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AF9 (MLLT3) and ENL (MLLT1) are members of the YEATS family (named after the five proteins first shown to contain this domain: Yaf9, ENL, AF9, Taf14, Sas5) defined by the presence of a YEATS domain. The YEATS domain is an epigenetic reader that binds to acetylated and crotonylated lysines, unlike the bromodomain which can only bind to acetylated lysines. All members of this family have been shown to be components of various complexes with roles in chromatin remodeling, histone modification, histone variant deposition, and transcriptional regulation. MLLT3 is a critical regulator of hematopoiesis with a role in maintaining the hematopoietic stem or progenitor cell (HSPC) population. Approximately 10% of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) patients harbor a translocation involving MLL (mixed lineage leukemia). In the context of MLL fusion patients with AML and ALL, MLL-AF9 and MLL-ENL fusions are observed in 34 and 31% of the patients, respectively. The intrinsically disordered C-terminal domain of MLLT3 (AHD, ANC1 homology domain) undergoes coupled binding and folding upon interaction with partner proteins AF4, DOT1L, BCOR, and CBX8. Backbone dynamics studies of the complexes suggest a role for dynamics in function. Inhibitors of the interaction of the intrinsically disordered AHD with partner proteins have been described, highlighting the feasibility of targeting intrinsically disordered regions. MLLT1 undergoes phase separation to enhance recruitment of the super elongation complex (SEC) and drive transcription. Mutations in MLLT1 observed in Wilms tumor patients enhance phase separation and transcription to drive an aberrant gene expression program., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells., (© 2020 by The American Society of Hematology.)

Background: Hippo-Yes-associated protein (YAP) signaling is a key regulator of organ size and tumorigenesis, yet the underlying molecular mechanism is still poorly understood. At present, the significance of the Hippo-YAP pathway in diffuse large B-cell lymphoma (DLBCL) is ill-defined., Methods: The expression of YAP in DLBCL was determined in public database and clinical specimens. The effects of YAP knockdown, CRISPR/Cas9-mediated YAP deletion, and YAP inhibitor treatment on cell proliferation and the cell cycle were evaluated both in vitro and in vivo. RNA sequencing was conducted to detect dysregulated RNAs in YAP-knockout DLBCL cells. The regulatory effects of insulin-like growth factor-1 receptor (IGF-1R) on Hippo-YAP signaling were explored by targeted inhibition and rescue experiments., Results: High expression of YAP was significantly correlated with disease progression and poor prognosis. Knockdown of YAP expression suppressed cell proliferation and induced cell cycle arrest in DLBCL cells. Verteporfin (VP), a benzoporphyrin derivative, exerted an anti-tumor effect by regulating the expression of YAP and the downstream target genes, CTGF and CYR61. In vitro and in vivo studies revealed that deletion of YAP expression with a CRISPR/Cas9 genome editing system significantly restrained tumor growth. Moreover, downregulation of IGF-1R expression led to a remarkable decrease in YAP expression. In contrast, exposure to IGF-1 promoted YAP expression and reversed the inhibition of YAP expression induced by IGF-1R inhibitors., Conclusions: Our study highlights the critical role of YAP in the pathogenesis of DLBCL and uncovers the regulatory effect of IGF-1R on Hippo-YAP signaling, suggesting a novel therapeutic strategy for DLBCL.

The Hippo pathway and its effector protein YAP (a transcriptional coactivator) have been identified as important in the biology of both hepatocellular carcinoma and cholangiocarcinoma. First identified as a tumor suppressor pathway in Drosophila , the understanding of the mammalian YAP signaling and its regulation continues to expand. In its "on" function, the canonical regulatory Hippo pathway, a well-described serine/threonine kinase module, regulates YAP function by restricting its subcellular localization to the cytoplasm. In contrast, when the Hippo pathway is "off," YAP translocates to the nucleus and drives cotranscriptional activity. Given the role of Hippo/YAP signaling in hepatic malignancies, investigators have sought to target these molecules; however, standard approaches have not been successful based on the pathways' negative regulatory role. More recently, additional regulatory mechanisms, such as tyrosine phosphorylation, of YAP have been described. These represent positive regulatory events that may be targetable. Additionally, several groups have identified potentiating feed-forward signaling for YAP in multiple contexts, suggesting other experimental therapeutic approaches to interrupt these signaling loops. Herein we explore the current data supporting alternative YAP regulatory pathways, review the described feed-forward signaling cascades that are YAP dependent, and explore targeting strategies that have been employed in preclinical models of hepatic malignancies.

Targeted disruption of the murine Hoxd10 gene (ΔHoxd10) leads to a high frequency of localized (gland-to-gland or regionally within a gland) lactation impairment in homozygous mutant mice as a single gene mutation. The effect of Hoxd10 disruption was enhanced by simultaneous disruption of Hoxd9 (ΔHoxd9/d10), a mutation shown previously to have no effect on mammary function as a single gene alteration. Mammary glands of homozygous ΔHoxd10 and ΔHoxd9/d10 females were indistinguishable from those of wild type littermate and age-matched control mice in late pregnancy. However, in lactation, 47% of homozygous ΔHoxd10 females, and 100% of homozygous ΔHoxd9/d10 females, showed localized or complete failure of two or more glands to undergo lactation-associated morphological changes and to secrete milk. Affected regions of ΔHoxd10 and ΔHoxd9/d10 mutants showed reduced prolactin receptor expression, reduced signal transducer and activator transcription protein 5 (STAT5) phosphorylation, reduced expression of downstream milk proteins, mislocalized glucose transporter 1 (GLUT1), increased STAT3 expression and phosphorylation, recruitment of leukocytes, altered cell cycle status, and increased apoptosis relative to unaffected regions and wild type control glands. Despite these local effects on alveolar function, transplantation results and hormone analysis indicate that Hoxd10 primarily has systemic functions that confer attenuated STAT5 phosphorylation on both wild type and ΔHoxd10 transplants when placed in ΔHoxd10 hosts, thereby exacerbating an underlying propensity for lactation failure in C57Bl/6 mice.

Hippo signaling functions to limit cellular growth, but the aberrant nuclear accumulation of its downstream YAP1 leads to carcinogenesis. YAP1/TEAD complex activates the oncogenic downstream transcription, such as CTGF and c-Myc. How YAP1 is protected in the cytoplasm from ubiquitin-mediated degradation remains elusive. In this study, a member of Angiomotin (Motin) family, AMOTL1 (Angiomotin Like 1), was screened out as the only one to promote YAP1 nuclear accumulation by several clinical cohorts, which was further confirmed by the cellular functional assays. The interaction between YAP1 and AMOTL1 was suggested by co-immunoprecipitation and immunofluorescent staining. The clinical significance of the AMOTL1-YAP1-CTGF axis in gastric cancer (GC) was analyzed by multiple clinical cohorts. Moreover, the therapeutic effect of targeting the oncogenic axis was appraised by drug-sensitivity tests and xenograft-formation assays. The upregulation of AMOTL1 is associated with unfavorable clinical outcomes of GC, and knocking down AMOTL1 impairs its oncogenic properties. The cytoplasmic interaction between AMOTL1 and YAP1 protects each other from ubiquitin-mediated degradation. AMOTL1 promotes YAP1 translocation into the nuclei to activate the downstream expression, such as CTGF. Knocking down AMOTL1, YAP1, and CTGF enhances the therapeutic efficacies of the first-line anticancer drugs. Taken together, AMOTL1 plays an oncogenic role in gastric carcinogenesis through interacting with YAP1 and promoting its nuclear accumulation. A combination of AMOTL1, YAP1, and CTGF expression might serve as a surrogate of Hippo activation status. The co-activation of the AMOTL1/YAP1-CTGF axis is associated with poor clinical outcomes of GC patients, and targeting this oncogenic axis may enhance the chemotherapeutic effects.

