Complex In Breast Cancer Cells Research Articles

Development of a novel PROTAC using the nucleic acid aptamer as a targeting ligand for tumor selective degradation of nucleolin

PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and are poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs.

TRIB3 Modulates PPARγ-Mediated Growth Inhibition by Interfering with the MLL Complex in Breast Cancer Cells.

Aberrant expression or activity of proteins are amongst the best understood mechanisms that can drive cancer initiation and progression, as well as therapy resistance. TRIB3, a member of the Tribbles family of pseudokinases, is often dysregulated in cancer and has been associated with breast cancer initiation and metastasis formation. However, the underlying mechanisms by which TRIB3 contributes to these events are unclear. In this study, we demonstrate that TRIB3 regulates the expression of PPARγ, a transcription factor that has gained attention as a potential drug target in breast cancer for its antiproliferative actions. Proteomics and phosphoproteomics analyses together with classical biochemical assays indicate that TRIB3 interferes with the MLL complex and reduces MLL-mediated H3K4 trimethylation of the PPARG locus, thereby reducing PPARγ mRNA expression. Consequently, the overexpression of TRIB3 blunts the antiproliferative effect of PPARγ ligands in breast cancer cells, while reduced TRIB3 expression gives the opposite effect. In conclusion, our data implicate TRIB3 in epigenetic gene regulation and suggest that expression levels of this pseudokinase may serve as a predictor of successful experimental treatments with PPARγ ligands in breast cancer.

Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo.

Human EGF Receptor 2 (HER2) is an important oncogene driving aggressive metastatic growth in up to 20% of breast cancer tumors. At the same time, it presents a target for passive immunotherapy such as trastuzumab (TZM). Although TZM has been widely used clinically since 1998, not all eligible patients benefit from this therapy due to primary and acquired drug resistance as well as potentially lack of drug exposure. Hence, it is critical to directly quantify TZM–HER2 binding dynamics, also known as cellular target engagement, in undisturbed tumor environments in live, intact tumor xenograft models. Herein, we report the direct measurement of TZM–HER2 binding in HER2-positive human breast cancer cells and tumor xenografts using fluorescence lifetime Forster Resonance Energy Transfer (FLI-FRET) via near-infrared (NIR) microscopy (FLIM-FRET) as well as macroscopy (MFLI-FRET) approaches. By sensing the reduction of fluorescence lifetime of donor-labeled TZM in the presence of acceptor-labeled TZM, we successfully quantified the fraction of HER2-bound and internalized TZM immunoconjugate both in cell culture and tumor xenografts in live animals. Ex vivo immunohistological analysis of tumors confirmed the binding and internalization of TZM–HER2 complex in breast cancer cells. Thus, FLI-FRET imaging presents a powerful analytical tool to monitor and quantify cellular target engagement and subsequent intracellular drug delivery in live HER2-positive tumor xenografts.

Regulation of LCoR and RIP140 expression in cervical intraepithelial neoplasia and correlation with CIN progression and dedifferentiation

PurposeLigand-dependent corepressor (LCoR) and receptor-interacting protein 140 (RIP140/NRIP1) play an important role in the regulation of multiple oncogenic signaling pathways and the development of cancer. LCoR and RIP140 form a nuclear complex in breast cancer cells and are of prognostic value in further prostate and cervical cancer. The purpose of this study was to analyze the regulation of these proteins in the development of cervical intraepithelial neoplasia (CIN I–III).MethodsImmunohistochemical analysis was obtained to quantify RIP140 and LCoR expression in formalin-fixed paraffin embedded tissue sections of cervical intraepithelial neoplasia samples. Tissue (n = 94) was collected from patients treated in the Department of Gynecology and Obstetrics, Ludwig-Maximilians-University of Munich, Germany, between 2002 and 2014. Correlations of expression levels with clinical outcome were carried out to assess for prognostic relevance in patients with CIN2 progression. Kruskal–Wallis test and Mann–Whitney U test were used for data analysis.ResultsNuclear LCoR overexpression correlates significantly with CIN II progression. Nuclear RIP140 expression significantly increases and nuclear LCoR expression decreases with higher grading of cervical intraepithelial neoplasia. Cytoplasmic RIP140 expression is significantly higher in CIN III than in CIN I or CIN II.ConclusionA decrease of nuclear LCoR expression in line with an increase of dedifferentiation of CIN can be observed. Nuclear LCoR overexpression correlates with CIN II progression indicating a prognostic value of LCoR in cervical intraepithelial neoplasia. Nuclear and cytoplasmic RIP140 expression increases significantly with higher grading of cervical intraepithelial neoplasia underlining its potential role in the development of pre-cancerous lesions. These findings support the relevance of LCoR and RIP140 in the tumorigenesis indicating a possible role of LCoR and RIP140 as targets for novel therapeutic approaches in cervical intraepithelial neoplasia and cervical cancer.

