Cancer-associated fibroblasts promote cancer stemness by inducing expression of the chromatin-modifying protein CBX4 in squamous cell carcinoma.

The chromobox-containing protein CBX4 is an important regulator of epithelial cell proliferation and differentiation, and has been implicated in several cancer types. The cancer stem cell (CSC) population is a key driver of metastasis and recurrence. The undifferentiated, plastic state characteristic of CSCs relies on cues from the microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of the microenvironment that can influence the CSC population through the secretion of extracellular matrix and a variety of growth factors. Here we show CBX4 is a critical regulator of the CSC phenotype in squamous cell carcinomas of the skin and hypopharynx. Moreover, CAFs can promote the expression of CBX4 in the CSC population through the secretion of interleukin-6 (IL-6). IL-6 activates JAK/STAT3 signaling to increase ∆Np63α-a key transcription factor that is essential for epithelial stem cell function and the maintenance of proliferative potential that is capable of regulating CBX4. Targeting the JAK/STAT3 axis or CBX4 directly suppresses the aggressive phenotype of CSCs and represents a novel opportunity for therapeutic intervention.

ΔNp63α in cancer: importance and therapeutic opportunities.

Our understanding of cancer and the key pathways that drive cancer survival has expanded rapidly over the past several decades. However, there are still important challenges that continue to impair patient survival, including our inability to target cancer stem cells (CSCs), metastasis, and drug resistance. The transcription factor p63 is a p53 family member with multiple isoforms that carry out a wide array of functions. Here, we discuss the critical importance of the ΔNp63α isoform in cancer and potential therapeutic strategies to target ΔNp63α expression to impair the CSC population, as well as to prevent metastasis and drug resistance to improve patient survival.

BRD8 maintains glioblastoma by epigenetic reprogramming of the p53 network.

Inhibition of the tumour suppressive function of p53 (encoded by TP53) is paramount for cancer development in humans. However, p53 remains unmutated in the majority of cases of glioblastoma (GBM)-the most common and deadly adult brain malignancy1,2. Thus, how p53-mediated tumour suppression is countered in TP53 wild-type (TP53WT) GBM is unknown. Here we describe a GBM-specific epigenetic mechanism in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex. This mechanism causes a repressive chromatin state that prevents transactivation by p53 and sustains proliferation. Notably, targeting the bromodomain of BRD8 displaces H2AZ, enhances chromatin accessibility and engages p53 transactivation. This in turn enforces cell cycle arrest and tumour suppression in TP53WT GBM. In line with these findings, BRD8 is highly expressed with H2AZ in proliferating single cells of patient-derived GBM, and is inversely correlated with CDKN1A, a canonical p53 target that encodes p21 (refs. 3,4). This work identifies BRD8 as a selective epigenetic vulnerability for a malignancy for which treatment has not improved for decades. Moreover, targeting the bromodomain of BRD8 may be a promising therapeutic strategy for patients with TP53WT GBM.

EZH2 regulates a SETDB1/ΔNp63α axis via RUNX3 to drive a cancer stem cell phenotype in squamous cell carcinoma.

Enhancer of zeste homolog 2 (EZH2) and SET domain bifurcated 1 (SETDB1, also known as ESET) are oncogenic methyltransferases implicated in a number of human cancers. These enzymes typically function as epigenetic repressors of target genes by methylating histone H3 K27 and H3-K9 residues, respectively. Here, we show that EZH2 and SETDB1 are essential to proliferation in 3 SCC cell lines, HSC-5, FaDu, and Cal33. Additionally, we find both of these proteins highly expressed in an aggressive stem-like SCC sub-population. Depletion of either EZH2 or SETDB1 disrupts these stem-like cells and their associated phenotypes of spheroid formation, invasion, and tumor growth. We show that SETDB1 regulates this SCC stem cell phenotype through cooperation with ΔNp63α, an oncogenic isoform of the p53-related transcription factor p63. Furthermore, EZH2 is upstream of both SETDB1 and ΔNp63α, activating these targets via repression of the tumor suppressor RUNX3. We show that targeting this pathway with inhibitors of EZH2 results in activation of RUNX3 and repression of both SETDB1 and ΔNp63α, antagonizing the SCC cancer stem cell phenotype. This work highlights a novel pathway that drives an aggressive cancer stem cell phenotype and demonstrates a means of pharmacological intervention.

