Immune deficiency augments the prevalence of p53 loss of heterozygosity in spontaneous tumors but not bi-directional loss of heterozygosity in bone marrow progenitors.

p53 loss of heterozygosity (LOH) is a frequent event in tumors of somatic and Li-Fraumeni syndrome patients harboring p53 mutation. Here, we focused on resolving a possible crosstalk between the immune-system and p53 LOH. Previously, we reported that p53 heterozygous bone-marrow mesenchymal progenitor cells undergo p53 LOH in-vivo. Surprisingly, the loss of either the wild-type p53 allele or mutant p53 allele was detected with a three-to-one ratio in favor of losing the mutant allele. In this study, we examined whether the immune-system can affect the LOH directionality in bone marrow progenitors. We found that mesenchymal progenitor cells derived from immune-deficient mice exhibited the same preference of losing the mutant p53 allele as immune-competent matched cells, nevertheless, these animals showed a significantly shorter tumor-free survival, indicating the possible involvement of immune surveillance in this model. Surprisingly, spontaneous tumors of p53 heterozygous immune-deficient mice exhibited a significantly higher incidence of p53 LOH compared to that observed in tumors derived of p53 heterozygous immune-competent mice. These findings indicate that the immune-system may affect the p53 LOH prevalence in spontaneous tumors. Thus suggesting that the immune-system may recognize and clear cells that underwent p53 LOH, whereas in immune-compromised mice, those cells will form tumors with shorter latency. In individuals with a competent immune-system, p53 LOH independent pathways may induce malignant transformation which requires a longer tumor latency. Moreover, this data may imply that the current immunotherapy treatment aimed at abrogating the inhibition of cellular immune checkpoints may be beneficial for LFS patients.

Rescue of embryonic stem cells from cellular transformation by proteomic stabilization of mutant p53 and conversion into WT conformation.

p53 is a well-known tumor suppressor that is mutated in over 50% of human cancers. These mutations were shown to exhibit gain of oncogenic function compared with the deletion of the gene. Additionally, p53 has fundamental roles in differentiation and development; nevertheless, mutant p53 mice are viable and develop malignant tumors only on adulthood. We set out to reveal the mechanisms by which embryos are protected from mutant p53-induced transformation using ES cells (ESCs) that express a conformational mutant of p53. We found that, despite harboring mutant p53, the ESCs remain pluripotent and benign and have relatively normal karyotype compared with ESCs knocked out for p53. Additionally, using high-content RNA sequencing, we show that p53 is transcriptionally active in response to DNA damage in mutant ESCs and elevates p53 target genes, such as p21 and btg2. We also show that the conformation of mutant p53 protein in ESCs is stabilized to a WT conformation. Through MS-based interactome analyses, we identified a network of proteins, including the CCT complex, USP7, Aurora kinase, Nedd4, and Trim24, that bind mutant p53 and may shift its conformation to a WT form. We propose this conformational shift as a novel mechanism of maintenance of genomic integrity, despite p53 mutation. Harnessing the ability of these protein interactors to transform the oncogenic mutant p53 to the tumor suppressor WT form can be the basis for future development of p53-targeted cancer therapy.

Inactivation of myocardin and p16 during malignant transformation contributes to a differentiation defect.

Myocardin is known as an important transcriptional regulator in smooth and cardiac muscle development. Here we found that myocardin is frequently repressed during human malignant transformation, contributing to a differentiation defect. We demonstrate that myocardin is a transcriptional target of TGFbeta required for TGFbeta-mediated differentiation of human fibroblasts. Serum deprivation, intact contact inhibition response, and the p16ink4a/Rb pathway contribute to myocardin induction and differentiation. Restoration of myocardin expression in sarcoma cells results in differentiation and inhibition of malignant growth, whereas inactivation of myocardin in normal fibroblasts increases their proliferative potential. Myocardin expression is reduced in multiple types of human tumors. Collectively, our results demonstrate that myocardin is an important suppressive modifier of the malignant transformation process.

Various p53 mutant proteins differently regulate the Ras circuit to induce a cancer-related gene signature.

