Breast Cancer Metastasis Suppressor Research Articles

Cancer-specific promoters for expression-targeted gene therapy: ran, brms1 and mcm5.

To expand the library of promoters that can be used for expression-targeted gene delivery to cancer cells, the specificity and strength of expression of three cancer-related gene promoters was evaluated: RAS-related nuclear protein ((P) ran), breast cancer metastasis suppressor 1 ((P) brms1) and minichromosome maintenance complex component 5 ((P) mcm5). The expression of reporter genes under the control of these promoters demonstrated selectivity in cancer cell lines of breast, prostate and ovarian origins versus a panel of normal cell types. The (P) ran was next used to regulate the expression of a bioactive exon (a constitutively active form of human caspase 3) to induce apoptosis in cancer cells. Further evaluation was performed in an orthotopic model of murine bladder cancer. The average strengths of reporter expression had relative intensities of 99.8% ((P) ran), 87.7% ((P) brms1) and 55.8% ((P) mcm5) versus the strong (P) cmv-driven positive control. Comparisons of expression-targeted reporter gene expression for these three promoters versus the clinically interesting promoter for the human telomerase reverse transcriptase gene ((P) hTERT) yielded an improvement of two- to 15-fold. Following transfection, cell death was evident from morphologic observations and viability assays performed on the cancer cells lines, with little (if any) effects seen when the same genes were delivered to normal cells. Cell viability was reduced by up to 60% after one treatment, with cell death via apoptosis implied by caspase 3 detection. During the in vivo preclinical study, reduced tumor burden, lack of mineralization and decreased inflammation were demonstrated after only three treatments. The ran, brms1, and mcm5 promoters have the specificity and strength needed for cancer-specific expression-targeted gene therapy. (p) ran in particular produced exciting results when coupled with a version of the caspase 3 exon to treat bladder cancer. Copyright © 2016 John Wiley & Sons, Ltd.

MHP-1 inhibits cancer metastasis and restores topotecan sensitivity via regulating epithelial-mesenchymal transition and TGF-β signaling in human breast cancer cells

BackgroundCordyceps has long been used to treat cancer. However, its pharmacologically active components as well as the molecular mechanisms underlying its effects are still unclear. PurposeTo investigate the effect of MHP-1, a newly isolated polysaccharide from Mortierella hepialid (the asexual structure of C. sinensis), on breast cancer metastasis. Study designThe effect of MHP-1 on breast cancer cell migration, epithelial-mesenchymal transition (EMT) and TGF-β signaling were investigated in vitro and in vivo. The effect of MHP-1 against topotecan-resistant MCF-7 cells that developed an EMT-like phenotype was also examined. MethodsThe in vitro effect of MHP-1 on breast cancer cell proliferation and migration was evaluated by CCK8 and transwell assay. Morphological changes were observed and EMT markers were detected by western blot. The production of MMPs was measured by quantitative PCR and ELISA assay. To further investigate the mechanism that MHP-1 inhibited breast cancer EMT, western blot, ELISA, luciferase reporter gene assay, siRNA, quantitative PCR, immunohistochemistry, and xenograft tumor model were performed. ResultsMHP-1 inhibited breast cancer cell migration but did not cause any cytotoxicity. MHP-1 significantly surpressed breast cancer EMT, and slightly decreased MMP-9 secretion. TGF-β signaling was selectively inhibited after MHP-1 treatment, and other EMT-related pathways, like Wnt and Notch, were not affected. MHP-1 reduced the secretion of TGF-β1, but rarely affected other EMT-induced cytokines. Dual luciferase assay and Smad2/3 phosphorylation analysis indicated that MHP-1 suppressed TGF-β signaling. We further showed that MHP-1 restored sensitivity in topotecan (TPT)-resistant MCF-7 cells that developed an EMT-like phenotype. Similarly, the effect of TPT on resistant MCF-7 cells was also increased either by ALK5 (TGFβRI) siRNA or by a small molecular inhibitor of ALK5, SB-431542. MHP-1 inhibited breast cancer metastasis in the MDA-MB-231 xenograft model, and the immunohistochemical staining showed dramatically decreased expression of ALK5 and vimentin, and increased expression of E-cadherin. ConclusionMHP-1 significantly inhibited breast cancer metastasis and restored drug sensitivity in TPT-resistant cells via down-regulation of TGF-β signaling and EMT program. The combination of non-toxic agents like MHP-1 and current anti-cancer drugs should be considered in the future treatment of cancer.