BRD4 Regulates Transcription Factor ΔNp63α to Drive a Cancer Stem Cell Phenotype in Squamous Cell Carcinomas.

Bromodomain containing protein 4 (BRD4) plays a critical role in controlling the expression of genes involved in development and cancer. Inactivation of BRD4 inhibits cancer growth, making it a promising anticancer drug target. The cancer stem cell (CSC) population is a key driver of recurrence and metastasis in patients with cancer. Here we show that cancer stem-like cells can be enriched from squamous cell carcinomas (SCC), and that these cells display an aggressive phenotype with enhanced stem cell marker expression, migration, invasion, and tumor growth. BRD4 is highly elevated in this aggressive subpopulation of cells, and its function is critical for these CSC-like properties. Moreover, BRD4 regulates ΔNp63α, a key transcription factor that is essential for epithelial stem cell function that is often overexpressed in cancers. BRD4 regulates an EZH2/STAT3 complex that leads to increased ΔNp63α-mediated transcription. Targeting BRD4 in human SCC reduces ΔNp63α, leading to inhibition of spheroid formation, migration, invasion, and tumor growth. These studies identify a novel BRD4-regulated signaling network in a subpopulation of cancer stem-like cells, elucidating a possible avenue for effective therapeutic intervention. SIGNIFICANCE: This study identifies a signaling cascade driven by BRD4 that upregulates ΔNp63α to promote cancer stem-like properties, which has potential therapeutic implications for the treatment of squamous cell carcinomas.

p63-related signaling at a glance.

p63 (also known as TP63) is a transcription factor of the p53 family, along with p73. Multiple isoforms of p63 have been discovered and these have diverse functions encompassing a wide array of cell biology. p63 isoforms are implicated in lineage specification, proliferative potential, differentiation, cell death and survival, DNA damage response and metabolism. Furthermore, p63 is linked to human disease states including cancer. p63 is critical to many aspects of cell signaling, and in this Cell science at a glance article and the accompanying poster, we focus on the signaling cascades regulating TAp63 and ΔNp63 isoforms and those that are regulated by TAp63 and ΔNp63, as well the role of p63 in disease.

The potential impact of tumor suppressor genes on human gametogenesis: a case-control study.

PURPOSE: To study the incidence of tumor suppressor gene (TSG) mutations in men and women with impaired gametogenesis. METHODS: Gene association analyses were performed on blood samples in two distinct patient populations: males with idiopathic male infertility and females with unexplained diminished ovarian reserve (DOR). The male study group consisted of men with idiopathic azoospermia, oligozoospermia, asthenozoospermia, or teratozoospermia. Age-matched controls were men with normal semen analyses. The female study group consisted of women with unexplained DOR with anti-Müllerian hormone levels ≤ 1.1 ng/mL. Controls were age-matched women with normal ovarian reserve (> 1.1 ng/mL). RESULTS: Fifty-seven male cases (mean age = 38.4; mean sperm count = 15.7 ± 12.1; mean motility = 38.2 ± 24.7) and 37 age-matched controls (mean age = 38.0; mean sperm count = 89.6 ± 37.5; mean motility = 56.2 ± 14.3) were compared. Variants observed in CHD5 were found to be enriched in the study group (p = 0.000107). The incidence of CHD5 mutation c.*3198_*3199insT in the 3'UTR (rs538186680) was significantly higher in cases compared to controls (p = 0.0255). 72 DOR cases (mean age = 38.7; mean AMH = 0.5 ± 0.3; mean FSH = 11.7 ± 12.5) and 48 age-matched controls (mean age = 37.6; mean AMH = 4.1 ± 3.0; mean FSH = 7.1 ± 2.2) were compared. Mutations in CHD5 (c.-140A>C), RB1 (c.1422-18delT, rs70651121), and TP53 (c.376-161A>G, rs75821853) were found at significantly higher frequencies in DOR cases compared to controls (p ≤ 0.05). In addition, 363 variants detected in the DOR patients were not present in the control group. CONCLUSION: Unexplained impaired gametogenesis in both males and females may be associated with genetic variation in TSGs. TSGs, which play cardinal roles in cell-cycle control, might also be critical for normal spermatogenesis and oogenesis. If validated in larger prospective studies, it is possible that TSGs provide an etiological basis for some patients with impaired gametogenesis.

Different regulation of limb development by p63 transcript variants.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

The Chromodomain Helicase DNA-Binding Chromatin Remodelers: Family Traits that Protect from and Promote Cancer.