Localization of GPSM2 in the Nucleus of Invasive Breast Cancer Cells Indicates a Poor Prognosis.

Purpose: GPSM2 (G protein signaling modulator 2) was reported to be involved in the cell division of breast cancer cells. Additionally, cytoplasmic dynein may mediate the transport process of GPSM2. DYNC1I1 (Cytoplasmic dynein 1 intermediate chain 1) is the most common cargo-binding subunit of dynein. However, the relationship between GPSM2 and DYNC1I1 and its clinical value is unclear.Methods: Immunohistochemical staining was performed for assessment of GPSM2 and DYNC1I1 expression. Immunoprecipitation analysis was used to assess the interaction between GPSM2 and DYNC1I1.Results: GPSM2 was correlated with clinical characteristics of breast cancer patients and is an unfavorable independent prognostic factor. In addition, nuclear expression of GPSM2 is an unfavorable independent prognostic factor (HR = 2.658, 95% CI = 1.490–4.741, p = 0.001). GPSM2 and DYNC1I1 are known to form a complex in breast cancer cells. Patients who were positive for expression of both DYNC1I1 and GPSM2 presented with shorter recurrence-free survival than other patients. Importantly, patients with GPSM2 nuclear expression showed higher DYNC1I1 expression.Conclusion: GPSM2 was an independent prognostic factor in breast cancer and nuclear expression of GPSM2 was significantly associated with poor prognosis, which was related to the positive expression of DYNC1I1. Examination of both GPSM2 and DYNC1I1 is necessary to establish a prognosis in breast cancer patients.

Synthesis and biological assessment of a ruthenium(II) cyclopentadienyl complex in breast cancer cells and on the development of zebrafish embryos

Ruthenium-based complexes currently attract great attention as they hold promise to replace platinum-based drugs as a first line cancer treatment. Whereas ruthenium arene complexes are some of the most studied species for their potential anticancer properties, other types of ruthenium complexes have been overlooked for this purpose. Here, we report the synthesis and characterization of Ru(II) cyclopentadienyl (Cp), Ru(II) cyclooctadienyl (COD) and Ru(III) complexes bearing anastrozole or letrozole ligands, third-generation aromatase inhibitors currently used for the treatment of estrogen receptor positive (ER +) breast cancer. Among these complexes, Ru(II)Cp 2 was the only one that displayed a high stability in DMSO and in cell culture media and consequently, the only complex for which the in vitro and in vivo biological activities were investigated. Unlike anastrozole alone, complex 2 was considerably cytotoxic in vitro (IC50 values < 1 μM) in human ER + breast cancer (T47D and MCF7), triple negative breast cancer (TNBC) (MBA-MB-231), and in adrenocortical carcinoma (H295R) cells. Theoretical (docking simulation) and experimental (aromatase catalytic activity) studies suggested that an interaction between 2 and the aromatase enzyme was not likely to occur and that the bulkiness of the PPh3 ligands could be an important factor preventing the complex to reach the active site of the enzyme. Exposure of zebrafish embryos to complex 2 at concentrations around its in vitro cytotoxicity IC50 value (0.1–1 μM) did not lead to noticeable signs of toxicity over 96 h, making it a suitable candidate for further in vivo investigations. This study confirms the potential of Ru(II)Cp complexes for breast cancer therapy, more specifically against TNBCs that are usually not responsive to currently used chemotherapeutic agents.

The Effects of Thiamine on Breast Cancer Cells.