Activator protein (AP)-1 transcription factors are essential elements of the pro-oncogenic functions of transforming growth factor-β (TGFβ)-SMAD signaling. Here we show that in multiple HER2+ and/or EGFR+ breast cancer cell lines these AP-1-dependent tumorigenic properties of TGFβ critically rely on epidermal growth factor receptor (EGFR) activation and expression of the ΔN isoform of transcriptional regulator p63. EGFR and ΔNp63 enabled and/or potentiated the activation of a subset of TGFβ-inducible invasion/migration-associated genes, e.g., ITGA2, LAMB3, and WNT7A/B, and enhanced the recruitment of SMAD2/3 to these genes. The TGFβ- and EGF-induced binding of SMAD2/3 and JUNB to these gene loci was accompanied by p63-SMAD2/3 and p63-JUNB complex formation. p63 and EGFR were also found to strongly potentiate TGFβ induction of AP-1 proteins and, in particular, FOS family members. Ectopic overexpression of FOS could counteract the decrease in TGFβ-induced gene activation after p63 depletion. p63 is also involved in the transcriptional regulation of heparin binding (HB)-EGF and EGFR genes, thereby establishing a self-amplification loop that facilitates and empowers the pro-invasive functions of TGFβ. These cooperative pro-oncogenic functions of EGFR, AP-1, p63, and TGFβ were efficiently inhibited by clinically relevant chemical inhibitors. Our findings may, therefore, be of importance for therapy of patients with breast cancers with an activated EGFR-RAS-RAF pathway.

Breast cancer is the second leading cause of cancer-related deaths among women, largely due to the progression of a significant fraction of primary tumours to the metastatic stage. Here, we show that zinc-finger protein 750 (ZNF750) opposes the migration and invasion of breast cancer cells by repressing a prometastatic transcriptional programme, which includes genes involved in focal adhesion and extracellular matrix interactions, such as LAMB3 and CTNNAL1. Mechanistically, ZNF750 recruits the epigenetic modifiers KDM1A and HDAC1 to the promoter regions of LAMB3 and CTNNAL1, influencing histone marks and transactivating these genomic sites. Gene expression analysis in cancer patient datasets indicated that ZNF750 and its targets were negative prognostic factors in breast cancer. Together, our findings shed light on the molecular mechanism by which ZNF750 regulates cell migration and invasion, suggesting a role in breast cancer metastasis.

Hydrated magnesium silicate (or "talc" particles) is a sclerosis agent commonly used in the management of malignant pleural effusions, a common symptom of metastatic diseases, including lung cancers. However, the direct effects of talc particles to lung carcinoma cells, which can be found in the malignant pleural effusion fluids from patients with lung cancer, are not fully understood. Here, we report a study of the signaling pathways that can modulate the cell death and IL-6 secretion induced by talc particles in human lung carcinoma cells. We found that talc-sensitive cells have higher mRNA and protein expression of PI3K catalytic subunits α and β. Further experiments confirmed that modulation (inhibition or activation) of the PI3K pathway reduces or enhances cellular sensitivity to talc particles, respectively, independent of the inflammasome. By knocking down specific PI3K isoforms, we also confirmed that both PI3Kα and -β mediate the observed talc effects. Our results suggest a novel role of the PI3K pathway in talc-induced cell death and IL-6 secretion in lung carcinoma cells. These cellular events are known to drive fibrosis, and thus further studies of the PI3K pathway may provide a better understanding of the mechanisms of talc sclerosis in the malignant pleural space.

A growing number of studies suggest a tumor suppressor role for the SWI/SNF complex involved in the remodeling of chromatin. Alterations of this complex have been found in many tumors of different origins, with topographic, morphologic and phenotypic diversity. Notably, they define 2 types of thoracic tumors: SMARCA4-deficient non-small cell lung carcinoma and SMARCA4-deficient sarcoma. Some clinical features appear to be common to both, such as intrathoracic localization, smoking exposure, male predominance and poor prognosis. However, the histological distinction between these two entities is sometimes difficult and it is not excluded that these entities belong to the same tumor spectrum with different degrees of differentiation. The therapy of these tumors is not yet codified. These tumors do not seem associated with oncogenic driver mutations allowing the prescription of targeted therapy, but immunotherapy has been shown to be effective in rare reported cases. More specific treatments using EZH2 inhibitors also seem promising in SMARCA4 deficient sarcomas., (Copyright © 2019 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Myocardin-related transcription factor B (MRTFB) is a candidate tumor-suppressor gene identified in transposon mutagenesis screens of the intestine, liver, and pancreas. Using a combination of cell-based assays, in vivo tumor xenograft assays, and Mrtfb knockout mice, we demonstrate here that MRTFB is a human and mouse colorectal cancer (CRC) tumor suppressor that functions in part by inhibiting cell invasion and migration. To identify possible MRTFB transcriptional targets, we performed whole transcriptome RNA sequencing in MRTFB siRNA knockdown primary human colon cells and identified 15 differentially expressed genes. Among the top candidate tumor-suppressor targets were melanoma cell adhesion molecule (MCAM), a known tumor suppressor, and spindle apparatus coiled-coil protein 1 (SPDL1), which has no confirmed role in cancer. To determine whether these genes play a role in CRC, we knocked down the expression of MCAM and SPDL1 in human CRC cells and showed significantly increased invasion and migration of tumor cells. We also showed that Spdl1 expression is significantly down-regulated in Mrtfb knockout mouse intestine, while lower SPDL1 expression levels are significantly associated with reduced survival in CRC patients. Finally, we show that depletion of MCAM and SPDL1 in human CRC cells significantly increases tumor development in xenograft assays, further confirming their tumor-suppressive roles in CRC. Collectively, our findings demonstrate the tumor-suppressive role of MRTFB in CRC and identify several genes, including 2 tumor suppressors, that act downstream of MRTFB to regulate tumor growth and survival in CRC patients., Competing Interests: The authors declare no competing interests.