A plethora of mutations in chromatin regulators in diverse human cancers is emerging, attesting to the pivotal role of chromatin dynamics in tumorigenesis. A recurrent theme is inactivation of the chromodomain helicase DNA-binding (CHD) family of proteins-ATP-dependent chromatin remodelers that govern the cellular machinery's access to DNA, thereby controlling fundamental processes, including transcription, proliferation, and DNA damage repair. This review highlights what is currently known about how genetic and epigenetic perturbation of CHD proteins and the pathways that they regulate set the stage for cancer, providing new insight for designing more effective anti-cancer therapies.

Deletions linked to TP53 loss drive cancer through p53-independent mechanisms.

Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a 'loss of heterozygosity' deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.1, produces a greater effect on lymphoma and leukaemia development than Trp53 deletion. Mechanistically, the effect of 11B3 loss on tumorigenesis involves co-deleted genes such as Eif5a and Alox15b (also known as Alox8), the suppression of which cooperates with Trp53 loss to produce more aggressive disease. Our results imply that the selective advantage produced by human chromosome 17p deletion reflects the combined impact of TP53 loss and the reduced dosage of linked tumour suppressor genes.

Architects of the genome: CHD dysfunction in cancer, developmental disorders and neurological syndromes.

Chromatin is vital to normal cells, and its deregulation contributes to a spectrum of human ailments. An emerging concept is that aberrant chromatin regulation culminates in gene expression programs that set the stage for the seemingly diverse pathologies of cancer, developmental disorders and neurological syndromes. However, the mechanisms responsible for such common etiology have been elusive. Recent evidence has implicated lesions affecting chromatin-remodeling proteins in cancer, developmental disorders and neurological syndromes, suggesting a common source for these different pathologies. Here, we focus on the chromodomain helicase DNA binding chromatin-remodeling family and the recent evidence for its deregulation in diverse pathological conditions, providing a new perspective on the underlying mechanisms and their implications for these prevalent human diseases.

The tumor suppressor Chd5 is induced during neuronal differentiation in the developing mouse brain.

Epigenetic regulation of gene expression orchestrates dynamic cellular processes that become perturbed in human disease. An understanding of how subversion of chromatin-mediated events leads to pathologies such as cancer and neurodevelopmental syndromes may offer better treatment options for these pathological conditions. Chromodomain Helicase DNA-binding protein 5 (CHD5) is a dosage-sensitive tumor suppressor that is inactivated in human cancers, including neural-associated malignancies such as neuroblastoma and glioma. Here we report a detailed analysis of the temporal and cell type-specific expression pattern of Chd5 in the mammalian brain. By analyzing endogenous Chd5 protein expression during mouse embryogenesis, in the neonate, and in the adult, we found that Chd5 is expressed broadly in multiple brain regions, that Chd5 sub-cellular localization undergoes a switch from the cytoplasm to the nucleus during mid-gestation, and that Chd5 expression is retained at high levels in differentiated neurons of the adult. These findings may have important implications for defining the role of CHD5-mediated chromatin dynamics in the brain and for elucidating how perturbation of these epigenetic processes leads to neuronal malignancies, neurodegenerative diseases, and neurodevelopmental syndromes.

FGFR2 signaling underlies p63 oncogenic function in squamous cell carcinoma.

Oncogenic transcription factors drive many human cancers, yet identifying and therapeutically targeting the resulting deregulated pathways has proven difficult. Squamous cell carcinoma (SCC) is a common and lethal human cancer, and relatively little progress has been made in improving outcomes for SCC due to a poor understanding of its underlying molecular pathogenesis. While SCCs typically lack somatic oncogene-activating mutations, they exhibit frequent overexpression of the p53-related transcription factor p63. We developed an in vivo murine tumor model to investigate the function and key transcriptional programs of p63 in SCC. Here, we show that established SCCs are exquisitely dependent on p63, as acute genetic ablation of p63 in advanced, invasive SCC induced rapid and dramatic apoptosis and tumor regression. In vivo genome-wide gene expression analysis identified a tumor-survival program involving p63-regulated FGFR2 signaling that was activated by ligand emanating from abundant tumor-associated stroma. Correspondingly, we demonstrate the therapeutic efficacy of extinguishing this signaling axis in endogenous SCCs using the clinical FGFR2 inhibitor AZD4547. Collectively, these results reveal an unanticipated role for p63-driven paracrine FGFR2 signaling as an addicting pathway in human cancer and suggest a new approach for the treatment of SCC.

