PAH2 Domain Research Articles

The Sin3A/MAD1 Complex, through Its PAH2 Domain, Acts as a Second Repressor of Retinoic Acid Receptor Beta Expression in Breast Cancer Cells

Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARβ 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 RARβ 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 RARβ 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 RARβ 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 RARβ from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARβ 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 RARβ promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARβ activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARβ expression. These data suggest that SIN3A/MAD1 acts as a second RARβ repressor and may be involved in fine-tuning retinoid sensitivity.

Invasive phenotype in triple negative breast cancer is inhibited by blocking SIN3A–PF1 interaction through KLF9 mediated repression of ITGA6 and ITGB1

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.

Interactions, structural aspects and evolutionary perspectives of the yeast 'START'-regulatory network.

Molecular signal transduction networks, which conduct transcription at the G1 to S phase transition of the eukaryotic cell division cycle have been identified in diverse taxa from mammals to baker's yeast with analogous functional organization. However, regarding some network components, such as the transcriptional regulators STB1 and WHI5, only few orthologs exist, which are confined to individual Saccharomycotina species. While Whi5 has been characterized as yeast analog of human Rb protein, in the particular case of Stb1 (Sin three binding protein 1) identification of functional analogs emerges as difficult because to date its exact functionality still remains obscured. By aiming to resolve Stb1's enigmatic role this Perspective article especially surveys works covering relations between Cyclin/CDKs, the heteromeric transcription factor complexes SBF (Swi4/Swi6) and MBF (Mbp1/Swi6), as well as additional coregulators (Whi5, Sin3, Rpd3, Nrm1) which are collectively associated with the orderly transcription at 'Start' of the Saccharomyces cerevisiae cell cycle. In this context, interaction capacities of the Sin3-scaffold protein are widely surveyed because its four PAH domains (Paired Amphiphatic Helix) represent a 'recruitment-code' for gene-specific targeting of repressive histone deacetylase activity (Rpd3) via different transcription factors. Here, Stb1 plays a role in Sin3's action on transcription at the G1/S-boundary. Through bioinformatic analyses a potential Sin3-interaction domain (SID) was detected in Stb1, and beyond that, connections within the G1/S-regulatory network are discussed in structural and evolutionary context thereby providing conceptual perspectives.

Structural Insight into the Binding of TGIF1 to SIN3A PAH2 Domain through a C-Terminal Amphipathic Helix.

TGIF1 is a transcriptional repressor playing crucial roles in human development and function and is associated with holoprosencephaly and various cancers. TGIF1-directed transcriptional repression of specific genes depends on the recruitment of corepressor SIN3A. However, to date, the exact region of TGIF1 binding to SIN3A was not clear, and the structural basis for the binding was unknown. Here, we demonstrate that TGIF1 utilizes a C-terminal domain (termed as SIN3A-interacting domain, SID) to bind with SIN3A PAH2. The TGIF1 SID adopts a disordered structure at the apo state but forms an amphipathic helix binding into the hydrophobic cleft of SIN3A PAH2 through the nonpolar side at the holo state. Residues F379, L382 and V383 of TGIF1 buried in the hydrophobic core of the complex are critical for the binding. Moreover, homodimerization of TGIF1 through the SID and key residues of F379, L382 and V383 was evidenced, which suggests a dual role of TGIF1 SID and a correlation between dimerization and SIN3A-PAH2 binding. This study provides a structural insight into the binding of TGIF1 with SIN3A, improves the knowledge of the structure–function relationship of TGIF1 and its homologs and will help in recognizing an undiscovered SIN3A-PAH2 binder and developing a peptide inhibitor for cancer treatment.

Cooption of polyalanine tract into a repressor domain in the mammalian transcription factor HoxA11.

An enduring problem in biology is explaining how novel functions of genes originated and how those functions diverge between species. Despite detailed studies on the functional evolution of a few proteins, the molecular mechanisms by which protein functions have evolved are almost entirely unknown. Here, we show that a polyalanine tract in the homeodomain transcription factor HoxA11 arose in the stem-lineage of mammals and functions as an autonomous repressor module by physically interacting with the PAH domains of SIN3 proteins. These results suggest that long polyalanine tracts, which are common in transcription factors and often associated with disease, may tend to function as repressor domains and can contribute to the diversification of transcription factor functions despite the deleterious consequences of polyalanine tract expansion.

Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3S HDAC complex in C. elegans.

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.

Sin3A recruits Tet1 to the PAH1 domain via a highly conserved Sin3-Interaction Domain

The Sin3A complex acts as a transcriptional hub, integrating the function of diverse transcription factors with histone modifying enzymes, notably, histone deacetylases (HDAC) 1 and 2. The Sin3A protein sits at the centre of the complex, mediating multiple simultaneous protein-protein interactions via its four paired-amphipathic helix (PAH) domains (PAH1-4). The PAH domains contain a conserved four helical bundle, generating a hydrophobic cleft into which the single-helix of a Sin3-interaction domain (SID) is able to insert and bind with high affinity. Although they share a similar mode of interaction, the SIDs of different repressor proteins bind to only one of four potential PAH domains, due to the specific combination of hydrophobic residues at the interface. Here we report the identification of a highly conserved SID in the 5-methylcytosine dioxygenase, Tet1 (Tet1-SID), which interacts directly with the PAH1 domain of Sin3A. Using a combination of NMR spectroscopy and homology modelling we present a model of the PAH1/Tet1-SID complex, which binds in a Type-II orientation similar to Sap25. Mutagenesis of key residues show that the 11-amino acid Tet1-SID is necessary and sufficient for the interaction with Sin3A and is absolutely required for Tet1 to repress transcription in cells.

Sertraline, chlorprothixene, and chlorpromazine characteristically interact with the REST-binding site of the corepressor mSin3, showing medulloblastoma cell growth inhibitory activities

Dysregulation of repressor-element 1 silencing transcription factor REST/NRSF is related to several neuropathies, including medulloblastoma, glioblastoma, Huntington’s disease, and neuropathic pain. Inhibitors of the interaction between the N-terminal repressor domain of REST/NRSF and the PAH1 domain of its corepressor mSin3 may ameliorate such neuropathies. In-silico screening based on the complex structure of REST/NRSF and mSin3 PAH1 yielded 52 active compounds, including approved neuropathic drugs. We investigated their binding affinity to PAH1 by NMR, and their inhibitory activity toward medulloblastoma cell growth. Interestingly, three antidepressant and antipsychotic medicines, sertraline, chlorprothixene, and chlorpromazine, were found to strongly bind to PAH1. Multivariate analysis based on NMR chemical shift changes in PAH1 residues induced by ligand binding was used to identify compound characteristics associated with cell growth inhibition. Active compounds showed a new chemo-type for inhibitors of the REST/NRSF-mSin3 interaction, raising the possibility of new therapies for neuropathies caused by dysregulation of REST/NRSF.

SIN3B promotes integrin αV subunit gene transcription and cell migration of hepatocellular carcinoma.

Paired amphipathic helix protein (SIN3B) is a transcription corepressor for many genes. Here we show a different regulation mechanism of integrin αV gene expression by SIN3B in human hepatocellular carcinoma (HCC). We first observed a close relationship between Integrin αV and SIN3B expressions in HCC patients and tumor cell lines with different metastatic potentials. Overexpression of SIN3B significantly accelerated the cell migration rate of SMMC-7721, but failed when integrin αV expression was silenced. Interestingly, SIN3B stimulated integrin αV subunit promoter activity only in the presence of sulfatide. Importantly, SIN3B was identified in the complex with sulfatide by mass spectrometry. Fat blot assay indicated that SIN3B specifically interacted with sulfatide. Molecular modeling suggested that sulfatide induced the conformational change of SIN3B from compacted α-helices to a relaxed β-sheet in PAH2 domain. The data of immunoprecipitation and ChIP assay indicated that altered SIN3B lost the binding affinity with MAD1 and HDAC2, which reduced the recruitment of HDAC2 on integrin αV gene promoter and prevented the deacetylation of the histone 3. In conclusion, this study demonstrated that SIN3B promoted the transcriptional activation of the integrin αV subunit gene promoter by reducing interaction with HDAC2.