(1) Background: Thiamine is an important cofactor for multiple metabolic processes. Its role in cancer has been debated for years. Our aim is to determine if thiamine can convert the cellular metabolic state of breast cancer cells from anaerobic to aerobic, thus reducing their growth. (2) Methods: Breast cancer (MCF7) and non-tumorigenic (MCF10A) cell lines were treated with various doses of thiamine and assessed for changes in cell growth. The mechanism of this relationship was identified through the measurement of enzymatic activity and metabolic changes. (3) Results: A high dose of thiamine reduced cell proliferation in MCF7 (63% decrease, p < 0.0001), but didn’t affect apoptosis and the cell-cycle profile. Thiamine had a number of effects in MCF7; it (1) reduced extracellular lactate levels in growth media, (2) increased cellular pyruvate dehydrogenase (PDH) activities and the baseline and maximum cellular oxygen consumption rates, and (3) decreased non-glycolytic acidification, glycolysis, and glycolytic capacity. MCF10A cells preferred mitochondrial respiration instead of glycolysis. In contrast, MCF7 cells were more resistant to mitochondrial respiration, which may explain the inhibitory effect of thiamine on their proliferation. (4) Conclusions: The treatment of MCF7 breast cancer cells with 1 μg/mL and 2 μg/mL of thiamine for 24 h significantly reduced their proliferation. This reduction is associated with a reduction in glycolysis and activation of the PDH complex in breast cancer cells.

Abstract 3303: Survivin negatively-regulates autophagy through interference with the formation of Atg5-Atg12-Atg16L complex in breast cancer cells

Abstract Survivin, a member of the inhibitors-of-apoptosis proteins family, is highly expressed in different cancers and its expression is correlated with tumorigenesis and tumor metastasis. At the cellular and molecular levels, Survivin is a dual functions protein that promotes mitosis and also inhibits apoptosis. In this study, we found that Survivin is a novel negative-regulator of autophagy. Molecular analysis and computational modeling revealed that Survivin negatively regulates the formation of Atg5-Atg12 conjugates, which plays important roles in the process of autophagosome elongation, in breast cancer cells. It also negatively regulates the expression and activity of cathepsin L in cells. Interestingly, we found that Survivin binds to p62/SQSTM1 and LC3B and is also an autophagic substrate protein, in which its expression is in part directly regulated by autophagy at the protein level. Finally, mechanistic study revealed YM155, a Survivin inhibitor that was originally developed to inhibit Survivin gene transcription, downregulates Survivin expression in part through autophagic protein degradation. Taken together, findings of this study provide new mechanistic insights into both molecular functions and regulations of Survivin and the molecular mechanism of actions of YM155, a drug currently undergoing clinical trials for cancer treatment. Citation Format: Hsiu-Han Chan, Mohane Selvaraj Coumar, Siao-Muk Cheng, Shing-Ling Tsai, Chun-Hui Lin, Shang-Hung Chen, Euphemia Leung, Chun Hei Antonio Cheung. Survivin negatively-regulates autophagy through interference with the formation of Atg5-Atg12-Atg16L complex in breast cancer cells [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 3303. doi:10.1158/1538-7445.AM2017-3303

Calmodulin Binding to Death Receptor 5-mediated Death-Inducing Signaling Complex in Breast Cancer Cells.

Activation of death receptor-5 (DR5) leads to the formation of death-inducing signaling complex (DISC) for apoptotic signaling. TRA-8, a DR5 specific agonistic antibody, has demonstrated significant cytotoxic activity in vitro and in vivo without inducing hepatotoxicity. Calmodulin (CaM) that is overexpressed in breast cancer plays a critical role in regulating DR5-mediated apoptosis. However, the mechanism of CaM in regulating DR5-mediated apoptotic signaling remains unknown. In this study, we characterized CaM binding to DR5-mediated DISC for apoptosis in TRA-8 sensitive breast cancer cell lines using co-immunoprecipitation, fluorescence microscopic imaging, caspase signaling analysis, and cell viability assay. Results show that upon DR5 activation, CaM was recruited into DR5-mediated DISC in a calcium dependent manner. CaM antagonist, trifluoperazine (TFP), inhibited CaM recruitment into the DISC and attenuated DISC formation. DR5 oligomerization is critical for DISC formation for apoptosis. TFP decreased TRA-8 activated DR5 oligomerization, which was consistent with TFP's effect on DR5-mediated DISC formation. TFP and Ca2+ chelator, EGTA, impeded TRA-8-activated caspase-dependent apoptotic signaling, and TFP decreased TRA-8-induced cell cytotoxicity. These results demonstrated CaM binding to DR5-mediated DISC in a calcium dependent manner and may identify CaM as a key regulator of DR5-mediated DISC formation for apoptosis in breast cancer. J. Cell. Biochem. 118: 2285-2294, 2017. © 2017 Wiley Periodicals, Inc.

