Expression Of P53 Target Genes Research Articles

Abstract PS19-21: Tak1 signaling regulates p53 through a mechanism involving ribosomal stress

Abstract Triple-negative breast cancer (TNBC) is among the most aggressive forms of breast cancer with limited therapeutic options. TAK1 is implicated in aggressive behavior of TNBC, while means are not fully understood. Here, we report that pharmacological blockade of TAK1 signaling hampered ribosome biogenesis (RBG) by reducing expression of RBG regulators such as RRS1, while not changing expression of ribosomal core proteins. Importantly, TAK1 blockade upregulated expression of p53 target genes in cell lines carrying wild type (wt) TP53 but not in p53-mutant cells. By examining involvement of the ribosomal stress response, we found that p53 activation by blockade of TAK1 was prevented by depletion of ribosomal protein RPL11. Further, siRNA-mediated depletion of TAK1 or RELA resulted in activation of p53 signaling and this response was dependent on RPL11. Knockdown of RRS1 disrupted nucleolar organization and resulted in activation of p53. Genomic TCGA data showed that TNBCs express high levels of ribosome biogenesis regulators, and elevated RRS1 levels correlate with unfavorable prognosis. Cytotoxicity data showed that TNBC cell lines are more sensitive to TAK1 inhibitor compared to luminal and HER2+ cell lines. Together, the data indicate that TAK1 regulates p53 activation by controlling ribosome biogenesis factors, and the TAK1-ribosome axis is a potential therapeutic target in TNBC. Citation Format: Andrei Bakin, Justin Zonneville. Tak1 signaling regulates p53 through a mechanism involving ribosomal stress [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-21.

MDM4 inhibition: a novel therapeutic strategy to reactivate p53 in hepatoblastoma

Hepatoblastoma (HB) is the most common pediatric liver malignancy. High-risk patients have poor survival, and current chemotherapies are associated with significant toxicities. Targeted therapies are needed to improve outcomes and patient quality of life. Most HB cases are TP53 wild-type; therefore, we hypothesized that targeting the p53 regulator Murine double minute 4 (MDM4) to reactivate p53 signaling may show efficacy. MDM4 expression was elevated in HB patient samples, and increased expression was strongly correlated with decreased expression of p53 target genes. Treatment with NSC207895 (XI-006), which inhibits MDM4 expression, or ATSP-7041, a stapled peptide dual inhibitor of MDM2 and MDM4, showed significant cytotoxic and antiproliferative effects in HB cells. Similar phenotypes were seen with short hairpin RNA (shRNA)-mediated inhibition of MDM4. Both NSC207895 and ATSP-7041 caused significant upregulation of p53 targets in HB cells. Knocking-down TP53 with shRNA or overexpressing MDM4 led to resistance to NSC207895-mediated cytotoxicity, suggesting that this phenotype is dependent on the MDM4-p53 axis. MDM4 inhibition also showed efficacy in a murine model of HB with significantly decreased tumor weight and increased apoptosis observed in the treatment group. This study demonstrates that inhibition of MDM4 is efficacious in HB by upregulating p53 tumor suppressor signaling.

The autism-related protein CHD8 contributes to the stemness and differentiation of mouse hematopoietic stem cells

Chromodomain helicase DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin-remodeling factor that is encoded by the most frequently mutated gene in individuals with autism spectrum disorder. CHD8 is expressed not only in neural tissues but also in many other organs; however, its functions are largely unknown. Here, we show that CHD8 is highly expressed in and maintains the stemness of hematopoietic stem cells (HSCs). Conditional deletion of Chd8 specifically in mouse bone marrow induces cell cycle arrest, apoptosis, and a differentiation block in HSCs in association with upregulation of the expression of p53 target genes. A colony formation assay and bone marrow transplantation reveal that CHD8 deficiency also compromises the stemness of HSCs. Furthermore, additional ablation of p53 rescues the impaired stem cell function and differentiation block of CHD8-deficient HSCs. Our results thus suggest that the CHD8-p53 axis plays a key role in regulation of the stemness and differentiation of HSCs.

Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

Glioblastoma is the most lethal brain tumor and its pathogenesis remains incompletely understood. KDM4C is a histone H3K9 demethylase that contributes to epigenetic regulation of both oncogene and tumor suppressor genes and is often overexpressed in human tumors, including glioblastoma. However, KDM4C’s roles in glioblastoma and the underlying molecular mechanisms remain unclear. Here, we show that KDM4C knockdown significantly represses proliferation and tumorigenesis of glioblastoma cells in vitro and in vivo that are rescued by overexpressing wild-type KDM4C but not a catalytic dead mutant. KDM4C protein expression is upregulated in glioblastoma, and its expression correlates with c-Myc expression. KDM4C also binds to the c-Myc promoter and induces c-Myc expression. Importantly, KDM4C suppresses the pro-apoptotic functions of p53 by demethylating p53K372me1, which is pivotal for the stability of chromatin-bound p53. Conversely, depletion or inhibition of KDM4C promotes p53 target gene expression and induces apoptosis in glioblastoma. KDM4C may serve as an oncogene through the dual functions of inactivation of p53 and activation of c-Myc in glioblastoma. Our study demonstrates KDM4C inhibition as a promising therapeutic strategy for targeting glioblastoma.

BCL7C suppresses ovarian cancer growth by inactivating mutant p53.

B-cell CLL/lymphoma 7 protein family member C (BCL7C) located at chromosome 16p11.2 shares partial sequence homology with the other two family members, BCL7A and BCL7B. Its role in cancer remains completely unknown. Here, we report our finding of its tumor-suppressive role in ovarian cancer. Supporting this is that BCL7C is downregulated in human ovarian carcinomas, and its underexpression is associated with unfavorable prognosis of ovarian cancer as well as some other types of human cancers. Also, ectopic BCL7C restrains cell proliferation and invasion of ovarian cancer cells. Consistently, depletion of BCL7C reduces apoptosis and promotes cell proliferation and invasion of these cancer cells. Mechanistically, BCL7C suppresses mutant p53-mediated gene transcription by binding to mutant p53, while knockdown of BCL7C enhances the expression of mutant p53 target genes in ovarian cancer cells. Primary ovarian carcinomas that sustain low levels of BCL7C often show the elevated expression of mutant p53 target genes. In line with these results, BCL7C abrogates mutant p53-induced cell proliferation and invasion, but had no impact on proliferation and invasion of cancer cells with depleted p53 or harboring wild-type p53. Altogether, our results demonstrate that BCL7C can act as a tumor suppressor to prevent ovarian tumorigenesis and progression by counteracting mutant p53 activity.

Ubiquitin ligase DTX3 empowers mutant p53 to promote ovarian cancer development

The deltex family protein DTX3 is believed to possess E3 ubiquitin ligase activity, as it contains a classic RING finger domain. However, its biological role and the underlying mechanism in cancer remain largely elusive. Here, we identified DTX3 as a novel mutant p53-interacting protein in ovarian carcinoma. Mechanistically, DTX3 mediated mutant p53 ubiquitination and stabilization by perturbing the MDM2-mutant p53 interaction, consequently leading to activation of diverse mutant p53 target genes. Importantly, a positive correlation between the expression of DTX3 and mutant p53 target genes was further validated in ovarian carcinomas. Ectopic DTX3 promoted, while depletion of DTX3 suppressed, ovarian cancer cell proliferation and invasion. Remarkably, the pro-tumorigenic effect of DTX3 is dependent on mutant p53, because ablation of mutant p53 significantly impaired DTX3-induced gene expression and ovarian cancer cell growth and propagation. Furthermore, DTX3 elevated the expression of mutant p53 target genes and boosted ovarian tumor growth in vivo. Finally, DTX3 was amplified and overexpressed in ovarian carcinomas, which is significantly associated with unfavorable prognosis. Altogether, our findings unveil the oncogenic role of DTX3 in ovarian cancer development by bolstering mutant p53 activity.

