Tumor-suppressive functions of 4-MU on breast cancer cells of different ER status: Regulation of hyaluronan/HAS2/CD44 and specific matrix effectors
Theodoros T. Karalisa, Paraskevi Heldinb, Demitrios H. Vyniosa, Thomas Neillc, Simone Buraschic, Renato V. Iozzoc, Nikos K. Karamanosa, Spyros S. Skandalisa, *
a- Biochemistry, Biochemical Analysis & Matrix Pathobiology Res. Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26110 Patras, Greece
b- Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582 SE- 751 23 Uppsala, Sweden
c- Department of Pathology, Anatomy, and Cell Biology and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, United States
*Correspondence to Dr. Spyros S. Skandalis: [email protected]
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Highlights
Enhanced hyaluronan synthesis promotes the malignant phenotype of various cancers,
including breast cancer
4-MU inhibits hyaluronan synthesis and deposition in ECM as well as within breast cancer
cells, especially in cells lacking ERα
4-MU modulates the expression of HAS2 and HYALs -1, -2, and induces the substantial
loss of hyaluronan receptor CD44 from cell protrusions
Differential effects are evoked by 4-MU in breast cancer cell functional properties
depending on the absence or presence of ERα
4-MU opposes breast cancer progression by regulating matrix-degrading enzymes and
inflammatory mediators with tumor-promoting functions
4-MU could represent a promising therapeutic candidate for specific breast cancer
subtypes with regard to their ER status
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Abstract
The malignant phenotype of various cancers is linked to enhanced expression of hyaluronan, a pro-angiogenic glycosaminoglycan whose expression is suppressed by 4-methylumbelliferone (4-MU), a non-toxic oral agent used as a dietary supplement to improve health and combat prostate cancer. However, little is known about the effects of hyaluronan inhibition by 4-MU on breast cancer cells. In this study, we investigated the role of 4-MU in mammary carcinoma cells with distinct malignant phenotypes and estrogen receptor (ER) status, a major prognostic factor in the clinical management of breast cancers. We focused on two breast cancer cell lines, the low metastatic and ER+ MCF-7 cells, and the highly-aggressive and ER- MDA-MB-231 cells. Treatment with 4-MU caused a dose-dependent decrease of hyaluronan accumulation in the extracellular matrix as well as within the breast cancer cells, most prevalent in cells lacking ER. This decrease in hyaluronan was accompanied by suppression of Hyaluronan Synthase 2 (HAS2), the major enzyme responsible for the synthesis of hyaluronan, and by induction of hyaluronidases (HYALs) -1 and -2. Moreover, 4-MU induced intense phenotypic changes and substantial loss of CD44, a major hyaluronan receptor, from cell protrusions. Importantly, 4-MU evoked differential effects depending on the absence or presence of ER. Only the ER+ cells showed signs of apoptosis, as determined by cleaved PARP-1, and anoikis as shown by concurrent loss of E-cadherin and β-catenin. Interestingly, 4-MU significantly reduced migration, adhesion and invasion of ER- breast cancer cells, and concurrently reduced the expression and activity of several matrix degrading enzymes and pro-inflammatory molecules with tumor- promoting functions. Collectively, our findings suggest that 4-MU could represent a novel therapeutic for specific breast cancer subtypes with regard to their ER status via suppression of hyaluronan synthesis and regulation of HAS2, CD44, matrix-degrading enzymes and inflammatory mediators.
Introduction
Breast cancer is the most frequently diagnosed cancer in women characterized by high genotypic and phenotypic diversity. Estrogen receptor (ER) status is the most important discriminator of breast cancers, which are divided into two major categories: ER-positive and ER- negative depending on the expression of ERα [1]. Interestingly, ERα, PgR and HER2 are the three mandatory prognostic and predictive factors in breast cancer currently used in clinical practice and drive treatment decisions [2]. With regard to the tumor microenvironment, breast cancers can be classified on the basis of the differential expression of extracellular matrices (ECMs) giving rise to four ECM subtypes, which are associated with different clinical outcomes [3, 4].
Hyaluronan, a prominent component of ECMs, is a linear glycosaminoglycan exhibiting a simple structure but extremely complex functions. Hyaluronan is a ubiquitous component of connective, epithelial and neural tissues found predominately in the extracellular space but also intracellularly [5, 6]. It is produced by three membrane-associated independently regulated hyaluronan synthases (HAS1, 2 and 3), which synthesize hyaluronan molecules of distinct molecular sizes by repeatedly adding glucuronic acid (GlcUA) and N-acetyl-glucosamine (GlcNAc) to the nascent polysaccharide as it is extruded into the ECMs [7]. On the other hand, hyaluronan is degraded by endogenous hyaluronidases (HYAL -1, -2, -3, and PH-20), mechanical forces, as well as by oxidative stress (reactive oxygen species-mediated cleavage). Hyaluronan catabolism results in the generation of bioactive oligomers/fragments giving rise to contrasting size-dependent functions of hyaluronan [8, 9].
Hyaluronan plays crucial roles in the onset and progression of malignant tumors, including breast cancer [3, 10, 11]. The contribution of hyaluronan to tumor progression relies on its metabolism, including the regulation of HASs and HYALs that define the amount and size of hyaluronan molecules, and on the binding of hyaluronan to its cellular receptors, mainly CD44 and RHAMM, activating various oncogenic signaling pathways and modulating cellular functions. Moreover, hyaluronan-CD44 interactions play a role in both maintaining proper stem cell niches and in the expansion of cancer-initiating cells [6, 12-19].
HASs can be regulated by multiple mechanisms, such as ubiquitination, O-GlcNAcylation, phosphorylation as well as epigenetic mechanisms, thus affecting hyaluronan production. As a consequence, alterations in hyaluronan size and amounts have substantial impact on various cellular functions like migration, proliferation and differentiation, but also cell invasion and epithelial-to-mesenchymal transition (EMT). The latter function regulates both physiological and pathological processes including angiogenesis, inflammation and cancer [20-31]. The multiple contributions of hyaluronan to inflammation, autoimmunity, and tumor growth and metastasis, have generated great interest in identifying pharmacologic tools that would inhibit hyaluronan synthesis and accumulation. One agent that has received much attention is 4- methylumbelliferone (4-MU), which is a coumarin derivative that primarily acts through regulation
of UDP-glucose dehydrogenase (UGDH), an enzyme required for both hyaluronan and sulfated- glycosaminoglycan production [32].
Notably, 4-MU is an already approved oral drug and it has been used to treat biliary spasm [32] and more recently to treat prostate cancers in preclinical studies [33].
4-MU can inhibit hyaluronan synthesis dose-dependently in both normal and cancer cells [34- 37]. Two possible mechanisms have been proposed for the 4-MU-mediated inhibition of hyaluronan. First, 4-MU reduces the expression of HASs but the exact underlying mechanisms remain unclear. Second, 4-MU functions as a competitive substrate for UDP- glucuronosyltransferase (UGT), an enzyme involved in the generation of the UDP-precursors needed for hyaluronan synthesis, by covalently binding to GlcUA. Consequently, UDP-GlcUA concentration declines in the cytosol resulting in reduced hyaluronan synthesis [38].
