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Malignant ovarian ascites are rich in cellular components, membrane-bound vesicles, and soluble proteins. This study focused on the structure of membrane-bound vesicles and their ability to promote invasion in cultured malignant ovarian epithelium. Membrane vesicles were derived from women with stage I–IV malignant ovarian ascites and from nonmalignant gynecologic ascites. Isolated vesicles were characterized by immunofluorescence and Western blot analysis. Using gel zymography for matrix metalloproteinase (MMP) detection and a colorimetric assay for urokinase-type plasminogen activator (uPA) analysis, we analyzed the proteinase activities of MMP-2, MMP-9, and uPA from the prepared vesicles, whole cells isolated from ascites, and the cell-free ultracentrifuged supernatant. The invasiveness of established cultured malignant ovarian epithelium on addition of ascites-derived vesicles was tested using a Matrigel-based invasion assay. Fractionation of malignant ascites revealed that extracellular matrix-degrading proteinases including MMPs and uPA are localized preferentially in membrane vesicles. All malignant vesicles tested, regardless of cancer stage, stimulated invasion. Furthermore, the combination of ovarian cancer cells and membrane vesicles resulted in greater uPA activation than that of cells or vesicles alone. Membrane vesicles from malignant ascites were also found to contain activated MMP-2, MMP-9, and uPA. Our data suggest that vesicle-stimulated proteinase activation leads to increased extracellular matrix degradation, which may facilitate tumor cell invasion and metastasis.

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 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.

Triple Negative Breast cancer (TNBC) is an aggressive subtype of breast cancer associated with early recurrence and poor prognosis. The treatment options are limited due to lack of expression of common drug targets: estrogen receptor (ER), Progesterone receptor (PR) and Epidermal growth factor receptor 2 (Her2). Epigenetic programs can generate aberrant transcription contributing to TNBC progression; however the dynamic and reversible nature of epigenetic changes offers the possibility to reprogram cancer cells to re-express targets that can render TNBC sensitive to targeted therapies like tamoxifen. Envisioning such ‘epidrugs’, we previously published that targeting PAH2 domain of the master transcriptional scaffold Sin3 by stable expression of 13-mer peptide corresponding to a specific motif called SID (mSin3A interaction domain) disrupts its interaction with a small group of SID-containing transcription factors. This interference reverts the expression of important breast cancer-associated genes and impairs tumor growth in vivo. We have now extended our study towards the evaluation of a cell penetrating SID peptide (pSID) in in vitro and in vivo models to establish parameters for the design of targeted epigenetic therapy for TNBC. pSID co-localizes with Sin3A and interference with PAH2-mediated Sin3A functions by pSID is shown by disruption of Sin3A-MAD1 interactions in Co-IP and Duo-Link assays. pSID treatment in MDA-MB 231 cells results in functional re-expression of CDH1 and ER along with increased H3K4 and decreased H3K27 methylation on their promoters. We also show reduction in the tumorsphere formation by pSID-pretreated MDA-MB-231 cells indicating possible epigenetic reprogramming of tumor initiating stem cells towards a differentiated phenotype. Support to this hypothesis is added by the 50% reduction in tumor growth and re-expression of CDH1 observed in FVB mice injected with pSID-pretreated MMTV-myc cells. Moreover, microarray expression analysis indicates pSID-induced EMT reversal, increased cell adhesion and reduced cell migration. Intriguingly, upon further dissection of the mechanism of epigenetic regulation by pSID we show dissociation of two important chromatin readers/modifiers from the Sin3 complex: histone H3K4Me3/2 demethylase JARID1B and H3K4Me0 binding PHD-like domain containing protein PF1; both with significantly correlated overexpression in invasive breast carcinoma. We also observe loss of recruitment of JARID1B but not Sin3A from the CDH1 promoter. Currently studies are underway to understand the cooperative role between JARID1B and PF1 in potentiating the aberrant transcription regulation by Sin3 at important breast cancer-associated promoters that can be selectively reprogrammed by SID decoys. We believe this selectivity can limit the otherwise adverse affects that may be observed by the use of generic HDAC inhibitors and demethylating agents. Citation Format: Rossitza Christova, Kevin Petrie, Nidhi Bansal, Boris Leibovitch, Louise Howell, Veronica Gil, Ming-Ming Zhou, Edgardo Ariztia, Eduardo Farias, Arthur Zelent, Samuel Waxman. Targeted PF1, JARID1B inhibition induces epigenetic reprogramming in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 411. doi:10.1158/1538-7445.AM2014-411

The Sin3 A/B adapter proteins function as structural scaffolds for repressor/activator complexes that regulate transcription through the specific association with histone modifying enzymes and a number of transcription factors. Sin3 contains four paired amphipathic α-helices (PAH domains). We have reported earlier that targeted disruption of the PAH2 domain with a SID (Sin3 Interaction Domain) peptide decoy in triple negative (TN) breast cancer cells leads to cytoskeletal reorganization, loss of anchorage independent growth and 3D invasive morphology and decreased cell adhesion and invasion. There is epigenetic reprogramming of silenced genes such as CDH1, ESR1 and RARA which are re-expressed and together contribute to a SID decoy induced switch from basal to a more differentiated luminal phenotype (Farias, et. al., PNAS, 2010, 107:11811-6). Computerized screening coupled with such assays as Duolink, GST pull downs and mammalian two hybrid identified small molecule inhibitors (SMI) that mimic the effects of the SID decoy peptide. SMI inhibit cellular invasion at nanomolar range and in in vivo mouse myc TN breast cancer prolong latency, decrease local invasion and metastasis. Tumors recovered showed evidence of re-expression of E-cadherin and estrogen receptor. Early effects of SID decoy in TN breast cancer cells include inhibition of invasion that is associated with a significant decrease in Src phosphorylation within 2-4hr of treatment. Recovery of phosphorylation after SID decoy washout is coupled with recovered invasion at 24hr. These effects occurred prior to measurable increase in E-cadherin expression, suggesting a non-transcriptional effect. In Drosophila larval breast cancer models with activated Src/Ras, overexpression of SID inhibited (60%) tumor growth in the eye imaginal disc, and was eradicated by the addition of a MEK inhibitor (AZD-6244), indicating a strong synergy between SID and AZD-6244. In the adult fly SID expression greatly inhibited RetMEN2B induced eye tumors (90%). These data demonstrate that SID decoys have a potential to be effective agents in the treatment of TN breast cancer by promoting basal phenotype reversal, inhibiting invasion and metastasis, and could be synergistic with specific inhibitors of signal transduction targets. Moreover, SID decoys can overcome profound oncogenic stimulus such as Ras, Src and Ret suggesting that they have a potential greater role than just in the treatment of TN breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1826. doi:1538-7445.AM2012-1826