Loss of expression of context-specific tumor suppressors is a critical event that facilitates the development of prostate cancer. Zinc finger and BTB domain containing transcriptional repressors, such as ZBTB7A and ZBTB16, have been recently identified as tumor suppressors that play important roles in preventing prostate cancer progression. In this study, we used combined ChIP-seq and RNA-seq analyses of prostate cancer cells to identify direct ZBTB7A-repressed genes, which are enriched for transcriptional targets of E2F, and identified that the androgen receptor (AR) played a critical role in the transcriptional suppression of these E2F targets. AR recruitment of the retinoblastoma protein (Rb) was required to strengthen the E2F-Rb transcriptional repression complex. In addition, ZBTB7A was rapidly recruited to the E2F-Rb binding sites by AR and negatively regulated the transcriptional activity of E2F1 on DNA replication genes. Finally, ZBTB7A suppressed the growth of castration-resistant prostate cancer (CRPC) in vitro and in vivo , and overexpression of ZBTB7A acted in synergy with high-dose testosterone treatment to effectively prevent the recurrence of CRPC. Overall, this study provides novel molecular insights of the role of ZBTB7A in CRPC cells and demonstrates globally its critical role in mediating the transcriptional repression activity of AR. SIGNIFICANCE: ZBTB7A is recruited to the E2F-Rb binding sites by AR and negatively regulates the transcriptional activity of E2F1 on DNA replication genes., (©2019 American Association for Cancer Research.)

T cell acute lymphoblastic leukaemia (T-ALL) cases include subfamilies that overexpress the TAL1/LMO, TLX1/3 and HOXA transcription factor oncogenes. While it has been shown that TAL1/LMO transcription factors induce self-renewal of thymocytes, whether this is true for other transcription factor oncogenes is unknown. To address this, we have studied NUP98-HOXD13-transgenic (NHD13-Tg) mice, which overexpress HOXA transcription factors throughout haematopoiesis and develop both myelodysplastic syndrome (MDS) progressing to acute myeloid leukaemia (AML) as well as T-ALL. We find that thymocytes from preleukaemic NHD13-Tg mice can serially transplant, demonstrating that they have self-renewal capacity. Transcriptome analysis shows that NHD13-Tg thymocytes exhibit a stem cell-like transcriptional programme closely resembling that induced by Lmo2, including Lmo2 itself and its critical cofactor Lyl1. To determine whether Lmo2/Lyl1 are required for NHD13-induced thymocyte self-renewal, NHD13-Tg mice were crossed with Lyl1 knockout mice. This showed that Lyl1 is essential for expression of the stem cell-like gene expression programme in thymocytes and self-renewal. Surprisingly however, NHD13 transgenic mice lacking Lyl1 showed accelerated T-ALL and absence of transformation to AML, associated with a loss of multipotent progenitors in the bone marrow. Thus multiple T cell oncogenes induce thymocyte self-renewal via Lmo2/Lyl1; however, NHD13 can also promote T-ALL via an alternative pathway.

Radiotherapy is the mainstay and primary curative treatment modality in nasopharyngeal carcinoma (NPC), whose efficacy is limited by the development of intrinsic and acquired radioresistance. Thus, deciphering new molecular targets and pathways is essential for enhancing the radiosensitivity of NPC. SALL4 is a vital factor in the development and prognosis of various cancers, but its role in radioresistance remains elusive. This study aimed to explore the association of SALL4 expression with radioresistance of NPC. It was revealed that SALL4 expression was closely correlated with advanced T classification of NPC patients. Inhibition of SALL4 reduced proliferation and sensitized cells to radiation both in vitro and in vivo. Furthermore, SALL4 silencing increased radiation-induced DNA damage, apoptosis, and G2/M arrest in CNE2 and CNE2R cells. Moreover, knockdown of SALL4 impaired the expression of p-ATM, p-Chk2, p-p53, and anti-apoptosis protein Bcl-2, while pro-apoptosis protein was upregulated. These findings indicate that SALL4 could induce radioresistance via ATM/Chk2/p53 pathway and its downstream proteins related to apoptosis. Targeting SALL4 might be a promising approach for the development of novel radiosensitizing therapeutic agents for radioresistant NPC patients., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Metastasis is the main cause of cancer mortality. However, the triggering mechanisms and regulation of epithelial-mesenchymal transition (EMT) factors in the commitment of metastasis have not been well characterized. Spermatogenic Zip 1 (SPZ1) acts as a proto-oncogene and an upstream regulator of EMT during tumorigenesis. Here we report that the HIV-1 Tat-interacting protein 60 kDa (Tip60) acetyltransferase mediates acetylation at lysine residues of SPZ1 at positions 369 and 374, and of TWIST1 at positions 73 and 76, which are required for SPZ1-TWIST1 complex formation and cancer cell migration in vitro and in vivo. Ectopic SPZ1 and TWIST1 expression, but not that of TWIST1 alone, enhanced vascular endothelial growth factor (VEGF) expression via the recruitment of bromodomain-containing protein 4 (BRD4), thus enhancing RNA-Pol II-dependent transcription and inducing metastasis. Neutralization of VEGF using humanized monoclonal antibodies such as Avastin, effectively abrogated the EMT and oncogenesis induced by the acetylated SPZ1-TWIST1 complex. Our findings highlight the importance of acetylation signaling in the SPZ1-TWIST1-BRD4 axis in the mediation of EMT and its regulation during tumor initiation and metastasis.

Metastatic clear cell renal cell carcinoma (CCC) remains incurable despite advances in the development of anti-angiogenic targeted therapies and the emergence of immune checkpoint inhibitors. We have previously shown that the sonic hedgehog-Gli signaling pathway is oncogenic in CCC allowing us to identify the developmental Lim1 transcription factor as a Gli target and as a new oncogene in CCC regulating cell proliferation and apoptosis, and promoting tumor growth. In this previous study, preliminary in vitro results also suggested that Lim1 may be implicated in metastatic spread. Here we investigated the potential pro-metastatic role of Lim1 in advanced CCC (1) in vitro using a panel of CCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene either naturally or by gene transfer and (2) ex vivo in 30 CCC metastatic tissues, including lymph nodes, lung, skin, bone, and adrenal metastases, and (3) in vivo, using a metastatic model by intravenous injection of siRNA-transfected cells into Balb/c nude. Our in vitro results reveal that Lim1 knockdown time-dependently decreased CCC cell motility, migration, invasion, and clonogenicity by up to 50% regardless of their VHL status. Investigating the molecular machinery involved in these processes, we identified a large panel of Lim1 targets known to be involved in cell adhesion (paxillin and fibronectin), epithelial-mesenchymal transition (Twist1/2 and snail), invasion (MMP1/2/3/8/9), and metastatic progression (CXCR4, SDF-1, and ANG-1). Importantly, Lim1 was found constitutively expressed in all metastatic tissues. The H-score in metastatic tissues being significantly superior to the score in the corresponding primary tumor tissues (P value = 0.009). Furthermore, we showed that Lim1 silencing decreases pulmonary metastasis development in terms of number and size in the in vivo metastatic model of human CCC. Taken together, these experiments strengthen the potential therapeutic value of Lim1 targeting as a promising novel approach for treating metastatic human CCC.

Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca 2+ entry by enhancing store-operated Ca 2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca 2+ -induced Lats1/2 activation. Ca 2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca 2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca 2+ is a crucial intracellular cue that regulates the Hippo pathway and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.

Epithelial tissues rely on a highly coordinated balance between self-renewal, proliferation, and differentiation, disruption of which may drive carcinogenesis. The epigenetic regulator KMT2D ( MLL4 ) is one of the most frequently mutated genes in all cancers, particularly epithelial cancers, yet its normal function in these tissues is unknown. Here, we identify a novel role for KMT2D in coordinating this fine balance, as depletion of KMT2D from undifferentiated epidermal keratinocytes results in reduced proliferation, premature spurious activation of terminal differentiation genes, and disorganized epidermal stratification. Genome-wide, KMT2D interacts with p63 and is enriched at its target enhancers. Depletion of KMT2D results in a broad loss of enhancer histone modifications H3 Lys 4 (H3K4) monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac) as well as reduced expression of p63 target genes, including key genes involved in epithelial development and adhesion. Together, these results reveal a critical role for KMT2D in the control of epithelial enhancers and p63 target gene expression, including the requirement of KMT2D for the maintenance of epithelial progenitor gene expression and the coordination of proper terminal differentiation., (© 2018 Lin-Shiao et al.; Published by Cold Spring Harbor Laboratory Press.)

MYC is a major cancer driver but is documented to be a difficult therapeutic target itself. Here, we report on the biological activity, the structural basis, and therapeutic effects of the family of multitargeted compounds that simultaneously disrupt functions of two critical MYC-mediating factors through inhibiting the acetyllysine binding of BRD4 and the kinase activity of PI3K. We show that the dual-action inhibitor impairs PI3K/BRD4 signaling in vitro and in vivo and affords maximal MYC down-regulation. The concomitant inhibition of PI3K and BRD4 blocks MYC expression and activation, promotes MYC degradation, and markedly inhibits cancer cell growth and metastasis. Collectively, our findings suggest that the dual-activity inhibitor represents a highly promising lead compound for the development of novel anticancer therapeutics.

Most clear cell renal carcinomas (ccRCCs) are initiated by somatic inactivation of the VHL tumor suppressor gene. The VHL gene product, pVHL, is the substrate recognition unit of an ubiquitin ligase that targets the HIF transcription factor for proteasomal degradation; inappropriate expression of HIF target genes drives renal carcinogenesis. Loss of pVHL is not sufficient, however, to cause ccRCC. Additional cooperating genetic events, including intragenic mutations and copy number alterations, are required. Common examples of the former are loss-of-function mutations of the PBRM1 and BAP1 tumor suppressor genes, which occur in a mutually exclusive manner in ccRCC and define biologically distinct subsets of ccRCC. PBRM1 encodes the Polybromo- and BRG1-associated factors-containing complex (PBAF) chromatin remodeling complex component BRG1-associated factor 180 (BAF180). Here we identified ccRCC lines whose ability to proliferate in vitro and in vivo is sensitive to wild-type BAF180, but not a tumor-associated BAF180 mutant. Biochemical and functional studies linked growth suppression by BAF180 to its ability to form a canonical PBAF complex containing BRG1 that dampens the HIF transcriptional signature., Competing Interests: W.G.K. receives consulting income and equity from Peloton Therapeutics, which is developing HIF2 inhibitors for treatment of kidney cancer.

Objective: To investigate the function and mechanism of zinc finger protein 750 (ZNF750) in esophageal squamous cell carcinoma (ESCC). Methods: Xenograft in nude mice was applied to detect the tumorigenesis of ZNF750-depleted ESCC cells. Western blot was performed to observe the expression of downstream target protein of ZNF750 in ESCC cell lines and xenograft tumor tissues in which ZNF750 was knocked down. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay was used to determine the proliferation of ZNF750 stably depleted cells after restoration of its target protein. Results: The tumor weight of blank control, negative control and ZNF750 knockdown groups was 137±26 mg, 161±31 mg and 463±89 mg, respectively, with a statistically significant difference ( P <0.01). The expressions of Kruppel-like factor 4 (KLF4) in ZNF750-depleted ESCC cells and its derived tumor tissue xenograft in nude mice were significantly down-regulated. Restoration of KLF4 in ZNF750 stably depleted cells significantly inhibited the cell proliferation ( P <0.01). Conclusions: ZNF750 may be a new tumor suppressor in the tumorigenesis of ESCC, and the inhibition of cell proliferation induced by ZNF750 may be partially through the regulation of KLF4 expression.

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that plays a pivotal role in cancer and inflammatory diseases. BRD4 binds and stays associated with chromatin during mitosis, bookmarking early G1 genes and reactivating transcription after mitotic silencing. BRD4 plays an important role in transcription, both as a passive scaffold via its recruitment of vital transcription factors and as an active kinase that phosphorylates RNA polymerase II, directly and indirectly regulating transcription. Through its HAT activity, BRD4 contributes to the maintenance of chromatin structure and nucleosome clearance. This review summarizes the known functions of BRD4 and proposes a model in which BRD4 actively coordinates chromatin structure and transcription., (© Society for Leukocyte Biology.)

Cervical cancer is one of the most common female cancers worldwide. Although the therapeutic outcomes of patients with early-stage cervical cancer have been significantly improved in the past decades, tumor metastasis and recurrence remain the major causes of cervical cancer-related deaths. In cervical squamous cell carcinoma (SCC), the aberrant activation of epithelial-mesenchymal transition (EMT), a crucial process in invasion and metastasis of epithelial cancer, could promote lymph nodal metastasis and recurrence, and predicts poor prognosis. In this study, we show that the expression levels of EMT markers, β-catenin and Vimentin, are associated with the p63 isoform ΔNp63α in SCC by using immunohistochemistry staining and analysis. Compared to the control SiHa cells (SiHa-NC), the expression of E-cadherin and β-catenin are upregulated, while Vimentin and ZEB1 are downregulated in the constructed SiHa cell line with stable ΔNp63α overexpression (SiHa-ΔNp63α). Besides, the migration and invasion abilities are also suppressed in SiHa-ΔNp63α cells with a typical epithelial morphology with cobblestone-like shape, suggesting that ΔNp63α is a vital EMT repressor in SCC cells. In addition, the involvement of miR-205/ZEB1 axis in the inhibition effect of ΔNp63α on EMT program is revealed by a miRNA array and confirmed by the subsequent transfection of the miR-205 mimic and antagomir. Moreover, SCC patients with low ΔNp63α expression and high EMT level show more frequent metastasis and recurrence as well as reduced overall survival. Therefore, EMT program and its vital repressor ΔNp63α could be used as biomarkers for tumor metastasis and recurrence in cervical cancer.