Concomitant expression of mutant p53 and oncogenic Ras, leading to cellular transformation, is well documented. However, the mechanisms by which the various mutant p53 categories cooperate with Ras remain largely obscure. From this study we suggest that different mutant p53 categories cooperate with H-Ras in different ways to induce a unique expression pattern of a cancer-related gene signature (CGS). The DNA-contact p53 mutants (p53(R248Q) and p53(R273H)) exhibited the highest level of CGS expression by cooperating with NFκB. Furthermore, the Zn(+2) region conformational p53 mutants (p53(R175H) and p53(H179R)) induced the CGS by elevating H-Ras activity. This elevation in H-Ras activity stemmed from a perturbed function of the p53 transcription target gene, BTG2. By contrast, the L3 loop region conformational mutant (p53(G245S)) did not affect CGS expression. Our findings were further corroborated in human tumor-derived cell lines expressing Ras and the aforementioned mutated p53 proteins. These data might assist in future tailor-made therapy targeting the mutant p53-Ras axis in cancer.

Mutant p53R273H attenuates the expression of phase 2 detoxifying enzymes and promotes the survival of cells with high levels of reactive oxygen species.

Uncontrolled accumulation of reactive oxygen species (ROS) causes oxidative stress and induces harmful effects. Both high ROS levels and p53 mutations are frequent in human cancer. Mutant p53 forms are known to actively promote malignant growth. However, no mechanistic details are known about the contribution of mutant p53 to excessive ROS accumulation in cancer cells. Herein, we examine the effect of p53(R273H), a commonly occurring mutated p53 form, on the expression of phase 2 ROS-detoxifying enzymes and on the ability of cells to readopt a reducing environment after exposure to oxidative stress. Our data suggest that p53(R273H) mutant interferes with the normal response of human cells to oxidative stress. We show here that, upon oxidative stress, mutant p53(R273H) attenuates the activation and function of NF-E2-related factor 2 (NRF2), a transcription factor that induces the antioxidant response. This effect of mutant p53 is manifested by decreased expression of phase 2 detoxifying enzymes NQO1 and HO-1 and high ROS levels. These findings were observed in several human cancer cell lines, highlighting the general nature of this phenomenon. The failure of p53(R273H) mutant-expressing cells to restore a reducing oxidative environment was accompanied by increased survival, a known consequence of mutant p53 expression. These activities are attributable to mutant p53(R273H) gain of function and might underlie its well-documented oncogenic nature in human cancer.

Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts.

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Chemotherapeutic agents induce the expression and activity of their clearing enzyme CYP3A4 by activating p53.

Cytochrome P450 (P450) enzymes are abundantly expressed in the human liver where they hydroxylate organic substrates. In a microarray screen performed in human liver cells, we found a group of eleven P450 genes whose expression was induced by p53 (CYP3A4, CYP3A43, CYP3A5, CYP3A7, CYP4F2, CYP4F3, CYP4F11, CYP4F12, CYP19A1, CYP21A2 and CYP24A1). The mode of regulation of four representative genes (CYP3A4, CYP3A7, CYP4F2 and CYP4F3) was further characterized. The genes were induced in a p53-dependent manner in HepG2 and Huh6 cells (both are cancer-derived human liver cells) and in primary liver cells isolated from human donors. Furthermore, p53 was found to bind to p53-responsive elements in the genes' DNA-regulatory regions and to enhance their transcription in a reporter gene assay. Importantly, when p53 was activated following the administration of either of three different anticancer chemotherapeutic agents (cisplatin, etoposide or doxorubicin), it was able to induce CYP3A genes, which are the main factors in systemic clearance of these agents. Finally, the p53-dependent induction of P450 genes following either Nutlin or chemotherapy treatment led to enhanced P450 enzymatic activity. Thus, in addition to the well-established role of p53 at the tumor site, our data unravels a novel function of hepatic p53 in inducing P450 enzymes and position p53 as a major factor in the hepatic response to xenobiotic and metabolic signals. Importantly, this study reveals a novel pathway for the induction of CYP3As by their substrates through p53, warranting the need for careful consideration when designing systemically administered chemotherapeutic regimens.

p53, a novel regulator of lipid metabolism pathways.

