Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate and has been shown in various cancers to promote metastasis, angiogenesis, osteolysis, and chemoresistance. Although heparanase is thought to act predominantly extracellularly or within the cytoplasm, it is also present in the nucleus, where it may function in regulating gene transcription. Using myeloma cell lines, we report here that heparanase enhances chromatin accessibility and confirm a previous report that it also upregulates the acetylation of histones. Employing the Multiple Myeloma Research Foundation CoMMpass database, we demonstrate that patients expressing high levels of heparanase display elevated expression of proteins involved in chromatin remodeling and several oncogenic factors compared to patients expressing low levels of heparanase. These signatures were consistent with the known function of heparanase in driving tumor progression. Chromatin opening and downstream target genes were abrogated by inhibition of heparanase. Enhanced levels of heparanase in myeloma cells led to a dramatic increase in phosphorylation of PTEN, an event known to stabilize PTEN, leading to its inactivity and loss of tumor suppressor function. Collectively, this study demonstrates that heparanase promotes chromatin opening and transcriptional activity, some of which likely is through its impact on diminishing PTEN tumor suppressor activity.

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Identification of a Novel E2F3 Product Suggests a Mechanism for Determining Specificity of Repression by Rb Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3626-3632.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3626-3632 ◽

Cited By ~ 110

Author(s):

Gustavo Leone ◽

Faison Nuckolls ◽

Seiichi Ishida ◽

Monique Adams ◽

Rosalie Sears ◽

...

Keyword(s):

Cell Growth ◽

Tumor Suppressor ◽

Target Genes ◽

Transcriptional Repressor ◽

Tumor Suppressor Function ◽

Proliferating Cells ◽

Quiescent Cells ◽

Suppressor Function ◽

Intronic Promoter

ABSTRACT The tumor suppressor function of Rb is intimately related to its ability to interact with E2F and repress the transcription of E2F target genes. Here we describe a novel E2F product that specifically interacts with Rb in quiescent cells. This novel E2F, which we term E2F3b, is encoded by a unique mRNA transcribed from an intronic promoter within the E2F3 locus. The E2F3b RNA differs from the previously characterized E2F3 RNA, which we now term E2F3a, by the utilization of a unique coding exon. In contrast to the E2F3a product that is tightly regulated by cell growth, the E2F3b product is expressed equivalently in quiescent and proliferating cells. But, unlike the E2F4 and E2F5 proteins, which are also expressed in quiescent cells and form complexes with the p130 protein, the E2F3b protein associates with Rb and represents the predominant E2F-Rb complex in quiescent cells. Thus, the previously described specificity of Rb function as a transcriptional repressor in quiescent cells coincides with the association of Rb with this novel E2F product.

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Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre–B ALL

Journal of Experimental Medicine ◽

10.1084/jem.20160049 ◽

2017 ◽

Vol 214 (3) ◽

pp. 793-814 ◽

Cited By ~ 15

Author(s):

Hilde Schjerven ◽

Etapong F. Ayongaba ◽

Ali Aghajanirefah ◽

Jami McLaughlin ◽

Donghui Cheng ◽

...

Keyword(s):

Tumor Suppressor ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Suppressor Gene ◽

Tumor Suppressor Function ◽

Gene Encoding ◽

Specific Expression ◽

Suppressor Function ◽

Genome Wide ◽

Conserved Gene

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre–B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre–B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre–B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre–B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre–B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage–specific expression of multiple genes through chromatin compaction at its target genes.

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The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1

Molecular Cancer ◽

10.1186/s12943-021-01439-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Beatriz Sanchez-Solana ◽

Dunrui Wang ◽

Xiaolan Qian ◽

Parthibane Velayoudame ◽

Dhirendra K. Simanshu ◽

...

Keyword(s):

Tumor Suppressor ◽

Molecular Mechanisms ◽

Structural Model ◽

Suppressor Gene ◽

Lipid Transfer ◽

Tumor Suppressor Function ◽

Suppressor Activity ◽

Caveolin 1 ◽

Suppressor Function ◽

Start Domain

Abstract Background DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown. Methods Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START. Results We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties. Conclusion These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.

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Consequences of Zmat3 loss in c-MYC- and mutant KRAS-driven tumorigenesis

Cell Death and Disease ◽

10.1038/s41419-020-03066-9 ◽

2020 ◽

Vol 11 (10) ◽

Author(s):

Sarah A. Best ◽

Cassandra J. Vandenberg ◽

Etna Abad ◽

Lachlan Whitehead ◽

Laia Guiu ◽

...