Triple-negative breast cancers (TNBC) comprise a heterogeneous subgroup of tumors with a generally poor prognosis. Subclassification of TNBC based on genomic analyses shows that basal-like TNBCs, specifically the basal A or BL2 subtype, are characterized by the expression of ΔNp63, a transcription factor that has been attributed a variety of roles in the regulation of proliferation, differentiation, and cell survival. To investigate the role(s) of p63 in basal-like breast cancers, we used HCC1806 cells that are classified as basal A/BL2. We show that these cells endogenously express p63, mainly as the ΔNp63α isoform. TP63 gene knockout by CRISPR resulted in viable cells that proliferate more slowly and adhere less tightly, with an increased rate of migration. Analysis of adhesion-related gene expression revealed a complex set of alterations in p63-depleted cells, with both increased and decreased adhesion molecules and adhesion substrates compared to parental cells expressing p63. Examination of the phenotype of these cells indicated that endogenous p63 is required to suppress the expression of luminal markers and maintain the basal epithelial phenotype, with increased levels of both CK8 and CK18 and a reduction in N-cadherin levels in cells lacking p63. On the other hand, the level of CK5 was not decreased and ER was not increased, indicating that p63 loss is insufficient to induce full luminal-type differentiation. Taken together, these data demonstrate that p63 exerts multiple pro-oncogenic effects on cell differentiation, proliferation and adhesion in basal-like breast cancers.

Cancer stem cells (CSCs) are responsible for cancer progression and patient prognosis. Tripartite motif 16 (TRIM16) is a proteasome coactivator that regulates proteolytic activity in eukaryotic cells. Abundant evidence has shown that TRIM16 is lowly expressed in a number of human carcinomas. In a previous study, we demonstrated that TRIM16 suppressed cancer malignancy and that TRIM16 expression levels were associated with favorable prognostic parameters of patients with cancer. However, the precise role of this motif in the pathogenesis of breast cancer remains unknown. In the present study, we examined 29 human breast cancer specimens, and found that TRIM16 was lowly expressed in breast cancers; thus, TRIM16 expression is negatively correlated with metastasis in breast cancer patients. Moreover, we showed that TRIM16 suppressed CSC properties in a population of breast cancer cells. TRIM16 depletion resulted in an increased proportion of CSCs relative to breast cancer cells when several assays were used to assess CSC properties. Finally, we demonstrated that TRIM16 directly regulated the degradation of Gli‑1 protein via the ubiquitin‑proteasome pathway. In conclusion, we propose that inhibition of CSC properties may be one of the functions of TRIM16 as a suppressor of breast cancer progression.

The epithelial-mesenchymal-transition (EMT) represents a morphogenetic program involved in developmental processes such as gastrulation and neural crest formation. The EMT program is co-opted by epithelial tumor cells and endows them with features necessary for spreading to distant sites, such as invasion, migration, apoptosis resistance and stemness. Thereby, EMT facilitates metastasis formation and therapy resistance. A growing number of transcription factors has been implicated in the regulation of EMT. These include EMT-inducing transcription factors (EMT-TFs), the most prominent being SNAIL, SLUG, ZEB1, ZEB2 and TWIST, and negative regulators of EMT, such as p53. Furthermore, a growing number of microRNAs, such as members of the miR-200 and miR-34 family, have been characterized as negative regulators of EMT. EMT-TFs and microRNAs, such as ZEB1/2 and miR-200 or SNAIL and miR-34, are often engaged in double-negative feedback loops forming bistable switches controlling the transitions from epithelial to the mesenchymal cell states. Within this chapter, we will provide a comprehensive overview over the transcription factors and microRNAs that have been implicated in the regulation of EMT in colorectal cancer. Furthermore, we will highlight the regulatory connections between EMT-TFs and miRNAs to illustrate common principles of their interaction that regulate EMTs.

Low oxygen tension (hypoxia) is a common characteristic of solid tumors and strongly correlates with poor prognosis and resistance to treatment. In response to hypoxia, cells initiate a cascade of transcriptional events regulated by the hypoxia inducible factor-1 (HIF-1) heterodimer. Since the oxygen-sensitive HIF-1α subunit is stabilized during hypoxia, it functions as the regulatory subunit of the protein. To date, while the mechanisms governing HIF-1α protein stabilization and function have been well studied, those governing HIF1A gene expression are not fully understood. However, recent studies have suggested that methylation of a HIF-1 binding site in the HIF1A promoter prevents its autoregulation. Here we report that the POZ-ZF transcription factor Kaiso modulates HIF1A gene expression by binding to the methylated HIF1A promoter in a region proximal to the autoregulatory HIF-1 binding site. Interestingly, Kaiso's regulation of HIF1A occurs primarily during hypoxia, which is consistent with the finding that Kaiso protein levels peak after 4 h of hypoxic incubation and return to normoxic levels after 24 h. Our data thus support a role for Kaiso in fine-tuning HIF1A gene expression after extended periods of hypoxia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Liver cancer stem cells (CSCs) have been identified and shown to have self-renewal and differentiation properties; however, the biology of these hepatic CSCs remains largely unknown. Here, we analyzed transcriptome gene expression profiles of liver CSCs and non-CSCs from hepatocellular carcinoma (HCC) cells lines and found that the transcription factor (TF) ZIC2 is highly expressed in liver CSCs. ZIC2 was required for the self-renewal maintenance of liver CSCs, as ZIC2 depletion reduced sphere formation and xenograft tumor growth in mice. We determined that ZIC2 acts upstream of the TF OCT4 and that ZIC2 recruits the nuclear remodeling factor (NURF) complex to the OCT4 promoter, thereby initiating OCT4 activation. In HCC patients, expression levels of the NURF complex were consistent with clinical severity and prognosis. Moreover, ZIC2 and OCT4 levels positively correlated to the clinicopathological stages of HCC patients. Altogether, our results indicate that levels of ZIC2, OCT4, and the NURF complex can be detected and used for diagnosis and prognosis prediction of HCC patients. Moreover, these factors may be potential therapeutic targets for eradicating liver CSCs.

Cell identity signals influence the invasive capability of tumor cells, as demonstrated by the selection for programs of epithelial-to-mesenchymal transition (EMT) during malignant progression. Breast cancer cells retain canonical epithelial traits and invade collectively as cohesive groups of cells, but the signaling pathways critical to their invasive capabilities are still incompletely understood. Here we report that the transcription factor ΔNp63α drives the migration of basal-like breast cancer (BLBC) cells by inducing a hybrid mesenchymal/epithelial state. Through a combination of expression analysis and functional testing across multiple BLBC cell populations, we determined that ΔNp63α induces migration by elevating the expression of the EMT program components Slug and Axl. Interestingly, ΔNp63α also increased the expression of miR-205, which can silence ZEB1/2 to prevent the loss of epithelial character caused by EMT induction. In clinical specimens, co-expression of various elements of the ΔNp63α pathway confirmed its implication in motility signaling in BLBC. We observed that activation of the ΔNp63α pathway occurred during the transition from noninvasive ductal carcinoma in situ to invasive breast cancer. Notably, in an orthotopic tumor model, Slug expression was sufficient to induce collective invasion of E-cadherin-expressing BLBC cells. Together, our results illustrate how ΔNp63α can drive breast cancer cell invasion by selectively engaging promigratory components of the EMT program while, in parallel, still promoting the retention of epithelial character., (©2015 American Association for Cancer Research.)