Little is known on the effects of hyaluronan inhibition by 4-MU in breast cancer cells [36, 39-41]. Thus, we have investigated the role of 4-MU on two breast cancer cell models of distinct ER status: the MCF-7/ER+ cells with low metastatic potential and the highly-aggressive MDA-MB-231/ER- cells. Our findings suggest that 4-MU might represent a promising therapeutic candidate for specific breast cancer subtypes with regard to their ER status, a major classification and predictive marker in breast cancers, via suppression of hyaluronan/HAS2/CD44, and regulation of matrix-degrading enzymes and inflammatory mediators.
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Results
Inhibition of hyaluronan synthesis by 4-MU in breast cancer cells
Hyaluronan synthesis is induced not only by the cross-talk between stromal and tumor cells, but also by the tumor cells themselves promoting their aggressive potential [42-46]. In previous studies, silencing of HAS2 suppressed the malignant phenotype of invasive breast cancer cells both in anchorage-dependent and anchorage-independent cultures, while antisense inhibition of HAS2 in MDA-MB-231 cells inhibited the in vivo formation of tumors [47, 48]. To explore the effects of 4-MU on hyaluronan production, we quantified total hyaluronan released by the two ER+ and ER- cells following a 24-h incubation with increasing concentrations of 4-MU. The results showed that ER+ cells synthesized nearly undetectable levels of hyaluronan, and thus there was no appreciable effect by the drug (Fig. 1A). In contrast, the ER- cells showed a robust production of hyaluronan under basal conditions and a significant dose-dependent inhibition of hyaluronan by 4-MU. Regarding the intracellular hyaluronan synthesis and accumulation, immunofluorescence analysis of MCF-7 cells revealed a weak intracellular and cell membrane- associated staining for hyaluronan that was further reduced upon 4-MU treatment. On the other hand, a strong cell-associated staining for hyaluronan was observed for MDA-MB-231 cells, which was substantially reduced in the presence of increasing concentrations of 4-MU in a dose- dependent manner (Fig. 1B).
4-MU modulates actin cytoskeleton and CD44 expression in breast cancer cells
The significant reduction in hyaluronan content, especially intracellularly, prompted us to examine the distribution and relative expression of CD44, the main hyaluronan receptor, upon 4- MU treatment. We used a combination of antibodies against CD44 and Phalloidin-iFluor488 to visualize filamentous actin (F-actin). We found that increasing concentrations of 4-MU evoked changes in the ER+ cells. Specifically, 4-MU caused a significant reduction and disorganization in actin cytoskeleton and overall cell morphology (Fig. 2A). Notably, the ER+ showed minimal and patchy expression of CD44 with no significant differences between control and 4-MU-treated cells (Fig. 2A). In ER- cells, 4-MU also induced disorganization of the actin cytoskeleton (Fig. 2B). However, and in marked contrast to the cells expressing ER the ER- cells showed markedly-elevated immunoreactivity for the hyaluronan receptor CD44 (Fig. 2B). Under baseline conditions, CD44 clustered to the leading edges of the tumor cells (arrows, Fig. 2B). Notably, CD44 signal remained strong in 4-MU-treated cells, but appeared more diffuse in the cytosol (Fig. 2B).
Collectively, these results suggest that 4-MU suppresses hyaluronan synthesis and secretion in the more aggressive/ER- cells with a concurrent re-distribution of CD44 but without significantly affecting the levels of the hyaluronan receptor.
4-MU inhibits breast cancer cell migration
To evaluate the effects of 4-MU treatment on cell properties, we performed wound-healing assays with increasing concentrations of 4-MU. Notably, the migratory potential of ER cells was not appreciably affected (Fig. 3A). In contrast, the ER- cells showed a dose-dependent inhibition of migration (p<0.001, Fig. 3B), which reached its highest levels within 24 h after 4- MU treatment and maintained at these levels until 72 h.
Next, we performed immunofluorescence analyses on the ER cells using anti-CD44 and Phalloidin-iFluor488 (Fig. 3C). We choose 24 h, a time where the maximal inhibition of migration was observed, especially at higher 4-MU concentrations (Fig. 3B). We found actin-rich cell membrane protrusions in the vehicle-treated ER cells (arrowheads, Fig. 3C). Notably, 4-MU (1 mM) caused perturbation of the actin cytoskeleton and a substantial loss of CD44 from cell protrusions (Fig. 3C).
To further investigate these observations, we performed similar experiments using ERcells in the presence or absence of Hermes-1 mAb, which specifically blocks hyaluronan-CD44 interactions [49], or exogenous high molecular weight (HMW) hyaluronan. We discovered that Hermes-1 significantly reduced cell migration but to a lesser extent than 4-MU (~30% and ~57%, respectively), compared to vehicle (DMSO)-treated cells (Fig. 3D). Combination of Hermes-1 and 4-MU further reduced cell motility to ~75% of (Fig. 3D), suggesting a role for CD44 as well as its interactions with newly synthesized hyaluronan in 4-MU-mediated changes in cell motility. Notably, exogenous HMW hyaluronan did not rescue the suppressed migration evoked by either 4-MU alone or in combination with Hermes-1 (Fig. 3D). These findings suggest that cell associated hyaluronan bound to CD44 rather than extracellular hyaluronan would favor migration-related signals in ER cells.
Finally, although Hermes-1 alone slightly affected the production and secretion of hyaluronan, the co-treatment with 1 mM 4-MU further reduced secreted hyaluronan by ~54%, vis-a-vis 4-MU-treated cells (~40%) (Fig. 3E). These results suggest an involvement of CD44 in the synthesis and/or extrusion of hyaluronan from the ER- breast cancer cells.
4-MU inhibits breast cancer cell invasion and enhances cell adhesion
To investigate the role of 4-MU on cancer invasion, we performed cell invasion assays through collagen Type I matrix. We found that ER cells minimally invaded the collagen matrix and that only the highest concentration of 4-MU (2 mM) efficiently suppressed cell invasiveness (Fig. 4A) as well as the total invaded area (Fig. 4B). In contrast, the ER cells exhibited a much higher invasive capacity than ER cells (p<0.001, Fig. 4A) and a much more robust inhibition of invasiveness (Fig. 4A) and of total invaded area (Fig 4B) at all concentrations of 4-MU. These results suggest that the newly-synthesized HA may promote breast cancer cell invasiveness.
To evaluate the role of hyaluronan-CD44 interactions in cell invasion, we incubated ER
cells with Hermes-1 mAb in the presence and absence of 1 mM 4-MU. We discovered that Hermes-1 caused the highest reduction (~66%) in cell invasion (Fig. 4C) and invaded area (Fig. 4D), but their combination did not result in any additional effect. These data suggest a crucial role for CD44 and newly synthesized hyaluronan in evoking breast cancer cell invasion.
Given the significant inhibitory effects of 4-MU and Hermes-1 mAb on the invasiveness of ER cells, we next examined their adhesiveness to collagen Type I matrices. Exposure to either 4-MU or Hermes-1 alone significantly induced cell adhesion (p<0.01, Fig. 5A), which was further enhanced by combining the two effectors (p<0.001, Fig. 5A). These findings suggest that breast cancer cell adhesiveness involves interactions between CD44 and the newly synthesized hyaluronan pericellular coat.
