The hyaluronan receptors CD44 and Rhamm (CD168) form complexes with ERK1,2 that sustain high basal motility in breast cancer cells

CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a nonintegral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture. Here we describe an autocrine mechanism utilizing cell surface Rhamm-CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines that requires endogenous hyaluronan synthesis and the formation of Rhamm-CD44-ERK1,2 complexes. Motile/invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit more elevated basal activation of ERK1,2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm, and ERK1,2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Combinations of anti-CD44, anti-Rhamm antibodies, and a MEK1 inhibitor (PD098059) had less-than-additive blocking effects, suggesting the action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent autocrine mechanism to coordinate sustained signaling through ERK1,2, leading to high basal motility of invasive breast cancer cells. Therefore, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, such as cell surface Rhamm.     

(−)‑Oleocanthal inhibits proliferation and migration by modulating Ca2+ entry through TRPC6 in breast cancer cells

Triple negative breast cancer is an aggressive type of cancer that does not respond to hormonal therapy and current therapeutic strategies are accompanied by side effects due to cytotoxic actions on normal tissues. Therefore, there is a need for the identification of anti-cancer compounds with negligible effects on non-tumoral cells. Here we show that (−)‑oleocanthal (OLCT), a phenolic compound isolated from olive oil, selectively impairs MDA-MB-231 cell proliferation and viability without affecting the ability of non-tumoral MCF10A cells to proliferate or their viability. Similarly, OLCT selectively impairs the ability of MDA-MB-231 cells to migrate while the ability of MCF10A to migrate was unaffected. The effect of OLCT was not exclusive for triple negative breast cancer cells as we found that OLCT also attenuate cell viability and proliferation of MCF7 cells. Our results indicate that OLCT is unable to induce Ca²⁺ mobilization in non-tumoral cells. By contrast, OLCT induces Ca²⁺ entry in MCF7 and MDA-MB-231 cells, which is impaired by TRPC6 expression silencing. We have found that MDA-MB-231 and MCF7 cells overexpress the channel TRPC6 as compared to non-tumoral MCF10A and treatment with OLCT for 24–72 h downregulates TRPC6 expression in MDA-MB-231 cells. These findings indicate that OLCT impairs the ability of breast cancer cells to proliferate and migrate via downregulation of TRPC6 channel expression while having no effect on the biology of non-tumoral breast cells.     

ANXA2 could act as a moderator of EGFR-directed therapy resistance in triple negative breast cancer

Triple negative breast cancer (TNBC) patients cannot benefit from EGFR-targeted therapy even though the EGFR is highly expressed, because patients exhibit resistance to these drugs. Unfortunately, the molecular mechanisms remain relatively unknown. ANXA2, highly expressed in invasive breast cancer cells, is closely related with poor prognosis, and acts as a molecular switch to EGFR activation. In this study, MDA-MB-231 cells and MCF7 cells were used. Our results showed that ANXA2 expression is inversely correlated with cell sensitivity to gefitinib. Knockdown of ANXA2 expression in MDA-MB-231 cells increased the gefitinib induced cell death. When ANXA2 was overexpressed in MCF7 cells, the gefitinib induced cell death was decreased. Furthermore, we demonstrated that phosphorylation of ANXA2 at Tyr23 is negatively correlated with the sensitivity of TNBC to gefitinib. Altogether, our results suggest a new role of ANXA2 in regulating sensitivity of TNBC MDA-MB-231 cells to the EGFR inhibitor gefitinib.     

Behavioral remodeling of normal and cancerous epithelial cell lines with differing invasion potential induced by substrate elastic modulus

ABSTRACT

The micro-environment of cancer cells in the body is mechanically stiffer than that of normal cells. We cultured three breast cell lines of MCF10A-normal, MCF7-noninvasive, and MDA-MB-231-invasive on PDMS substrates with different elastic moduli and different cellular features were examined.Effects of substrate stiffness on cell behavior were evident among all cell lines. Cancerous cells were more sensitive to substrate stiffness for cell behaviors related to cell motility and migration which are necessary for invasion. The invasive cancerous cells were the most motile on substrates with moderate stiffness followed by non-invasive cancerous cells. Gene markers alterations were generally according to the analyzed cell movement parameters. Results suggest that alterations in matrix stiffness may be related to cancer disease and progression.     