Abstract 31: Targeted dissociation of PF1 from SIN3A chromatin regulator complex inhibits tumor growth and metastasis in triple-negative breast cancer

Abstract Triple negative breast cancer (TNBC) is characterized by an aggressive, poorly differentiated phenotype associated with early recurrence. Chemotherapy is the only treatment option due to the lack of an effective molecular target. Targeting reversible epigenetic alterations in TNBC to reprogram the TNBC phenotype may increase chemosensitivity and identify effective targeted therapies. The SIN3 chromatin modifier complex is a multidomain scaffold protein which plays a key role in multiple cellular functions including epigenetic gene expression regulation, making SIN3 a potential therapeutic target. The PAH-2 domain of SIN3 binds with different affinities to a group of proteins that contain variations of an amino acid sequence known as the SIN3 interaction domain (SID). We previously reported the effects of blocking PAH-2 interaction with MAD-1, a SID sequence containing protein by using SID peptides and small molecule inhibitors (SMI) as decoys. We now report the effects of blocking the PAH-2 binding of PF1 and TGIF1, two other Sin3 interacting proteins with different amino acid sequence and PAH-2 binding affinities. Treatment with MAD-1 SID peptide and SMI revert the epithelial to mesenchymal transition (EMT) process, inhibit cancer stem cells expansion, cell invasion, tumor growth and metastasis development in human and mouse models of TNBC. TGIF1 interaction with SIN3 is associated with the invasive phenotype and EMT regulation by modulating wnt signaling and inhibiting beta catenin nuclear localization which is reverted by TGIF1 knockdown. PF1, which binds MRG15 and KDM5A/B, an epigenetic modifier, is involved in the regulation of cancer stem cell compartment expansion, DNA damage control, senescence, apoptosis and the expression of genes involved in metastatic progression. Interestingly, decrease in H3K4me3 is found in stem cell and EMT genes following 72 hour SID peptide treatment. PF1 dissociation from Sin3 by transfecting MDA-MB-231 and 4T1 cells with a PF1-SID expressing vector grown in 3D morphogenesis cultures display the formation of small organoids in the MDA-MB-231PF1-SID and tubular morphogenesis in the 4T1PF1-SID cells. There is increased activated caspase-3 and γH2AX, reduced Ki67, tenascin-C and marked cortical actin reorganization. This is accompanied by small organized colonies with loss of invasive and proliferative phenotype. In vivo, 4T1PF1-SID barely form tumors and have a 95% decrease in lung metastasis. These results show that decoys can be designed to block specific proteins that bind to the PAH-2 domain resulting in different outcomes which contribute to a common effect on inhibition of tumor progression and metastasis dissemination. Therefore, this strategy opens a potential therapeutic alternative for TNBC patients for whom there are no other therapeutic options besides chemotherapy. Citation Format: Rama Kadamb, Nidhi Bansal, Boris A Leibovitch, Yeon-Jin Kwon, Ming-Ming Zhou, Eduardo Farias, Samuel Waxman. Targeted dissociation of PF1 from SIN3A chromatin regulator complex inhibits tumor growth and metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 31.

Sin3a-Tet1 interaction activates gene transcription and is required for embryonic stem cell pluripotency.

Sin3a is a core component of histone-deacetylation-activity-associated transcriptional repressor complex, playing important roles in early embryo development. Here, we reported that down-regulation of Sin3a led to the loss of embryonic stem cell (ESC) self-renewal and skewed differentiation into mesendoderm lineage. We found that Sin3a functioned as a transcriptional coactivator of the critical Nodal antagonist Lefty1 through interacting with Tet1 to de-methylate the Lefty1 promoter. Further studies showed that two amino acid residues (Phe147, Phe182) in the PAH1 domain of Sin3a are essential for Sin3a–Tet1 interaction and its activity in regulating pluripotency. Furthermore, genome-wide analyses of Sin3a, Tet1 and Pol II ChIP-seq and of 5mC MeDIP-seq revealed that Sin3a acted with Tet1 to facilitate the transcription of a set of their co-target genes. These results link Sin3a to epigenetic DNA modifications in transcriptional activation and have implications for understanding mechanisms underlying versatile functions of Sin3a in mouse ESCs.