ZFPM2, encoding a zinc finger protein and abundantly expressed in the brain, uterus and smooth muscles, plays important roles in cardiac and gonadal development. Abnormal expression of ZFPM2 in ovarian tumors and neuroblastoma has been reported but hitherto its genetic association with cancer and effects on gliomas have not been studied. In the present study, the hexamer insertion-deletion polymorphism rs71305152, located within a large haplotype block spanning intron 1 to intron 3 of ZFPM2, was genotyped in Chinese cohorts of glioma (n = 350), non-glioma cancer (n = 354) and healthy control (n = 463) by direct sequencing and length polymorphism in gel electrophoresis, and ZFPM2 expression in glioma tissues (n = 69) of different grades was quantified by real-time RT-PCR. Moreover, potential natural selection pressure acting on the gene was investigated. Disease-association analysis showed that the overall genotype of rs71305152 was significantly associated with gliomas (P = 0.016), and the heterozygous genotype compared to the combined homozygous genotypes was less frequent in gliomas than in controls (P = 0.005) or non-glioma cancers (P = 0.020). ZFPM2 mRNA expression was negatively correlated with the grades of gliomas (P = 0.002), with higher expression levels in the low-grade gliomas. In the astrocytoma subtype, higher ZFPM2 expression was also correlated with the rs71305152 heterozygous genotype (P = 0.028). In addition, summary statistics tests gave highly positive values, demonstrating that the gene is under the influence of balancing selection. These findings suggest that ZFPM2 is a glioma susceptibility gene, its genotype and expression showing associations with incidence and severity, respectively. Moreover, the balancing selection acting on ZFPM2 may be related to the important roles it has to play in multiple organ development or associated disease etiology.

Background: Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer (NSCLC). Current staging methods do not adequately predict outcome for this disease. EMX2 is a homeo-domain containing transcription factor known to regulate a key developmental pathway. This study assessed the significance of EMX2 as a prognostic and predictive marker for resectable lung SCC., Methods: Two independent cohorts of patients with lung SCC undergoing surgical resection were studied. EMX2 protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. EMX2 expression levels in tissue specimens were scored and correlated with patient outcomes. Chemo-sensitivity of lung SCC cell lines stably transfected with EMX2 shRNAs to cisplatin, carboplatin, and docetaxel was examined in vitro., Results: EMX2 expression was down-regulated in lung SCC tissue samples compared to their matched adjacent normal tissues. Positive EMX2 expression was significantly associated with improved overall survival in stage I lung SCC patients, and in stage II/IIIA lung SCC patients receiving adjuvant chemotherapy. EMX2 expression was also associated with expression of EMT markers in both lung SCC cell lines and tissue samples. Knock-down of EMX2 expression in lung SCC cells promoted chemo-resistance and cell migration., Conclusions: EMX2 expression is down-regulated in lung SCC and its down-regulation is associated with chemo-resistance in lung SCC cells, possibly through regulation of Epithelial-to-Mesenchymal Transition (EMT). EMX2 may serve as a novel prognostic marker for stage I lung SCC patients and a prediction marker for stage II/IIIA lung SCC patients receiving adjuvant chemotherapy.

Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis-free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis.

The cell fate determination factor Dachshund (DACH1) functions as a novel suppressor in the progression of various neoplasms. Previous study has suggested that hypermethylation of promoter region was responsible for the reduction of DACH1 expression in hepatocellular carcinoma (HCC), and associated with the progression of HCC, but the clinical significance and the exact molecular mechanisms of DACH1 in the progression of HCC remain unclear. In this study, we employed public microarray data analysis and tissue microarrays (TMAs) technologies and showed that DACH1 expression was reduced in HCC even at early stage and associated with the tumor progression. Notably, Kaplan-Meier analysis further indicated DACH1 could be an independent prognostic factor for the overall survival of HCC. Further, mechanistic studies revealed that overexpression of DACH1 inhibited the growth and migration of HCC cell line, which were dependent in part on the inactivation of Wnt pathway via phosphorylation of GSK3β to suppress β-catenin. In agreement, the abundance of DACH1 was inversely correlated with several Wnt target genes. Collectively, our study indicated β-catenin is a novel target of DACH1 in HCC.

Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration, invasion, and drug resistance in several types of cancer. In this study, our aim was to clarify microRNAs (miRNAs)-related mechanisms underlying EMT followed by acquired resistance to chemotherapy in glioblastoma (GBM). We used multiple methods to achieve our goal including microarray analysis, qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, wound-healing assay and invasion assay. We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87). Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance. SNAI2 was identified as a direct target gene of miR-203. The knockdown of SNAI2 by short hairpin RNA (shRNA) inhibited EMT and drug resistance. In GBM patients, miR-203 expression was inversely related to SNAI2 expression, and those tumors with low expression of miR-203 experienced poorer clinical outcomes. Our findings indicate that re-expression of miR-203 or targeting SNAI2 might serve as potential therapeutic approaches to overcome chemotherapy resistance in GBM.

The transcription factor p63 is a member of the p53 gene family that plays a complex role in cancer due to its involvement in epithelial differentiation, cell cycle arrest and apoptosis. MicroRNAs are a class of small, non-coding RNAs with an important regulatory role in various cellular processes, as well as in the development and progression of cancer. A number of microRNAs have been shown to function as transcriptional targets of p63. Conversely, microRNAs also can modulate the expression and activity of p63. However, the p63-microRNA regulatory circuit has not been addressed in depth so far. Here, computational genomic analysis was performed using miRtarBase, Targetscan, microRNA.ORG, DIANA-MICROT, RNA22-HSA and miRDB to analyze miRNA binding to the 3'UTR of p63. JASPAR (profile score threshold 80%) and TFSEARCH datasets were used to search transcriptional start sites for p53/p63 response elements. Remarkably, these data revealed 63 microRNAs that targeted p63. Furthermore, there were 39 microRNAs targeting p63 that were predicted to be regulated by p63. These analyses suggest a crosstalk between p63 and microRNAs. Here, we discuss the crosstalk between p63 and the microRNA network, and the role of their interactions in cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive carcinoma with a poor prognosis. To date, there is no effective treatment for this fatal disease. The manipulation of epigenetic proteins, such as BRD4, has recently emerged as an alternative therapeutic strategy. Our objective was to analyze the effect of BRD4 on the cell progression and chemoresistance of PDAC and the novel mechanisms involved. In the present study, we firstly revealed that the expression of BRD4 was significantly upregulated in PDAC cell lines, compared to that in human pancreatic duct epithelial cells. An in vitro assay showed that the suppression of BRD4 impaired PDAC cell viability and proliferation. Similarly, the tumor growth rate was also decreased in vivo after silencing of BRD4. Furthermore, we showed that the expression of BRD4 was increased after treatment with gemcitabine (GEM). Combination treatment of GEM and BRD4 silencing had a synergistic effect on the chemotherapeutic efficacy in the PANC-1 and MIAPaCa-2 cell lines, and significantly promoted apoptosis. In particular, we demonstrated that BRD4 activated the Sonic hedgehog (Shh) signaling pathway members in a ligand-independent manner in the PDAC cells. Together, our results indicate the important role of BRD4 in PDAC cell proliferation and chemoresistance and suggests that BRD4 is a promising target directed against the transcriptional program of PDAC.

