The LCC15-MB Human Breast Cancer Cell Line Expresses Osteopontin and Exhibits an Invasive and Metastatic Phenotype

We have characterized the LCC15-MB cell line which was recently derived from a breast carcinoma metastasis resected from the femur of a 29-year-old woman. LCC15-MB cells are vimentin (VIM) positive, exhibit a stellate morphology in routine cell culture, and form penetrating colonies when embedded in three-dimensional gels of Matrigel or fibrillar collagen. They show high levels of activity in the Boyden chamber chemomigration and chemoinvasion assays, and like other invasive human breast cancer (HBC) cell lines, LCC15-MB cells activate matrix-metalloproteinase-2 in response to treatment with concanavalin A. In addition, these cells are tumorigenic when implanted subcutaneously in nude mice and recolonize bone after arterial injection. Interestingly, both the primary lesion and the bone metastasis from which LCC15-MB were derived, as well as the resultant cell line, abundantly express the bone matrix protein osteopontin (OPN). OPN is also expressed by the highly metastatic MDA-MB-435 cells, but not other invasive or noninvasive HBC cell lines. Expression of OPN is retained in the subcutaneous xenograft and intraosseous metastases of LCC15-MB as detected by immunohistochemistry. Both VIM and OPN expression have been associated with breast cancer invasion and metastasis, and their expression by the LCC15-MB cell line is consistent with its derivation from a highly aggressive breast cancer. These cells provide a useful model for studying molecular mechanisms important for breast cancer metastasis to bone and, in particular, the implication(s) of OPN and VIM expression in this process.     

Studies of the interactions between melatonin and 2 Hz, 0.3 mT PEMF on the proliferation and invasion of human breast cancer cells

Interactions between the hormone melatonin at pharmacological concentrations (10(-3) M) and 2 Hz, 0.3 mT pulsed electromagnetic fields (PEMF) on the proliferation and invasion of human breast cancer cells were studied in vitro. Three types of human breast cancer cells were used in this study: MDA-MB-435, MDA-MB-231, and MCF-7. Results showed that cellular growth of MDA-MB-231 cells, which were reported to be lowly metastatic, and MCF-7 cells, which were reported to be nonmetastatic, were both significantly reduced by melatonin regardless of the presence of the field. Results also showed that MDA-MB-435 and MDA-MB-231 cells were invasive, with MDA-MB-231 cells being more invasive than the MDA-MB-435 cells for both unexposed and experimental-PEMF groups. In addition, invasion studies showed that MCF-7 cells were not invasive and that melatonin did not have any effects on the invasion of these cells, with or without the PEMF. It is also suggested that since metastasis requires growth and invasion into tissue, anti-invasion agents can be used in conjunction with melatonin to prevent formation of secondary metastases. The overall studies suggest that PEMF at 2 Hz, 0.3 mT does not influence cancer metastasis; while having clinical merit in the healing of soft tissue injury, this field has shown no influence on cancer cells as 60 Hz power line fields have.     

Norepinephrine promotes the β1-integrin-mediated adhesion of MDA-MB-231 cells to vascular endothelium by the induction of a GROα release

The migratory activity of tumor cells and their ability to extravasate from the blood stream through the vascular endothelium are important steps within the metastasis cascade. We have shown previously that norepinephrine is a potent inducer of the migration of MDA-MB-468 human breast carcinoma cells and therefore investigated herein, whether the interaction of these cells as well as MDA-MB-231 and MDA-MB-435S human breast carcinoma cells with the vascular endothelium is affected by this neurotransmitter as well. By means of a flow-through assay under physiologic flow conditions, we show that norepinephrine induces an increase of the adhesion of the MDA-MB-231 cells, but not of MDA-MB-468 and MDA-MB-435S cells to human pulmonary microvascular endothelial cells (HMVEC). The adhesion of MDA-MB-231 cells was based on a norepinephrine-mediated release of GROα from HMVECs. GROα caused a β1-integrin-mediated increase of the adhesion of MDA-MB-231 cells. Most interestingly, this effect of norepinephrine, similar to the aforementioned induction of migration in MDA-MB-468 cells, was mediated by β-adrenergic receptors and therefore abrogated by β-blockers. In conclusion, norepinephrine has cell line-specific effects with regard to certain steps of the metastasis cascade, which are conjointly inhibited by clinically established β-blockers. Therefore, these results may deliver a molecular explanation for our recently published retrospective data analysis of patients with breast cancer which shows that β-blockers significantly reduce the development of metastases.     