Decreased HCRP1 promotes breast cancer metastasis by enhancing EGFR phosphorylation

Previous study showed that hepatocellular carcinoma related protein 1 (HCRP1) is decreased in breast cancer. HCRP1 expression is inversely related to epithelial growth factor receptor (EGFR) in breast cancer tissues, and patients with breast cancer expressing lower HCRP1 tended to suffer a shorter life expectancy. However, the detailed biological functions of HCRP1 in breast cancer as well as the interaction between HCRP1 and EGFR remain unexplored. In this study, we examined HCRP1 expression in breast cancer tissues and cell lines by western blot. Thereafter, we performed transwell migration and matrigel invasion assays after siRNA interference and lentiviral vector of HCRP1 infection. To further investigate the interaction between HCRP1 downregulation and EGFR signaling pathway, we evaluated the phosphorylation status of EGFR, Erk1/2 and Akt by western blot following HCRP1-siRNA transfection. Moreover, we investigated the in vivo functions of HCRP1 using a breast cancer xenograft model. We found that HCRP1 depletion significantly promoted breast cancer migration and invasion while HCRP1 overexpression produced an opposite effect. In addition, HCRP1 depletion decreased EGFR degradation and enhanced phosphorylation of EGFR. Interestingly, HCRP1 depletion also led to insensitivity to EGFR inhibitors treatment. The in vivo experiment confirmed the metastasis inhibition function of HCRP1. The present data indicate that HCRP1 inhibits breast cancer metastasis through downregulating EGFR phosphorylation.

MicroRNA-320a inhibits breast cancer metastasis by targeting metadherin.

Dysregulated microRNAs play important pathological roles in carcinogenesis that are yet to be fully elucidated. This study was performed to investigate the biological functions of microRNA-320a (miR-320a) in breast cancer and the underlying mechanisms. Function analyses for cell proliferation, cell cycle, and cell invasion/migration, were conducted after miR-320a silencing and overexpression. The specific target genes of miR-320a were predicted by TargetScan algorithm and then determined by dual luciferase reporter assay and rescue experiment. The relationship between miR-320a and its target genes was explored in human breast cancer tissues. We found that miR-320a overexpression could inhibit breast cancer invasion and migration abilities in vitro, while miR-320a silencing could enhance that. In addition, miR-320a could suppress activity of 3′-untranslated region luciferase of metadherin (MTDH), a potent oncogene. The rescue experiment revealed that MTDH was a functional target of miR-320a. Moreover, we found that MTDH was negatively correlated with miR-320a expression, and it was related to clinical outcomes of breast cancer. Further xenograft experiment also showed that miR-320a could inhibit breast cancer metastasis in vivo. Our findings clearly demonstrate that miR-320a suppresses breast cancer metastasis by directly inhibiting MTDH expression. The present study provides a new insight into anti-oncogenic roles of miR-320a and suggests that miR-320a/MTDH pathway is a putative therapeutic target in breast cancer.

Cucurbitacin B inhibits breast cancer metastasis and angiogenesis through VEGF-mediated suppression of FAK/MMP-9 signaling axis

Available breast cancer therapeutic strategies largely target the primary tumor but are ineffective against tumor metastasis and angiogenesis. In our current study, we determined the effect of Cucurbitacin B (CuB), a plant triterpenoid, on the metastatic and angiogenic potential of breast cancer cells. CuB was found to inhibit cellular proliferation and induce apoptosis in breast cancer cells in a time- and dose-dependent manner. Further, CuB-treatment significantly inhibited the migratory and invasive potential of highly metastatic breast cancer MDA-MB-231 and 4T1 cells at sub-IC50 concentrations, where no significant apoptosis was observed. CuB was also found to inhibit migratory, invasive and tube-forming capacities of HUVECs in vitro. In addition, inhibition of pre-existing vasculature in chick embryo chorioallantoic membrane ex vivo further supports the anti-angiogenic effect of CuB. CuB-mediated anti-metastatic and anti-angiogenic effects were associated with the downregulation of VEGF/FAK/MMP-9 signaling, which has been validated by using FAK-inhibitor (FI-14). CuB-treatment resulted in a significant inhibition of VEGF-induced phosphorylation of FAK and MMP-9 expressions similar to the action of FI-14. CuB was also found to decrease the micro-vessel density as evidenced by the decreased expression of CD31, a marker for neovasculature. Further, CuB-treatment inhibited tumor growth, lung metastasis and angiogenesis in a highly metastatic 4T1-syngeneic mouse mammary cancer. Collectively, our findings suggest that CuB inhibited breast cancer metastasis and angiogenesis, at least in part, through the downregulation of VEGF/FAK/MMP-9 signaling.

Ferulic acid exerts antitumor activity and inhibits metastasis in breast cancer cells by regulating epithelial to mesenchymal transition.