Background: Transformer (Tra) 2β is a member of the serine/arginine-rich (SR)-like protein family that regulates alternative splicing of numerous genes in a concentration-dependent manner. Several types of cancer cells up-regulate Tra2β expression, while the regulatory mechanism of Tra2β expression remains to be elucidated. In this study, we examined the transcriptional regulation and possible functions of Tra2β in human colon cancer cells., Methods: We cloned 959 bp-upstream of the human TRA2β 5'-flank into luciferase constructs. Chromatin immunoprecipitation (ChIP) was employed to identify crucial cis element(s) and trans activator(s) of the TRA2β promoter. Tra2β expression in the human colon and colon cancer tissues was examined by immunohistochemistry., Results: In response to sodium arsenite, colon cancer cells (HCT116) increased levels of TRA2β1 mRNA encoding a functional, full-length Tra2β with a peak around 6 h without changing its mRNA stability. Transient expression assays using a reporter gene driven by serially truncated TRA2β promoters and Chip assay demonstrated that an Ets1-binding site present at -64 to -55 bp was crucial for basal transcription, while three heat shock elements (HSEs) located at -145 to -99 bp mediated the oxidant-induced transactivation of TRA2β. Tra2β knockdown caused apoptosis of HCT116 cells. Tra2β were preferentially expressed in proliferative compartment of normal human colonic glands and adenocarcinomas, where Ets1 and heat shock factor 1 were also highly expressed., Conclusions: Our results suggest that oxidative stress-responsive Tra2β may play an important role in colon cancer growth.

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21(WAF1). ZNF313 ubiquitinates p21(WAF1) and also destabilizes p27(KIP1) and p57(KIP2), three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16(INK4A) and p15(INK4B). ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21(WAF1)-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21(WAF1), whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

Epidermal differentiation and stratification, crucial for barrier formation, are regulated by a complex interplay of transcription factors, including the evolutionarily conserved Grainyhead-like 3 (Grhl3/Get1); Grhl3-deleted mice exhibit impaired epidermal differentiation and decreased expression of multiple differentiation genes. To test whether Grhl3 regulates epidermal genes indirectly by controlling the expression of specific microRNAs (miRs), we performed miR profiling and identified 11 miRs that are differentially regulated in Grhl3(-/-) skin, one of which is miR-21, previously shown to be upregulated in diseased skin, including in psoriasis and squamous cell skin cancer. We found that miR-21 is normally expressed in the post-mitotic suprabasal layers of the epidermis, overlapping with Grhl3. The miR-21 promoter is bound and repressed by Grhl3 indicating that these two factors are involved in a regulatory loop maintaining homeostasis in the epidermis. Although miR-21 overexpression in normal keratinocytes had mild effects on the expression of several known miR-21 targets, an enhanced downregulation of the miR-21 tumor-related targets, including MSH2, was observed in Ras-transformed keratinocytes. The increased sensitivity of transformed keratinocytes to miR-21's effects occurs in part through downregulation of the RNA-binding protein DND1 during the transformation process. Additionally, we observed increased tumorigenesis in mice subcutaneously injected with transformed keratinocytes lacking Grhl3. These findings indicate that decreased Grhl3 expression contributes to tumor progression and upregulation of the oncomir miR-21 in squamous cell carcinoma of the skin.

Aberrant transcriptional programs in combination with abnormal proliferative signaling drive leukemic transformation. These programs operate in normal hematopoiesis where they are involved in hematopoietic stem cell (HSC) proliferation and maintenance. Ets Related Gene (ERG) is a component of normal and leukemic stem cell signatures and high ERG expression is a risk factor for poor prognosis in acute myeloid leukemia (AML). However, mechanisms that underlie ERG expression in AML and how its expression relates to leukemic stemness are unknown. We report that ERG expression in AML is associated with activity of the ERG promoters and +85 stem cell enhancer and a heptad of transcription factors that combinatorially regulate genes in HSCs. Gene expression signatures derived from ERG promoter-stem cell enhancer and heptad activity are associated with clinical outcome when ERG expression alone fails. We also show that the heptad signature is associated with AMLs that lack somatic mutations in NPM1 and confers an adverse prognosis when associated with FLT3 mutations. Taken together, these results suggest that transcriptional regulators cooperate to establish or maintain primitive stem cell-like signatures in leukemic cells and that the underlying pattern of somatic mutations contributes to the development of these signatures and modulate their influence on clinical outcome.

The origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia-retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34(+) cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes. Transcription factor-binding sites (eg, the c-myc-binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα-binding sites were also protected from aberrant DNA methylation in APL cells. Consistent with this, myeloid cells from preleukemic PML-RARα knock-in mice did not show altered DNA methylation and the expression of PML-RARα in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. Treatment of APL blasts with all-trans retinoic acid also did not result in immediate DNA methylation changes. The results of the present study suggest that aberrant DNA methylation is associated with leukemia phenotype but is not required for PML-RARα-mediated initiation of leukemogenesis.

High-grade conventional osteosarcoma is a malignant tumour predominantly affecting adolescents and, despite multimodal intensive therapy, lethal for one third of the patients. Although there is currently detailed knowledge of normal skeletal development, this has not been integrated into research on the genesis of osteosarcoma. Recently we showed that the canonical Wnt pathway is not active in osteosarcoma and that its reactivation is disadvantageous to osteosarcoma cells. Since Wnt is regulating normal skeletogenesis together with other pathways, here we report on the activities of the bone morphogenic protein (BMP), the transforming growth factor beta (TGFβ) and the hedgehog (Hh) pathways in osteosarcoma. Human osteosarcoma samples (n=210), benign bone tumours of osteoblastic lineage called osteoblastoma (n=25) and osteosarcoma cell lines (n=19) were examined. For pathway activity luciferase transcriptional reporter assays and gene and protein expression analyses were performed. Immunohistochemical analysis of phosphorylated Smad1 and Smad2, the intracellular effectors of BMP and TGFβ, respectively, showed nuclear expression of both proteins in 70% of the osteosarcoma samples at levels comparable to osteoblastoma. Interestingly cases with lower expression showed significantly worse disease free survival. This may imply that drugs restoring impaired signalling pathways in osteosarcoma might change the tumour's aggressive clinical course, however targeted pathway modulation in vitro did not affect cell proliferation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5'long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5'LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5'LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches.

Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1-/- cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis.

The Yes-associated protein (YAP), an oncoprotein in the Hippo tumor suppressor pathway, regulates tumorigenesis and has been found in a variety of tumors, including breast, ovarian, and hepatocellular cancers. Although YAP functions through its WW domains, the YAP WW domain-binding partners have not yet been completely determined. With this study, we demonstrate that YAP functions partially through its binding to KLF5, a transcription factor that promotes breast cell proliferation and survival. YAP interacted with the KLF5 PY motif through its WW domains, preventing the E3 ubiquitin ligase WWP1 from ubiquitinating KLF5. Overexpression of the wild-type YAP but not the WW domain-mutated YAP up-regulated KLF5 protein levels and mRNA expression levels of KLF5 downstream target genes, including FGFBP1 (alias FGF-BP) and ITGB2. In addition, knockdown of YAP decreased expression levels of KLF5, FGF-BP, and ITGB2. Depletion of either YAP or KLF5 decreased breast cell proliferation and survival in MCF10A and SW527 breast cell lines, and stable knockdown of either YAP or KLF5 suppressed SW527 xenograft growth in mice. The YAP upstream kinase LATS1 suppressed the KLF5-FGF-BP axis, as well as cell growth through YAP signaling. Both YAP and KLF5 are coexpressed in estrogen receptor ERα-negative breast cell lines. These findings suggest that KLF5 could be an important transcription factor partner for YAP and may contribute to the Hippo pathway., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

BM stromal cells (BMSCs) are key players in the microenvironmental support of multiple myeloma (MM) cell growth and bone destruction. A spliced form of the X-box-binding protein-1 (XBP1s), a major proximal effector of unfolded protein response signaling, is highly expressed in MM cells and plays an indispensable role in MM pathogenesis. In the present study, we found that XBP1s is induced in the BMSCs of the MM microenvironment. XBP1s overexpression in healthy human BMSCs enhanced gene and/or protein expression of VCAM-1, IL-6, and receptor activator of NF-κB ligand (RANKL), enhancing BMSC support of MM cell growth and osteoclast formation in vitro and in vivo. Conversely, deficiency of XBP1 in healthy donor BMSCs displayed a range of effects on BMSCs that were opposite to those cells with overexpression of XBP1s. Knock-down of XBP1 in MM patient BMSCs greatly compromised their increased VCAM-1 protein expression and IL-6 and RANKL secretion in response to TNFα and reversed their enhanced support of MM-cell growth and osteoclast formation. Our results demonstrate that XBP1s is a pathogenic factor underlying BMSC support of MM cell growth and osteoclast formation and therefore represents a therapeutic target for MM bone disease.

Breast cancers are highly heterogeneous but can be grouped into subtypes based on several criteria, including level of expression of certain markers. Claudin-low breast cancer (CLBC) is associated with early metastasis and resistance to chemotherapy, while gene profiling indicates it is characterized by the expression of markers of epithelial-mesenchymal transition (EMT) - a phenotypic conversion linked with metastasis. Although the epigenetic program controlling the phenotypic and cellular plasticity of EMT remains unclear, one contributor may be methylation of the E-cadherin promoter, resulting in decreased E-cadherin expression, a hallmark of EMT. Indeed, reduced E-cadherin often occurs in CLBC and may contribute to the early metastasis and poor patient survival associated with this disease. Here, we have determined that methylation of histone H3 on lysine 9 (H3K9me2) is critical for promoter DNA methylation of E-cadherin in three TGF-β-induced EMT model cell lines, as well as in CLBC cell lines. Further, Snail interacted with G9a, a major euchromatin methyltransferase responsible for H3K9me2, and recruited G9a and DNA methyltransferases to the E-cadherin promoter for DNA methylation. Knockdown of G9a restored E-cadherin expression by suppressing H3K9me2 and blocking DNA methylation. This resulted in inhibition of cell migration and invasion in vitro and suppression of tumor growth and lung colonization in in vivo models of CLBC metastasis. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also paves a way for the development of new treatment strategies for CLBC.

Objective: Pancreatic cancer is characterised by invasive tumour spread and early metastasis formation. During epithelial-mesenchymal transition, loss of the cell adhesion molecule E-cadherin is frequent and can be caused by genetic or epigenetic modifications, recruitment of transcriptional activators/repressors or post-translational modifications. A study was undertaken to investigate how E-cadherin expression in human pancreatic adenocarcinoma and pancreatic cancer cell lines is regulated., Methods: In 25 human pancreatic cancer resection specimens, the coding region of the E-cadherin gene (CDH1) was sequenced for somatic mutations. The tumour samples and 11 established human pancreatic cancer cell lines were analysed by immunohistochemistry, western blot analysis, chromatin immunoprecipitation and methylation-specific PCR. The role of specific histone deacetylase inhibitors (HDACi) on pancreatic tumour cell migration and proliferation was studied in vitro., Results: Neither somatic mutations nor CDH1 promoter hypermethylation were found to be responsible for downregulation of E-cadherin in pancreatic cancer. In the transcriptionally active CDH1 promoter, acetylation of histones H3 and H4 was detected whereas HDAC1 and HDAC2 were found attached only to a silent promoter. Expression of ZEB1, a transcription factor known to recruit HDACs, was seen in E-cadherin-deficient cell lines in which ZEB1/HDAC complexes were found attached to the CDH1 promoter. Moreover, knockdown of ZEB1 prevented HDAC from binding to the CDH1 promoter, resulting in histone acetylation and expression of E-cadherin. HDACi treatment attenuated tumour cell migration and proliferation., Conclusions: These findings imply an important role for histone deacetylation in the downregulation of E-cadherin in human pancreatic cancer. Recruitment of HDACs to the CDH1 promoter is regulated by the transcription factor ZEB1, and inhibition of HDACs may be a promising antitumour therapy for pancreatic cancer.

Basonuclins 1 and 2 are nuclear proteins specific to vertebrates. They have recently been shown to be the orthologs of the DISCO proteins of insects. All of these proteins have an essential function in embryonic development. It is likely that they are transcription factors with multiple gene targets, some of which are transcribed by DNA polymerase I, and others by polymerase II. It remains to be found which targets are common to the two basonuclins and even to the DISCO proteins, and why certain cell types possess either basonuclin 1 or basonuclin 2, while others possess both. The implication of the basonuclins in several human diseases adds to their interest as regulatory proteins., (© 2012 médecine/sciences – Inserm / SRMS.)

Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in TGF-β-mediated senescence, epithelial-to-mesenchymal transition (EMT), and cancer stem cell functions. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here, we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like 1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1-enriched EMT-competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage-independent growth and tumor formation in nude mice. RNA interference experiments suggested the involvement of ZEBs in anchorage-independent colony formation, invasion, and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation were recapitulated upon Notch inhibition by DNMAML1 in organotypic three-dimensional culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of esophageal squamous cancers., (©2011 AACR.)