Inhibition of breast cancer cell adhesion and bone metastasis by the extracellular adherence protein of Staphylococcus aureus

Bone metastasis is a common sequelae of breast cancer and the interaction of alpha v beta3-integrin with osteopontin (OPN) found in the extracellular matrix of mineralized tissues is implicated in this process. The integrin-dependent proadhesive and promigratory functions of OPN are particularly attributed to the 40 kD N-terminal fragment that derives upon matrix metalloproteinase (MMP) cleavage. Based on the broad repertoire of interactions between Staphylococcus aureus extracellular adherence protein (Eap) and host components, we here characterized Eap to specifically interact with recombinant full-length OPN and the 40 kD N-terminal MMP cleavage fragment, but not with the 32 kD or the 25 kD C-terminal fragments of OPN. Eap thereby prevented the OPN/alpha v beta3-integrin interaction, as well as the alpha v beta3-integrin-dependent adhesion of MDA-MB-231 breast cancer cells to full-length OPN or to the 40 kD fragment and the migration of these cells towards OPN. Furthermore, Eap treatment markedly impaired the development of osseous metastasis of human MDA-MB-231 cells in vivo. Taken together, Eap may represent an attractive novel treatment for the prevention of breast cancer bone metastasis.     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

Fascin, an actin-bundling protein, promotes breast cancer progression in vitro

Fascin, an actin-cross-linking protein, is up-regulated in breast cancer and correlates with a more aggressive disease. This study was conducted to elucidate the effects of manipulating fascin in breast cancer cells on the metastasis-associated events, including proliferation, adhesion, invasion, epithelial-mesenchymal transition (EMT) and enrichment of a CD44(+) /CD24(-) subpopulation that show some stem/progenitor cell properties. Western blot analysis of a panel of breast cancer cell lines revealed high expression of fascin in MDA-MB-435 and MDA-MB-231 cells but revealed no or low expression in MDA-MB-453, Her-18 and T47D. Gain-of-function and loss-of-function studies in breast cancer cells demonstrated that forced expression of fascin promoted cell proliferation assessed by the MTT assay, decreased cellular adhesion to fibronectin and potentiated the invasive capacity in the Transwell chamber invasion assay. Conversely, down-regulation of fascin via small interfering RNA increased cell adhesion and facilitated cell proliferation and invasion. In addition, fascin participated in the EMT and modulated the proportion of the CD44(+) /CD24(-) subpopulation in breast cancer cells. In conclusion, our data highlight an important role for fascin in breast cancer progression in vitro through orchestrating a variety of cellular events associated with metastasis, and thus, targeting this gene might have therapeutic implications.     