Metastasis, which frequently occurs in breast cancer, is the major cause of mortality; therefore, new treatment strategies are urgently needed. Ferulic acid, isolated from Ferulafoetida, a perennial herb, has shown antineoplastic activity in various types of cancers, such as colon and lung cancer, and central nervous system tumors. However, its potential role in suppressing breast cancer metastasis has not been fully understood. In the present study, we evaluated the antitumor activity of ferulic acid in breast cancer cell line-based invitro and invivo models. We first showed that ferulic acid treatment resulted in decreased viability, increased apoptosis and suppression of metastatic potential in breast cancer cell line MDA-MB-231. Furthermore, it was demonstrated that the antitumor activity of ferulic acid and its role in suppressing metastasis were regulated by the reversal of epithelial-mesenchymal transition (EMT). Consistent with our findings invitro, the antitumor potential of ferulic acid was also verified in an MDA-MB-231 xenograft mouse model where significantly decreased tumor volume, weight and increased apoptosis were observed. Taken together, these results indicate that ferulic acid may be used as an effective therapeutic agent against breast cancer.

Addition of vasopressin synthetic analogue [V4Q5]dDAVP to standard chemotherapy enhances tumour growth inhibition and impairs metastatic spread in aggressive breast tumour models

[V(4)Q(5)]dDAVP is a novel 2nd generation vasopressin analogue with robust antitumour activity against metastatic breast cancer. We recently reported that, by acting on vasopressin V2r membrane receptor present in tumour cells and microvascular endothelium, [V(4)Q(5)]dDAVP inhibits angiogenesis and metastatic progression of the disease without overt toxicity. Despite chemotherapy remaining as a primary therapeutic option for aggressive breast cancer, its use is limited by low selectivity and associated adverse effects. In this regard, we evaluated potential combinational benefits by adding [V(4)Q(5)]dDAVP to standard-of-care chemotherapy. In vitro, combination of [V(4)Q(5)]dDAVP with sub-IC50 concentrations of paclitaxel or carmustine resulted in a cooperative inhibition of breast cancer cell growth in comparison to single-agent therapy. In vivo antitumour efficacy of [V(4)Q(5)]dDAVP addition to chemotherapy was first evaluated using the triple-negative MDA-MB-231 breast cancer xenograft model. Tumour-bearing mice were treated with i.v. injections of [V(4)Q(5)]dDAVP (0.3μg/kg, thrice weekly) in combination with weekly cycles of paclitaxel (10mg/kg i.p.). After 6weeks of treatment, combination regimen resulted in greater tumour growth inhibition compared to monotherapy. [V(4)Q(5)]dDAVP addition was also associated with reduction of local aggressiveness, and impairment of tumour invasion and infiltration of the skin. Benefits of combined therapy were confirmed in the hormone-independent and metastatic F3II breast cancer model by combining [V(4)Q(5)]dDAVP with carmustine (25mg/kg i.p.). Interestingly, [V(4)Q(5)]dDAVP plus cytotoxic agents severely impaired colony forming ability of tumour cells and inhibited breast cancer metastasis to lung. The present study shows that [V(4)Q(5)]dDAVP may complement conventional chemotherapy by modulating metastatic progression and early stages of microtumour establishment, and thus supports further preclinical testing of the compound for the management of aggressive breast cancer.

CK2α' Drives Lung Cancer Metastasis by Targeting BRMS1 Nuclear Export and Degradation.

Breast cancer metastasis suppressor 1 (BRMS1) is decreased in non-small cell lung cancer (NSCLC) and other solid tumors, and its loss correlates with increased metastases. We show that BRMS1 is posttranslationally regulated by TNF-induced casein kinase 2 catalytic subunit (CK2α') phosphorylation of nuclear BRMS1 on serine 30 (S30), resulting in 14-3-3ε-mediated nuclear exportation, increased BRMS1 cytosolic expression, and ubiquitin-proteasome-induced BRMS1 degradation. Using our in vivo orthotopic mouse model of lung cancer metastases, we found that mutation of S30 in BRMS1 or the use of the CK2-specific small-molecule inhibitor CX4945 abrogates CK2α'-induced cell migration and invasion and decreases NSCLC metastasis by 60-fold. Analysis of 160 human NSCLC specimens confirmed that tumor CK2α' and cytoplasmic BRMS1 expression levels are associated with increased tumor recurrence, metastatic foci, and reduced disease-free survival. Collectively, we identify a therapeutically exploitable posttranslational mechanism by which CK2α-mediated degradation of BRMS1 promotes metastases in lung cancer. Cancer Res; 76(9); 2675-86. ©2016 AACR.