Quantum dot-based cell motility assay

Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.     

Cytoskeletal remodeling by C3G to induce neurite-like extensions and inhibit motility in highly invasive breast carcinoma cells

Cytoskeletal remodeling is responsible for cell plasticity and facilitates differentiation, motility and adherence related functions. C3G (RAPGEF1), an exchange factor for Ras family of small GTPases, regulates cytoskeletal reorganization to induce filopodia in epithelial cells and neurite growth in neuroblastoma cells. Here we show that C3G overexpression induces neurite-like extensions (NLE) in MDA-MB-231 and BT549 breast carcinoma cells and not in a variety of other cancer cell lines examined. These processes were actin-rich with nodes, branches and microspikes. C3G associates with the cytoskeleton and its expression enabled stabilization of microtubules. NLE formation was dependent on Rap, Rac and Cdc42. C3G expression was associated with a decrease in cellular β-catenin levels specifically in MDA-MB-231 and BT549 cells. β-Catenin stabilization induced by GSK-3β inhibition, or coexpression of β-catenin, reduced C3G induced NLE formation. Time lapse analysis showed reduced motility of C3G expressing cells compared to GFP expressing cells. Our results suggest that C3G overexpression can induce phenotypic characteristics of neuronal cells in highly invasive breast cancer cells and inhibit their motility.     

The fibroblast growth factor-inducible 14 receptor is highly expressed in HER2-positive breast tumors and regulates breast cancer cell invasive capacity

Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor-inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor-like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor-negative (HER2(+)/ER(-)) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER(-) breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-kappaB (NF-kappaB) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-kappaB signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference-mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. Expression profiling of the Fn14-depleted cells revealed deregulation of NF-kappaB activity. Our findings support a role for Fn14-mediated NF-kappaB pathway activation in breast tumor invasion and metastasis.     

Phosphorylation of DARPP-32 regulates breast cancer cell migration downstream of the receptor tyrosine kinase DDR1

Cell migration plays a central role in processes such as development, wound healing and cancer metastasis. Here we describe a novel interaction between DDR1, a receptor tyrosine kinase activated by collagen, and the phosphoprotein DARPP-32 in mammary epithelial cells. DARPP-32 expression was readily detected in non-transformed mammary cell lines, but was strongly reduced or even absent in breast tumor cell lines, such as MCF7. Transfection of MCF7 cells with DARPP-32 resulted in severely impaired cell migration, while DARPP-32 transfection into the DDR1-deficient breast cancer cell line MDA-MB-231 did not alter migration. Co-expression of both DDR1 and DARPP-32 in MDA-MB-231 cells inhibited migration, thereby supporting a critical role of the DDR1/DARPP-32 complex in motility. Mutational substitution of the phosphorylation sites Thr-34 or Thr-75 on DARPP-32 revealed that phosphorylation of Thr-34 is necessary for the ability of DARPP-32 to impair breast tumor cell migration. Thus, DARPP-32 signaling downstream of DDR1 is a potential new target for effective anti-metastatic breast cancer therapy.     