UCH-L1在乳腺癌中的表达及其与转移关系的研究

目的研究泛素羧基末端水解酶L1(UCH-L1)与磷酸化p38(p-p38)在乳腺癌组织、细胞系中的表达情况、两种蛋白的表达与临床病理特征的关系以及UCH-L1与乳腺癌侵袭转移的关系。方法用免疫组织化学方法检测乳腺癌组织中UCH-L1与p-p38蛋白的表达情况,用Western Blot方法检测乳腺癌组织以及细胞系中UCH-L1与p-p38蛋白的表达情况。应用UCH-L1特异性抑制剂作用于乳腺癌高侵袭高转移细胞系MDA-MB-435s后,用Western Blot观察UCH-L1与p-p38蛋白表达改变的情况,用Transwell实验检测MDA-MB-435s细胞侵袭潜能的改变。结果 UCH-L1和p-p38蛋白在乳腺浸润性导管癌中的表达高于其在癌旁正常乳腺组织中的表达(P=0.012,P=0.001),二者呈正相关(r=0.397,P=0.000),并与乳腺癌的TNM分期(P=0.017,P=0.010)、淋巴结转移情况(P=0.033,P=0.021)相关。乳腺上皮细胞系MCF-10A、乳腺癌低侵袭低转移细胞系MCF-7和乳腺癌高侵袭高转移细胞系MDA-MB-435s中两种蛋白表达水平呈递增趋势(P均<0.05)。UCH-L1特异性抑制剂可以浓度依赖性地下调MDA-MB-435s细胞系中p-p38蛋白的表达水平(P均<0.05),并能抑制乳腺癌细胞的侵袭转移潜能。结论 UCH-L1、p-p38过表达与乳腺癌的TMN分期、淋巴结转移有关。UCH-L1可能通过上调p-p38介导乳腺癌转移。     

Progranulin stimulated by LPA promotes the migration of aggressive breast cancer cells

Activator and inhibitor roles for the 88-kDa-secreted glycoprotein progranulin (PGRN) have been demonstrated in ovarian cancer cells. Here, we investigated the effects of PGRN in breast cancer migration. Testing MCF7, MDA-MB-453, and MDA-MB-231 human breast cancer cells and the MCF10A breast epithelial cell line, we demonstrate that LPA-induced PGRN stimulation led to a significant increase in cell invasion of MDA-MB-453 and MDA-MB-231 cells only (p<0.05). Moreover, incubation with an anti-PGRN antibody, an inhibitor of the ERK pathway (PD98059) or both in combination inhibited the ability of MDA-MB-231 cells to invade. Furthermore, the expression of focal adhesion kinases promoted by LPA-induced PGRN was also inhibited by PD98059 alone or in combination with an anti-PGRN antibody (p<0.05). Taken together, these results suggest that the LPA activation of PGRN involving the ERK pathway is critical to promote MDA-MB-231 breast cancer cell invasion.     

Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins (“delRGD” or “RGE”) supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.     

Gangliosides expressed on breast cancer cells are E-selectin ligands

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe^x) and sialyl Lewis A (sLe^a) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin–Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin–Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.     

Progranulin Stimulated by LPA Promotes the Migration of Aggressive Breast Cancer Cells

Abstract

Activator and inhibitor roles for the 88-kDa-secreted glycoprotein progranulin (PGRN) have been demonstrated in ovarian cancer cells. Here, we investigated the effects of PGRN in breast cancer migration. Testing MCF7, MDA-MB-453, and MDA-MB-231 human breast cancer cells and the MCF10A breast epithelial cell line, we demonstrate that LPA-induced PGRN stimulation led to a significant increase in cell invasion of MDA-MB-453 and MDA-MB-231 cells only (p < 0.05). Moreover, incubation with an anti-PGRN antibody, an inhibitor of the ERK pathway (PD98059) or both in combination inhibited the ability of MDA-MB-231 cells to invade. Furthermore, the expression of focal adhesion kinases promoted by LPA-induced PGRN was also inhibited by PD98059 alone or in combination with an anti-PGRN antibody (p < 0.05). Taken together, these results suggest that the LPA activation of PGRN involving the ERK pathway is critical to promote MDA-MB-231 breast cancer cell invasion.     

Atp6v1c1 May Regulate Filament Actin Arrangement in Breast Cancer Cells

Previous studies have shown that the rate of breast cancer metastasis correlates with the expression of vacuolar H⁺-ATPases (V-ATPases). However, how V-ATPase is involved in breast cancer metastasis remains unknown. Our previous study showed that Atp6v1c1-depleted osteoclasts did not form organized actin rings and that Atp6v1c1 co-localizes with F-actin. In this study, we found that the normal arrangement of filamentous actin is disrupted in Atp6v1c1-depleted 4T1 mouse breast cancer cells and in the ATP6V1C1-depleted human breast cancer cell lines MDA-MB-231 and MDA-MB-435s. We further found that Atp6v1c1 co-localizes with F-actin in 4T1 cells. The results of our study suggest that high expression of Atp6v1c1 affects the actin structure of cancer cells such that it facilitates breast cancer metastasis. The findings also indicate that Atp6v1c1 could be a novel target for breast cancer metastasis therapy.     

