Probing into the chemopreventive properties of synthetic 1,3,6-tri-O-galloyl-α-D-glucose (α-TGG) against glioblastoma and triple-negative breast cancer-derived cell models.

Cancer Metastasis Is Accelerated through Immunosuppression during Snail-Induced EMT of Cancer Cells

Epithelial to mesenchymal transition (EMT) is a cellular program that converts non-motile epithelial cells into invasive mesenchymal cells. EMT is implicated in cancer metastasis, chemo-resistance, cancer progression, and generation of cancer stem cells (CSCs). Inducing mesenchymal to epithelial transition (MET), the reverse phenomenon of EMT, is proposed as a novel strategy to target triple negative and tamoxifen-resistant breast cancer. Triple negative breast cancer (TNBC) is characterized by the loss of hormone receptors, a highly invasive mesenchymal phenotype, and a lack of targeted therapy. Estrogen receptor-positive breast cancer can be targeted by tamoxifen, an ER antagonist. However, these cells undergo EMT over the course of treatment and develop resistance. Thus, there is an urgent need to develop therapeutic interventions to target these aggressive cancers. In this study, we examined the role of novel diphenylamine analogs in converting the mesenchymal phenotype of MDA-MB-231 TNBC cells to a lesser aggressive epithelial phenotype. Using analog-based drug design, a series of diphenylamine analogs were synthesized and initially evaluated for their effect on E-cadherin protein expression and changes incell morphology, which was quantified by measuring the spindle index (SI) value. Selected compound 1 from this series increases the expression of E-cadherin, a primary marker for epithelial cells, and decreases the mesenchymal markers SOX2, ZEB1, Snail, and vimentin. The increase in epithelial markers and the decrease in mesenchymal markers are consistent with a phenotypic switch from spindle-like morphology to cobblestone-like morphology. Furthermore, Compound 1 decreases spheroid viability, cell migration, and cell proliferation in triple negative BT-549 and tamoxifen-resistant MCF-7 breast cancer cells.

Background: Triple negative breast cancer (TNBC) is known for its aggressive behavior, poor prognosis and still remains as a difficult disease since treatment options are limited. Despite some success in PARP inhibition in BRCA gene mutation patients or platinating agents that may offer superior outcomes in a subset of TNBC patients (pts), currently, there are no targeted therapies for TNBC available. Specific biomarkers are urgently needed for developing effective treatments to predict which patients will respond to the given therapy. In this regard, circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options. Since TNBC is closely related to epithelial-mesenchymal transition (EMT), a stem cell phenotype and, in addition, androgen receptor (AR) expression has been detected in up to a third of TNBC pts, we here established a multi-marker gene panel for the characterization of CTCs in TNBC pts and compared these findings with CTC characteristics in non-TNBC pts. Methods: 2x5 ml blood of 30 TNBC pts before and/or after neoadjuvant therapy and 30 non- TNBC pts (E+/PR+: n=23; ER+/PR-: n=4; HER2+: n=1; HER2+/ER+: n=1; HER2+/ER+/PR+: n=1) before therapy were analyzed for CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Subsequently, cDNA was gene specifically pre-amplified using TaqMan PreAmp Master Mix according to in house designed assays. Establishment of a 19 gene qPCR panel was performed for the markers PI3K, AKT2, ERCC1, Aurka, HER2, HER3, EGFR, ALK, AR (androgene receptor), BRCA1, c-KIT, c-MET, KRT5, mTOR, NOTCH1, PARP1, SRC1, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taken the false positive rate in healthy donors into account and defined as Ct(cutoff)-Ct(sample)-[Ct(CD45cutoff)-Ct(CD45sample)]. Results: In general, the distribution of the markers across all patients was highly variable. However, different expression patterns were found when CTCs of TNBC pts were compared with those of non-TNBC pts. In TNBC pts, SRC1 was the gene that was predominantly expressed, followed by c-Kit, HER3, BRCA1 and AURKA expression, before as well as after therapy. Interestingly, AKT2, EGFR, ERCC1 and PARP1 expression could not be detected at any time point studied. In addition, ALK, AR, c-Met, HER2 and KRT5 were only detected before but not after therapy. All other genes were expressed below 15%. In contrast, in non-TNBC pts, AKT2 was the gene that was predominantly expressed, followed by c-MET, HER3 and PI3K whereas c-KIT, ERCC1, mTOR and NOTCH1 were never found. All other genes were expressed below 10%. Conclusion: We successfully established a gene panel for the detection of the heterogeneous CTC population and demonstrated that CTCs in TNBC pts and non-TNBC pts show different genetic profiles. Although these data have to be confirmed in a bigger patient cohort, the knowledge about the individual target gene expression profile might efficiently help to predict a personalized targeted therapy for these pts in the future. Citation Format: Bittner A-K, Hoffmann O, Hauch S, Kimmig R, Kasimir-Bauer S. Circulating tumor cells in triple-negative and non-triple negative breast cancer patients show different genetic profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-05.