Immunofluorescence analysis of the cells grown on collagen Type I matrices showed that 4- MU evoked cell rounding and actin cytoskeleton reorganization in a dose-dependent manner (Fig. 5B, Supplementary Fig. 1A). Treatment with Hermes-1 mAb or in combination with 4-MU further enhanced these changes characterized by the intense presence of filamentous actin at cell periphery (arrows, Fig. 5B). In addition, treatment of cells with either 4-MU or Hermes-1 or both showed a remarkable redistribution of CD44, which was found condensed in the perinuclear zone (Fig. 5C, Supplementary Fig. 1B). Collectively, these results indicate that 4-MU and Hermes-1 critically affect the invasive potential and adhesiveness of ER cells to collagen Type I matrices in a similar but opposite manner as both strongly inhibit cell invasion while promoting cell adhesion.
Differential effect of 4-MU on breast cancer cell viability and proliferation
Next, we tested whether 4-MU would also affect cell viability and proliferation. To this end, we exposed both ER and ER cells to increasing concentrations of 4-MU for different time periods (24, 48, 72h). We found a dose- and time-dependent reduction in cell proliferation and viability in both cell types (Fig. 6A). This reduction was more pronounced in the ER+ both at 48 and 72 h (Fig. 6A). However, ER cells already reacted at 24 h to smaller concentrations of 4-MU than ER cells (1 mM) (Fig. 6A).
To further investigate these observations, we examined apoptosis induction by immunoblotting for PARP-1 protein, a key component of the DNA single-strand break repair pathway [50]. Interestingly, 4-MU evoked PARP-1 cleavage, a hallmark of apoptosis, as shown by the generation of the 29-kDa PARP-1 fragment (cPARP-1) in a dose-dependent manner exclusively in ER cells (Fig. 6B). In addition, immunoblotting analyses revealed a substantial decrease of E-cadherin and β-catenin protein levels, two key components in adherens junctions, in 4-MU-treated ER+ cells (Fig. 6C).
4-MU downregulates the expression of HAS2, CD44, matrix-degrading enzymes and inflammatory mediators
Next, we investigated whether 4-MU would affect the expression of key components involved in breast cancer invasion and metastasis, including, the three hyaluronan synthases (HAS1, 2 and 3), various isoforms of CD44 (CD44s, v3, v6 and v9) and two key enzymes that degrade hyaluronan (HYAL-1, -2) and heparanase. In ER cells, 4-MU induced HAS1 and inhibited HAS2, but evoked no changes in HAS3 (Fig. 7A). In contrast, the ER cells expressed ~180- fold more HAS2 than ER cells and its levels were significantly suppressed by 4-MU (p<0.001, Fig. 7B and supplementary Fig. 2A).
In the ER cells, where CD44 variants predominate, CD44 mRNA levels were all reduced by 4-MU (Fig. 7C and Supplementary Fig. 2B). In contrast, the ER cells primarily expressed the standard isoform of CD44 (CD44s), which was between 70 and 150 higher than the other isoforms (Fig. 7D and Supplementary Fig. 2B). Their levels were all markedly reduced by 4-MU (Fig. 7D).
Notably, 4-MU significantly modulated the expression of multiple matrix-degrading enzymes including HYAL-1 and HYAL-2, with HYAL-2 being the main hyaluronidase in both breast cancer cell types (Fig. 7E-F, Supplementary Fig. 2C). 4-MU also caused down-regulation of heparanase in ER cells (p<0.5, Fig. 7G), which was dramatic in ER cells (p<0.001, Fig. 7H).
Treatment with 4-MU caused a significant reduction of the mRNA expression levels of several proteases, including: MMP-2, MMP-7, MMP-9, MT1-MMP/MMP-14 as well as those of MMP endogenous inhibitors TIMP-1 and TIMP-2, with the exception of MMP-1 and MT1-MMP that were up-regulated in ER and ER cells, respectively (Fig. 8A). In addition, 4-MU treatment showed a reverse effect in the expression of the components of the plasminogen activation system, which catalyze the enzymatic conversion of plasminogen to active plasmin that can degrade most ECM proteins and activate MMPs. Specifically, 4-MU significantly down-regulated the expression of uPA in ER cells but not in the ER cells where it was induced, as well as it affected the expression levels of tPA in an opposite manner in these cells (p<0.001, Fig. 8B). On the other hand, PAI-1 was down-regulated in both breast cancer cell lines (Fig. 8B).
To assess the enzymatic activities of secreted MMP-2 and MMP-9, we performed functional assays using gelatin zymography. The results showed that 4-MU reduced MMP-2 activity in both cells, while reduced that of MMP-9 in ER cells but with no detectable enzymatic activity in ER cells (Fig. 8A). Casein zymography revealed a significant reduction of MMP-7 (Fig. 8A) and uPA (Fig. 8B) activities in ER cells, with low or no detectable activities in ER cells. We note that there is a good correlation between the gene expression data and their respective enzymatic activities.
Finally, 4-MU significantly down-regulated the expression of inflammatory effectors such as IL-6, at both mRNA and protein levels (Fig. 9A,B), IL-8 (CXCL8; Fig. 9C) and CXCL-1 (Fig. 9D) in
ER cells, which constitutively express high levels of pro-inflammatory mediators, while no apparent changes were observed in ER cells apart from the induction in IL-8 (Fig. 9C).
Collectively, these results indicate that the effects of 4-MU are quite vast and multifactorial. However, our results point to a suppression of enzymes involved in matrix digestion and stimulation of enzymes that could counteract cancer progression.
Discussion
Given the complex nature of breast cancer, we designed this study in order to extend our current knowledge on the therapeutic potential of 4-MU. We hypothesized that estrogen receptor, a major prognostic factor in the clinical evolution and therapeutic management of breast cancer, would provide a differential effect on the biology of breast cancer subtypes vis-à-vis 4-MU treatment. Numerous studies have shown an inter-dependence of hyaluronan system (i.e. hyaluronan, HASs, HYALs, CD44) with ER in both ligand (estrogen)-dependent and independent manners. For instance, it is known that estrogen induces hyaluronan production in the skin [51, 52], while hyaluronan/CD44 interactions promote ERtranscriptional activation during ovarian cancer progression [53] and may contribute to the silencing of estrogen-driven genes in breast carcinoma [54]. On the other hand, the abundant expression of HAS2 and CD44 in the hormone-negative breast cancer cells might have a significant clinical relevance in controlling basal-like breast cancer distant metastasis [55]. High levels of HAS protein, both in stromal and carcinoma cells, are associated with poor differentiation, HER-positivity and poor patient outcome [56]. Notably, HYAL-1 has been found to be selectively repressed by estrogens in ER breast cancer cells indicating HYAL-1 as an ER target gene [57].