Identification of a novel aspartic-like protease differentially expressed in human breast cancer cell lines

Four different human breast cancer cell lines were examined to search for genes associated with tumor growth and metastasis. Each of these cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and MDA-MB-435, displays different phenotypic characteristics ranging from poorly to highly tumorigenic and metastatic. The differences in gene expression profiles of these cell lines generated by differential display technique should allow one to identify candidates as putative oncogenes or tumor/metastasis suppressor genes. A novel cDNA expressed in the highly tumorigenic and metastatic cell line, MDA-MB-435, was identified and isolated by this approach. The function for this gene, designated ALP56 (aspartic-like protease 56 kDa), in tumor progression is suggested by the homology of the encoded protein to aspartic proteases, such as cathepsin D. The amino acid residues in two catalytic domains of this family are highly conserved in those domains of ALP56. Northern hybridization indicated that the expression of ALP56 is associated with growth and metastasis of MDA-MB-435 tumors in immunodeficient mice. In situ hybridization of biopsies from breast cancer and colon cancer patients indicated that ALP56 is upregulated in human primary tumors and liver metastasis. These results suggest that this novel gene correlates with human tumor progression.     

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.     

Characterization of Spontaneous and TGF-β-Induced Cell Motility of Primary Human Normal and Neoplastic Mammary Cells In Vitro Using Novel Real-Time Technology

The clinical complications derived from metastatic disease are responsible for the majority of all breast cancer related deaths. Since cell migration and invasion are a prerequisite for metastasis their assessment in patient cancer cells in vitro may have prognostic value for the tumor''s metastatic capacity. We employed real-time cell analysis (RTCA) on the xCELLigence DP system to determine in vitro motility of patient-derived primary human breast cancer epithelial cells (HBCEC). Initially, the RTCA assay was validated using established human breast cancer cell lines with either an invasive (MDA-MB-231, MDA-MB-435s) or a non-invasive phenotype (MCF-7, MDA-MB-468), and primary NSCLC cells (Tu459). Previous standard assays of cell migration/invasion revealed that only MDA-MB-231, −435s, and Tu459 cells exhibited spontaneous and TGF-β1-stimulated migration and invasion through a Matrigel barrier. In the present study, the TGF-β1-stimulated activities could be blocked by SB431542, a potent kinase inhibitor of the TGF-β type I receptor ALK5. Application of the RTCA assay to patient-derived tumor cells showed that 4/4 primary HBCEC and primary NSCLC cells, but not normal human mammary epithelial cells (HMEC), displayed high spontaneous migratory and invasive activity which correlated with higher MMP-2 expression and uPA protein levels in HBCEC compared to HMEC. Upon treatment with TGF-β1, HBCEC exhibited morphologic and gene regulatory alterations indicative of epithelial-to-mesenchymal transition. However, exclusively the invasive but not the migratory activity of HBCEC was further enhanced by TGF-β1. This indicates the requirement for molecular, e.g. integrin interactions with Matrigel components in HBCEC in order to become responsive to pro-invasive TGF-β effects. Together, these results show for the first time that tumorigenic HBCEC but not normal HMEC possess a strong basal migratory as well as a basal and TGF-β1-inducible invasive potential. These findings qualify the RTCA assay as an in vitro migration/invasion testing system for patient-specific primary breast cancer cells.     

CD44v4 is a major E-selectin ligand that mediates breast cancer cell transendothelial migration

Background

Endothelial E-selectin has been shown to play a pivotal role in mediating cell–cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. However, the counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown.

