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1.
Transl Oncol ; 16: 101320, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34968869

RESUMO

SIN3A, a scaffold protein has regulatory functions in tumor biology. Through its Paired amphipathic helix (PAH2) domain, SIN3A interacts with PHF12 (PF1), a protein with SIN3 interaction domain (SID) that forms a complex with MRG15 and KDM5A/B. These components are often overexpressed in cancer. In the present study, we evaluated the role of SIN3A and its interacting partner PF1 in mediating inhibition of tumor growth and invasion in triple negative breast cancer (TNBC). We found profound inhibition of invasion, migration, and induction of cellular senescence by specific disruption of the PF1/SIN3A PAH2 domain interaction in TNBC cells expressing PF1-SID transcript or peptide treatment. Genome-wide transcriptomic analysis by RNA-seq revealed that PF1-SID downregulates several gene sets and pathways linked to invasion and migration. Integrin α6 (ITGA6) and integrin ß1 (ITGB1) and their downstream target proteins were downregulated in PF1-SID cells. We further determined increased presence of SIN3A and transcriptional repressor, KLF9, on promoters of ITGA6 and ITGB1 in PF1-SID cells. Knockdown of KLF9 leads to re-expression of ITGA6 and ITGB1 and restoration of the invasive phenotype, functionally linking KLF9 to this process. Overall, these data demonstrate that specific disruption of PF1/SIN3A, inhibits tumor growth, migration, and invasion. Also, PF1-SID not only inhibits tumor growth by senescence induction and reduced proliferation, but it also targets cancer stem cell gene expression and blocks mammosphere formation. Overall, these data demonstrate a mechanism whereby invasion and metastasis of TNBC can be suppressed by inhibiting SIN3A-PF1 interaction and enhancing KLF9 mediated suppression of ITGA6 and ITGB1.

2.
Cells ; 11(7)2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35406744

RESUMO

Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARß is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RARß has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RARß expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RARß induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RARα and RARß from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARß expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RARα agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RARß promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARß activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARß expression. These data suggest that SIN3A/MAD1 acts as a second RARß repressor and may be involved in fine-tuning retinoid sensitivity.


Assuntos
Neoplasias da Mama , Proteínas de Ciclo Celular , Receptores do Ácido Retinoico , Complexo Correpressor Histona Desacetilase e Sin3 , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina , Feminino , Humanos , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/genética
3.
Mol Cell Biol ; 22(17): 6148-57, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12167709

RESUMO

The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This chromatin structure is required for heat shock-inducible expression and depends on two (CT)n*(GA)n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT)n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.


Assuntos
Cromatina/ultraestrutura , Proteínas de Ligação a DNA , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Proteínas de Choque Térmico/genética , Proteínas de Homeodomínio/fisiologia , TATA Box/genética , Fatores de Transcrição TFII/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sítios de Ligação , Cromatina/genética , DNA/genética , DNA/metabolismo , DNA Recombinante/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Larva , Substâncias Macromoleculares , Modelos Genéticos , Mutagênese , Mutação Puntual , Ligação Proteica , Fator de Transcrição TFIID
4.
Oncotarget ; 8(51): 88421-88436, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29179446

RESUMO

Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poorer survival in triple negative breast cancer (TNBC) patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need in the treatment of cancer. We reported that the selective inhibition of the PAH2 domain of SIN3A protein function markedly suppressed metastatic dissemination to the lungs in TNBC xenograft bearing mice. Here, we show that TNBC cell lines treated with Sin3 interaction domain (SID) decoy peptides that bind to PAH2 display a strong in vitro inhibition of transwell invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin deposition and downregulation of known Wnt target genes that are associated with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Wnt pathway inhibition by SID decoy peptide was confirmed by decreased Wnt reporter activity and altered cytoplasmic localization of nuclear ß-catenin. TGIF1, a transcription factor that modulates Wnt signaling and known to interact with the PAH2 domain of SIN3A, can be dissociated from the SIN3A complex by SID decoys. TGIF1 knockdown inhibits WNT target genes and in vitro cell invasion suggesting that TGIF1 might be a key target of the SID decoys to block tumor invasion. Taken together, targeting SIN3 function using SID decoys is a novel strategy to reverse invasion and the EMT program in TNBC translating into the inhibition of metastasis dissemination and eradication of residual disease.

