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Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

Introduction: The re-emergence of the tumour growth factor-beta (TGF-beta)-related embryonic morphogen Nodal has recently been reported in several different human cancers. In this study, we examined the expression of Nodal in a series of benign and malignant human breast tissues to determine the clinical significance of this expression and whether Nodal could represent a potential therapeutic target in breast cancer., Methods: Tissue sections from 431 therapeutically naive patients diagnosed with benign or malignant breast disease were stained for Nodal by immunohistochemistry and analysed in a blinded manner. The degree of Nodal staining was subsequently correlated with available clinical data, such as diagnoses and disease stage. These tissue findings were further explored in breast cancer cell lines MDA-MB-231 and MDA-MB-468 treated with a Nodal blocking antibody to determine biological effects for target validation., Results: A variable degree of Nodal staining was detected in all samples. The intensity of Nodal staining was significantly greater in undifferentiated, advanced stage, invasive breast cancer compared with benign breast disease or early stage breast cancer. Treatment of human breast cancer cells in vitro with Nodal blocking antibody significantly reduced proliferation and colony-forming ability in soft agar, concomitant with increased apoptosis., Conclusions: These data suggest a potential role for Nodal as a biomarker for disease progression and a promising target for anti-Nodal therapy in breast cancer.

Breast carcinoma cells and embryonic progenitors similarly implement stem cell-associated signaling pathways to sustain continued growth and plasticity. Indeed, recent studies have implicated signaling pathways, including those associated with the Notch, and Transforming Growth Factor-Beta (TGF-beta) superfamilies, as instrumental to both embryological development and breast cancer progression. In particular, Nodal, an embryonic morphogen belonging to the TGF-beta superfamily, and its co-receptor, Cripto-1, are requisite to both embryogenesis and mammary gland maturation. Moreover, these developmental proteins have been shown to promote breast cancer progression. Here, we review the role of Nodal and its co-receptor Cripto-1 during development and we describe how this signaling pathway may be involved in breast cancer tumorigenesis. Moreover, we emphasize the potential utility of this signaling pathway as a novel target for the treatment and diagnosis of breast cancer.

The development and progression of cancer is controlled by gene expression, often regulated through chromatin packaging. Heterochromatin protein 1(Hsalpha) (HP1(Hsalpha)), one of three human HP1 family members, participates in heterochromatin formation and gene regulation. HP1(Hsalpha) possesses an amino-terminal chromodomain, which binds methylated lysine 9 of histone H3 (meK9 H3), and a carboxyl-terminal chromoshadow domain (CSD) that is required for dimerization and interaction with partner proteins. HP1(Hsalpha) is down-regulated in invasive metastatic breast cancer cells compared with poorly invasive nonmetastatic breast cancer cells. Expression of EGFP-HP1(Hsalpha) in highly invasive MDA-MB-231 cells causes a reduction in in vitro invasion, without affecting cell growth. Conversely, knock-down of HP1(Hsalpha) levels in the poorly invasive breast cancer cell line MCF-7 increased invasion, without affecting cell growth. To determine whether functions of the CSD were required for the regulation of invasion, mutant forms of HP1(Hsalpha) were expressed in MDA-MB-231 cells. A W174A mutation that disrupts interactions between HP1(Hsalpha) and PXVXL-containing partner proteins reduced invasion similar to that of the wild type protein. In contrast, an I165E mutation that disrupts dimerization of HP1(Hsalpha) did not decrease invasion. No gross changes in localization and abundance of HP1(Hsbeta), HP1(Hsgamma), and meK9 H3 were observed upon expression of wild type and mutant forms of HP1(Hsalpha) in MDA-MB-231 cells. Taken together, these data demonstrate that modulation of HP1(Hsalpha) alters the invasive potential of breast cancer cells through mechanisms requiring HP1 dimerization, but not interactions with PXVXL-containing proteins.

We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with beta-aminopropionitrile (betaAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of betaAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with betaAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on betaAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer.