We have previously reported that site-specific disruption of the Sin3 complex results in targeted epigenetic reprogramming and differentiation in TN breast cancer (Farias et al., PNAS 2010, 107:11811-6). The Sin3 A/B are multidomain adapter proteins part of a multisubunit corepressor scaffold that regulates transcription via the recruitement of HDAC and BP2/JARID1 A. Sin3 (A/B) contain four paired amphipathic α-helices known as PAH domains, a central HID (HDAC interaction domain) also serves as docking platform for a variety of corepressors, a C-terminal domain is a highly conserved region. We have targeted the disruption of the PAH2 domain binding to SID (mSin3A interaction domain) containing transcription factors such as MAD1, REST, KLF-9, -10, -11, -13 and -16, using a peptide decoy containing the SID motif. These studies have been carried out in several breast cancer cell lines including 5 TN, 2 estrogen receptor positive (ER+) and a non-transformed mammary cell line. Transfection with a plasmid expressing minimal SID or treatment with a 13 amino-acid peptide (SID peptide) disrupts transcription factor binding to the PAH2 domain resulting in extreme changes in morphology, cytoskeletal organization, adhesion, decreased extracellular proteolytic activity (MMP-9 and uPA), loss of invasive capacity and anchorage independent growth. There was re-expression of functional E-cadherin, estrogen and retinoic acid receptors. Effects in vivo xenograft also included inhibition of tumor growth and metastasis in FVB mice. 3D cultures in Matrigel have revealed a SID decoy dependent switch from a basal to a more luminal phenotype. These effects of SID decoy occurred in TN but not in ER+ or non-transformed breast cancer cell lines. Taken together, these results support the hypothesis in which SID decoy interference with PAH2 domain binding can revert Epithelial/Mesenchymal Transition in TN breast cancer cells and results in a less malignant phenotype. Fourteen candidate small molecule inhibitors, from a computerized screen of 115,000 compounds, were tested in a mammalian two hybrid assay and some of them have been tested in cellular invasion, proliferation and morphogenesis assays. One of them, compound 14 (C14) has shown no toxicity, induces E-cadherin expression and is effective in inhibiting Matrigel invasion of MDA-MB-231 at 100 nM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2826. doi:10.1158/1538-7445.AM2011-2826

Our recent work in understanding silenced retinoic acid response genes in breast cancer led us to explore the role of transcription repressor complexes in gene silencing in breast cancer. To this end we constructed a set of tagged vectors that contain a specific MAD1 motif called SID (mSin3A interaction domain), which binds with high affinity to block the function of the Sin3. Sin3A/B serve as multisubunit co-repressor scaffold protein that regulate gene transcription by recruiting histone deacetylase and histone demethylase activities to sequence-specific transcriptional repressors which are aberrant in breast cancer. The PAH2 domain of Sin3A/B binds with high affinity to a small number of transcription factors, and offers a more specific epigenetic target which contributes to the development of breast cancer. PAH-2 domain a specific component of a transcriptional repressor complex that plays an important role in modulating a small number of transcription factors containing the Sin3 PAH-2 interaction domain (SID). Here we demonstrated that in both human and mouse breast cancer cells, the targeted disruption of Sin3 function by introduction with their partners by the expression of SID transcript or peptide decoy interfered with PAH2 binding to SID-containing partner proteins as measured by co-immunoprecipitation and mammalian two-hybrid assays, reverteds the silencing of several genes involved in cell growth and differentiation. We observed that the SID decoy induced clear signs of differentiation in both human and mouse breast cancer cellsIn particular, the which include theSID decoys led to acinar morphogenesis in 3D cultures, increased adherence to collagen type-IV and laminin, reduced invasive phenotype and impaired tumor growth in vivo (>75%). This was associated with epigenetic reprogramming characterized by a marked increase in H3K4 2/3 methylation and a modest increase in H3 acetylation in the promoter region, promoter DNA demethylation and re-expression of the important breast cancer-associated silenced genes encoding E-cadherin, and, estrogen receptor α (ERα) and retinoic acid receptor β (RARβ)and impairment in tumor growth in vivo. There was increased expression of E-cadherin, CRBP1 and p27 known RAR response genes. The re-expression of ERα and RARβ in the “triple negative” MDA-MB-231 breast cancer cell line is functional since there was significant growth inhibition by tamoxifen after stimulation with 17b-estradiol and RAR activation by atRA and AM580. Therefore, the development of small molecules that mimic the 13 amino acid SID peptide and block interactions between PAH2 and SID-containing proteins This offers a new novel approach for treating this type of breast cancer and may also provide wider therapeutic implicationstriple negative breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 572.