Synthesis and Evaluation of Piperazine-Tethered Derivatives of Alepterolic Acid as Anticancer Agents

Alepterolic acid is a natural diterpenoid isolated from Aleuritopteris argentea with potential anti-cancer activity. In this study, alepterolic acid was modified to construct a series of arylformyl piperazinyl derivatives ( 3a – 3p ). The synthesized derivatives were fully characterized with HRMS, NMR, and IR. Four compounds with inhibition rate higher than 30 % at 10 μM ( 3f , 3n , 3g and 3k ) were further measured to obtain the IC₅₀ values against four cancer cell lines, including hepatoma cell lines HepG2, lung cancer cell lines A549, estrogen receptor-positive cell lines MCF7, and triple-negative breast cancer (TNBC) cell lines MDA-MB-231 by MTT assay. It was found that these compounds were more effective to HepG2 and MDA-MB-231 cells, while less toxic to A549 and MCF7 cells, and compound 3n as the most toxic derivatve against MDA-MB-231 cell lines, with IC₅₀ value of 5.55±0.56 μM. Trypan blue staining and colony formation assay showed that compound 3n inhibited the growth of MDA-MB-231 cells and prevented colony formation. Hoechst staining, flow cytometry and western blot analysis revealed that compound 3n induced caspase-dependent apoptosis in MDA-MB-231 cells. Conclusively, compound 3n was demonstrated to be a potential anti-cancer lead compound for further investigation.     

Selenium Nanoparticles Induce the Chemo-Sensitivity of Fluorouracil Nanoparticles in Breast and Colon Cancer Cells

Drug resistance is a major challenge of breast and colon cancer therapies leading to treatment failure. The main objective of the current study is to investigate whether selenium nanoparticles (nano-Se) can induce the chemo-sensitivity of 5-fluorouracil (FU)-encapsulated poly (D, L-lactide-co-glycolide) nanoparticles (nano-FU) in breast and colon cancer cell lines. Nano-Se and nano-FU were synthesized and characterized, then applied individually or in combination upon MCF7, MDA-MB-231, HCT 116, and Caco-2 cancerous cell lines. Cytotoxicity, cellular glucose uptake, and apoptosis, as well as malondialdehyde (MDA), nitric oxide (NO), and zinc (Zn) levels, were investigated upon the different treatments. We have resulted that nano-FU induced cell death in MCF7 and Caco-2 more effectively than MDA-MB-231 and HCT 116 cell lines. Moreover, nano-FU plus nano-Se potentiate MCF7 and Caco-2 chemo-sensitivity were higher than MDA-MB-231 and HCT 116 cancerous cell lines. It is relevant to note that Se and FU nano-formulations inhibited cancer cell bioenergetics via glucose uptake slight blockage. Furthermore, nano-FU increased the levels of NO and MDA in media over cancer cells, while their combinations with nano-Se rebalance the redox status with Zn increment. We noticed that MCF7 cell line is sensitive, while MDA-MB-231 cell line is resistant to Se and nano-Se. This novel approach could be of great potential to enhance the chemo-sensitivity in breast and colon cancer cells.

     

Livin promotes progression of breast cancer through induction of epithelial–mesenchymal transition and activation of AKT signaling

The inhibitor of apoptosis proteins (IAP) are closely correlated with proliferation, apoptosis, motility, and metastasis. Livin is the most recently identified IAP, and its role in breast progression remains unknown. In our study, analyses of 50 patients with breast cancer revealed that the positive expression rate of Livin was higher in breast cancer tissues (62%) relative to that in adjacent (35%) and normal tissues (25%). Livin expression in breast cancer correlated with the clinical stage and axillary lymph node metastasis and could be used as a prognostic marker. Our in vitro experiment revealed that Livin was highly expressed in high-invasive MDA-MB-231 cells as compared to low-invasive cells (MCF-7). Suppression of Livin by short-hairpin RNA reduced the Livin expression of MDA-MB-231 cells and subsequently inhibited tumor cell growth, proliferation, and colony formation and induced tumor cell apoptosis, motility, migration, and invasion. Overexpression of Livin in MCF7 cells resulted in increased migration and invasion capabilities of the cells without affecting proliferation and apoptosis. In addition, epithelial–mesenchymal transition (EMT) was induced by Livin expression in breast cancer cell lines. The high level of phosphorylated AKT in MDA-MB-231 cells was suppressed by Livin knockdown. Further, Livin-induced migration and invasion could be abolished by either the application of the phosphoinositide-3-kinase inhibitor LY294002 or knockdown of AKT expression using small-interfering RNA. In conclusion, Livin serves as an independent prognostic indicator for breast cancer. Livin expression promotes breast cancer metastasis through the activation of AKT signaling and induction of EMT in breast cancer cells both in vitro and in vivo.     