Gallic acid abolishes the EGFR/Src/Akt/Erk-mediated expression of matrix metalloproteinase-9 in MCF-7 breast cancer cells

Several studies have revealed that natural compounds are valuable resources to develop novel agents against dysregulation of the EGF/EGFR-mediated matrix metalloproteinase-9 (MMP-9) expression in cancer cells. In view of the findings that EGF/EGFR-mediated MMP-9 expression is closely related to invasion and metastasis of breast cancer. To determine the beneficial effects of gallic acid on the suppression of breast cancer metastasis, we explored the effect of gallic acid on MMP-9 expression in EGF-treated MCF-7 breast cancer cells. Treatment with EGF up-regulated MMP-9 mRNA and protein levels in MCF-7 cells. EGF treatment induced phosphorylation of EGFR and elicited Src activation, subsequently promoting Akt/NFκB (p65) and ERK/c-Jun phosphorylation in MCF-7 cells. Activation of Akt/p65 and ERK/c-Jun was responsible for the MMP-9 up-regulation in EGF-treated cells. Gallic acid repressed the EGF-induced activation of EGFR and Src; furthermore, inactivation of Akt/p65 and ERK/c-Jun was a result of the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. Over-expression of constitutively active Akt and MEK1 or over-expression of constitutively active Src eradicated the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. A chromosome conformation capture assay showed that EGF induced a chromosomal loop formation in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun activation. Treatment with gallic acid, EGFR inhibitor, or Src inhibitor reduced DNA looping. Taken together, our data suggest that gallic acid inhibits the activation of EGFR/Src-mediated Akt and ERK, leading to reduced levels of p65/c-Jun-mediated DNA looping and thus inhibiting MMP-9 expression in EGF-treated MCF-7 cells.

BRMS1 and HPA as Progression, Clinical Biological Behaviors, and Poor Prognosis-related Biomarkers for Gallbladder Adenocarcinoma.

To study the expression of breast cancer metastasis suppressor 1 (BRMS1) and heparanase (HPA) and evaluate their clinicopathologic significance and relationship in benign and malignant lesions of the gallbladder. EnVision immunohistochemical method for determining the expression of BRMS1 and HPA was used in routinely paraffin-embedded sections of surgical resected specimens from gallbladder adenocarcinoma, peritumoral tissues, polyp, and chronic cholecystitis. The positive rate of BRMS1 expression was significantly lower in gallbladder adenocarcinoma than that in peritumoral tissues (P<0.01), polyp (P<0.01), and chronic cholecystitis (P<0.01). The positive rate of HPA expression was significantly higher in gallbladder adenocarcinoma than that in peritumoral tissues (P<0.01), polyp (P<0.05), and chronic cholecystitis (P<0.01). The positive expression of BRMS1 and negative expression of HPA were significantly associated with differentiation, lymph node metastasis, and invasion of adenocarcinoma (P<0.05 or P<0.01). Unitivariate Kaplan-Meier analysis showed that decreased expression of BRMS1 (P=0.008) or increased expression of HPA (P=0.016) was associated with decreased overall survival. Multivariate Cox regression analysis showed that decreased expression of BRMS1 (P=0.011) and/or increased expression of HPA (P=0.019) was an independent bad prognostic predictor in gallbladder adenocarcinoma. The expression of BRMS1 and/or HPA might be closely related to the carcinogenesis, clinical biological behaviors, and prognosis of gallbladder adenocarcinoma.

Baicalein suppresses metastasis of breast cancer cells by inhibiting EMT via downregulation of SATB1 and Wnt/&amp;beta;-catenin pathway

BackgroundThe flavonoid baicalein, a historically used Chinese herbal medicine, shows a wide range of biological and pharmaceutical effects, among which its potent antitumor activity has raised great interest in recent years. However, the molecular mechanism involved in the antimetastatic effect of baicalein remains poorly understood. This study aimed to verify the inhibitory effects of baicalein on metastasis of MDA-MB-231 human breast cancer cells both in vitro and in vivo, as well as to investigate the related mechanisms.MethodsMTT assay was used to examine the inhibition of baicalein on proliferation of MDA-MB-231 cells. Wound healing assay and the in vitro invasion assay was carried out to investigate the effects of baicalein on migration and invasion of MDA-MB-231 cells, respectively. In order to explore the effects of baicalein on tumor metastasis in vivo, xenograft nude mouse model of MDA-MB-231 cells was established. Animals were randomly divided into four groups (control, therapy group, and low-dose and high-dose prevention group, n=6), and treated with baicalein as designed. Following sacrifice, their lungs and livers were collected to examine the presence of metastases. qRT-PCR and Western blot were performed to study the effects of baicalein on expression of SATB1, EMT-related molecules, and Wnt/β-catenin signaling components of MDA-MB-231 cells as well as the metastatic tissue. Effects of baicalein on the expression of target proteins in vivo were also analyzed by immunohistochemistry.ResultsOur results indicated that baicalein suppressed proliferation, migration, and invasion of MDA-MB-231 cells in a time- and dose-dependent manner. Based on assays carried out in xenograft nude mouse model, we found that baicalein inhibited tumor metastasis in vivo. Furthermore, baicalein significantly decreased the expression of SATB1 in MDA-MB-231 cells. It suppressed the expression of vimentin and SNAIL while enhancing the expression of E-cadherin. Baicalein also downregulated the expression of Wnt1 and β-catenin proteins and transcription level of Wnt/β-catenin-targeted genes.ConclusionOur results demonstrate that baicalein has the potential to suppress breast cancer metastasis, possibly by inhibition of EMT, which may be attributed to downregulation of both SATB1 and the Wnt/β-catenin pathway. Taken together, baicalein may serve as a promising drug for metastasis treatment of breast cancer.