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium.

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5' sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.

An allelic series of Trp63 mutations defines TAp63 as a modifier of EEC syndrome.

Human Ectrodactyly, Ectodermal dysplasia, Clefting (EEC) syndrome is an autosomal dominant developmental disorder defined by limb deformities, skin defects, and craniofacial clefting. Although associated with heterozygous missense mutations in TP63, the genetic basis underlying the variable expressivity and incomplete penetrance of EEC is unknown. Here, we show that mice heterozygous for an allele encoding the Trp63 p.Arg318His mutation, which corresponds to the human TP63 p.Arg279His mutation found in patients with EEC, have features of human EEC. Using an allelic series, we discovered that whereas clefting and skin defects are caused by loss of Trp63 function, limb anomalies are due to gain- and/or dominant-negative effects of Trp63. Furthermore, we identify TAp63 as a strong modifier of EEC-associated phenotypes with regard to both penetrance and expressivity.

FANCD2 activates transcription of TAp63 and suppresses tumorigenesis.

Fanconi anemia (FA) is a rare genetic disorder characterized by an increased susceptibility to squamous cell cancers. Fifteen FA genes are known, and the encoded proteins cooperate in a common DNA repair pathway. A critical step is the monoubiquitination of the FANCD2 protein, and cells from most FA patients are deficient in this step. How monoubiquitinated FANCD2 suppresses squamous cell cancers is unknown. Here we show that Fancd2-deficient mice are prone to Ras-oncogene-driven skin carcinogenesis, while Usp1-deficient mice, expressing elevated cellular levels of Fancd2-Ub, are resistant to skin tumors. Moreover, Fancd2-Ub activates the transcription of the tumor suppressor TAp63, thereby promoting cellular senescence and blocking skin tumorigenesis. For FA patients, the reduction of FANCD2-Ub and TAp63 protein levels may account for their susceptibility to squamous cell neoplasia. Taken together, Usp1 inhibition may be a useful strategy for upregulating TAp63 and preventing or treating squamous cell cancers in the general non-FA population.

Chd5 requires PHD-mediated histone 3 binding for tumor suppression.

Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.

ΔNp63α is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell proliferation and tumorigenesis.

The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor-suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene-induced senescence to drive tumorigenesis in vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin-remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation and suggest that Lsh-mediated chromatin-remodeling events are critical to this process.

Throwing the cancer switch: reciprocal roles of polycomb and trithorax proteins.

The discovery that cancer can be governed above and beyond the level of our DNA presents a new era for designing therapies that reverse the epigenetic state of a tumour cell. Understanding how altered chromatin dynamics leads to malignancy is essential for controlling tumour cells while sparing normal cells. Polycomb and trithorax group proteins are evolutionarily conserved and maintain chromatin in the 'off' or 'on' states, thereby preventing or promoting gene expression, respectively. Recent work highlights the dynamic interplay between these opposing classes of proteins, providing new avenues for understanding how these epigenetic regulators function in tumorigenesis.

A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias.

The human congenital syndromes ectrodactyly ectodermal dysplasia-cleft lip/palate syndrome, ankyloblepharon ectodermal dysplasia clefting, and split-hand/foot malformation are all characterized by ectodermal dysplasia, limb malformations, and cleft lip/palate. These phenotypic features are a result of an imbalance between the proliferation and differentiation of precursor cells during development of ectoderm-derived structures. Mutations in the p63 and interferon regulatory factor 6 (IRF6) genes have been found in human patients with these syndromes, consistent with phenotypes. Here, we used human and mouse primary keratinocytes and mouse models to investigate the role of p63 and IRF6 in proliferation and differentiation. We report that the DeltaNp63 isoform of p63 activated transcription of IRF6, and this, in turn, induced proteasome-mediated DeltaNp63 degradation. This feedback regulatory loop allowed keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate. Importantly, mutations in either p63 or IRF6 resulted in disruption of this regulatory loop: p63 mutations causing ectodermal dysplasias were unable to activate IRF6 transcription, and mice with mutated or null p63 showed reduced Irf6 expression in their palate and ectoderm. These results identify what we believe to be a novel mechanism that regulates the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation and links the pathogenesis of 2 genetically different groups of ectodermal dysplasia syndromes into a common molecular pathway.