Clotrimazole preferentially inhibits human breast cancer cell proliferation, viability and glycolysis

Background

Clotrimazole is an azole derivative with promising anti-cancer effects. This drug interferes with the activity of glycolytic enzymes altering their cellular distribution and inhibiting their activities. The aim of the present study was to analyze the effects of clotrimazole on the growth pattern of breast cancer cells correlating with their metabolic profiles.

Methodology/Principal Findings

Three cell lines derived from human breast tissue (MCF10A, MCF-7 and MDA-MB-231) that present increasingly aggressive profiles were used. Clotrimazole induces a dose-dependent decrease in glucose uptake in all three cell lines, with K_i values of 114.3±11.7, 77.1±7.8 and 37.8±4.2 µM for MCF10A, MCF-7 and MDA-MB-231, respectively. Furthermore, the drug also decreases intracellular ATP content and inhibits the major glycolytic enzymes, hexokinase, phosphofructokinase-1 and pyruvate kinase, especially in the highly metastatic cell line, MDA-MB-231. In this last cell lineage, clotrimazole attenuates the robust migratory response, an effect that is progressively attenuated in MCF-7 and MCF10A, respectively. Moreover, clotrimazole reduces the viability of breast cancer cells, which is more pronounced on MDA-MB-231.

Conclusions/Significance

Clotrimazole presents deleterious effects on two human breast cancer cell lines metabolism, growth and migration, where the most aggressive cell line is more affected by the drug. Moreover, clotrimazole presents little or no effect on a non-tumor human breast cell line. These results suggest, at least for these three cell lines studied, that the more aggressive the cell is the more effective clotrimazole is.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

Effects of liposome-entrapped annamycin in human breast cancer cells: interference with cell cycle progression and induction of apoptosis

The effects of liposome-encapsulated annamycin (L-Ann) were investigated in two human breast cancer cell lines, MCF7 and MDA-MB-435. For comparative purposes, doxorubicin (Dx) was used throughout the study. A 4-hour treatment with L-Ann was significantly more active in MDA-MB-435 than in MCF7 cells (IC(50) values of 0.03 and 0.08 microg/ml, respectively), whereas Dx was equally active in the two cell lines (IC(50) 0.12 microg/ml). L-Ann induced an accumulation of cells in G2M phases which was dose-dependent in MDA-MB-435 but not in MCF7 cells. Dx also caused a dose-dependent increase of G2M cell fraction in MDA-MB-435 cells, whereas a G2M cell accumulation was observed only after treatment with the highest Dx concentration in MCF7 cells. G2M phase cell accumulations induced in MCF7 cells by L-Ann or Dx were accompanied by a decrease in cdc2 kinase activity and in cyclin B1 and cdc2 expression. Conversely, in MDA-MB-435 cells exposed to L-Ann or Dx, cdc2 kinase activity, cyclin B1 and cdc2 expression increased in parallel to the increase in the number of cells accumulated in the G2M phase. L-Ann and Dx induced apoptosis in MDA-MB-435 but not in MCF7 cells. In MDA-MB-435 cells exposed to L-Ann or Dx, no change was observed in the expression of bax, but there was a p53-independent increase in p21(waf1) expression. In MCF7 cells, treatment with L-Ann or Dx induced an increase in p53 expression with a consequent transactivation of p21(waf1) and bax. Our results indicate that L-Ann is more cytotoxic than Dx in breast cancer cells and is able to induce apoptosis through p53-independent mechanisms.