STARD13-correlated ceRNA network inhibits EMT and metastasis of breast cancer.

Competing endogenous RNAs (ceRNAs) network has been correlated with the initiation and development of cancer. Here, we identify CDH5, HOXD1, and HOXD10 as putative STARD13 ceRNAs and they display concordant patterns with STARD13 in different metastatic potential breast cancer cell lines and tissues. Notably, 3’UTRs of these genes suppress breast cancer metastasis via inhibiting epithelial-mesenchymal transition (EMT) in vitro and in vivo, which are activated through the crosstalk between STARD13 and its ceRNAs in 3’UTR- and miRNA-dependent manners. In addition, Kaplan-Meier survival analysis reveals that mRNA level of STARD13 and its ceRNAs is remarkably associated with survival of breast cancer patients. These results suggest that 3’UTRs of CDH5, HOXD1, and HOXD10 inhibit breast cancer metastasis via serving as STARD13 ceRNAs.

MiR-148a functions to suppress metastasis and serves as a prognostic indicator in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) presents a major challenge in the clinic due to its lack of reliable prognostic markers and targeted therapies. Accumulating evidence strongly supports the notion that microRNAs (miRNAs) are involved in tumorigenesis and could serve as biomarkers for diagnostic purposes. To identify miRNAs that functionally suppress metastasis of TNBC, we employed a concerted approach with selecting miRNAs that display differential expression profiles from bioinformatic analyses of breast cancer patient databases and validating top candidates with functional assays using breast cancer cell lines and mouse models. We have found that miR-148a exhibits properties as a tumor suppressor as its expression is inversely correlated with the ability of both human and mouse breast cancer cells to colonize the lung in mouse xenograft tumor models. Mechanistically, miR-148a appears to suppress the extravasation process of cancer cells, likely by targeting two genes WNT1 and NRP1 in a cell non-autonomous manner. Importantly, lower expression of miR-148a is detected in higher-grade tumor samples and correlated with increased likelihood to develop metastases and poor prognosis in subsets of breast cancer patients, particularly those with TNBC. Thus, miR-148a is functionally defined as a suppressor of breast cancer metastasis and may serve as a prognostic biomarker for this disease.

RAGE: A novel target for breast cancer growth and metastasis.