Methodology/Principal Findings

By assessing migration of various breast cancer cells across TNF-α pre-activated human umbilical vein endothelial cells (HUVECs), we found that breast cancer cells migrated across HUVEC monolayers differentially and that transmigration was E-selectin dependent. Cell surface labeling with the E-selectin extracellular domain/Fc chimera (exE-selectin/Fc) showed that the transmigration capacity of breast cancer cells was correlated to both the expression level and localization pattern of E-selectin binding protein(s) on the tumor cell surface. The exE-selectin/Fc strongly bound to metastatic MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells, but not non-metastatic MCF-7 and T47D cells. Binding of exE-selectin/Fc was abolished by removal of tumor cell surface sialyl lewis x (sLe^x) moieties. Employing an exE-selectin/Fc affinity column, we further purified the counterreceptor of E-selectin from metastatic breast cancer cells. The N-terminal protein sequence and cDNA sequence identified this E-selectin ligand as a ∼170 kD human CD44 variant 4 (CD44v4). Purified CD44v4 showed a high affinity for E-selectin via sLe^x moieties and, as expected, MDA-MB-231 cell adhesion to and migration across HUVEC monolayers were significantly reduced by down-regulation of tumor cell CD44v4 via CD44v4-specific siRNA.

Conclusions/Significance

We demonstrated, for the first time, that breast cancer cell CD44v4 is a major E-selectin ligand in facilitating tumor cell migration across endothelial monolayers. This finding offers new insights into the molecular basis of E-selectin–dependent adhesive interactions that mediate breast cancer cell transendothelial metastasis.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Adhesion of nonmetastatic and highly metastatic breast cancer cells to endothelial cells exposed to shear stress

The mechanical stimulus of shear stress has to date been neglected when studying the adhesion of cancer cells to the endothelium. Confluent monolayers of endothelial cells were subjected to either 4 or 15 hours of arterial shear stress. Adhesion of nonmetastatic (MCF-7) and highly metastatic (MDA-MB-435) human breast cancer cells was then quantified using a detachment assay carried out inside the parallel plate flow chamber. Four hours of shear stress exposure had no effect on adhesion. However, 15 hours of shear stress exposure led to marked changes in the ability of the endothelial monolayer to bind human breast cancer cells. An increase in adhesive strength was observed for nonmetastatic MCF-7 cells, while a decrease in adhesive strength was observed for highly metastatic MDA-MB-435 cells. Hence, endothelial shear stress stimulation does influence the adhesion of cancer cells to the endothelium and can have different effects on the adhesion of cancer cells with different metastatic potentials. Furthermore, adhesion of nonmetastatic and highly metastatic human breast cancer cells may be controlled by two different endothelial cell adhesion molecules that are differentially regulated by shear stress. Immunohistochemistry confirmed that shear stress did in fact differentially regulate endothelial cell adhesion molecule expression.     

Enhanced expression of LKB1 in breast cancer cells attenuates angiogenesis, invasion, and metastatic potential

LKB1 (also known as STK11) is a recently identified tumor suppressor gene whose mutation can lead to Peutz-Jeghers syndrome, which is characterized by gastrointestinal polyps and cancers of different organ systems. Approximately 30% of sporadic breast cancer samples express low levels of LKB1. This suggests that the LKB1 gene may be related to the tumorigenesis of breast cancer. We reintroduced LKB1 into MDA-MB-435 breast cancer cells that lack the LKB1 gene to investigate how overexpression of LKB1 affects tumor invasiveness and metastasis. Overexpression of the LKB1 protein in breast cancer cells resulted in significant inhibition of in vitro invasion. In vivo, LKB1 expression reduced tumor growth in the mammary fat pad, microvessel density, and lung metastasis. LKB1 overexpression was associated with down-regulation of matrix metalloproteinase-2, matrix metalloproteinase-9, vascular endothelial growth factor, and basic fibroblast growth factor mRNA and protein levels. Overexpression of the LKB1 protein in human breast cancer is significantly associated with a decrease in microvessel density. Our results indicate that LKB1 plays a negative regulatory role in human breast cancer, a finding that may lead to a new therapeutic strategy.