RACK1 stabilises the activity of PP2A to regulate the transformed phenotype in mammary epithelial cells

Conflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. RACK1 has been identified as a key regulator downstream of growth factor and adhesion signalling and as a direct binding partner of PP2A. Our objective was to further characterise the interaction between PP2A and RACK1 and to advance our understanding of this complex in breast cancer cells. We examined how the PP2A holoenzyme is assembled on the RACK1 scaffold in MCF-7 cells. We used immobilized peptide arrays representing the entire PP2A-catalytic subunit to identify candidate amino acids on the C subunit of PP2A that might be involved in binding of RACK1. We identified the RACK1 interaction sites on PP2A. Stable cell lines expressing PP2A with FR69/70AA, R214A and Y218F substitutions were generated and it was confirmed that the RACK1/PP2A interaction is essential to stabilise PP2A activity. We used Real-Time Cell Analysis and a series of assays to demonstrate that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of breast cancer cells and plays a role in maintenance of the cancer phenotype. This work has significantly advanced our understanding of the RACK1/PP2A complex and suggests a pro-carcinogenic role for the RACK1/PP2A interaction. This work suggests that approaches to target the RACK1/PP2A complex are a viable option to regulate PP2A activity and identifies a novel potential therapeutic target in the treatment of breast cancer.

Paxillin, a novel controller in the signaling of estrogen to FAK/N-WASP/Arp2/3 complex in breast cancer cells

Breast cancer is the major cause of cancer-related death in women. Its treatment is particularly difficult when metastasis occurs. The ability of cancer cells to move and invade the surrounding environment is the basis of local and distant metastasis. Cancer cells are able to remodel the actin cytoskeleton, which requires the recruitment of numerous structural and regulatory proteins that modulate actin filaments dynamics, including Paxillin or the Neural Wiskott-Aldrich Syndrome Protein (N-WASP). We show that 17-β estradiol (E2) induces phosphorylation of Paxillin and its translocation toward membrane sites where focal adhesion complexes are assembled. This cascade is triggered by a Gαi1/Gβ protein-dependent signaling of estrogen receptor α (ERα) to c-Src, focal adhesion kinase (FAK) and Paxillin. Within this complex, activated Paxillin recruits the small GTPase Cdc42, which triggers N-WASP phosphorylation. This results in the redistribution of Arp2/3 complexes at sites where membrane structures related to cell movement are formed. Recruitment of Paxillin, Cdc42 and N-WASP is necessary for cell adhesion, migration and invasion induced by E2 in breast cancer cells. In parallel, we investigated whether Raloxifene (RAL), a selective estrogen receptor modulator (SERMs), could inhibit or revert the effects of E2 in breast cancer cell movement. We found that, in the presence of E2, RAL acts as an ER antagonist and displays an inhibitory effect on estrogen-promoted cell adhesion and migration via FAK/Paxillin/N-WASP. Our findings identify an original mechanism through which estrogen regulates breast cancer cell motility and invasion via Paxillin. These results may have clinical relevance for the development of new therapeutic strategies for cancer treatment.

Abstract 3292: Epsin promotes breast cancer progression and metastasis by controlling nf-κb activation

Abstract Epsins are a family of endocytic clathrin adaptors. We have previously reported that epsin deficiency inhibits tumor growth by enhancing VEGF signaling in vascular endothelial cells. However, the pattern of tumor-intrinsic expression of epsin and its influence on cancer growth and metastasis is still unclear. Here, we demonstrate that epsin is upregulated in human cancers, including breast cancer, where its high expression correlates with poor relapse-free survival. Epsin promotes cell proliferation and invasion in vitro, and breast tumorigenesis and metastasis in vivo. Mechanistically, epsin interacts with and stabilizes TNFR1 signaling complex in breast cancer cells and enhances NF-κB signaling. Our findings suggest an important role of epsin in contributing to breast cancer progression and metastasis. Citation Format: Xiaofeng Cai, Hong Chen. Epsin promotes breast cancer progression and metastasis by controlling nf-κb activation. [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 3292. doi:10.1158/1538-7445.AM2014-3292

PDZ and LIM domain protein 1(PDLIM1)/CLP36 promotes breast cancer cell migration, invasion and metastasis through interaction with α-actinin