BACKGROUND & AIMS: In this study we aimed at characterizing the regulation of hepatic metabolic pathways by the p53 transcription factor. METHODS: Analysis of gene expression following alteration of p53 status in several human- and mouse-derived cells using microarray analysis, quantitative real-time PCR, chromatin immunoprecipitation, and reporter gene assays. A functional assay was performed to determine lipid transfer activity. RESULTS: We identified a novel role for the p53 protein in regulating lipid and lipoprotein metabolism, a process not yet conceived as related to p53, which is known mainly for its tumor suppressive functions. We revealed a group of 341 genes whose expression was induced by p53 in the liver-derived cell line HepG2. Twenty of these genes encode proteins involved in many aspects of lipid homeostasis. The mode of regulation of three representative genes (Pltp, Abca12, and Cel) was further characterized. In addition to HepG2, the genes were induced following activation of p53 in human primary hepatic cells isolated from liver donors. p53-dependent regulation of these genes was evident in other cell types namely Hep3B cells, mouse hepatocytes, and fibroblasts. Furthermore, p53 was found to bind to the genes' promoters in designated p53 responsive elements and thereby increase transcription. Importantly, p53 augmented the activity of secreted PLTP, which plays a major role in lipoprotein biology and atherosclerosis pathology. CONCLUSIONS: These findings expose another facet of p53 functions unrelated to tumor suppression and render it a novel regulator of hepatic lipid metabolism and consequently of systemic lipid homeostasis and atherosclerosis development.

Transcriptional activity of ATF3 in the stromal compartment of tumors promotes cancer progression.

Compelling evidences have rendered the tumor microenvironment a crucial determinant in cancer outcome. Activating transcription factor 3 (ATF3), a stress response transcription factor, is known to have a dichotomous role in tumor cells, acting either as a tumor suppressor or an oncogene in a context-dependent manner. However, its expression and possible role in the tumor microenvironment are hitherto unknown. Here we show that ATF3 is upregulated in the stromal compartment of several types of cancer. Accordingly, Cancer-associated fibroblasts (CAFs) ectopically expressing ATF3 proliferated faster as indicated by increased colony-forming capacity and promoted the growth of adjacent tumor cells when co-injected into nude mice. Utilizing a genome-wide profiling approach, we unraveled a robust gene expression program induced by ATF3 in CAFs. Focusing on a specific subset of genes, we found that the ability of stromal ATF3 to promote cancer progression is mediated by transcriptional repression of CLDN1 and induction of CXCL12 and RGS4. In addition, regulation of LIF, CLDN1, SERPINE2, HSD17B2, ITGA7 and PODXL by ATF3 mediated the increased proliferation capacity of CAFs. In sum, our findings implicate ATF3 as a novel stromal tumor promoter and suggest that targeting ATF3 pathway might be beneficial for anticancer therapy.

Modulated expression of WFDC1 during carcinogenesis and cellular senescence.

Fibroblasts located adjacent to the tumor [cancer-associated fibroblasts (CAFs)] that constitute a large proportion of the cancer-associated stroma facilitate the transformation process. In this study, we compared the biological behavior of CAFs that were isolated from a prostate tumor to their normal-associated fibroblast (NAF) counterparts. CAFs formed more colonies when seeded at low cell density, exhibited a higher proliferation rate and were less prone to contact inhibition. In contrast to the general notion that high levels of alpha-smooth muscle actin serve as a marker for CAFs, we found that prostate CAFs express it at a lower level compared with prostate NAFs. Microarray analysis revealed a set of 161 genes that were altered in CAFs compared with NAFs. We focused on whey acidic protein four-disulfide core domain 1 (WFDC1), a known secreted protease inhibitor, and found it to be downregulated in the CAFs. WFDC1 expression was also dramatically downregulated in highly prolific mesenchymal cells and in various cancers including fibrosarcomas and in tumors of the lung, bladder and brain. Overexpression of WFDC1 inhibited the growth rate of the fibrosarcoma HT1080 cell line. Furthermore, WFDC1 level was upregulated in senescent fibroblasts. Taken together, our data suggest an important role for WFDC1 in inhibiting proliferation of both tumors and senescent cells. Finally, we suggest that the downregulation of WFDC1 might serve as a biomarker for cellular transformation.

Prostate stromal cells produce CXCL-1, CXCL-2, CXCL-3 and IL-8 in response to epithelia-secreted IL-1.

It is well accepted that tumor microenvironment is essential for tumor cells survival, cancer progression and metastasis. However, the mechanisms by which tumor cells interact with their surrounding at early stages of cancer development are largely unidentified. The aim of this study was to identify specific molecules involved in stromal-epithelial interactions that might contribute to early stages of prostate tumor formation. Here, we show that conditioned medium (CM) from immortalized non-transformed prostate epithelial cells stimulated immortalized prostate stromal cells to express cancer-related molecules. CM obtained from epithelial cells triggered stromal cells to express and secrete CXCL-1, CXCL-2, CXCL-3 and interleukin (IL)-8 chemokines. This effect was predominantly mediated by the cytokines of the IL-1 family secreted by the epithelial cells. Thus, prostate epithelial cells induced the secretion of proinflammatory and cancer-promoting chemokines by prostate stromal cells. Such interactions might contribute to prostatic inflammation and progression at early stages of prostate cancer formation.