Expression of the SIN3 homologue from banana, MaSIN3, suppresses ABA responses globally during plant growth in Arabidopsis

The SIN3 family of co-repressors is a family of highly conserved eukaryotic repressor proteins that regulates diverse functions in yeasts and animals but remains largely uncharacterized functionally even in plants like Arabidopsis. The sole SIN3 homologue in banana, MaSIN3, was identified as a 1408 amino acids, nuclear localized protein conserved to other SIN3s in the PAH, HID and HCR domains. Interestingly, MaSIN3 over-expression in Arabidopsis mimics a state of reduced ABA responses throughout plant development affecting growth processes such as germination, root growth, stomatal closure and water loss, flowering and senescence. The reduction in ABA responses is not due to reduced ABA levels but due to suppression of expression of several transcription factors mediating ABA responses. Transcript levels of negative regulators of germination (ABI3, ABI5, PIL5, RGL2 and RGL3) are reduced post-imbibition while those responsible for GA biosynthesis are up-regulated in transgenic MaSIN3 over-expressers. ABA-associated transcription factors are also down-regulated in response to ABA treatment. The HDAC inhibitors, SAHA and sodium butyrate, in combination with ABA differentially suppress germination in control and transgenic lines suggesting the recruitment by MaSIN3 of HDACs involved in suppression of ABA responses in different processes. The studies provide an insight into the ability of MaSIN3 to specifically affect a subset of developmental processes governed largely by ABA.

Abstract 2083: Development of novel targeted adjuvant therapy for triple negative breast cancer

Abstract There is an unmet clinical need for targeted adjuvant therapy in Triple Negative Breast Cancer (TNBC) to overcome its poor prognosis, short disease-free interval and metastatic dissemination. We previously reported that blocking interactions between the PAH2 domain of chromatin regulator Sin3 and Sin3 interaction domain (SID) containing proteins like PF1 and TGIF1 by SID decoys (peptides and small molecule, C16) decreased the cancer stem cell population, invasion, EMT, and metastases. This, programmed upregulation of retinoid signaling that sensitized TNBC cells to AM80, a novel clinically available RARα-specific agonist. Here we report preclinical investigations on effects of SID decoys and AM80 treatments on cellular heterogeneity, primary tumors, metastatic dissemination, minimum residual disease (MRD) and host microenvironment. Using CyTOF2, we made single cell measurements of markers of differentiation, proliferation and stemness in CSC-enriched 4T1 tumorspheres. Treatment with SID peptide decreased cell populations expressing nanog, sox2, vimentin and β-catenin with increase in γH2AX. Addition of AM80 in combination with C16, resulted in populations with increased expression of differentiation marker CD24 with decrease in vimentin, β-catenin and Ki-67. To interrogate the neo-adjuvant effects of C16-AM80 treatments, primary 4T1 tumors in Balb/c mice were treated with C16 and AM80 alone or in combination. Compared to DMSO, ~40 % decrease in tumor weight, 60% decrease in ALDH activity and 60% decrease in lung metastasis was seen in mice treated with C16-AM80 combination. In post-surgical adjuvant settings, in both 4T1 and MMTV-myc xenografts, we observed 100% disease-free survival and absence of macrometastasis in mice receiving minimally toxic adjuvant therapy with the C16-AM80 combination for 90 days. However, MRD was found consisting of a small number of single CK8+ cells which failed to form colonies when recovered from the bone marrow and selectively cultured in vitro. Upon stopping the treatments, 20-40% animals developed macrometastases within the first three months. To test the influence of C16-AM80 to condition the host microenvironment to prevent macrometastases mice received only pretreatment with C16 and AM80 followed by 4T1 cells injected in the tail vein. The percentage of parenchyma occupied by metastatic nodules were: DMSO = 50%; AM80 = 25%; C16 >10% and C16-AM = <<10%; the mitotic features were greatly reduce from 0-5 in DMSO to 0-1 per 400x/field in the C16-AM80 combination. The predominant effects were observed with C16 treatment, which was enhanced to a small degree in combination with AM80. These results suggest that C16 can potentially induce changes in the host microenvironment to prevent the colonization and metastatic growth of cancer cells in TNBC. Altogether, our preclinical studies merit expansion of a pre-clinical program for development of C16-AM80 combination as a targeted adjuvant therapy to treat TNBC. Citation Format: Nidhi Bansal, Eduardo Farias, Veronica Gonzalez, Garry Nolan, Samuel Waxman. Development of novel targeted adjuvant therapy for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2017-2083

Targeted interference of SIN3A-TGIF1 function by SID decoy treatment inhibits Wnt signaling and invasion in triple negative breast cancer cells.