Background: Epithelial to Mesenchymal transition (EMT) is an important and coordinated series of events associated with tumor metastasis and invasion. Recent studies had shown the importance of CCN5 (also known as WISP-2,Wnt-1-induced signaling protein-2) in the regulation of various carcinomas including the breast cancer. Recent studies had showed that ectopic expression of CCN5 can reverse Epithelial-Mesenchymal transition (EMT) and inhibit cancer metastasis. Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been extensively studied as a bioactive dietary component against various types of carcinomas through multiple mechanisms such as anti-oxidation, induction of apoptosis, inhibition of angiogenesis and metastasis. However, the mechanism of action of EGCG in breast carcinoma is uncertain. Objective: The objective of the present study is to determine whether CCN5 plays any significant role in EGCG-mediated cytotoxicity in triple negative breast cancer cells. Results: Exposure of triple negative human breast cancer (TNBC) cells, MDA-MB-231 and HCC70 to EGCG resulted in a dose-dependent inhibition of proliferation after 72h and the IC50 was observed around 75μM for MDA-MB-231 cells and 50μM for HCC70 respectively. We found EGCG-treatment effectively induces MET and inhibits the in vitro migration parallel with the induction of CCN5 expression in TNBC cells in a dose-dependent fashion. Furthermore, consistent with in vitro findings, tumor progression was drastically inhibited in EGCG-treated MDA-MB-231-tumor xenograft in nude mouse model. Conclusion: EGCG imparts its anti-cancer activity in both TNBC cells as well as MB-231-tumor xenografts via induction of CCN5. Citation Format: Amlan Das, Snigdha Banerjee, Archana De, Inamul Haque, Gargi Maity, Matt McEwen, Sushanta K. Banerjee. The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4385. doi:10.1158/1538-7445.AM2013-4385

Triple-negative breast cancers (TNBC) are a subset of breast cancer that is highly aggressive and has a poor prognosis. The TNBC subtype has no effective molecular targeted therapies. Gene expression studies have identified a subset of TNBC that is enriched with androgen receptors (AR) and androgen receptor signaling. AR levels are most abundant in the luminal AR (LAR) molecular subtype of TNBC, but other non-LAR molecular TNBC subtypes also display high levels of AR expression and activity. Interference with androgen signaling in TNBC with AR-inhibiting drugs have shown a reduction in epithelial-to-mesenchymal transition (EMT), which is a process by which epithelial cells lose their cell polarity and cell-cell adhesion and gain migratory and invasive properties to become mesenchymal-like stem cells. Approximately one-third of TNBC expressed AR, and evaluation of AR-positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. Previous studies have shown that AR inhibition or AR knockdown significantly reduces migration and invasion and EMT in different TNBC cell lines. γ-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer effects and have little or no effect on normal cell viability. Studies show that treatment with 0-7 µM γ-tocotrienol reduced AR levels, proliferation, migration and invasion in a dose-responsive manner in TNBC MDA-MB-231 and MDA MB-453 cell lines. Results also show that treatment with 5 µM (MDA-MB-231) and 7 µM (MDA-MB-453) γ-tocotrienol induced a reversal in EMT cell biomarkers, as well as reversal in EMT morphological and behavioral characteristics in these TNBC cell lines. Western blot analysis show that similar treatment with γ-tocotrienol significantly decreased DHT-induced N-cadherin (mesenchymal cell biomarker) and increased expression in cytokeratin 18 and E-cadherin (epithelial cell biomarkers) in these cells. AR signaling is known to play an important role in stimulating DHT-induced EMT by stimulating the STAT3/Snail signaling pathway in MDA-MB231. However, results in the present study shows that γ-tocotrienol treatment significantly inhibited activation of STAT3 and Snail proteins. Additional immunocytochemistry experiments showed that γ-tocotrienol treatment significantly inhibited DHT-induced cytoskeleton changes in TNBC MDA-MB-231 and MDA MB-453 cell lines. In conclusion, these results demonstrated that γ-tocotrienol treatment inhibits AR expression and DHT-dependent EMT and cytoskeleton changes in TNBC cells. These findings suggest that AR may be a potential therapeutic target for treating both LAR and non-LAR TNBC subtypes. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Nayef Aldabaan, Tasmin A. Sultana, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 967.