Identification of gene networks associated with erythroid differentiation.

Erythropoiesis is a multistep process involving a large number of genes, which balance between proliferation, differentiation and survival of the erythroid cells. To understand the molecular mechanisms of erythropoiesis and related pathological aberrations, we analyzed three stages of in vitro differentiating human erythroid cells by expression profiling. We identified distinct clusters of genes, each with a unique expression pattern during differentiation. As JAK2 was shown to play a central role in myeloproliferative disorders, we focused on one cluster which includes JAK2 and other genes with high correlation to JAK2 expression. These genes had a low expression at the early erythroblast which increased in the intermediate stage and further slightly increased in the last stage of differentiation. Our results indicate that gene networks may associate with JAK2 expression in erythroid differentiation. It is intriguing to determine whether the pathogenesis of polycythemia vera (PV), harboring a common or uncommon JAK2 mutation, involves alterations in independent gene pathways that underlie the normal erythropoietic process.

p53-Repressed miRNAs are involved with E2F in a feed-forward loop promoting proliferation.

Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild-type p53. These miRNAs are repressed by p53 in an E2F1-mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi-gene transcriptional and post-transcriptional feed-forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network.

Correction: Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides.

[This corrects the article DOI: 10.18632/oncotarget.10516.].

Mutant p53-dependent mitochondrial metabolic alterations in a mesenchymal stem cell-based model of progressive malignancy.

It is well accepted that malignant transformation is associated with unique metabolism. Malignant transformation involves a variety of cellular pathways that are associated with initiation and progression of the malignant process that remain to be deciphered still. Here we used a mouse model of mutant p53 that presents a stepwise progressive transformation of adult Mesenchymal Stem Cells (MSCs). While the established parental p53Mut-MSCs induce tumors, the parental p53WT-MSCs that were established in parallel, did not. Furthermore, tumor lines derived from the parental p53Mut-MSCs (p53Mut-MSC-TLs), exhibited yet a more aggressive transformed phenotype, suggesting exacerbation in tumorigenesis. Metabolic tracing of these various cell types, indicated that while malignant transformation is echoed by a direct augmentation in glycolysis, the more aggressive p53Mut-MSC-TLs demonstrate increased mitochondrial oxidation that correlates with morphological changes in mitochondria mass and function. Finally, we show that these changes are p53Mut-dependent. Computational transcriptional analysis identified a mitochondrial gene signature specifically downregulated upon knock/out of p53Mut in MSC-TLs. Our results suggest that stem cells exhibiting different state of malignancy are also associated with a different quantitative and qualitative metabolic profile in a p53Mut-dependent manner. This may provide important insights for cancer prognosis and the use of specific metabolic inhibitors in a personalized designed cancer therapy.

Mutant p53 protects cells from 12-O-tetradecanoylphorbol-13-acetate-induced death by attenuating activating transcription factor 3 induction.

Mutations in p53 are ubiquitous in human tumors. Some p53 mutations not only result in loss of wild-type (WT) activity but also grant additional functions, termed "gain of function." In this study, we explore how the status of p53 affects the immediate response gene activating transcription factor 3 (ATF3) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-protein kinase C (PKC) pathway. We show that high doses of TPA induce ATF3 in a WT p53-independent manner correlating with PKCs depletion and cell death. We show that cells harboring mutant p53 have attenuated ATF3 induction and are less sensitive to TPA-induced death compared with their p53-null counterparts. Mutagenesis analysis of the ATF3 promoter identified the regulatory motifs cyclic AMP-responsive element binding protein/ATF and MEF2 as being responsible for the TPA-induced activation of ATF3. Moreover, we show that mutant p53 attenuates ATF3 expression by two complementary mechanisms. It interacts with the ATF3 promoter and influences its activity via the MEF2 site, and additionally, it attenuates transcriptional expression of the ATF3 activator MEF2D. These data provide important insights into the molecular mechanisms that underlie mutant p53 gain of function.

hTERT-immortalized prostate epithelial and stromal-derived cells: an authentic in vitro model for differentiation and carcinogenesis.

Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate-specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.

A Mutant p53-Dependent Embryonic Stem Cell Gene Signature Is Associated with Augmented Tumorigenesis of Stem Cells.

Mutations in the tumor suppressor p53 are the most frequent alterations in human cancer. These mutations include p53-inactivating mutations as well as oncogenic gain-of-function (GOF) mutations that endow p53 with capabilities to promote tumor progression. A primary challenge in cancer therapy is targeting stemness features and cancer stem cells (CSC) that account for tumor initiation, metastasis, and cancer relapse. Here we show that in vitro cultivation of tumors derived from mutant p53 murine bone marrow mesenchymal stem cells (MSC) gives rise to aggressive tumor lines (TL). These MSC-TLs exhibited CSC features as displayed by their augmented oncogenicity and high expression of CSC markers. Comparative analyses between MSC-TL with their parental mutant p53 MSC allowed for identification of the molecular events underlying their tumorigenic properties, including an embryonic stem cell (ESC) gene signature specifically expressed in MSC-TLs. Knockout of mutant p53 led to a reduction in tumor development and tumorigenic cell frequency, which was accompanied by reduced expression of CSC markers and the ESC MSC-TL signature. In human cancer, MSC-TL ESC signature-derived genes correlated with poor patient survival and were highly expressed in human tumors harboring p53 hotspot mutations. These data indicate that the ESC gene signature-derived genes may serve as new stemness-based prognostic biomarkers as well as novel cancer therapeutic targets.Significance: Mesenchymal cancer stem cell-like cell lines express a mutant p53-dependent embryonic stem cell gene signature, which can serve as a potential prognostic biomarker and therapeutic target in cancer. Cancer Res; 78(20); 5833-47. ©2018 AACR.

Various stress stimuli rewire the profile of liver secretome in a p53-dependent manner.

Liver is an important secretory organ that consistently manages various insults in order to retain whole-body homeostasis. Importantly, it was suggested that the tumor-suppressor p53 plays a role in a variety of liver physiological processes and thus it is being regarded as a systemic homeostasis regulator. Using high-throughput mass spectrometric analysis, we identified various p53-dependent liver secretome profiles. This allowed a global view on the role of p53 in maintaining the harmony of liver and whole-body homeostasis. We found that p53 altered the liver secretome differently under various conditions. Under physiological conditions, p53 controls factors that are related mainly to lipid metabolism and injury response. Upon exposure to various types of cancer therapy agents, the hepatic p53 is activated and induces the secretion of proteins related to additional pathways, such as hemostasis, immune response, and cell adhesion. Interestingly, we identified a possible relationship between p53-dependent liver functions and lung tumors. The latter modify differently liver secretome profile toward the secretion of proteins mainly related to cell migration and immune response. The notion that p53 may rewire the liver secretome profile suggests a new non-cell autonomous role of p53 that affect different liver functions and whole organism homeostasis.

Mutant p53 gain of function underlies high expression levels of colorectal cancer stem cells markers.

Emerging notion in carcinogenesis ascribes tumor initiation and aggressiveness to cancer stem cells (CSCs). Specifically, colorectal cancer (CRC) development was shown to be compatible with CSCs hypothesis. Mutations in p53 are highly frequent in CRC, and are known to facilitate tumor development and aggressiveness. Yet, the link between mutant p53 and colorectal CSCs is not well-established. In the present study, we set to examine whether oncogenic mutant p53 proteins may augment colorectal CSCs phenotype. By genetic manipulation of mutant p53 in several cellular systems, we demonstrated that mutant p53 enhances colorectal tumorigenesis. Moreover, mutant p53-expressing cell lines harbor larger sub-populations of cells highly expressing the known colorectal CSCs markers: CD44, Lgr5, and ALDH. This elevated expression is mediated by mutant p53 binding to CD44, Lgr5, and ALDH1A1 promoter sequences. Furthermore, ALDH1 was found to be involved in mutant p53-dependent chemotherapy resistance. Finally, analysis of ALDH1 and CD44 in human CRC biopsies indicated a positive correlation between their expression and the presence of oncogenic p53 missense mutations. These findings suggest novel insights pertaining the mechanism by which mutant p53 enhances CRC development, which involves the expansion of CSCs sub-populations within CRC tumors, and underscore the importance of targeting these sub-populations for CRC therapy.