Rucaparib Treatment Alters p53 Oscillations in Single Cells to Enhance DNA-Double-Strand-Break-Induced Cell Cycle Arrest

DNA double strand breaks induce oscillatory expression of the transcription factor p53 that is dependent on ataxia telangiectasia mutated (ATM) activity and the rate of double strand break resolution. Although p53 dynamics are known to play a role in the regulation of cell fate determination, the consequences of the variability in dynamics associated with differences in repair rates and utilized repair pathways are unknown. Using single-cell time-lapse microscopy, we found that disruption of specific repair pathways has distinct impacts on p53 dynamics. The small-molecule rucaparib, an inhibitor of the alternative end-joining-associated protein poly (ADP-ribose) polymerase (PARP), increased p53 pulse duration, altering the temporal expression of multiple p53 target genes. As a result, combination treatments of the radiomimetic drug neocarzinostatin with rucaparib drove prolonged growth arrest beyond that of DNA damage alone. This study highlights how pharmacological manipulation of DNA repair pathways may be used to alter p53 dynamics to enhance therapeutic regimens.

Repression of P53 Function by SIRT5-Mediated Desuccinylation at Lysine 120

p53 is a classic tumor suppressor that functions in maintaining genome stability by inducing either cell arrest for damage repair or cell apoptosis to eliminate damaged cells in response to different types of stress. Posttranslational modifications (PTMs) of p53 are thought to be the most effective way for modulating of p53 activation. Here, we show that SIRT5 interacts with p53 and suppresses its transcriptional activity. Using mass spectrometric analysis, we identify a previously unknown PTM of p53, namely, succinylation of p53 at Lysine 120 (K120), which occurs after DNA damage. SIRT5 catalyzes desuccinylation of p53 at K120, resulting in the suppression of p53 activation. Moreover, using double knockout mice, we validate that the suppression of p53 target gene expression and cell apoptosis upon DNA damage is dependent on cellular p53. Our study identifies a novel PTM of p53 that regulates its activation as well as reveals a new target of SIRT5 acting as a desuccinylase.

Abstract 2468: Phosphorylation of Thr55 mediates the tumor suppression activities of p53

Abstract P53 is a transcription factor that plays a central role in the regulation of cell cycle, apoptosis and senescence. In unstressed cells, p53 is kept at low level under the strict control of its negative regulators. Upon DNA damage or oncogenic stress, p53 is activated via post-translational modifications. The phosphorylation of serine and threonine, such as Ser15 and Ser46, play important roles in p53 regulation. However, data for Thr55 (T55) phosphorylation is limited and functions of T55 phosphorylation remain elusive. We have previously defined an intramolecular interaction in p53 which modulates DNA binding. The observation of altered intramolecular interactions in T55A and T55D mutants leads to the hypothesis that T55 phosphorylation might affect p53 functions. We generate a T55 phospho-specific antibody to investigate the regulation through this site. The results show T55 phosphorylation is induced by DNA damage compounds. We induce the expression of wild type and T55A mutant p53 in p53 null H1299 cells via the transduction of doxycycline-inducible lentiviral gene expression plasmids. T55A mutant p53 exhibits significantly weaker inhibition of cell viability compared with wild type p53 containing cell in the existence of DNA damaging compounds. Consistently, T55A mutant p53 induces lower mRNA expression level of several important apoptosis related genes and higher MDM2 level in comparison with wild type p53. Overall, our results reveal that phosphorylation of Thr55 is induced by several DNA damaging compounds and alters the expression of p53 target genes, leading to an enhanced tumor suppression. Citation Format: Fan He, Lihong Chen, Jiandong Chen. Phosphorylation of Thr55 mediates the tumor suppression activities of p53 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2468.