International audience

Deciphering molecular mechanisms that control epithelial-to-mesenchymal transition (EMT) contributes to our understanding of how tumor cells become invasive and competent for intravasation. We have established that transforming growth factor β activates Smad proteins, which induce expression of the embryonic factor high mobility group A2 (HMGA2), which causes mesenchymal transition. HMGA2 associates with Smad complexes and induces expression of an established regulator of EMT, the zinc finger transcription factor Snail. We now show that HMGA2 can also induce expression of a second regulator of EMT, the basic helix-loop-helix transcription factor Twist. Silencing of endogenous Twist demonstrated that this protein acts in a partially redundant manner together with Snail. Double silencing of Snail and Twist reverts mesenchymal HMGA2-expressing cells to a more epithelial phenotype when compared with single silencing of Snail or Twist. Furthermore, HMGA2 can directly associate with A:T-rich sequences and promote transcription from the Twist promoter. The new evidence proposes a model whereby HMGA2 directly induces multiple transcriptional regulators of the EMT program and, thus, is a potential biomarker for carcinomas displaying EMT during progression to more advanced stages of malignancy.

Background/Rational: Patients with triple negative breast cancers (TNBC) have limited therapeutic options beyond conventional chemotherapy. Unfortunately, high-risk for metastatic recurrence and chemotherapy resistant diseases cause the worst 5-year survival rate in patients with TNBC, which have been significant clinical challenges. Novel therapeutic targets or strategies to combat metastasis and chemotherapy resistance are necessary to improve quality of life and outcomes for patients with high risk TNBC. Epithelial-to-mesenchymal transition (EMT) and anoikis resistance are processes recognized as contributing to enhanced metastatic potential and treatment resistance. A subset of TNBC exhibits mesenchymal gene signatures and phenotypes that may be associated with high metastatic recurrence, chemotherapy resistance and immunosuppression. In a functional genomic screen, we identified several candidates as novel regulators of EMT and anoikis sensitivity of TNBC cells. We have focused on roles of one highly validated candidate, protein tyrosine kinase 6 (PTK6) on EMT, anoikis resistance and metastatic capacity in TNBC. Methods: We analyzed expression of PTK6 and mesenchymal markers in patient triple negative tumors by immunohistochemistry. In breast epithelial and TNBC cell lines, the levels of PTK6 were genetically modulated, and determined effects on growth, migration and EMT. In vivo mouse models were used to show effects of PTK6 inhibition on metastatic capacity of TNBC cells. We have also validated effects of PTK6 specific small molecule inhibitor on TNBC growth and metastases. In order to dissect specific mechanisms by which PTK6 inhibition regulates TNBC mesenchymal phenotypes, we used a siRNA library screening and identified novel E3 ligases that may be responsible for PTK6 inhibition-induced EMT regulation. Results: Overexpression of PTK6 in MCF10A cells is sufficient to promote an EMT; promotes migration, suppresses epithelial markers (E-cadherin/claudin-1) and increases mesenchymal markers (N-cadherin and fibronectin). In contrast, PTK6 inhibition either PTK6 shRNAs or treatment with a specific kinase inhibitor enhances E-cadherin expression and suppresses migration, anoikis resistance and lung colonization of TNBC cells. PTK6-dependent E-cadherin regulation is specifically dependent on levels of SNAIL, a transcriptional repressor that is associated with poor TNBC patient prognosis. SNAIL down-regulation by PTK6 inhibition is directly responsible for the modulation of anoikis sensitivity, which is in turn causally linked to lung colonization potential. PTK6 inhibition promotes the proteasome-dependent degradation of SNAIL via a novel mechanism independent of GSK3β/β-TRCP pathway or Fbox E3ligases (FBXO5, FBXO11, FBXL14) that are known to regulate SNAIL ubiquitination. Using a siRNA library screening approach, we identified novel E3 ligase candidates that may be responsible for SNAIL ubiquitination and degradation downstream of PTK6 inhibition. Conclusion/Future direction: PTK6 is a representative novel regulator of EMT and anoikis resistance that can be targeted to prevent metastases of TNBC. Modulation of mesenchymal phenotypes of TNBC cells may be able to regulate chemotherapy resistance and/or immunosuppressive microenvironment. Citation Format: Ito K, Park SH, Nayak A, Byerly J, Irie HY. Targeting PTK6 to treat mesenchymal triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-02.