Our study demonstrates that 4-MU, an orally bioavailable nontoxic dietary supplement and an established inhibitor of hyaluronan synthesis, displays onco-suppressive properties in breast cancer, in line with its established effects on various types of malignant tumors [34, 37, 39, 40, 58-62]. According to our working model (Fig. 10), treatment of breast cancer cells with 4-MU causes a dose-dependent decrease of hyaluronan accumulation in the extracellular space and, importantly, within the cells (mainly in ER cells) followed by the down-regulation of HAS2 and the up-regulation of HYAL-1 and HYAL-2. The inhibitory effect of 4-MU on HAS2 expression might be explained by the possible reduction of UDP-GlcUA availability due to active glucuronidation of 4-MU (4-MU-glucuronide), which can be responsible for the HAS2 down-regulation [28, 63]. However, this remains to be elucidated in this cancer cell model. On the other hand, the induction of HYALs by 4-MU implies an additional mode of action of 4- MU in breast cancer cells also at the hyaluronan degradation level. Our novel findings further suggest that different growth- and apoptosis-related mechanisms are evoked by 4-MU in breast
cancer cells. In MCF-7/ER+ cells, 4-MU induces apoptosis and/or anoikis and suppresses cell invasiveness, whereas in MDA-MB-231/ER- cells, 4-MU inhibits proliferation, migration, and invasion, while it promotes cell adhesion (Fig. 10).
The marked decrease of intracellular hyaluronan in ER cells could partially explain the 4- MU-mediated suppression of proliferation, as one of the established roles of intracellular hyaluronan is to favor mitosis [64]. On the other hand, the detection of cleaved PARP-1 in ER cells but not in ER cells, implies the induction of specific proteases (such as caspases) that could lead to cell death via apoptosis [65]. Interestingly, PARP-1 has emerged as a major target in synthetic lethality that has led to the development of PARP inhibitors (PARPi) [66]. Inhibition of PARP-1 activity compromises base excision repair and induces synthetic lethality in cancer cells with homologous recombination (HR) defects in contrast to the normal cells, which retain the ability to repair DNA through HR and are therefore resistant to PARPi [67].
The concurrent loss of E-cadherin and β-catenin evoked by 4-MU, together with the actin cytoskeleton changes in ER cells, suggests a remodeling of the multimolecular E-cadherin-β- catenin-actin junctional complexes resulting in the disorganization and impairment of these structures. Therefore, the 4-MU-induced cell apoptosis observed in ER cells could be also due to the disruption of E-cadherin-mediated adhesion, an event that has been proposed as a critical checkpoint in the modification of cell adhesion and the onset of anoikis [68]. However, this mechanism does not occur in ER cells, which expressed negligible E-cadherin while -catenin was not significantly affected by 4-MU. Since it has been shown that cross-talk exists between the hyaluronan matrix and focal adhesions, the observed changes in the proliferative phenotype of ERα- cells might involve the disruption of the cytoskeleton and focal adhesions after 4-MU treatment as previously shown for esophageal carcinoma cells [69]. Notably, the early reaction (already at 24 h) of ER cells to smaller concentrations of 4-MU compared to ER cells, indicates a susceptibility of these cells to low 4-MU concentrations, which can be important for in vivo studies in the future and is in line with the other experiments in this study.
The migratory potential of both cell types was also differentially affected by 4-MU since ER cells were much more affected than ER cells. The significant inhibitory effect of 4- MU on the motility of ER cells within the first 24 h after 4-MU treatment could be associated with their marked morphological changes induced by 4-MU in the same time frame (24 h). Importantly, the 4-MU-evoked redistribution of CD44, which was lost from cell protrusions (probably invadopodia), and its diffusely distributed pattern imply a critical role of CD44 in 4-MU- mediated effects. This concept is further reinforced by a significant retardation in wound closure after Hermes-1 and/or 4-MU treatment. These observations are in agreement with other reports, which demonstrated that CD44 is a crucial component in protrusions/invadopodia of ER cells where it interacts with MT1-MMP, a central player in mediating invadopodia-dependent ECM
degradation, to promote proteolytic activities and metastasis [70]. Accordingly, CD44 and MT1- MMP mRNA expression levels were decreased in 4-MU-treated ER cells further supporting the hypothesis of the impairment of CD44/MT1-MMP functions in cell migration and invasion.
Our findings related to cell invasion and adhesion suggest an opposite role for newly synthesized hyaluronan in these processes that possibly involves CD44 as shown also by the striking differences in CD44 distribution and microfilament network re-organization observed after 4-MU and/or Hermes-1 treatments. However, the possibility that other hyaluronan receptors, such as RHAMM, are also involved in these events cannot be excluded since RHAMM exerts motogenic/invasive functions similarly to CD44 and can sometimes function in a compensatory manner [71-74]. Moreover, the absence of cell adherent hyaluronan as well as the alteration of CD44 distribution could favor the collagen-integrin interactions and therefore ER cell adhesion properties. Importantly, the finding that exogenously added HMW hyaluronan did not rescue the 4-MU-mediated effect on the ER cell motility is in agreement with the study focused on breast cancer [39] but it is inconsistent with a study conducted in prostate cancer [37], suggesting cancer type-specific mechanisms of actions for 4-MU.
Our results indicate that the onco-suppressive properties of 4-MU on breast cancer cells, can be in part due to modulation of ECM-degrading enzyme, targeting both protein and complex carbohydrates, such as hyaluronan and heparan sulfate. Indeed, 4-MU-mediated down- regulation of heparanase, which drives tumor cell proliferation, metastasis and angiogenesis [75- 78], and MMP-7 that is anchored on heparan sulfate chains of cell surface receptors, such as syndecans and specific CD44v (i.e. CD44v3), could eliminate their shedding as well as the shedding and activation of membrane-anchored ligands of EGFR. The shedding inhibition of such ligands, including heparin-binding EGF (HB-EGF), TGF-, and amphiregulin, most likely results in the reduced breast cancer cell metastatic potential [75, 79, 80]. In addition, the expression of MMP-2 and MMP-9, critical regulators of cell invasion and motility, is induced by CD44-hyaluronan interactions [81, 82]. Therefore, their significant repression by 4-MU, especially MMP-2, in breast cancer cells could contribute to their reduced migratory and invasive potential upon 4-MU treatment.
The significant down-regulation of uPA and PAI-1 in 4-MU-treated ER cells is of general interest as these molecules are established prognostic indicators in breast cancer and comprise efficient predictors of distant metastases in a subset of early node-negative breast cancer patients [83, 84]. Thus, we propose that 4-MU could represent an effective therapeutic agent in specific breast cancer subtypes, especially given our results showing 4-MU-mediated differential effect on the expression and activity of uPA in ER and ER cells. A similar differential effect of 4-MU was also observed for inflammatory IL-8, which is known to be highly expressed in ER breast cancers but it increases invasiveness and metastatic potential of both ERα- and ERα+ breast cancer cells. In our study, ER cells expressed much higher IL-8 than ER cells,
which was significantly down-regulated by 4-MU. However, a significant increase (4-fold) of IL-8 was observed in ER cells indicating opposite effects of 4-MU in the expression of specific matrix effectors in different breast cancer subtypes.