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 β-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.

Abstract 4115: Inhibition of triple negative breast cancer cell invasion by the targeted interference of Sin3A function affecting Wnt and TGFβ signaling

Abstract Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poor survival in TNBC patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need for TNBC treatment and for other tumor types. We reported that decoys with the SID sequence designed to bind and inhibit the function of PAH-2 domain of Sin3A protein markedly prolong survival in the adjuvant setting due to inhibition of metastatic dissemination to the lungs and bone marrow in TNBC mouse models. Here, we show that TNBC cell lines treated with SID decoys (peptides) display a strong in vitro inhibition of migration and invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin, and inhibition of proteolytic enzymes (MMP9; MT-MMP1 and uPA) involved in extracellular matrix degradation. DNA microarray and Ingenuity pathway analysis (IPA) showed that the SID decoys inhibit Wnt and TGFβ signaling that is associated with epithelial to mesenchymal transition (EMT). Treatment with SID decoy peptide downregulated WNT/β-catenin-driven transactivation as measured by decreased promoter H3K4me3 and decreased expression of Wnt target genes like LEF1 and TCF7L2. We also show that SID decoys induce translocation of nuclear β-catenin to the cytoplasm in TNBC at 24 hours. Wnt/β-catenin is critical for EMT, cancer stem cell self-renewal, and early invasion in TNBC. TGIF1, a transcription factor that modulates TGFβ and Wnt signaling pathways and known to to interact with the PAH2 domain of Sin3A, can be dissociated from Sin3A complex by SID decoy treatment as measured by co-immunoprecipitation (Co-IP) and proximity linked assay. DNA microarray of SID peptide treated TNBC cells shows inhibition of TGFβ signaling evidenced by downregulation of MMP9, MT1-MMP and PLAU, known target genes of this pathway. This is in line with inhibition of the EMT program predicted by the IPA analysis in SID peptide treated TNBC. Taken together, the results indicate that SID decoys have potential value as therapeutic agents to revert the EMT program in TNBC that should translate into the inhibition of metastasis dissemination and eradication of residual disease in TNBC. To test this in clinic future investigations will involve the use of our previously identified small molecule mimetic of SID peptide, selamectin that is also a FDA approved drug. Use of a recently constructed cyclic stapled peptide that inhibits PAH-2 binding and invasion at <10nM is also anticipated. Citation Format: Yeon-Jin Kwon, Boris A. Leibovitch, Nidhi Bansal, Lutecia Pereira, Edgardo V. Ariztia, Kevin Petrie, Arthur Zelent, Ming-Ming Zhou, Eduardo F. Farias, Samuel Waxman. Inhibition of triple negative breast cancer cell invasion by the targeted interference of Sin3A function affecting Wnt and TGFβ signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4115.

Blocking the PAH2 domain of Sin3A inhibits tumorigenesis and confers retinoid sensitivity in triple negative breast cancer.

Triple negative breast cancer (TNBC) frequently relapses locally, regionally or as systemic metastases. Development of targeted therapy that offers significant survival benefit in TNBC is an unmet clinical need. We have previously reported that blocking interactions between PAH2 domain of chromatin regulator Sin3A and the Sin3 interaction domain (SID) containing proteins by SID decoys result in EMT reversal, and re-expression of genes associated with differentiation. Here we report a novel and therapeutically relevant combinatorial use of SID decoys. SID decoys activate RARα/β pathways that are enhanced in combination with RARα-selective agonist AM80 to induce morphogenesis and inhibit tumorsphere formation. These findings correlate with inhibition of mammary hyperplasia and a significant increase in tumor-free survival in MMTV-Myc oncomice treated with a small molecule mimetic of SID (C16). Further, in two well-established mouse TNBC models we show that treatment with C16-AM80 combination has marked anti-tumor effects, prevents lung metastases and seeding of tumor cells to bone marrow. This correlated to a remarkable 100% increase in disease-free survival with a possibility of “cure” in mice bearing a TNBC-like tumor. Targeting Sin3A by C16 alone or in combination with AM80 may thus be a promising adjuvant therapy for treating or preventing metastatic TNBC.