Metastasis is a major cause of mortality in Breast Cancer (BC) patients in part due to lack of clinically established targeted therapies. Among the different types of BC, triple negative BC (TNBC) (ER-, PR-, HER2-) has been associated the most with poor prognosis and survival due to early metastasis to other organs and a lack of clinically established targeted therapies. Hence, elucidating novel mechanisms that regulate metastasis would lead to the development of targeted therapies and new treatments for TNBC and metastatic breast cancers. It is now well accepted that solid tumors, including those in the breast, have an inflammatory microenvironment. Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules which has been associated with chronic inflammation, which in turn enhances the progression of various cancers [1]. We have shown that RAGE is expressed in a panel of aggressive BC cell lines and TNBC tissues [3]. High RAGE expression was also observed in lymph node and distant metastases patient samples. In addition, we observed that high RAGE expression was associated with poor prognosis in breast cancer [3]. RAGE has also been shown to play important role in various cancers including pancreatic cancer. Under hypoxic environment, RAGE was shown to interact directly to mutant KRAS and upregulate HIF1α that leads to the development of pancreatic cancer [2]. We have shown that RAGE deficiency inhibits the growth of murine breast cancer tumor cells [3]. RAGEdeficient mice have shown less chemically-induced inflammation [1]. Furthermore, we have shown that RAGE neutralizing antibody treatment significantly inhibited breast cancer metastasis in an intracardial mouse model [3]. RAGE is a multi-ligand receptor and binds to several inflammatory ligands such as advanced glycation end products (AGE), high mobility group box 1 peptide (HMGB-1), amyloid-β peptide and the S100 family of proteins. Our mechanistic investigation has revealed that RAGE mediates its functional effects in breast cancer by binding to S100A7 [3]. S100A7 is a small molecular weight calcium-binding protein [4]. Although a number of putative functions have been proposed for S100A7, its biological role, particularly in BC, remains to be defined [4]. Phylogenetic analyses have shown the mouse ancestor mS100a7a15 to be most related to human S100A7 [5]. It has been shown that mS100a7a15 is up-regulated during carcinogen-induced mammary tumorigenesis. However, the direct functional role of mS100a7a15 in disease progression is not well-characterized. We have shown that mS100a7a15 overexpression induced hyperplasia in mammary glands of these transgenic mice [5]. Upon binding to ligands, RAGE activates its downstream signaling mechanisms that augment and maintain chronic inflammatory conditions [1]. We have also shown that S100A7 enhances NF-kB activation and its nuclear translocation in TNBC cells. These features of RAGE make it an ideal candidate for therapeutic strategies against inflammation induced cancers. The link between inflammation and cancer has been postulated for a long time because of the presence of inflammatory cells in the tumor microenvironment, including myeloid cells and tumor associated macrophages (TAMs) [6]. These cells have been shown to significantly enhance tumor growth and the metastasis of various tumors and are key inhibitors of anti-tumor immunity [6]. In addition, collaborative interactions of tumor cells with TAMs have been associated with poor prognosis in BC [6]. TAMs have also been shown to play an important role in enhancing metastasis and drug resistance [6]. Therefore, inhibition of TAM accumulation and recruitment is a promising strategy for enhancing the effectiveness of immune-based therapies against tumors [6]. We and others have shown that RAGE is also expressed on immune cells [3, 5, 7]. RAGE expression was detected in different cell types present within the tumor microenvironment including macrophages. In addition, RAGE has been shown to be present in the microglia in the gliomas. Chen et al (2014) have shown that RAGE signaling in glioma TAMs regulates angiogenesis most likely by MMP9 [8]. We also showed in breast cancer mouse model that blocking of RAGE signaling significantly inhibited tumor growth, metastasis and angiogenesis through inhibition of MMP9+ TAMs [3]. In addition, RAGE also binds to S100A8/A9 to recruit myeloid derived suppressor cells (MDSCs) and thereby enhance cancer growth and metastasis [7]. These studies suggest that RAGE could enhance tumor growth and metastasis through regulating tumor microenvironment. Overall, our results indicate that RAGE could be used as a novel therapeutic target and biomarker for TNBCs. Furthermore RAGE/S100A7 signaling through paracrine and endocrine mechanisms in epithelial as well as TAMs plays an important role in linking inflammation to breast cancer development and metastasis (Fig. ​(Fig.1).1). In addition, RAGE also binds to S100A8/A9 to recruit myeloid derived suppressor cells (MDSCs) and thereby enhance breast cancer growth and metastasis. Figure 1 Role of S100A7/RAGE-signaling axis in breast cancer growth and metastasis

Muscleblind-like 1 suppresses breast cancer metastatic colonization and stabilizes metastasis suppressor transcripts.

Post-transcriptional deregulation is a defining feature of metastatic cancer. While many microRNAs have been implicated as regulators of metastatic progression, less is known about the roles and mechanisms of RNA-binding proteins in this process. We identified muscleblind-like 1 (MBNL1), a gene implicated in myotonic dystrophy, as a robust suppressor of multiorgan breast cancer metastasis. MBNL1 binds the 3' untranslated regions (UTRs) of DBNL (drebrin-like protein) and TACC1 (transforming acidic coiled-coil containing protein 1)-two genes that we implicate as metastasis suppressors. By enhancing the stability of these genes' transcripts, MBNL1 suppresses cell invasiveness. Consistent with these findings, elevated MBNL1 expression in human breast tumors is associated with reduced metastatic relapse likelihood. Our findings delineate a post-transcriptional network that governs breast cancer metastasis through RNA-binding protein-mediated transcript stabilization.

Abstract P1-06-03: Epithelial to mesenchymal transition (EMT) regulates the spontaneous generation of GD2+ breast cancer stem-like cells through NFκB activation