Keyword(s):

Tumor Suppressor ◽

Lung Adenocarcinoma ◽

Malignant Disease ◽

Target Genes ◽

Lung Tumor ◽

Tumor Development ◽

Neoplastic Transformation ◽

Cell Cycle Regulators ◽

Tumor Suppressor Function ◽

Downstream Target

Abstract TP53 is a critical tumor suppressor that is mutated in approximately 50% of human cancers. Unveiling the downstream target genes of TP53 that fulfill its tumor suppressor function is an area of intense investigation. Zmat3 (also known as Wig-1 or PAG608) is one such downstream target of p53, whose loss in hemopoietic stem cells lacking the apoptosis and cell cycle regulators, Puma and p21, respectively, promotes the development of leukemia. The function of Zmat3 in tumorigenesis however remains unclear. Here, to investigate which oncogenic drivers co-operate with Zmat3 loss to promote neoplastic transformation, we utilized Zmat3 knockout mice in models of c-MYC-driven lymphomagenesis and KrasG12D-driven lung adenocarcinoma development. Interestingly, unlike loss of p53, Zmat3 germline loss had little impact on the rate of tumor development or severity of malignant disease upon either the c-MYC or KrasG12D oncogenic activation. Furthermore, loss of Zmat3 failed to rescue KrasG12D primary lung tumor cells from oncogene-induced senescence. Taken together, we conclude that in the context of c-MYC-driven lymphomagenesis or mutant KrasG12D-driven lung adenocarcinoma development, additional co-occurring mutations are required to resolve Zmat3 tumor suppressive activity.

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Protein Phosphatase 1 Regulates the Tumor Suppressor Function of Ikaros and Radiation Resistance in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2465.2465 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2465-2465

Author(s):

Sinsa Dovat ◽

Kimberly J Payne ◽

Chunhua Song

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Tumor Suppressor ◽

Dna Binding ◽

Binding Affinity ◽

Chromatin Remodeling ◽

Target Genes ◽

Radiation Treatment ◽

Lymphoblastic Leukemia ◽

Suppressor Activity ◽

Dna Binding Affinity

Abstract Abstract 2465 The Ikaros gene encodes a DNA-binding zinc finger protein that acts as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Ikaros exerts its tumor suppressor activity by binding to the upstream regulatory regions of its target genes resulting in chromatin remodeling and transcriptional repression of the target gene. We have previously reported that Ikaros is a substrate for Protein Phosphatase 1 (PP1), and that PP1 regulates the DNA-binding affinity of Ikaros and its subcellular localization to pericentromeric heterochromatin. PP1 has been postulated to possess tumor suppressor activity, although the mechanisms were unknown. We hypothesize that PP1 positively regulates the tumor suppressor function of Ikaros in ALL. In this report, we studied the role of PP1 in regulating Ikaros function as a transcriptional regulator of its target genes in acute lymphoblastic leukemia (ALL), and in controlling the sensitivity of leukemia cells to radiation. An Ikaros construct with a mutated PP1 interaction site (IK 465/7A) was tested for: 1) association with histone deacetylase (HDAC) chromatin remodeling complex by co-immunoprecipitation; 2) in vivo DNA-binding to promoter regions of known Ikaros target genes by quantitative chromatin immunoprecipitation (qChIP); and 3) direct transcriptional repression of Ikaros target genes, by transient luciferase reporter assay. Our results demonstrate that the loss of interaction with PP1 results in hyperphosphorylation of Ikaros protein resulting in an inability of Ikaros to interact with the HDAC chromatin remodeling complex. The loss of PP1 interaction impaired Ikaros' ability to function as transcriptional repressor due to poor DNA-binding affinity toward the promoters of Ikaros target genes. The introduction of phosphoresistant (alanine) mutations at CK2 kinase phosphorylation sites on the Ikaros protein (IK 465/7A+A11 mutant) restored Ikaros' ability to bind the histone deacetylase complex (including HDAC1 and HDAC2 proteins), as well as its transcriptional repressor function and DNA-binding affinity toward promoters of its target genes. These data strongly suggest that dephosphorylation of Ikaros by PP1 is essential for its function in chromatin remodeling and regulation of gene expression. To study the role of PP1 in ALL, we treated primary ALL cells with specific inhibitors of PP1 and studied the impact PP1 inhibition on ALL cells. Our data demonstrate that the inhibition of PP1 activity results in decreased sensitivity of ALL cells to radiation treatment, and that these changes correlate with a decrease in Ikaros' DNA-binding affinity (as evidenced by qChIP), and a loss of Ikaros function as a direct regulator of target gene transcription. These studies identified PP1 as an important signal transduction pathway that controls the proliferation of ALL cells. Our results suggest that PP1-mediated dephosphorylation and CK2 kinase-mediated phosphorylation are two opposing signaling pathways that regulate Ikaros function as a tumor suppressor in ALL, as well as the resistance of leukemia cells to radiation treatment. Disclosures: No relevant conflicts of interest to declare.

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