Abstract 299: TAK1 signaling regulates p53 through a mechanism involving ribosomal stress

Abstract Triple-negative breast cancer (TNBC) is among the most aggressive forms of breast cancer with limited therapeutic options. TAK1 is implicated in aggressive behavior of TNBC, while means are not fully understood. Here, we report that pharmacological blockade of TAK1 signaling hampered ribosome biogenesis (RBG) by reducing expression of RBG regulators such as RRS1, while not changing expression of ribosomal core proteins. Importantly, TAK1 blockade upregulated expression of p53 target genes in cell lines carrying wild type (wt) TP53 but not in p53-mutant cells. By examining involvement of the ribosomal stress response, we found that p53 activation by blockade of TAK1 was prevented by depletion of ribosomal protein RPL11. Further, siRNA-mediated depletion of TAK1 or RELA resulted in activation of p53 signaling and this response was dependent on RPL11. Knockdown of RRS1 disrupted nucleolar organization and resulted in activation of p53. Genomic TCGA data showed that TNBCs express high levels of ribosome biogenesis regulators, and elevated RRS1 levels correlate with unfavorable prognosis. Cytotoxicity data showed that TNBC cell lines are more sensitive to TAK1 inhibitor compared to luminal and HER2+ cell lines. Together, the data indicate that TAK1 regulates p53 activation by controlling ribosome biogenesis factors, and the TAK1-ribosome axis is a potential therapeutic target in TNBC. Citation Format: Andrei V. Bakin, Justin Zonneville, Vincent Wong. TAK1 signaling regulates p53 through a mechanism involving ribosomal stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 299.

Abstract 3426: A synthetic lethality approach to eradicate AML via synergistic activation of pro-apoptotic p53 by MDM2 and BET inhibitors

Abstract Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation and minimize toxicity via MDM2i, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. By integrating the results of these two screens, we found inhibition of BRD4 activates p53 and its target genes. BRD4 (Bromodomain-containing protein 4) is a member of the bromodomain and extraterminal (BET) family proteins, which has typically been reported to activate genes, such as c-MYC, BCL2 and CDK4/6. However, we unexpectedly reveal that BRD4 acts as a transcriptional repressor of p53 target genes. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type p53 in vitro, against two mouse models of AML in vivo, and against several primary human patient blasts in vitro. Synergistic cell killing was associated with synergistic activation of p53 target genes, and cell killing and p53 target gene expression. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, targetable wild-type p53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML. Citation Format: Sha Li, Anne Latif, Ashley Newcombe, Kathryn Gilroy, Neil Robertson, Xue Lei, Darren Finlay, Helen Stewart, Karina Barbosa, Brian Higgins, Tim Chevassut, Xu Huang, Mhairi Copland, Karen Keeshan, Ani Deshpande, Peter Adams. A synthetic lethality approach to eradicate AML via synergistic activation of pro-apoptotic p53 by MDM2 and BET inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3426.

Interaction with p53 explains a pro-proliferative function for VHL in cancer.

The von Hippel-Lindau (VHL) protein binds and degrades hypoxia-inducible factors (HIF) hydroxylated by prolyl-hydroxylases under normoxia. Although originally described as a tumor suppressor, there is growing evidence that VHL may paradoxically promote tumor growth. The significance of its described interactions with many other proteins remains unclear. We found that VHL interacts with p53, preventing its tetramerization, promoterbinding and expression of its target genes p21, PUMA, and Bax. VHL limited the decrease in proliferation and increase in apoptosis caused by p53 activation, independent of prolyl-hydroxylation and HIF activity, and its presence in tumors caused a resistance to p53-inducing chemotherapy in vivo. We propose that VHL has both anti-tumor function, via HIF degradation, and a new pro-tumor function via p53 target (p21, PUMA, Bax) inhibition. Because p53 plays a critical role in tumor biology, isactivated by many chemotherapies, and because VHL levels vary among different tumors and its function can even be lost by mutations in some tumors, our results have important clinical applications. KEY MESSAGES: VHL and p53 physically interact and VHL inhibits p53 activity by limiting the formation of p53 tetramers. VHL attenuates the expression of p53 target genes in response to p53 stimuli. The inhibition of p53 by VHL is independent of HIF and prolyl-hydroxylation.