‘<scp>TWIST</scp> and switch’ metastasis

e13106 Background: Chemotherapy resistance remains a significant barrier in the effective treatment of patients with triple negative breast cancer (TNBC). TGF-β signaling is a well characterized oncogenic pathway in TNBC that promotes chemoresistance by inducing epithelial to mesenchymal transition (EMT) and tumor stemness. Aurora-A kinase (AURKA) is a serine/threonine kinase responsible for chromosomal stability during mitosis. Significantly, aberrant expression of AURKA induces breast cancer progression. We hypothesized that aberrant activation of TGF-β and AURKA signaling pathways contributes to chemoresistance in TNBC by promoting EMT and tumor stemness and that dual-targeting of these oncoproteins will enhance chemosensitivity. Methods: RNA-Seq data were analyzed from patient derived xenografts (PDx) established from patients with Stage I-III TNBC who received pre-operative taxane and anthracycline based chemotherapy. Chemoresistance was defined as an RCB score of 1-3, and chemosensitivity was defined as an RCB score of 0. Unique TNBC cell lines developed from brain metastasis PDxs (TNBC M14, TNBC M25) were treated in vitro with 10nM docetaxel (DTX), 50nM LY2157299 (TGF-β inhibitor) and 50nM MLN8237 (AURKA inhibitor). EMT reprogramming was determined by measuring the expression of vimentin, ALDH activity and mammosphere growth. Apoptotic cells following drug treatment were labeled with Red Annexin-V marker and monitored in real time using the IncuCyte instrument. Results: RNA-Seq revealed that there was no baseline difference in expression of AURKA between chemoresistant and chemosensitive TNBC PDxs. However, there was a 2.7-fold increase in AURKA expression in the post-treatment PDx models compared to pre-treatment PDx models. In vitro treatment of the M14 and M25 cell lines with DTX demonstrated no significant increase in apoptotic cells compared to control, whereas treatment with the combination of DTX, LY2157299 and MLN8237 resulted in a two-fold increase in apoptosis compared to treatment with DTX alone (p = 0.0068 M14, p = 0.003 M25). Dual-targeting with LY2157299 and MLN8237 reduced the expression of vimentin and ALDH activity. Conclusions: TGF-β and AURKA play a central role inducing EMT and a stem-cell-like phenotype in TNBC that confers chemoresistance. AURKA is up-regulated after exposure to chemotherapy in chemoresistant TNBC PDx models. LY2157299 and MLN8237 reduce TNBC stemness in M14 and M25 cell lines and enhance DTX chemosensitivity. Dual-targeting of TGF-β and AURKA is a potentially promising approach in chemoresistant TNBC.