Increased CLP36 expression has been found to be closely associated with breast cancer progression. However, whether and how it contributes to malignant behavior of breast cancer cells were not known. We show here that CLP36 is critical for promoting breast cancer cell migration and invasion in vitro and metastasis in vivo, whereas it is dispensable for breast cell proliferation and anchorage independent growth in vitro and tumor growth in vivo. CLP36 interacted with both α-actinin-1 and -4 in breast cancer cells. Depletion of either α-actinin-1 or -4 inhibited breast cancer cell migration. Furthermore, mutations inhibiting the α-actinin-binding activity abolished the ability of CLP36 to promote breast cancer cell migration. Finally, depletion of CLP36 or disruption of the CLP36-α-actinin complex in breast cancer cells substantially inhibited Cdc42 activation, cell polarization and migration. Our results identify CLP36 as an important regulator of breast cancer cell migration and metastasis, and shed light on how increased CLP36 expression contributes to progression of breast cancer.

Tissue Factor as a Novel Target for Treatment of Breast Cancer

Tissue factor (TF), a 47-kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa), is expressed in several tumor types. TF has been shown to play a role in cell signaling, inflammation, angiogenesis, as well as tumor growth and metastasis. Activation of the TF signaling pathway has been implicated in mediating the function of many tumor cell types and has led to TF as a potential target in the treatment of several malignancies. Formation of the TF-FVIIa complex in breast cancer cells has been shown to exert an antiapoptotic effect and play a key role in tumor growth and metastasis. Breast cancer growth is suppressed by inhibition of TF-mediated PAR2 signaling, and deficiency in PAR2 delays spontaneous breast cancer development in mice. TF is expressed in triple-negative breast cancer (TNBC), an aggressive type of breast cancer in which there is currently a paucity of available targets. Various methods of targeting TF have been investigated and include immunoconjugates or icons, anti-TF antibodies, TF pathway inhibitors, targeted photodynamic therapy, and microRNAs. These investigations may give way to promising clinical therapies for breast cancer, especially in TNBC, for which there are relatively few effective treatment options.

The TCF4/β-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/β-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/β-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/β-catenin mRNA levels and activated TCF4/β-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/β-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and β-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/β-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/β-catenin complex activity.

Profilin1 facilitates staurosporine‐triggered apoptosis by stabilizing the integrin β1–actin complex in breast cancer cells

Profilin1 (Pfn1) functions as a tumour suppressor against malignant phenotypes of cancer cells. A minimum level of Pfn1 is critical for the differentiation of human epithelial cells, and its lower expression correlates with the tumourigenesis of breast cancer cells and tissues. However, the molecular mechanisms underlying its anti-tumour action remain largely unknown. In this study, we found that stable expression of ectopic Pfn1 sensitized the breast cancer cell line MDA-MB-468 to apoptosis induced by staurosporine, a widely used natural apoptosis-inducing agent. Pfn1 overexpression could also up-regulate the expression of integrin α5β1, which has been shown to inhibit the transformed phenotype of cancer cells. Furthermore, the Pfn1-facilitated apoptosis induced by staurosporine was blocked in cells attached to a supplementary fibronectin substrate, which serves as a ligand of integrin α5β1. These results suggest that the insufficient fibronectin caused by the integrin α5β1 up-regulation might activate a signalling pathway leading to an increase of cellular apoptosis. Moreover, Pfn1 that primarily functions to promote local superstructure formation involving actin filaments and integrin β1 may contribute to its promotion on apoptosis. Our study indicated a previously uncharacterized role of Pfn1 in mediating staurosporine-inducing apoptosis in breast cancer cells via up-regulating integrin α5β1, and suggested a new target for breast cancer therapy.

TWIST Represses Estrogen Receptor-alpha Expression by Recruiting the NuRD Protein Complex in Breast Cancer Cells

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.

Complex Formation and Interactions between Transcription Factors Essential for Human Prolactin Receptor Gene Transcription