Abstract 3511: Targeted epigenetic reprogramming reverts tumor progression in triple-negative breast cancer models by the activation of retinoic acid receptor alpha

Abstract INTRODUCTION: Triple negative breast cancer (TNBC) is associated with aggressiveness, early recurrence and poor prognosis and no other therapeutic alternative besides chemotherapy. The silencing of genes associated with cell differentiation in cancer is required for tumor progression. In this process the epigenetic modifications induced by aberrant control of the chromatin remodeling machinery in transformed cells plays a key role. Therefore, finding a mechanism to restore the expression of these genes by reverting the abnormal epigenetic modifications is an important task in the fight against cancer. We showed previously that the selective interference of the Sin3's PAH2 domain with Sin3 interaction domain (SID) mimicking peptides restored expression of E-cadherin, estrogen receptor and RARa target genes such as RARb and CRBP1 leading to cell differentiation and anti-tumor effect in TNBC cells. Here we studied whether the re-expression of retinoic acid receptors (RARs) induced by the small molecule C16 (SID decoy) can make TNBC cells sensitive to selective retinoid therapies. EXPERIMENTAL APPROACH: Human and mouse cell models of TNBC as well as MMTV-Myc oncomice were used to test the effects of the SID decoys on the induction of RAR signaling, cell proliferation, induction of differentiation in 3D cultures, expansion of the cancer stem cell compartment, atypical ductal hyperplasia (ADH), ductal carcinola in situ (DCIS), tumor development and metastatic dissemination. RESULTS: Our data show that C16 induced a differential expression of RARs, ∼10 fold upregulation of RARa2, RARbeta1/2, ∼10 fold downregulation of RARg1 and ∼30% increased production of retinoic acid (RA). The combination of C16 plus RARa agonists, AM80 (tamibarotene) or AM580 induced 75% inhibition of cell proliferation in 2D cultures, induced immature acini in 3D and impaired cancer stem cell proliferation inhibiting tumorsphere formation (∼65%) in vitro. In vivo, the combination of C16 and AM580 reduced 90% tumor growth and metastasis. Interestingly, the treatment of MMTV-Myc oncomice with C16 alone or in combination with AM80 prevented mammary gland hyperplasia, DCIS and delayed tumor development (80% tumor free survival, Kaplan-Meier analysis). CONCLUSION: As a whole, the epigenetic reprogramming of the TNBC cells is promising for design of therapies based on the use of RARa agonists to induce differentiation of the tumor cells; giving these patients a chance of an alternative or complementary therapy to chemotherapy regimens. Citation Format: Nidhi Bansal, Almudena Bosch, Boris A. Leibovitch, Keely Pierzchalski, Zhou Ming-Ming, Maureen Kane, Samuel Waxman, Eduardo Farias. Targeted epigenetic reprogramming reverts tumor progression in triple-negative breast cancer models by the activation of retinoic acid receptor alpha. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3511. doi:10.1158/1538-7445.AM2015-3511

Abstract 1989: Targeting Sin3-Pf1 complex: Novel site-specific epigenetic therapy for triple negative breast cancer