Background: Although triple-negative breast cancers (TNBCs) constitute 10%-20% of all breast cancers, they are aggressive tumors with higher recurrence and poorer prognosis than other breast cancer subtypes. TNBCs frequently exhibit features of epithelial to mesenchymal transition (EMT) and are enriched in breast tumor initiating cells (BTICs). Recent studies have demonstrated that aberrant activation of the EMT program induces stem cell features in breast cells, suggesting that blocking the cross-talk between EMT and stemness may be an effective treatment for TNBCs. WISP3/CCN6, a secreted matrix associated protein, is expressed in normal breast tissues and is decreased in aggressive breast cancer samples. We have shown that CCN6 knockdown in nontumorigenic breast cells triggers EMT and invasion. We hypothesize that CCN6 regulates the cross-talk between EMT and BTICs pathways in TNBCs. Methods: We overexpressed CCN6 in the TNBC cell lines, MDA-MB-231, SUM-159 and MDA-MB-436, which are aggressive and exhibit a mesenchymal-like phenotype and low endogenous CCN6 expression. We tested the effect of CCN6 overexpression in these cell lines using motility and invasion assays in vitro. Stem cell features were investigated by mammosphere assays, and the ALDEFLUOR assay by flow cytometry. We developed mammary xenografts in nude mice using serial dilutions to investigate the role of CCN6 in tumor initiation and metastasis. Results: Overexpression of CCN6 inhibits cell motility and invasion, and leads to phenotypic changes of mesenchymal-epithelial transition (MET), with downregulation of the mesenchymal cell marker vimentin, increased epithelial marker cytokeratin, and decreased protein levels of the EMT transcription factor Slug. CCN6 overexpression significantly decreased the ALDEFLUOR-positive population and mammosphere formation of TNBC cell lines. In vivo, CCN6 overexpression delayed tumor initiation and reduced metastasis in human breast cancer xenografts. Slug reconstitution was sufficient to rescue the effects of CCN6 on MET and stem cell properties of TNBCs. Conclusions: These data demonstrate that CCN6 regulates EMT and tumor initiating cell features in TNBC at least in part through Slug. Further, our study suggests that CCN6 may be a novel regulator of the EMT-stem cell cross talk in TNBC, with possible clinical application. Citation Format: Wei Huang, Kathy A. Toy, Celina G. Kleer. WISP3/CCN6 regulates epithelial-mesenchymal transition and tumor initiating cell properties in triple negative breast cancer cells through Slug. [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 1037. doi:10.1158/1538-7445.AM2014-1037

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Background: Triple negative breast cancer (TNBC) is a heterogeneous group of disease. TNBC is closely related to epithelial-mesenchymal transition (EMT) and breast cancer stem cell (BCSC) phenotype. Recent studies have shown that TNBC can be classified into six subtypes including basal-like, mesenchymal–like and mesenchymal stem-like. However, clinical significance of EMT phenotype in TNBC is not clear. Methods: We performed immunohistochemical analyses of EMT markers (expression of vimentin, smooth muscle actin, osteonectin and N-cadherin; loss of E-cadherin), BCSC markers (CD44+/CD24- and ALDH1) and EMT inducers (CD146 and ZEB1) in 173 TNBCs using tissue microarrays, and correlated their expressions with clinicopathologic features of the tumor. Results: Expression of vimentin, CD44+/CD24- and CD146 was significantly higher in basal-like TNBCs (TNBC with expression of CK5/6 and/or EGFR) than in non-basal-like TNBCs. Expression of EMT markers was commonly correlated with high histologic grade, CD44+/CD24- phenotype and metaplastic carcinoma. CD146 expression was related to the expression of EMT markers and CD44+/CD24- phenotype. ZEB1 expression showed an association with the expression of smooth muscle actin, but not with BCSC markers. And it was closely correlated with high histologic grade and metaplastic carcinoma. In survival analyses, although expression of EMT and BCSC makers was not associated with the survival of the patients, ZEB1 expression was found to be an independent prognostic factor for poor disease-free survival of the patients. Conclusion: EMT phenotype can be a signature of certain subgroup of TNBC. Especially, ZEB1 expression can be used as a potential biomarker to define a subgroup of TNBC associated with poor clinical outcome. Citation Format: Min Hye Jang, Hyun Jeong Kim, Eun Joo Kim, Hee Jin Lee, So Yeon Park. Epithelial-mesenchymal transition phenotype in triple negative breast cancer: ZEB1 as a potential biomarker for poor clinical outcome [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-39.