Abstract Breast cancer recurrence may be a consequence of persistent breast cancer stem-like cells (BCSCs) that survive chemo- or hormonal therapy. Therefore, targeting BCSCs could complement standard chemotherapy. We discovered that the ganglioside GD2 is expressed on and defines BCSCs (Battula et al., JCI, 2012), as consequence of activation of the enzyme GD3 synthase (GD3S). Inhibition of GD3S expression inhibited breast cancer metastasis to lung. We also observed that GD2- breast cancer cells spontaneously generate GD2+ cells in vitro. As induction of EMT generates a stem cell–like phenotype, we hypothesized that EMT regulates the generation of GD2+ breast cancer cells. To test this hypothesis, MDA-MB-231and SUM159 cells were cultured in vitro and the percentage of GD2+ cells was measured over time. Interestingly, the percentage and absolute number of GD2+ cells increased in a time-dependent manner, suggesting the spontaneous generation of GD2+ cells. Concomitantly, mesenchymal-related markers including vimentin, N-cadherin, and twist increased 3 to 6 - fold. To further investigate whether this process is operational in vivo, GFP+ MDA-MB-231 cells were transplanted into mammary fat pads of NOD/SCID mice. Each week, a group of mice was sacrificed, tumors were extracted and the number of GFP+GD2+ cells was determined by flow cytometry. In line with our in vitro results, we observed significant increases in GD2+ BCSCs with increasing tumor volume from 15.1%±4.6% to 37%±8.7% over a 6 week period, suggesting that breast cancer cells spontaneously undergo EMT during tumor progression and generate GD2+ BCSCs. To identify possible targets to inhibit EMT in breast cancer cells, proteomic analysis using Kinexus® antibody arrays revealed activation of NFκB and focal adhesion kinase (FAK) signaling in GD2+ breast cancer cells. The activation of NFκB (phospho p65) in GD2+ cells was validated by CyTOF mass cytometry using metal tagged antibodies. These data suggest that inhibition of NFκB signaling may inhibit GD2+ BCSC growth. Indeed, the IKK inhibitor BMS345541 reduced GD2+ cells by &amp;gt;95% and inhibited GD3S expression (determined by qRT-PCR) in a dose- and time-dependent fashion. In contrast, treatment with doxorubicin increased the percentage of GD2+ cells, from 13.5±2.5% to 21±2.6% in MDA-MB-231 cells, suggesting that GD2+ cells are resistant to doxorubicin. In addition, treatment with BMS345541 inhibited the ability of breast cancer cells to form mammospheres by &amp;gt;90% in vitro. In-vivo tumorigenesis assay demonstrated that BMS345541 induced a significant decrease (p &amp;lt;0.01) in tumor volume, and increased survival of tumor bearing mice: median survival was 78 days for BMS345541-treated mice vs. 58 days for controls (p&amp;lt;0.002). Conclusion: GD2+ BCSCs are spontaneously produced during tumor progression by EMT and NFκB and FAK mediated signaling might regulate this process. Inhibition of NFκB and FAK signaling pathways may inhibit the spread of BCSCs and reduce breast cancer metastases. Citation Format: Battula VL, Sun J, Nguyen K, Hortobagyi G, Andreeff M. Epithelial to mesenchymal transition (EMT) regulates the spontaneous generation of GD2+ breast cancer stem-like cells through NFκB activation. [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 P1-06-03.

Abstract A72: The E3-ligase E6AP represses breast cancer metastasis through regulation of ECT2-Rho-GTPases signaling

Abstract Metastatic disease is the major cause of breast cancer related death and despite many advances, current therapies are rarely curative. Tumor cell migration and invasion require actin cytoskeletal reorganisation, to endow cells with capacity to disseminate and initiate the formation of secondary tumors. However, it is still unclear how these migratory cells colonise distant tissues to form macrometastases. The E6-associated protein, E6AP, acts both as an E3 ubiquitin-protein ligase and as a coactivator of steroid hormone receptors. We report that E6AP suppresses breast cancer invasiveness, colonisation and metastasis in mice and in breast cancer patients, loss of E6AP associates with poor prognosis, particularly for with basal breast cancer. E6AP regulates actin cytoskeletal remodelling via regulation of Rho-GTPases, acting as a negative regulator of ECT2, a GEF required for activation of Rho-GTPases. E6AP promotes ubiquitination and proteasomal degradation of ECT2 for which high expression predicts poor prognosis in breast cancer patients. We conclude that E6AP suppresses breast cancer metastasis by regulating actin cytoskeleton remodelling through the control of ECT2 and Rho-GTPase activity. These findings establish E6AP as a novel suppressor of metastasis and provide a compelling rationale for inhibition of ECT2 as a therapeutic approach for patients with metastatic breast cancer. Note: This abstract was not presented at the conference. Citation Format: Mariam Mansour, Sue Haupt, Ai-Leen Chan, Nathan Godde, Alexandra Rizzitelli, Sherene Loi, Franco Caramia, Siddartha Deb, Elena A. Takano, Mark Bishton, Cameron Johnstone, Yarra Levav-Cohen, Yong-Hui Jiang, Alpha S. Yap, Stephen Fox, Ora Bernard, Robin Anderson, Ygal Haupt. The E3-ligase E6AP represses breast cancer metastasis through regulation of ECT2-Rho-GTPases signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A72.