In conclusion, clinical therapies that target the drivers of individual breast cancers have
improved the outcome of breast cancer patients, especially endocrine therapies for ER
luminal-type cancers and trastuzumab for HER2+ cancers [85]. However, ER basal-type breast cancers do not respond to hormonal therapies while they display short time from relapse to death [86]. Our study suggests that 4-MU, an already available oral drug, possesses distinct potent onco-suppressive and anti-inflammatory properties in breast cancer cells of different ER status through modulation of specific effectors of the tumor microenvironment like hyaluronan/HAS2/CD44, glycosaminoglycan-degrading enzymes, matrix proteases and pro- inflammatory mediators. Although these findings are supported by several studies that have demonstrated an inter-connection between ER and hyaluronan system, we cannot exclude the possibility that the observed differences in 4-MU effects on the two breast cancer cell lines used in this study could be partly due to their diverse differentiation and/or aggressiveness. The long and reassuring clinical track record of 4-MU, its oral route of delivery, and the promising in vitro and in vivo data in mice all support further exploration of this therapeutic strategy [32, 33, 87]. Therefore, 4-MU alone or 4-MU-containing combination therapies (with trastuzumab/Herceptin [40] or PARP inhibitors [67, 88, 89]) could hold a great potential as therapeutic schemes in breast cancers with regard to their ER status.
Materials and Methods
Cell culture and reagents
MCF-7 (low metastatic, ER+) and MDA-MB-231 (highly metastatic, ER-) breast cancer cell lines were obtained from the American Type Culture Collection (ATCC). Both cell lines were routinely cultured in complete medium [Dulbecco’s Modified Eagle’s Medium (DMEM, #LM- D1110/500, Biosera) supplemented with 10% fetal bovine serum (FBS, #FB-1000/500, Biosera) and a cocktail of antimicrobial agents (100 IU/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml gentamycin sulfate and 2,5 μg/ml amphotericin B)] at 37oC, 95% humidified air/5% CO2. Every two days, medium was replaced with fresh one. When approximately 80% cell confluency was reached, cells were trypsinized for 3 min with trypsin-EDTA 1x in PBS (#LM-T1706/500, Biosera) and seeded in new Petri dishes. All experiments were conducted in serum-free conditions. Handling and storage of all reagents was performed according to the manufacturers’ instructions. 4-methylumbelliferone (4-MU, #M1508) was purchased from Sigma-Aldrich (Merck). All other chemicals used were of the best commercially available grade.
Determination of secreted hyaluronan concentration
Secreted hyaluronan by breast cancer cells was determined by using a microtiter-based assay, as described previously [22]. This assay is based on the specific binding of hyaluronan to the G1 globular domain of aggrecan. In brief, conditioned media obtained after the desired treatments were added to a 96-well microtiter plate (MaxiSorp Nunc-Immuno plates, Nalge Nunc International) pre-coated with G1 protein. Highly purified hyaluronan (0–100 ng/ml, Q-Med, Uppsala, Sweden) was used as a standard. Samples were incubated for 1h at 37°C, followed by an additional incubation with biotinylated G1 protein for another hour; its binding to hyaluronan was then determined by incubation for 1 h with peroxidase-conjugated streptavidin (1:1600 diluted; RPN 1051, GE Healthcare), followed by washings and incubation for 15 min with 3,3’,5,5’-tetramethyl-benzidine liquid substrate for ELISA (Supersensitive; #T4444, Sigma). The reaction was terminated with 2 M sulfuric acid, and the absorbance was measured at 450 nm using an ELISA reader. The inter-variation of this assay was less than 10%.
Enzyme-linked immunosorbent assay (ELISA) for IL-6 protein detemination
96-well plates were coated with anti-IL-6 antibody (#21679068, Immuno Tools) diluted in PBS overnight at 4oC. Coated wells were blocked with 3% BSA in PBS at 37oC for 1 h under constant agitation. Cell culture supernatants were added and plates were incubated for 1 h. Next, plates were incubated with a biotin-conjugated anti-IL-6 antibody for 1 h followed by Streptavidin-HRP (Amersham) incubation for 30 min at 37oC under constant agitation. 3,3’,5,5’-tetramethyl- benzidine liquid substrate for ELISA (Supersensitive; #T4444, Sigma) was added for 1-3 min until the samples obtained a light blue color and equal volumes of 2 M sulfuric acid were added to the
wells in order to stop the reactions. Sample absorbance was measured at 450 nm. Between each step wells were washed three times with PBS-Tween 0.05%.
Morphology/Phase contrast microscopy
For the observation of cell morphological changes, 10x104 MCF-7 and 5x104 MDA-MB-231 cells per well were seeded in a 24-well plate in complete medium. After 24 h incubation, cells were serum starved for 16 h. Then, the tested agents were added according to the experimental plan in serum-free culture medium and photographs were captured at several time points utilizing a color digital camera (CMOS) mounted on a phase contrast microscope (OLYMPUS CKX41, QImaging Micro Publisher 3.3RTV) through a 10x objective.
Wound healing and collagen Type I invasion assays
30x104 MCF-7 and 25x104 MDA-MB-231 cells per well were seeded in a 12-well plate in complete medium. After 24 h incubation, cells were serum starved for 16 h. The cell monolayer was then scratched using a pipette tip and washed two times with Dulbecco’s Phosphate Buffered Saline (PBS, #LM-52041/500, Biosera) followed by the addition of serum free DMEM containing the desired agents plus cytarabine (Pfizer). The cells were then incubated and photographed at various time points using a color digital camera (CMOS) mounted on a phase contrast microscope (OLYMPUS CKX41, QImaging Micro Publisher 3.3RTV) through a 10x objective. The images were quantified by measuring the wound area with Image J 1.50b Launcher Symmetry Software.
For the invasion assay in collagen type I, we used a previously published method [90] with minor modifications. Briefly, 50x104 MCF-7and 30x104 MDA-MB-231 cells per well were seeded in a 6-well plate in complete medium and incubated for 24 h, followed by overnight (16-18 h) starvation with serum free medium. The medium was then replaced with serum free medium containing the desired agents at the appropriate concentrations and the cells were incubated for 24 h. The cells were dissociated using 4 mM EDTA in PBS, collected, centrifuged, reconstituted in serum free medium, and counted. 6x104 cells were seeded in each well of a 12-well plate containing collagen type I gel and incubated in the presence or absence of the desired agents. After 24 h, photographs were captured using a color digital camera (CMOS) mounted on a phase contrast microscope (OLYMPUS CKX41, QImaging Micro Publisher 3.3RTV) through a 10x objective. The image quantification was performed as described in [90].
Cell viability
This assay was carried out as previously described [91]. In brief, 10x104 MCF-7 and 5x104 MDA-MB-231 cells per well were seeded in a 24-well plate and incubated for 24 h. The medium was then replaced with serum free medium and the cells were starved overnight (16-18 hours). The next day, fresh serum free medium containing the appropriate concentrations of the desired
agents was added and the cells were incubated for various time points. At each time point, cells were washed twice with PBS and stained with 0.5% (w/v) crystal violet solution in 20% methanol/distilled water for 20 min at 37oC with 150 oscillations on a bench rocker. Staining solution was removed and the excess dye was washed away using tap water, followed by two washes with distilled water. Stained cells were left to dry at room temperature for 24 h. Next, methanol was added to each well and the cell-bound dye was retrieved after 20 min incubation of the plate at 150 oscillations on a bench rocker. Following the incubation, optical density of each well was measured at 570 nm using a TECAN photometer, utilizing Magellan 6.
