRO1 as a potential target should take the mutation status of p53 into consideration.
Spontaneous tumors in the dog offer a unique opportunity as models to study human cancer etiology and therapy. p53, the most commonly mutated gene in human cancers, is found to be altered in dog cancers. However, little is known about the role of p53 in dog tumorigenesis. Here, we found that on exposure to DNA damage agents or MDM2 inhibitor nutlin-3, canine p53 is accumulated and capable of inducing its target genes, MDM2 and p21. We also found that on DNA damage, canine p53 is accumulated in the nucleus, followed by MDM2 nuclear translocation and increased 53BP1 foci formation. In addition, we found that canine p63 and p73 are up-regulated by DNA damage agents. Furthermore, colony formation assay showed that canine tumor cells are sensitive to DNA damage agents and nutlin-3 in a p53-dependent manner. Surprisingly, canine p21 is expressed as two isoforms. Thus, we generated multiple canine p21 mutants and found that amino acids 129 to 142 are required, whereas amino acid 139 is one of the key determinants, for the expression of two p21 isoforms. Finally, we showed that although the full-length human p21 cDNA expresses one polypeptide, amino acid 139 seems to play a similar role as that in canine p21 for various migration patterns. Taken together, our results indicate that canine p53 family proteins have biological activities similar to human counterparts. These similarities make the dog an excellent outbred spontaneous tumor model, and the dog can serve as a translation model from benchtop to cage side and then to bedside.
Transcription factor p63, a member of the p53 family, shares a high degree of sequence similarity with p53. Because of transcription from two distinct promoters, the p63 gene encodes two isoforms, TAp63 and ?Np63. Although TAp63 acts as a tumor suppressor, ?Np63 functions as an oncogene and is often overexpressed in squamous cell carcinomas. Thus, therapeutic agents targeting ?Np63 might be used to manage tumors that overexpress ?Np63. Here we found that arsenic trioxide, a frontline agent for acute promyelocytic leukemia, inhibits ?Np63 but not TAp63 expression in time- and dose-dependent manners. In addition, we found that arsenic trioxide decreases the stability of ?Np63 protein via a proteasome-dependent pathway but has little effect on the level of ?Np63 transcript. Furthermore, we found that arsenic trioxide activates the Pirh2 promoter and consequently induces Pirh2 expression. Consistent with this, we found that knockdown of Pirh2 inhibits, whereas ectopic expression of Pirh2 enhances, arsenic-induced degradation of ?Np63 protein. Importantly, we found that knockdown of ?Np63 sensitizes, whereas ectopic expression of ?Np63 inhibits, growth suppression induced by arsenic. Together, these data suggest that arsenic degrades ?Np63 protein at least in part via Pirh2-dependent proteolysis and that inhibition of ?Np63 expression facilitates tumor cells to arsenic-induced death.
p63, a homolog of the tumor-suppressor p53, is essential for the development of the epidermis and limbs. p63 is highly expressed in the epithelial cell layer and acts as a molecular switch that initiates epithelial stratification. However, the mechanisms controlling p63 protein levels are still far from being fully understood. Here, we demonstrate a regulatory protein for p63 activity. We found that Pirh2 (p53-induced RING-H2) 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 ?Np63 protein, ectopic expression of Pirh2 promotes, whereas knockdown of Pirh2 inhibits, keratinocyte differentiation. Collectively, our data suggest that Pirh2 has a physiologically relevant role in keratinocyte differentiation through the posttranslational modification of p63 protein.
HuR, a RNA binding protein, is known to function as a tumor maintenance gene in breast cancer and associated with tumor growth and poor prognosis. However, the cellular function of this protein remains largely unknown in normal mammary epithelial cells. Here, we showed that in immortalized MCF10A mammary epithelial cells, HuR knockdown inhibits cell proliferation and enhances premature senescence. We also showed that in three-dimensional culture, MCF10A cells with HuR knockdown form abnormal acini with filled lumen and an aberrant expression pattern of the extracellular matrix protein laminin V. In addition, we showed that HuR knockdown increases ?Np63, but decreases wild-type p53, expression in MCF10A cells. Moreover, we showed that ?Np63 knockdown partially rescues the proliferative defect induced by HuR knockdown in MCF10A cells. Consistent with this, we identified two U-rich elements in the 3-untranslated region of p63 mRNA, to which HuR specifically binds. Finally, we showed that HuR knockdown enhances ?Np63 mRNA translation but has no effect on p63 mRNA turnover. Together, our data suggest that HuR maintains cell proliferation and polarity of mammary epithelial cells at least in part via ?Np63.
Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer.
Despite significant advancements in osteosarcoma research, the overall survival of canine and human osteosarcoma patients has remained essentially static over the past 2 decades. Post-operative limb-spare infection has been associated with improved survival in both species, yet a mechanism for improved survival has not been clearly established. Given that the majority of canine osteosarcoma patients experiencing post-operative infections were treated with fluoroquinolone antibiotics, we hypothesized that fluoroquinolone antibiotics might directly inhibit the survival and proliferation of canine osteosarcoma cells. Ciprofloxacin or enrofloxacin were found to inhibit p21(WAF1) expression resulting in decreased proliferation and increased S-G(2)/M accumulation. Furthermore, fluoroquinolone exposure induced apoptosis of canine osteosarcoma cells as demonstrated by cleavage of caspase-3 and PARP, and activation of caspase-3/7. These results support further studies examining the potential impact of quinolones on survival and proliferation of osteosarcoma.
The cyclin-dependent kinase inhibitor p21(Waf1/Cip1) is a major regulator of the cell cycle and plays an important role in many cellular processes, including differentiation, stress response, apoptosis, and tumorigenesis. We previously cloned the gene encoding dog p21 and found that unlike its human ortholog, dog p21 is expressed as two isoforms, one high molecular mass band of 23 kDa and one low molecular mass band of 19 kDa. In the current study, we found that the high molecular mass band is phosphorylated, whereas the low molecular mass band is hypophosphorylated. Moreover, by generating multiple mutants of dog p21, we found that serine 123 and proline 124, which form a consensus site for proline-directed phosphorylation, are required for expression of the high molecular mass p21 isoform through phosphorylation at serine 123. Most importantly, we showed that serine 123 phosphorylation inhibits ubiquitin-independent proteasomal degradation of p21 protein and subsequently, prolongs p21 protein half-life and enhances the ability of p21 to suppress cell proliferation. Taken together, these data reveal that serine 123 phosphorylation modulates p21 protein stability and activity by suppressing ubiquitin-independent proteasomal degradation.
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 ER?. 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 ER?, 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 ER?. Specifically, we found that knockdown of ER? decreases expression of p53 and its downstream targets, MDM2 and p21. In addition, we found that ER? activates p53 transcription via binding to estrogen response element half-sites within the p53 promoter. Moreover, we found that loss of ER? desensitizes, whereas ectopic expression of ER? 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 ER? and p53 and a biological role of p53 in the DNA damage response in ER-positive breast cancers.
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.
Although brain tumors are classified and treated based upon their histology, the molecular factors involved in the development of various tumor types remain unknown. In this study, we show that the type and order of genetic events directs the development of gliomas, central nervous system primitive neuroectodermal tumors, and atypical teratoid/rhabdoid-like tumors from postnatal mouse neural stem/progenitor cells (NSC/NPC). We found that the overexpression of specific genes led to the development of these three different brain tumors from NSC/NPCs, and manipulation of the order of genetic events was able to convert one established tumor type into another. In addition, loss of the nuclear chromatin-remodeling factor SMARCB1 in rhabdoid tumors led to increased phosphorylation of eIF2?, a central cytoplasmic unfolded protein response (UPR) component, suggesting a role for the UPR in these tumors. Consistent with this, application of the proteasome inhibitor bortezomib led to an increase in apoptosis of human cells with reduced SMARCB1 levels. Taken together, our findings indicate that the order of genetic events determines the phenotypes of brain tumors derived from a common precursor cell pool, and suggest that the UPR may represent a therapeutic target in atypical teratoid/rhabdoid tumors.
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 C(3)H(2)C(3) 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.