Previous studies from our laboratory and others have demonstrated that treatment of breast cancer cells with exogenous maspin led to a significant decrease in cell motility, and an increase in cell adhesion to human fibronectin. However, the signaling mechanisms by which maspin, a putative tumor suppressor gene, might regulate cell motility and adhesion have not been previously addressed. In this study, we hypothesized that maspin could inhibit cell motility through the Rho GTPase pathway, specifically by affecting Rac activity. To test this intriguing hypothesis we utilized an experimental approach where invasive and metastatic MDA-MB-231 breast cancer cells were either treated exogenously with recombinant maspin protein, or stably transfected with maspin. The data revealed decreased Rac1 activity within 4 h, and a decrease in the Rac1 effector, PAK1, within 12 h. In addition, an increase in PI3K and ERK1/2 activities within 1 h of recombinant maspin (rMaspin) treatment was observed, which returned to baseline level after 12 h. ERK activity was shown to be downstream of PI3K, as pretreatment with the PI3K inhibitor, LY294002, inhibited the stimulation of ERK activity by rMaspin. Furthermore, rMaspintreated cells displayed approximately a 30% increase in cell adhesion which was abrogated by pretreatment with LY294002. Increased focal adhesions and stress fibers were observed after 12 h of rMaspin treatment, when the cells were least motile and had reverted to a more epithelial-like phenotype. These data suggest that maspin may inhibit cell motility by regulating Rac1 and subsequently PAK1 activity, and promote cell adhesion via PI3K/ERK pathways. This study provides new insights into the diverse signaling pathways affected by maspin to suppress the metastatic phenotype, and could contribute to novel therapeutic approaches for the treatment of invasive and metastatic breast cancer.

We identified previously an up-regulation in lysyl oxidase (LOX) expression,an extracellular matrix remodeling enzyme, in a highly invasive/metastatic human breast cancer cell line, MDA-MB-231, compared with MCF-7, a poorly invasive/nonmetastatic breast cancer cell line. In this study, we demonstrate that the mRNA expression of LOX and other LOX family members [lysyl oxidase-like (LOXL), LOXL2, LOXL3, and LOXL4] was observed only in breast cancer cells with a highly invasive/metastatic phenotype but not in poorly invasive/nonmetastatic breast cancer cells. LOX and LOXL2 showed the strongest association with invasive potential in both highly invasive/metastatic breast cancer cell lines tested (MDA-MB-231 and Hs578T). To determine whether LOX is directly involved in breast cancer invasion, LOX antisense oligonucleotides were transfected into MDA-MB-231 and Hs578T cells, and found to inhibit invasion through a collagen IV/laminin/gelatin matrix in vitro compared with LOX sense oligonucleotide-treated and untreated controls. In addition, treatment of MDA-MB-231 and Hs578T cells with beta-aminopropionitrile (an irreversible inhibitor of LOX enzymatic activity) decreased invasive activity. Conversely, MCF-7 cells transfected with the murine LOX gene demonstrated a 2-fold increase in invasiveness that was reversible by the addition of beta-aminopropionitrile in a dose-dependent manner. In addition, endogenous LOX mRNA expression was induced when MCF-7 cells were cultured in the presence of fibroblast conditioned medium or conditioned matrix, suggesting a role for stromal fibroblasts in LOX regulation in breast cancer cells. Moreover, the correlation of LOX up-regulation and invasive/metastatic potential was additionally demonstrated in rat prostatic tumor cell lines, and human cutaneous and uveal melanoma cell lines. These results provide substantial new evidence that LOX is involved in cancer cell invasion.

Maspin is a 42kDa tumor suppressor protein that belongs to the serine protease inhibitor (serpin) family. It inhibits cell motility and invasion in vitro, and tumor growth and metastasis in nude mice; however, maspin's molecular mechanism of action has remained elusive. Maspin contains several tyrosine residues and we hypothesized that phosphorylation of maspin could play a role in its biological function. Our study reveals that maspin is phosphorylated on tyrosine moiety(ies) in normal mammary epithelial cells endogenously expressing maspin. In addition, transfection of the maspin gene, using either a stable or inducible system into maspin-deficient breast cancer cell lines, yields a protein product that is phosphorylated on tyrosine residue(s). Furthermore, recombinant maspin protein can be tyrosine-phosphorylated by the kinase domain from the epidermal growth factor receptor in vitro. These novel observations suggest that maspin, which deviates from the classical serpin, may be an important signal transduction molecule in its phosphorylated form.

We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsalpha expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells (poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsalpha, but not HP1Hsbeta or HP1Hsgamma, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsalpha staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsalpha localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsalpha expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsalpha protein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsalpha in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsalpha in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsalpha normally silences genes involved in breast cancer invasion and metastasis.