Collagen Type I cell adhesion assay
50x104 MCF-7 and 30x104 MDA-MB-231 cells per well were seeded in 6-well plates in complete medium and incubated for 24 h followed by overnight starvation. The medium was replaced with serum free medium containing the desired agents and the cells were incubated for 24 h. Afterwards, the cells were dissociated using 4 mM EDTA in PBS, collected, centrifuged, reconstituted in serum free medium containing 0.1% BSA and counted. 25x103 cells per well were seeded on a 96-well plate, which was pre-coated with 40 μg/ml collagen type I in PBS at 4o C overnight and blocked for 30 min with 1% BSA in PBS, and incubated for 30 min to adhere. Cells that did not adhere were removed with two washes with PBS and each well was stained with 0.5% (w/v) crystal violet in 20% methanol/distilled water for 20 min at 37oC at 150 oscillations on a bench rocker. After staining, the procedure was followed as described above in the crystal violet assay.
Immunofluorescence studies
50x104 MCF-7 and 30x104 MDA-MB-231 cells were seeded on glass coverslips in complete medium and incubated for 24 h followed by overnight starvation. The medium was replaced with serum free medium containing the desired agents and the cells were incubated for various time periods. Cells were fixed with 4% para-formaldehyde in PBS, permeabilized with 0.05% Triton X- 100/PBS-Tween 0.01% for 1 min and blocked with 5% Bovine Serum Albumin (BSA)/PBS- Tween 0.01% for 1 h at room temperature. Primary antibodies in 1% BSA/PBS-Tween 0.01% were then added and samples were incubated at 4oC overnight. Next day, the appropriate secondary antibodies in 1% BSA/PBS-Tween 0.01% were added and incubated for 1 h in the dark. Finally, cells were washed, stained and mounted with DAPI. Between each step after fixation, cells were washed 3 times with PBS-Tween 0.01%. Visualization was performed using a fluorescent phase contrast microscope (OLYMPUS CKX41, QImaging Micro Publisher 3.3RTV) at 60x. For hyaluronan staining, specimens were blocked with 5% BSA, egg whites (2) diluted in distilled water and 5% non-fat dry milk in PBS in order to block endogenous biotin and avidin. Slides were stained with the following antibodies and reagents: Hermes-3 (1 g/ml, generously provided by Dr S. Jalkanen), Phalloidin-iFluor™ 488 Conjugate AAT Bioquest, Inc. (1:40,
BIOTIUM CFTM488, #00042), Biotin-HABP (4 g/ml, Calbiochem, Millipore), Streptavidin-Alexa Fluor 488 or 594 (Sigma, #S11227), Alexa Fluor 594 or 488 goat anti-rabbit (Biotium, #PSF006), Alexa Fluor 594 or 488 goat anti-mouse (Biotium, #PSF006).
RNA isolation, cDNA synthesis and qPCR
140x104 MCF-7 and 80x104 MDA-MB-231 cells were seeded in 60-mm Petri dishes in
complete medium for 24 hours followed by overnight (16-18 h) starvation in serum free medium. Then, cells were treated with vehicle (DMSO) or 1 mM 4-MU for 24 h. After the treatment, cells were collected in a falcon tube and RNA isolation was carried out using the NucleoSpin® RNA, MACHEREY-NAGEL kit according to the manufacturer’s instructions. Isolated RNA was quantified by measuring absorbance at 260 nm. cDNA was synthesized using the PrimeScript™ RT reagent Kit (Perfect Real Time), TAKARA (#RR037A) kit according to the manufacturer’s instructions. Real time-PCR analysis was conducted in 20 μl reaction mixture, according to the manufacturer’s instructions. The amplification was performed utilizing Rotor Gene Q (Qiagen, USA). All reactions were performed in triplicate and a standard curve was always included for each pair of primers for assay validation. In addition, a melting curve analysis was always performed for detecting the SYBR Greenbased objective amplicon. To provide quantification, the point of product accumulation in the early logarithmic phase of the amplification plot was defined by assigning a fluorescence threshold above the background, defined as the threshold cycle (Ct) number. Relative expression of different gene transcripts was calculated by the ΔΔCt method. The Ct of any gene of interest was normalized to the Ct of the normalizer (GAPDH). Fold changes (arbitrary units) were determined as 2-ΔΔCt. Genes of interest and utilized primers are presented in Table 1.
Western Blot analysis
Cells treated with vehicle (DMSO) or increasing concentrations (0.5, 1, 2 mM) of 4-MU for 24 h were collected in a falcon tube, washed twice in PBS and stored in -80οC until use or lysed immediately using lysis buffer [25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 0.5 mM sodium orthovanadate (Sigma-Aldrich, #S6508) and protease inhibitor cocktail 1x (Chemicon, Millipore, CA, #20-201)] at 4oC for 30 min. Every 10 min the cell pellet was vortexed for 10 sec. Then, the samples were centrifuged at 10.000 rpm for 10 min at 4oC and the supernatants were collected and stored at -20oC. Total protein in each sample was quantified using Coomassie PlusTM (Bradford) assay (Thermo Scientific, #23236) according to manufacturer’s instructions. Samples of equal protein amounts were reduced with β- mercaptoethanol in Laemmli sample buffer and boiled at 100oC for 5 minutes. Next, samples were separated by SDS-PAGE in 10% poly-acrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Macherey Nagel, Germany). The membranes were blocked with 5% (w/v) non-fat dry milk in PBS pH 7.4 containing 0.05% Tween-20 (PBS-T) for 1 h at room
temperature and were then incubated with primary antibodies for 16 h at 4oC. After three washes with PBS-T, membranes were incubated with HRP-conjugated secondary goat anti-rabbit IgG (Sigma-Aldrich, #A0545,) or anti-mouse IgG (Sigma-Aldrich, #A4416) for 1 h at room temperature. Detection of the proteins was performed by Pierce ECL Western Blotting Substrate (Thermo Scientific, #32106), according to the manufacturer’s instructions. Primary antibodies used in immunoblotting analyses include: Hermes-3 (1 g/ml) anti-PARP antibody (1:500, Abcam, #ab6079), purified mouse anti-E-Cadherin (1:500, BD Transduction Laboratories, #610182), -catenin polyclonal antibody (1:500, Protein tech, #51067-2-AP), monoclonal anti-- tubulin, clone DM1A (1:500, Sigma-Aldrich, #T9026).
Casein zymography
Cells were treated with vehicle (DMSO) or 1 mM 4-MU for 24 h and the supernatants were collected and concentrated using centrifugal filters (Ultracel®-10K, Amicon® Ultra-4). Protein concentration in each sample was quantified using Coomassie PlusTM (Bradford) assay (Thermo Scientific, #23236) according to manufacturer’s instructions. Equal amounts of protein were mixed with Laemmli sample buffer and incubated for 30 min at 37oC. Samples were separated by SDS-PAGE in 10% poly-acrylamide gels containing 1 mg/ml casein and 1 g/ml plasminogen. Gels were washed twice with distilled water, followed by three washes with washing buffer (100 mM Tris-HCl, 2.5% (v/v) Triton X-100, 50 mM disodium EDTA, 0.1 mg/ml sodium azide, pH=8.0) for 30 min at room temperature. Then, gels were washed once with incubation buffer (100 mM Tris-HCl, 50 mM disodium EDTA, 0.1 mg/ml sodium azide, pH=8.0) for 30 min at room temperature and incubated with incubation buffer for 16-20 h at 37oC under constant agitation. Incubation buffer was removed and the gels were stained for 30 minutes at room temperature with 0.25% (w/v) Coomassie R-250 and destained with destaining buffer (50% methanol, 10% acetic acid and 40% distilled water). Gels were photographed and stored at 4oC in 7% CH3COOH.