Constitutive NF-?B activation by proinflammatory cytokines plays a major role in cancer progression. However, the underlying mechanism is still unclear. We report here that histone methyltransferase NSD2 (also known as MMSET or WHSC1), a target of bromodomain protein ANCCA/ATAD2, acts as a strong coactivator of NF-?B by directly interacting with NF-?B for activation of target genes, including those for interleukin-6 (IL-6), IL-8, vascular endothelial growth factor A (VEGFA), cyclin D, Bcl-2, and survivin, in castration-resistant prostate cancer (CRPC) cells. NSD2 is recruited to the target gene promoters upon induction and mediates NF-?B activation-associated elevation of histone H3K36me2 and H3K36me3 marks at the promoter, which involves its methylase activity. Interestingly, we found that NSD2 is also critical for cytokine-induced recruitment of NF-?B and acetyltransferase p300 and histone hyperacetylation. Importantly, NSD2 is overexpressed in prostate cancer tumors, and its overexpression correlates with NF-?B activation. Furthermore, NSD2 expression is strongly induced by tumor necrosis factor alpha (TNF-?) and IL-6 via NF-?B and plays a crucial role in tumor growth. These results identify NSD2 to be a key chromatin regulator of NF-?B and mediator of the cytokine autocrine loop for constitutive NF-?B activation and emphasize the important roles played by NSD2 in cancer cell proliferation and survival and tumor growth.
p73, a p53 family tumor suppressor, is expressed as TA and ?N isoforms. Due to the role of p73 in tumor suppression and neural development, its expression and activity are tightly regulated by multiple mechanisms, including transcription and posttranslational modifications. Here, we found that p73 mRNA stability is regulated by RNPC1, an RNA binding protein and a target of the p53 family. We also showed that a CU-rich element in the 3 untranslated region of p73 is recognized by and responsive to RNPC1. To explore the physiological significance of RNPC1-regulated p73 expression, we showed that the loss of RNPC1 in p53-null mouse embryonic fibroblasts leads to reduced expression of p73, along with decreased expression of p21, p130, and ?-H2A.X, and consequently a decreased number of senescent cells. Furthermore, we observed that knockdown of TAp73 or p21, another target of RNPC1, attenuates the inhibitory effect of RNPC1 on cell proliferation and premature senescence, whereas combined knockdown of TAp73 and p21 completely abolishes it. Due to the fact that RNPC1 is a target of p73, the mutual regulation between p73 and RNPC1 constitutes a novel feed-forward loop, which might be explored as a target for tumors without a functional p53.
Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.
p73 is expressed as TA and ?N isoforms, both of which are implicated in tumor suppression and/or promotion. To address how p73 possesses these opposing functions, we developed three-dimensional culture of MCF10A cells, which undergo cell morphogenesis to form polarized spheroids with hollow lumen similar to normal mammary acini in vivo. Here, we showed that upon knockdown of p73, particularly TAp73 but not ?Np73, MCF10A cells formed irregular and near-normal acini without hollow lumen in three-dimensional culture. We also found that upon knockdown of p73 or TAp73, but not ?Np73, MCF10A cells underwent epithelial-to-mesenchymal transition (EMT) via down-regulation of E-cadherin coupled with up-regulation of ?-catenin and laminin V. In addition, we found that Snail-1, Slug, and Twist, all of which are known to act as EMT inducers by repressing E-cadherin expression, were increased markedly upon knockdown of p73 and TAp73 but little if any by ?Np73. Furthermore, we showed that knockdown of p73 or TAp73 in MCF10A cells led to a marked increase in cell proliferation and migration. Together, our data suggest that TAp73 is necessary for maintaining normal cell polarity by suppressing EMT.
The RNA-binding protein HuR, a member of the embryonic lethal abnormal vision/Hu protein family, plays a critical role in many cellular processes, including cell proliferation, angiogenesis, and inflammatory response. Despite significant progresses in understanding how HuR functions, the mechanism by which HuR expression is controlled is still poorly understood. Here, we showed that RNA-binding protein RNPC1 post-transcriptionally regulates HuR expression via mRNA stability. Specifically, we showed that overexpression of RNPC1 increases, whereas knockdown or knock-out of RNPC1 decreases, the level of HuR transcript and protein. Moreover, we showed that RNPC1, but not mutant RNPC1 deficient in RNA binding, stabilizes HuR transcript via binding to its 3-untranslated region. Furthermore, to determine the biological significance of RNPC1-enhanced HuR expression, we showed that HuR, by repressing c-Myc expression, facilitates RNPC1-mediated growth suppression. Together, we have uncovered a novel mechanism by which HuR is regulated by RNPC1 via mRNA stability and HuR is a mediator of RNPC1-induced growth suppression.
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