5.
Oncotarget ; 6(33): 34087-105, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26460951

RESUMO

Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Feminino , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Estrutura Terciária de Proteína , Complexo Correpressor Histona Desacetilase e Sin3 , Esferoides Celulares , Fatores de Transcrição/genética , Células Tumorais Cultivadas
6.
Mol Cancer Ther ; 14(8): 1824-36, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26078298

RESUMO

Triple-negative breast cancers (TNBC) lacking estrogen, progesterone, and HER2 receptors account for 10% to 20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial-mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide, we performed an in silico screen for PAH2 domain-binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3-PAH2 interaction with MAD, induce expression of CDH1 and ESR1, and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes.


Assuntos
Ivermectina/análogos & derivados , Proteínas Repressoras/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antígenos CD , Antiparasitários/farmacologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ivermectina/química , Ivermectina/farmacologia , Camundongos , Modelos Moleculares , Conformação Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Blood ; 111(6): 3145-54, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18156491

RESUMO

MMSET, identified by its fusion to the IgH locus in t(4;14)-associated multiple myeloma, possesses domains found within chromatin regulators, including the SET domain. MMSET protein is overexpressed and highly associated with chromatin in myeloma cell lines carrying t(4;14). MMSET possesses methyltransferase activity for core histone H3 lysine 4 and histone 4 lysine 20, whereas MMSET made in cells only modified H4. Segments of MMSET fused to the Gal4 DNA binding domain repressed transcription of a chromatin-embedded Gal4 reporter gene. MMSET-mediated repression was associated with increased H4K20 methylation gene and loss of histone acetylation. Consistent with this repressive activity, MMSET could form a complex with HDAC1 and HDAC2, mSin3a, and the histone demethylase LSD1, suggesting that it is a component of corepressor complexes. Furthermore, MMSET coexpression enhances HDAC1- and HDAC2-mediated repression in transcriptional reporter assays. Finally, shRNA-mediated knockdown of MMSET compromised viability of a myeloma cell line, suggesting a biologic role for the protein in malignant cell growth. Collectively, these data suggest that, by acting directly as a modifier of chromatin as well as through binding of other chromatin-modifying enzymes, MMSET influences gene expression and potentially acts as a pathogenic agent in multiple myeloma.


Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica/genética , Animais , Catálise , Linhagem Celular , Núcleo Celular/enzimologia , Sobrevivência Celular , Cromossomos Humanos Par 4/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Melanoma/enzimologia , Melanoma/genética , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Proteínas Repressoras/química , Proteínas Repressoras/genética
8.
Science ; 303(5658): 669-72, 2004 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-14752161

RESUMO

Genes normally resident in euchromatic domains are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by position effect variegation (PEV). Loss-of-function mutations resulting in suppression of PEV have identified critical components of heterochromatin, including proteins HP1, HP2, and histone H3 lysine 9 methyltransferase. Here, we demonstrate that this silencing is dependent on the RNA interference machinery, using tandem mini-white arrays and white transgenes in heterochromatin to show loss of silencing as a result of mutations in piwi, aubergine, or spindle-E (homeless), which encode RNAi components. These mutations result in reduction of H3 Lys9 methylation and delocalization of HP1 and HP2, most dramatically in spindle-E mutants.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/metabolismo , Interferência de RNA , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Alelos , Animais , Proteínas Argonautas , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Genes de Insetos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Metilação , Mutação , Proteínas/genética , Proteínas/fisiologia , Complexo de Inativação Induzido por RNA , Transgenes
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