Gelatin zymography
Cells were treated with vehicle (DMSO) or 1 mM 4-MU for 24 h and the supernatants were collected and concentrated using centrifugal filters (Ultracel®-10K, Amicon® Ultra-4). Protein concentration in each sample was quantified using Coomassie PlusTM (Bradford) assay (Thermo Scientific, #23236) according to manufacturer’s instructions. Equal amounts of protein were mixed with Laemmli sample buffer and incubated for 30 min at 37 oC. Samples were separated by SDS-PAGE in 10% poly-acrylamide gels containing 1 mg/ml gelatin. Gels were washed twice with distilled water, followed by three washes with washing buffer (100 mM Tris-HCl, 5% (v/v) Triton X-100, pH=7.3) for 30 min at room temperature. Then, gels were washed once with incubation buffer (100 mM Tris-HCl, 0.1% (v/v) Triton X-100, 5 mM CaCl2, pH=7.3) for 30 min at room temperature and incubated with incubation buffer for 16-20 h at 37 oC under constant
agitation. Incubation buffer was removed and the gels were stained for 30 minutes at room temperature with 0.25% (w/v) Coomassie R-250 and destained with destaining buffer (50% methanol, 10% acetic acid and 40% distilled water). Gels were photographed and stored at 4 oC in 7% acetic acid.
Statistical analysis
Each experiment was performed in triplicate. Reported values are expressed as mean ± standard deviation. Statistical significant differences were evaluated using GraphPad Prism unpaired t test (95% confidence intervals).
Acknowledgments
This work was supported by the EU Marie Sklodovska-Curie grant no. 645756 “GLYCANC” (to N.K.K. and S.S.S.).
Key words: Hyaluronan,
CD44,
hyaluronan synthase, 4-methylumbelliferone,
breast cancer,
estrogen receptors
Abbreviations used:
ER, Estrogen receptor; ECMs, extracellular matrices; GlcUA, glucuronic acid; GlcNAc, N-acetyl- glucosamine; HAS, HA synthase; HYAL, hyaluronidase; 4-MU, 4-methylumbelliferone; UGT, UDP-glucuronosyltransferase; HMW, high molecular weight; MMP, matrix metalloproteinase;
MT1-MMP, membrane type 1-metalloproteinase; TIMP, tissue inhibitor of metalloproteinase, IL,
interleukin; uPA, urokinase plasminogen activator; tPA, tissue plasminogen activator PAI-1,
plasminogen activator inhibitor type-1; CXCL-1, chemokine (C-X-C motif) ligand-1; EGFR,
epidermal growth factor receptor; HB-EGF, heparin-binding EGF; PARP, poly(adenosine
diphosphate-ribose) polymerase; EMT, epithelial-to-mesenchymal transition; mAb, monoclonal
antibody
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References
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Table I. Primer sequences used for quantitative RT-PCR
Gene Primer Sequence (5’-3’) Tannealing
CD44s Sense:ATAATAAAGGAGCAGCACTTCAGGA
Anti-sense:ATAATTTGTGTCTTGGTCTCTGGTAGC 60OC
CD44v3 Sense:ATAATGGCTGGGAGCCAAATGAAGAAA
Anti-sense:ATAATCATCATCATCAATGCCTGATCCAGA 60OC
CD44v6 Sense:ATAATCAGAAGGAACAGTGGTTTGGCA Anti-sense:ATAATGTCTTCTTTGGGTGTTTGGCGA 60OC
CD44v9 Sense:ATAATGAGCTTCTCTACATCACATGAAGGC
Anti-sense: TAATGTCAGAGTAGAAGTTGTTGGATGGTC 60OC
HAS1 Sense:GGAATAACCTCTTGCAGCAGTTTC Anti-sense: GCCGGTCATCCCCAAAAG 60OC
HAS2 Sense:TCGCAACACGTAACGCAAT
Anti-sense:ACTTCTCTTTTTCCACCCCATTT 60OC
HAS3 Sense:AACAAGTACGACTCATGGATTTCCT Anti-sense:GCCCGCTCCACGTTGA 60OC
HYAL-1 Sense:GATTGCAGTGTCTTCGATGTGGTA
Anti-sense:GGGAGCTATAGAAAATTGTCATGTCA 60OC
HYAL-2 Sense:CTAATGAGGGTTTTGTGAACCAGAATAT Anti-sense:GCAGAATCGAAGCGTGGATAC 60OC
MMP-1 Sense:CCTCGCTGGGAGCAAACA
Anti-sense: TTGGCAAATCTGGCGTGTAA 60OC
MMP-2 Sense:CGTCTGTCCCAGGATGACATC Anti-sense:ATGTCAGGAGAGGCCCCATA 62OC
MMP-7 Sense:GCTGGCTCATGCCTTTGC
Anti-sense: TCCTCATCGAAGTGAGCATCTC 60OC
MMP-9 Sense:TTCCAGTACCGAGAGAAAGCCTAT Anti-sense: GGTCACGTAGCCCACTTGGT 60OC
MT1-MMP Sense:CATGGGCAGCGATGAAGTCT
Anti-sense: CCAGTATTTGTTCCCCTTGTAGAAGTA 60OC
TIMP-1 Sense: CGCTGACATCCGGTTCGT
Anti-sense: TGTGGAAGTATCCGCAGACACT 60OC
TIMP-2 Sense: GGGCACCAGGCCAAGTT
Anti-sense: CGCACAGGAGCCATCACT 60OC
uPA Sense: ACTACTACGGCTCTGAAGTCACCA
Anti-sense: GAAGTGTGAGACTCTCGTGTAGAC 60OC
tPA Sense: CAGGAAATCCATGCCCGATT
Anti-sense: GCTGCAACTTTTGACAGGCAC 60OC
PAI-1 Sense: CTGACTTCACGAGTCTTTCAGACC
Anti-sense: CCCATGAAAAGGACTGTTCCTGTG 60OC
Heparanase Sense: TCACCATTGACGCCAACCT
Anti-sense: CTTTGCAGAACCCAGGAGGAT 60OC
IL-6 Sense: TCCAGAACAGATTTGAGAGTAGTG Anti-sense: GCATTTGTGGTTGGGTCAGG 58OC
IL-8 Sense: CTCCAAACCTTTCCACCCC
Anti-sense: GATTCTTGGATACCACAGAGAATG 57OC
CXCL-1 Sense: CAAACCGAAGTCATAGCCAC Anti-sense: CTTCAGGAACAGCCACCAGT 57OC
GAPDH Sense: AGGCTGTTGTCATACTTCTCAT Anti-sense: GGAGTCCACTGGCGTCTT 60OC
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Fig. 1. Effects of 4-MU on secreted and intracellular hyaluronan in ΕR+ and ΕR- breast cancer cells. (A) Quantification of secreted hyaluronan amounts by a microtiter-based assay in conditioned media of breast cancer cells treated for 24 h with vehicle (DMSO) or increasing concentrations of 4-MU, as indicated. The values represent the mean ±SD of 3 independent experiments run in triplicate (*p<0.05, ***p<0.001). (B) Immunofluorescence analysis of intracellular hyaluronan was performed with biotin-HABP (red) in breast cancer cells treated for 24 h as in panel A. Nuclei are shown in blue (DAPI). Scale bars ~ 40 m.