Abstract B45: Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing

Abstract MDM2 undergoes complex alternative splicing and one of its spliced isoforms, MDM2-ALT1, comprised of coding exons 3 and 12, is highly expressed in pediatric rhabdomyosarcoma, and its expression correlates with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress, and this alternative splicing is conserved in the mouse Mdm2 gene. Furthermore, previous research from our lab has reported that the splicing factors SRSF1, FUBP1, and PTBP1 are involved in this regulation; nevertheless, the way that MDM2 splicing is controlled is not entirely understood. In this work, we explore the role of the splicing factor SRSF2/SC35 in the alternative splicing of MDM2, hypothesizing that this factor acts as a positive regulator, in both human and mouse, facilitating the production of the full-length MDM2 mature transcript. This splicing factor has been reported to be mutated in several cancers, including myelodysplastic syndromes and rhabdomyosarcoma. Moreover, using a minigene system that mimics the endogenous Mdm2 splicing in response to UV and cisplatinum-induced DNA damage and the CRISPR-Cas9 technology, we show that mutations in SRSF2 predicted binding sites alter Mdm2 alternative splicing. The CRISPR-engineered mutant cells that express this splice isoform show increased proliferation in an Arf/p19 null context. On the other hand, we show that by using antisense oligonucleotides working as splice-switching molecules that target the SRSF2 binding sites in the MDM2 pre-mRNA, we are able to shift the expression of the MDM2 splice isoforms to drive p53 target gene expression in p53 wild-type cells. Furthermore, in an effort to understand better the effect of the MDM2-ALT1 isoform in carcinogenesis, we performed the CRISPR-Cas9 technique to generate a mouse model expressing the cancer-associated spliced isoform. We expect the mouse to be a cancer-sensitized model that will give rise to RMS tumors and provide a preclinical model for testing splice-switching oligonucleotide therapies. Citation Format: Matias I. Montes, Daniel F. Comiskey, Safiya Khurshid, Dawn S. Chandler. Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B45.

Rearranged ERG confers robustness to prostate cancer cells by subverting the function of p53

ObjectiveERG rearrangements are frequent and early events in prostate cancer. The functional role of rearranged ERG, however, is still incompletely understood. ERG rearrangements are maintained during prostate cancer progression suggesting that they may confer a selective advantage. The molecular basis of this notion is the subject of this study. MethodsA variety of immunological methods were used to characterize the effects of rearranged ERG on p53. Consequences of an overexpression of N-terminally deleted ERG on p53 function were interrogated by measuring apoptosis and cellular senescence in the presence or absence of exogenous DNA damage. Effects of N-terminally deleted ERG on the transactivation function of p53 were analyzed by qRT-PCR. ResultsWe show that overexpression of ERG leads to an increased basal level of DNA damage and a stabilization of p53 that involves a sequestration of its E3 ubiquitin ligase, MDM2, into nucleoli. A higher p53 expression was also observed in vivo in an ERG-overexpressing prostatic intraepithelial neoplasia mouse model. The correlation between ERG and p53 expression was corroborated in 163 patients with prostate cancer. ERG overexpression was found to inhibit both apoptosis and cellular senescence induced by exogenous DNA damage. Mechanistically, this protective effect of ERG involved an abrogation of the DNA damage-induced expression of p53 target genes. ConclusionsBy protecting tumor cells from the antiproliferative consequences of genotoxic stress, ERG may allow the survival and proliferation of genomically unstable tumor cells. Targeting ERG may therefore represent a promising strategy to suppress such adverse features during prostate cancer progression.

Good Cop, Bad Cop: Defining the Roles of Δ40p53 in Cancer and Aging.

The tumour suppressor p53 is essential for maintaining DNA integrity, and plays a major role in cellular senescence and aging. Understanding the mechanisms that contribute to p53 dysfunction can uncover novel possibilities for improving cancer therapies and diagnosis, as well as cognitive decline associated with aging. In recent years, the complexity of p53 signalling has become increasingly apparent owing to the discovery of the p53 isoforms. These isoforms play important roles in regulating cell growth and turnover in response to different stressors, depending on the cellular context. In this review, we focus on Δ40p53, an N-terminally truncated p53 isoform. Δ40p53 can alter p53 target gene expression in both a positive and negative manner, modulating the biological outcome of p53 activation; it also functions independently of p53. Therefore, proper control of the Δ40p53: p53 ratio is essential for normal cell growth, aging, and responses to cancer therapy. Defining the contexts and the mechanisms by which Δ40p53 behaves as a “good cop or bad cop” is critical if we are to target this isoform therapeutically.