Abstract B29: Anti-breast cancer effects of the Vav/Rac inhibitor Ehop-016 in the tumor microenvironment

Abstract Metastatic breast cancer still lacks effective treatment, and remains the primary cause of mortality. Therefore, new effective strategies to inhibit breast cancer metastasis are needed. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that can promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activation by the oncogene Vav in metastatic cancer cells with an IC50 of 1 microM. Consequently, EHop-016 inhibits the activity of the Rac downstream effector p21-activated kinase (PAK) and cell migration in metastatic breast cancer cells; and significantly reduces tumor growth, metastasis, and angiogenesis in a mouse model of metastatic breast cancer. The reduced tumor angiogenesis in response to EHop-016 treatment may be attributed to the inhibition of the Vav/Rac activity of endothelial cells, as shown by the ability of EHop-016 to inhibit the Rac activation and capillary tube formation of endothelial cells, in vitro. Recent studies have also shown a pivotal role for tumor associated stromal cells in signaling to the cancer cells to leave the primary tumor to initiate metastasis. Because Ehop-016 acts as a specific inhibitor of the Vav/Rac interaction, we hypothesize that EHop-016 inhibits the Vav/Rac signaling in not only cancer cells, but also in fibroblasts and leukocytes. To determine whether EHop-016 treatment affects the tumor microenvironment (TME), athymic nude mice bearing mammary fatpad tumors were treated with vehicle or 30 mg/kg BW EHop-016 by IP, 3X a week, for 52 days. Immunofluorescence microscopy of mammary tumor tissue sections demonstrated a statistically significant 40% decrease in Ki-67 expression, a 60% decrease in capillary tubes as quantified by CD-31 staining, and a 33% decrease in F4/80 positive macrophages in the tumors from mice following EHop-016 treatment, compared to those treated with vehicle. To investigate the effect of EHop-016 on the cytokine milieu, plasma recovered from the mice in this study were subjected to a cytokine array for common inflammatory mediators. EHop-016 treatment demonstrated a statistically significant decrease in mouse plasma levels of the major inflammatory mediator interleukin-6 (IL-6) that has been implicated with cancer progression in the TME, as well as a trend in reduced monocyte chemoattractant protein and tumor necrosis factor alpha. A PCR array analysis of the mammary tumors from this study also demonstrated a decrease in IL-6 levels in tumor tissue. Reduced macrophages and IL-6 signaling in the TME, in response to EHop-016 treatment, is expected to decrease the cytokine-mediated malignant signaling cross talk between cancer cells and stromal cells. Therefore, inhibition of Vav/Rac signaling in the TME may additionally enhance the anti-metastatic effects of EHop-016. This study was supported by National Institute on Minority Health and Health Disparities of the National Institutes of Health (NIMHHD/NIH) U54MD008149 and the Puerto Rico Science and Technology Trust to SD; Title V PPOHA P031M10505 and Title V Cooperative P031S130068 from U.S. Department of Education, and RCMI 8G12MD007583 to UCC; and UPR RCM NIH/NIMHHD grants 5U54CA096297 andR25GM061838 to THB. Citation Format: Linette Castillo-Pichardo, Cristina Del Valle, Jessica Morales, Valance Washington, Krizia Rohena-Rivera, Magaly Martinez, Luis Cubano, Suranganie Dharmawardhane. Anti-breast cancer effects of the Vav/Rac inhibitor Ehop-016 in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B29.

ERα Mediates Estrogen-Induced Expression of the Breast Cancer Metastasis Suppressor Gene BRMS1.

Recently, estrogen has been reported as putatively inhibiting cancer cell invasion and motility. This information is in direct contrast to the paradigm of estrogen as a tumor promoter. However, data suggests that the effects of estrogen are modulated by the receptor isoform with which it interacts. In order to gain a clearer understanding of the role of estrogen in potentially suppressing breast cancer metastasis, we investigated the regulation of estrogen and its receptor on the downstream target gene, breast cancer metastasis suppressor 1 (BRMS1) in MCF-7, SKBR3, TTU-1 and MDA-MB-231 breast cancer cells. Our results showed that estrogen increased the transcription and expression of BRMS1 in the ERα positive breast cancer cell line, MCF-7. Additionally, the ERα specific agonist PPT also induced the transcription and expression of BRMS1. However, the two remaining estrogen receptor (ER) subtype agonists had no effect on BRMS1 expression. In order to further examine the influence of ERα on BRMS1 expression, ERα expression was knocked down using siRNA (siERα). Western blot analysis showed that siERα reduced estrogen-induced and PPT-induced BRMS1 expression. In summary, this study demonstrates estrogen, via its α receptor, positively regulates the expression of BRMS1, providing new insight into a potential inhibitory effect of estrogen on metastasis suppression.

Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers

BackgroundCREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer.MethodsQuantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis.ResultsThis study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression.ConclusionsOur data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0672-x) contains supplementary material, which is available to authorized users.