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Fig. 2. 4-MU induces changes in actin cytoskeleton and CD44 localization in breast cancer cells. Immunofluorescence analysis for F-actin (green) and CD44 (green) in (A) ΕR+ and (B) ΕR- breast cancer cells. Cells were treated with vehicle (DMSO) or increasing concentrations of 4-MU for 24 h. Nuclei are shown in blue (DAPI). Arrows in panel B point at loci of condensed CD44 expression in the leading edges of ΕR- cells. Scale bars ~ 20 m.
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Fig. 3. 4-MU inhibits the migration of ΕRα- breast cancer cells. (Α, B) Time-dependent migration of ΕR+ and ΕR- cells treated with vehicle (DMSO) or 0.5, 1 and 2 mM 4-MU. (C) Immunofluorescence analysis for CD44 (red) and F-actin (green) in ΕR- cells treated with either vehicle (DMSO) or 1 mM 4-MU for 24 h. Arrowheads point at cell protrusions positively stained for F-actin and CD44. Nuclei are shown in blue (DAPI). Scale bars, 200 m (upper panel) and 50m (lower panel). (D) Time-dependent migration of ΕR- cells treated with vehicle (DMSO) or Hermes-1 mAb and/or HMW hyaluronan in presence and absence of 1mM 4-MU. (E)
Determination of secreted hyaluronan by a microtiter-based assay in conditioned media of ΕR- cells treated with vehicle (DMSO) or Hermes-1 mAb in presence and absence of 1mM 4-MU for 24 h. The values represent the mean ± SD of 3 independent experiments run in triplicate (*p<0.05, **p<0.01, ***p<0.001).
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Fig. 4. 4-MU inhibits breast cancer cell invasiveness. Cell invasion assessed by (A) the percentage of invading cells and (B) invaded area after treatment of ΕR+ and ΕR- cells with vehicle (DMSO) or 0.5, 1 and 2 mM 4-MU. (C) Invading ΕR- cells and (D) invaded area after treatment with vehicle (DMSO) or Hermes-1 mAb in presence and absence of 1mM 4-MU for 24 h. The values represent the mean ± SD of 3 independent experiments run in triplicate (*p<0.05, **p<0.01, ***p<0.001).
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Fig. 5. 4-MU promotes the adhesiveness of ERα- breast cancer cells. (A) Adhesion of ER- cells on collagen type I matrices after treatment with vehicle (DMSO) or increasing concentrations of 4-MU in the presence or absence of Hermes-1 mAb. Immunofluorescence analysis of cells treated with vehicle (DMSO) or 1 mM 4-MU in the presence or absence of Hermes-1 mAb for (B) F-actin (green) and (C) CD44 (red). Nuclei are shown in blue (DAPI). Arrows point at cortical actin filaments at cell periphery. The values represent the mean ± SD of 3 independent experiments run in triplicate (*p<0.05, **p<0.01, ***p<0.001). Scale bars, 50 m.
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Fig. 6. 4-MU differentially affects breast cancer cell viability and proliferation. (A) Cell viability after exposure of ΕR+ and ΕR- breast cancer cells to vehicle (DMSO) or increasing concentrations of 4-MU for different time periods (24, 48, 72 h). Immunoblot analyses of (B) PARP-1/cPARP-1 and (C) E-cadherin/ -catenin in vehicle (DMSO) or 4-MU-treated ΕR+ and ΕR- cells (24 h post-treatment). The values represent the mean ± SD of 3 independent experiments run in triplicate (*p<0.05, **p<0.01, ***p<0.001).
Fig. 7. 4-MU regulates the expression of HAS2, CD44, and matrix-degrading enzymes in breast cancer cells. Quantitative qPCR analysis of (A, B) HASs (HAS1, HAS2, HAS3), (C, D) CD44 (CD44s, CD44v3, CD44v6, CD44v9), (E, F) HYALs (HYAL-1, HYAL-2) and (G, H) Heparanase in ΕR+ (A, C, E, G) and ΕR- (B, D, F, H) cells treated with vehicle (DMSO) or 1 mM 4-MU for 24 h. The values represent the mean ±SD of 3 independent experiments run in triplicate (*p<0.05, **p<0.01, ***p<0.001).
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Fig. 8. 4-MU regulates the expression and activity of matrix proteases in breast cancer cells. Quantitative qPCR analysis of (A) MMP-1, MMP-2, MMP-7, MMP-9, MT1-MMP/MMP-14, TIMP-1, TIMP-2, (B) PLAU/uPA, PLAT/tPA, SERPINE1/PAI-1 in ΕR+ and ΕR- cells treated with vehicle (DMSO) or 1 mM 4-MU for 24 h. (A) MMP-2 and MMP-9 gelatinolytic activities (assayed by gelatin zymography). (A, B) MMP-7 and uPA activities (assayed by casein zymography). The values represent the mean ± SD of 3 independent experiments run in triplicate (*p<0.05, **p<0.01, ***p<0.001). CB, Coomassie Blue staining.
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Fig. 9. 4-MU regulates the expression of inflammatory mediators in breast cancer cells. Quantitative qPCR analysis of (A) IL-6, (C) IL-8/CXCL-8, (D) CXCL-1 after treatment of ΕR+ and ΕR- cells with vehicle (DMSO) or 1 mM 4-MU for 24h. IL-6 protein levels (assessed by an ELISA assay) are also shown in (B). The values represent the mean ± SD of 3 independent experiments run in triplicate (*p<0.05, **p<0.01, ***p<0.001).
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Fig. 10. Proposed model of 4-MU-mediated onco-suppressive functions in breast cancer cells of different ER status. In ERα+ cells, 4-MU induces apoptosis and/or anoikis and suppresses cell invasiveness. In ERα- cells, 4-MU inhibits proliferation, migration, and invasion, while it promotes cell adhesion. Matrix effectors that are affected by 4-MU at mRNA and/or protein levels are also shown. For details, see text.
Supplementary Figures
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Supplementary Fig. 1. 4-MU promotes the adhesiveness of ERα- breast cancer cells.
Immunofluorescence analysis for (A) F-actin (green) and (B) CD44 (red) in ERα- cells grown on collagen type I matrices after treatment with vehicle (DMSO) or increasing concentrations of 4- MU for 24h. Nuclei are shown in blue (DAPI). Scale bars, 50 μm.
Supplementary Fig. 2. Relative gene expression of HASs, CD44 and HYALs in ERα+ and ERα- breast cancer cells. Quantitative qPCR analysis of (A) HAS1, HAS2, HAS3, (B) CD44s, CD44v3, CD44v6, CD44v9, (C) HYAL-1, HYAL-2 in ERα+ and ERα- cells. The values represent the mean ±SD of 3 independent experiments run in triplicate.
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