The protein association of estrogen receptor α ERα with DNA-bound SP1 and C/EBPβ is essential for the 17β-estradiol (E2)-induced activation of human prolactin receptor (hPRLR) gene transcription. Protein-protein interaction and complex formation at the hPIII promoter of hPRLR was investigated. The basic region and leucine zipper (bZIP) of C/EBPβ, zinc finger (ZF) motifs of SP1, and the DNA binding domain of ERα were identified as regions responsible for the interactions between transfactors. The E2-induced interaction was confirmed by bioluminescence resonance energy transfer (BRET) assays of live cells. The combination of BRET/bimolecular luminescence complementation assay revealed that ERα exists as a constitutive homodimer, and E2 induced a change(s) in ERα homodimer conformation favorable for its association with C/EBPβ and SP1. Chromatin immunoprecipitation and small interfering RNA knockdown of members of the complex in breast cancer cells demonstrated the endogenous recruitment of components of the complex onto the hPIII promoter of the hPRLR gene. SP1 is the preferred transfactor for the recruitment of ERα to the complex that facilitates the C/EBPβ association. The E2/ERα-induced hPRLR transcription was demonstrated in ERα-negative breast cancer cells. This study indicates that the enhanced complex formation of ERα dimer with SP1 and C/EBPβ by E2 has an essential role in the transcriptional activation of the hPRLR gene.

Abstract 2174: Progesterone receptor, AP-1 and Stat3 cooperative transcriptional complex in breast cancer

Abstract We have previously demonstrated that progestin induces the rapid activation of AP-1 transcription factor and its nuclear interaction with the progesterone receptor (PR) by tethering in C4HD cells an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice and in the human breast cancer line T47D. In this work, we demonstrated that MPA induces AP-1 transcriptional activation through the activation of MAPKs signaling pathways. Our recent findings have demonstrated that MPA induces the recruitment of Stat3 and PR to the GAS site in the cyclin D1 promoter. Here, we identified a novel PR/AP-1/Stat3 transcriptional complex in breast cancer cells. Cyclin D1 is a cancer-related protein whose promoter contains AP-1 binding sites (TRE sites) and Stat3 binding sites (GAS sites) in a very close proximity and lacks progesterone response elements (PRE sites). By using quantitative chromatin immunoprecipitation (ChIP) and sequential ChIP, we demonstrated that progestin induces the recruitment of c-jun, c-fos and PR to the TRE site and this recruitment is abolished when we pretreat the cells with the MAPKs inhibitor U0126. When cells were transfected with c-jun and c-fos Dominant Negatives Forms (TAM-67 and A-Fos respectively) none of these proteins are able to activate cyclin D1 promoter or are recruited to the TRE site and there is not MPA-induced Stat3 recruitment to the GAS site. In the same way when we pre-treat the cells with siRNAs against Stat3 neither c-jun, c-fos, PR or Stat3 are recruited to the AP-1 or GAS siteS in the cyclin D1 promoter. Inhibition of MPA-induced AP-1 transcriptional activation by transfection of breast cancer cells with TAM-67 and A-Fos resulted in complete abrogation of progestin-induced in vitro and in vivo breast cancer growth. Our findings reveal a novel MPA-induced PR/AP-1/Stat3 cooperative transcriptional complex in breast cancer cells. Interestingly the blockage of any of the members of the complex prevents its assembly and inhibits the MPA-induced cyclin D1 up-regulation and cell proliferation. 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 2174. doi:10.1158/1538-7445.AM2011-2174

Breast Tumor Kinase Phosphorylates p190RhoGAP to Regulate Rho and Ras and Promote Breast Carcinoma Growth, Migration, and Invasion

Breast tumor kinase (Brk), an Src-like nonreceptor tyrosine kinase, is overexpressed in breast cancer and several other cancer types. Our previous study indicates that Brk promotes cell migration and tumor invasion by phosphorylating the focal adhesion protein paxillin. Here, we report the identification of p190RhoGAP-A (p190) as a Brk substrate. Brk phosphorylates p190 at the Y(1105) residue both in vitro and in vivo, thereby promoting the association of p190 with p120RasGAP (p120). As a consequence, Brk stimulates p190 and attenuates p120 functions, leading to RhoA inactivation and Ras activation, respectively. In carcinoma cells expressing high levels of Brk, endogenous Brk functions as a key contributor to epidermal growth factor-induced p190 tyrosine phosphorylation. We present evidence showing that p190 phosphorylation plays essential roles in both migratory and proliferative effects of Brk. Furthermore, disruption of p190 phosphorylation-induced p190/p120 complex in breast cancer cells abolishes not only the abilities of Brk to regulate RhoA and Ras but also the stimulatory effects of Brk on proliferation, migration, invasion, transformation, and tumorigenicity. Together, our findings reveal a previously unknown function of Brk in regulating both RhoA and Ras by phosphorylating p190 and provide evidence for the crucial roles of this Brk-elicited signaling pathway in promoting breast malignancy.