Mdm4 controls ureteric bud branching via regulation of p53 activity

The antagonism between Mdm2 and its close homolog Mdm4 (also known as MdmX) and p53 is vital for embryogenesis and organogenesis. Previously, we demonstrated that targeted disruption of Mdm2 in the Hoxb7+ ureteric bud (Ub) lineage, which gives rise to the renal collecting system, causes renal hypodysplasia culminating in perinatal lethality. In this study, we examine the unique role of Mdm4 in establishing the collecting duct system of the murine kidney. Hoxb7Cre driven loss of Mdm4 in the Ub lineage (UbMdm4−/−) disrupts branching morphogenesis and triggers UB cell apoptosis. UbMdm4−/− kidneys exhibit abnormally dilated Ub tips while the medulla is hypoplastic. These structural alterations result in secondary depletion of nephron progenitors and nascent nephrons. As a result, newborn UbMdm4−/− mice have hypo-dysplastic kidneys. Transcriptional profiling revealed downregulation of the Ret-tyrosine kinase pathway components, Gdnf, Wnt11, Sox8, Etv4 and Cxcr4 in the UbMdm4−/− mice relative to controls. Moreover, the expression levels of the canonical Wnt signaling members Axin2 and Wnt9b are downregulated. Mdm4 deletion upregulated p53 activity and p53-target gene expression including Cdkn1a (p21), Gdf15, Ccng1, PERP, and Fas. Germline loss of p53 in UbMdm4−/− mice largely rescues kidney development and terminal differentiation of the collecting duct. We conclude that Mdm4 plays a unique and vital role in Ub branching morphogenesis and collecting system development.

HDAC3-ERα Selectively Regulates TNF-α-Induced Apoptotic Cell Death in MCF-7 Human Breast Cancer Cells via the p53 Signaling Pathway.

Tumor necrosis factor-α (TNF-α) plays a significant role in inflammation and cancer-related apoptosis. We identified a TNF-α-mediated epigenetic mechanism of apoptotic cell death regulation in estrogen receptor-α (ERα)-positive human breast cancer cells. To assess the apoptotic effect of TNF-α, annexin V/ propidium iodide (PI) double staining, cell viability assays, and Western blotting were performed. To elucidate this mechanism, histone deacetylase (HDAC) activity assay and immunoprecipitation (IP) were conducted; the mechanism was subsequently confirmed through chromatin IP (ChIP) assays. Finally, we assessed HDAC3–ERα-mediated apoptotic cell death after TNF-α treatment in ERα-positive human breast cancer (MCF-7) cells via the transcriptional activation of p53 target genes using luciferase assay and quantitative reverse transcription PCR. The TNF-α-induced selective apoptosis in MCF-7 cells was negatively regulated by the HDAC3–ERα complex in a caspase-7-dependent manner. HDAC3 possessed a p53-binding element, thus suppressing the transcriptional activity of its target genes. In contrast, MCF-7 cell treatment with TNF-α led to dissociation of the HDAC3–ERα complex and substitution of the occupancy on the promoter by the p53–p300 complex, thus accelerating p53 target gene expression. In this process, p53 stabilization was accompanied by its acetylation. This study showed that p53-mediated apoptosis in ERα-positive human breast cancer cells was negatively regulated by HDAC3–ERα in a caspase-7-dependent manner. Therefore, these proteins have potential application in therapeutic strategies.