Abstract Introduction: Dearth of clinically validated drug targets is a major challenge in Triple Negative Breast Cancer (TNBC) that limits the treatment options to chemotherapy with intense cytotoxic consequences. We previously identified PAH2 domain of Sin3 protein as potential therapeutic target in TNBC and shown that protein-protein interactions of Sin3 via its PAH-2 domain can be disrupted by decoys (Mad1-SID peptide/small molecule inhibitors) designed based on the Sin3 interaction domain (SID) of transcription factor Mad1. Chromatin regulator Pf1 shown to be overexpressed in breast cancer and interacting with PAH2 domain of Sin3 can also be dissociated from Sin3 complex by Mad1-SID. In this study we identify Pf1 to be a significant contributor to the oncogenic phenotype associated with TNBC. Methods: Pf1 knockdown lines were generated by stably transfecting MDA-MB-231 cells with Pf1-shRNA or scr-shRNA. Cells were cultured in 2D and assayed for cancer stem cell (CSC) markers and ALDH activity by flow cytometry. For colony morphogenesis cells were cultured in 3D Matrigel. qRT-PCR were performed to assay expression of Nanog, Sox2 and Oct4. Fluorescence anisotropy and co-immunoprecipitation assays were used to test Sin3-Mad1 and Sin3-Pf1 interaction in the presence or absence of Mad1-SID and/or Pf1-SID. Results: MDA-MB-231 cells transfected with Pf1-shRNA had over two fold reduced ability to form colonies in 3D Matrigel cultures, with ∼95% of colonies non-invasive in contrast to the invasive star-like colonies formed by cells transfected with Scr-shRNA. Additionally reduction in Pf1 level was accompanied by a ∼1.5-fold reduction (p<0.05) in the tumorsphere forming ability of MDA-MB-231 cells. Pf1 depletion also significantly reduced RNA and protein of Nanog, Oct4 and Sox2. Confocal imaging revealed reduced levels and nuclear accumulation of Nanog, Sox2 and Oct4 in cells transfected with Pf1-shRNA compared to Scr-shRNA. Pf1 knockdown resulted in a 2.5-fold decrease in ALDH1 positive cells (6.55% in Scr-shRNA vs 2.59% in Pf1-shRNA). The CD44low/CD24low/neg population was enriched 3 fold over cells transfected with Scr-shRNA. From a therapeutic perspective we have designed a linear peptide corresponding to the Sin3 interaction domain of Pf1 (Pf1-SID). By fluorescence anisotropy we show that in comparison to Mad1-SID (IC50 = 1.4±0.3 μM), several hundred times higher concentration of Pf1-peptide (IC50 = 826±162 μM) is required to dissociate Sin3-Mad1 interaction. Co-immunoprecipitation assays show that Pf1-SID can specifically dissociate Pf1 from Sin3 without affecting the binding of Mad1 with PAH2 domain of Sin3. Pf1-SID treated MDA-MB-231 cells also have reduced invasive capacity and CSC traits. Conclusion: PAH2 domain of Sin3 and its interaction with Pf1 is potential drug target and Pf-1SID-mediated disruption of Sin3-Pf1 complex as translation relevance for first site-specific epigenetic therapy for TNBC. Citation Format: Nidhi Bansal, Joanna Wexler, Yeon-jin Kwon, Elena C. Gil, Boris Leibovitch, Rajal Sharma, Arthur Zelent, Ming-Ming Zhou, Eduardo Farias, Samuel Waxman. Targeting Sin3-Pf1 complex: Novel site-specific epigenetic therapy for triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1989. doi:10.1158/1538-7445.AM2015-1989

Selective Inhibition of SIN3 Corepressor with Avermectins as a Novel Therapeutic Strategy in Triple-Negative Breast Cancer.

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.

PH Might play a role in regulating the function of paired amphipathic helices domains of human Sin3B by altering structure and thermodynamic stability

Human Sin3B (hSin3B), a transcription regulator, is a scaffold protein that binds to different transcription factors and regulates transcription. It consists of six conserved domains that include four paired amphipathic helices (PAH 1-4), histone deacetylase interaction domain (HID), and highly conserved region (HCR). Interestingly, the PAH domains of hSin3B are significantly homologous to each other, yet each one interacts with a specific set of unique transcription factors. Though various partners interacting with hSin3B PAH domains have been characterized, there is no structural information available on the individual PAH domains of hSin3B. Here we characterize the structure and stability of different PAH domains of hSin3B at both nuclear and physiological pH values by using different optical probes. We found that the native state structure and stability of different PAH domains are different at nuclear pH where hSin3B performs its biological function. We also found that PAH2 and PAH3 behave differently at both nuclear and physiological pH in terms of native state structure and thermodynamic stability, while the structural identity of PAH1 remains unaltered at both pH values. The study indicates that the structural heterogeneity of different PAH domains might be responsible for having a unique set of interacting transcription factors.