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.

Background: Patients with triple-negative breast cancer (TNBC), which lacks estrogen receptor and progesterone receptor and has low HER2 expression, have a very poor prognosis because the disease often metastasizes. Preventing metastasis, as well as inhibiting tumor growth, is crucial to improving the prognosis of patients with TNBC. We previously showed using reverse-phase protein array analysis that patients with ERK2-overexpressing TNBC had a higher risk of death than those with low-ERK2-expressing TNBC. TNBC is characterized by an epithelial-to-mesenchymal transition (EMT) phenotype, which has features in common with those of cancer stem cells. Cells undergoing EMT may contribute to the metastatic process and have increased ability to form mammospheres in vitro. The roles of the 2 ERK isoforms, ERK1 and ERK2, in TNBC are not well defined. We hypothesized that ERK2, but not ERK1promotes EMT and metastasis in TNBC. Methods &amp; Results: We compared parental SUM-149 TNBC cells and stable clones that constitutively expressed shERK1 or shERK2 and observed no differences in cell proliferation. However, reducing the expression of ERK2, but not reducing the expression of ERK1, changed the mesenchymal phenotype of TNBC to an epithelial phenotype, reduced formation of mammospheres in a dose-dependent manner, and reduced the stem cell subpopulation (CD44+/CD24-), which correlated with a reduction in migration and invasion. In addition, ERK2 knockdown inhibited anchorage-independent growth, an indicator of in vivo tumorigenicity. To determine which genes are involved in ERK2-induced EMT and metastasis, we performed transcriptional profiling using real-time PCR-based EMT and metastasis arrays. After normalization, we observed that mRNA levels of the epithelial markers KRT7 and KRT14 were increased by 4 and 1.8 fold, respectively, in ERK2-silenced cells as in control cells; mRNA levels of the mesenchymal markers Twist1, vimentin, and ZEB1 were reduced by 0.6, 0.1 and 0.2 fold respectively, in ERK2-silenced cells as in control cells; and mRNA levels of the metastasis-related genes MMP-2, MMP-7, MMP-13, IL1B, and COL4A2 were reduced by 0.3, 0.3, 0.6, 0.6 and 0.3 fold respectively, in ERK2-silenced cells as in control cells. Further, using an Affymetrix gene chip and the Inflammatory Breast Cancer World Consortium patient data set (n=389), we did a class comparison test in which the P value was calculated by Wilcoxon test. We observed significantly lower ERK1 mRNA expression in the TNBC group than the non-TNBC group (P &amp;lt; 0.0001) but significantly higher ERK2 mRNA expression in the TNBC group than in the non-TNBC group (P &amp;lt; 0.005). Conclusions: ERK2 but not ERK1 may promote tumorigenesis of the TNBC subgroup by enriching the cancer stem cell population and inducing EMT and metastasis. Our long-term goal is to confirm these findings in xenograft studies and develop ERK2-targeted therapy for TNBC. Citation Format: Chandra Bartholomeusz, Hitomi Saso, Ali Dadbin, Hiroko Masuda, Takahiro Kogawa, Steven Van Laere, François Bertucci, Gabriel N. Hortobagyi, Naoto T. Ueno. ERK2 rather than ERK1 contributes to EMT and metastatic potential in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 858. doi:10.1158/1538-7445.AM2013-858