Chronic exposure to excess iron promotes EMT and cancer via p53 loss in pancreatic cancer

Based on the evidence that hemochromatosis, an iron-overload disease, drives hepatocellular carcinoma, we hypothesized that chronic exposure to excess iron, either due to genetic or environmental causes, predisposes an individual to cancer. Using pancreatic cancer as our primary focus, we employed cell culture studies to interrogate the connection between excess iron and cancer, and combined in vitro and in vivo studies to explore the connection further. Ferric ammonium citrate was used as an exogenous iron source. Chronic exposure to excess iron induced epithelial-mesenchymal transition (EMT) in normal and cancer cell lines, loss of p53, and suppression of p53 transcriptional activity evidenced from decreased expression of p53 target genes (p21, cyclin D1, Bax, SLC7A11). To further extrapolate our cell culture data, we generated EL-KrasG12D (EL-Kras) mouse (pancreatic neoplastic mouse model) expressing Hfe+/+and Hfe−/− genetic background. p53 target gene expression decreased in EL-Kras/Hfe−/− mouse pancreas compared to EL-Kras/Hfe+/+ mouse pancreas. Interestingly, the incidence of acinar-to-ductal metaplasia and cystic pancreatic neoplasms (CPN) decreased in EL-Kras/Hfe−/− mice, but the CPNs that did develop were larger in these mice than in EL-Kras/Hfe+/+ mice. In conclusion, these in vitro and in vivo studies support a potential role for chronic exposure to excess iron as a promoter of more aggressive disease via p53 loss and SLC7A11 upregulation within pancreatic epithelial cells.

TAK1 signaling regulates p53 through a mechanism involving ribosomal stress

Triple-negative breast cancer (TNBC) is among the most aggressive forms of breast cancer with limited therapeutic options. TAK1 is implicated in aggressive behavior of TNBC, while means are not fully understood. Here, we report that pharmacological blockade of TAK1 signaling hampered ribosome biogenesis (RBG) by reducing expression of RBG regulators such as RRS1, while not changing expression of ribosomal core proteins. Notably, TAK1 blockade upregulated expression of p53 target genes in cell lines carrying wild type (wt) TP53 but not in p53-mutant cells, suggesting involvement of ribosomal stress in the response. Accordingly, p53 activation by blockade of TAK1 was prevented by depletion of ribosomal protein RPL11. Further, siRNA-mediated depletion of TAK1 or RELA resulted in RPL11-dependent activation of p53 signaling. Knockdown of RRS1 was sufficient to disrupt nucleolar structures and resulted in activation of p53. TCGA data showed that TNBCs express high levels of RBG regulators, and elevated RRS1 levels correlate with unfavorable prognosis. Cytotoxicity data showed that TNBC cell lines are more sensitive to TAK1 inhibitor compared to luminal and HER2+ cell lines. These results show that TAK1 regulates p53 activation by controlling RBG factors, and the TAK1-ribosome axis is a potential therapeutic target in TNBC.

The role of myoglobin in epithelial cancers: Insights from transcriptomics.

The muscle-associated respiratory protein myoglobin (MB) is expressed in multiple types of cancer, including breast and prostate tumors. In Kaplan-Meier analyses of the two tumor types, MB positivity is associated with favorable prognoses. Despite its well-characterized function in myocytes, the role of MB in cancer remains unclear. To study the impact of endogenous MB expression, small interfering RNA MB-knockdown cells were engineered using breast, prostate and colon cancer cell lines (MDA-MB468, LNCaP, DLD-1), and their transcriptomes were investigated using RNA-Seq at different oxygen levels. In MB-positive cells, increased expression of glycolytic genes was observed, which was possibly mediated by a higher activity of hypoxia-inducible factor 1α. In addition, the results of the gene set enrichment analysis suggested that MB contributed to fatty acid transport and turnover. MB-positive, wild-type-p53 LNCaP cells also exhibited increased expression of p53 target genes involved in cell cycle checkpoint control and prevention of cell migration. MB-positive cells expressing mutant p53 exhibited upregulation of genes associated with prolonged cancer cell viability and motility. Therefore, it was hypothesized that these transcriptomic differences may result from MB-mediated generation of nitric oxide or reactive oxygen species, thus employing established enzymatic activities of the globin. In summary, the transcriptome comparisons identified potential molecular functions of MB in carcinogenesis by highlighting the interaction of MB with key metabolic and regulatory processes.