During embryogenesis, the formation of primary vascular networks occurs via the processes of vasculogenesis and angiogenesis. In uveal melanoma, vasculogenic mimicry describes the 'embryonic-like' ability of aggressive, but not nonaggressive, tumor cells to form networks surrounding spheroids of tumor cells in three-dimensional culture; these recapitulate the patterned networks seen in patients' aggressive tumors and correlates with poor prognosis. The molecular profile of these aggressive tumor cells suggests that they have a deregulated genotype, capable of expressing vascular phenotypes. Similarly, the embryonic-like phenotype expressed by the aggressive human breast cancer cells is associated with their ability to express a variety of vascular markers. These studies may offer new insights for consideration in breast cancer diagnosis and therapeutic intervention strategies.

Pathology observational reports and experimental data suggest that keratin and vimentin intermediate filament (IF) coexpression in breast cancer confers a more aggressive "interconverted" phenotype, expressing both epithelial and mesenchymal markers. In this study, we extended previous observations by measuring the expression of keratin and vimentin, in relation to other selected biomarkers of disease progression, in postmenopausal women with breast cancer. Using immunohistochemical analysis of 54 archival, formalin-fixed, paraffin-embedded invasive breast cancers from a well-defined cohort, we examined relative IF (keratin and vimentin) expression in a semiquantitative fashion and compared these results with other biological markers and survival. By univariate analysis, we found that vimentin expression was inversely associated with keratin expression alone (P = 0.0089) and directly related to histological grade (P = 0.017), nuclear grade (P = 0.027), Ki67 growth fraction (P = 0.024), and epidermal growth factor receptor immunostaining (P = 0.019). The relative expression of keratin and vimentin in approximately similar amounts characterized tumors with the poorest prognosis, as compared with keratin-high/vimentin-negative or keratin-low/vimentin-positive tumors. These latter two groups demonstrated similar Kaplan-Meier survival curves; the former group (keratin and vimentin in approximately similar amounts) demonstrated a poorer survival, with a hazard ratio of 2.1 (95% confidence interval, 0.5-9.6). These data suggest that relative keratin and vimentin IF expression is more indicative of prognosis and tumor phenotype than either IF marker detected independently.

We have previously shown that human breast carcinoma cells demonstrating an interconverted phenotype, where keratin (epithelial marker) and vimentin (mesenchymal marker) intermediate filaments are both expressed, have an increased ability to invade a basement membrane matrix in vitro. This increase in invasive potential has been demonstrated in MDA-MB-231 cells, which constitutively express keratins and vimentin, and in MCF-7 cells transfected with the mouse vimentin gene (MoVi). However, vimentin expression alone is not sufficient to confer the complete metastatic phenotype in MoVi cells, as determined by orthotopic administration. Thus, in the present study, differential display analysis was utilized to identify genes that are associated with the invasive and/or metastatic phenotype of several human breast cancer cell lines. Forty-four of 84 PCR fragments were differentially expressed as assessed by Northern hybridization analysis of RNA isolated from MCF-7, MoVi, and MB-231 cell lines. Polyadenylated RNA from a panel of poorly invasive, invasive/non-metastatic, and invasive/metastatic breast carcinoma cell lines was used to differentiate between cell-specific gene expression and genes associated with the invasive and/or metastatic phenotype(s). We observed that lysyl oxidase and a zinc finger transcription factor were expressed only in the invasive and/or metastatic cell lines; whereas, a thiol-specific antioxidant and a heterochromatin protein were down-regulated in these cells. In contrast, tissue factor was expressed only in breast carcinoma cell lines having the highest invasive potential. These results suggest that specific genes involved in breast cancer invasion and metastasis can be separated by differential display methodology to elucidate the molecular basis of tumor cell progression.

The recently discovered tumor suppressor gene maspin has been shown to inhibit tumor cell motility, invasion, and metastasis in breast cancer by our laboratories. Nonetheless, the exploitation of maspin as a potential diagnostic and/or therapeutic tool has remained limited due to the lack of knowledge concerning its molecular and biological mechanism(s) of action. The work reported here demonstrates that recombinant maspin (rMaspin) has the ability to induce higher cell surface levels of alpha5- and alpha3-containing integrins and reduced levels of alpha2-, alpha4-, alpha6-, alpha(v)-, and some beta1-containing integrins in the metastatic human breast carcinoma cell line MDA-MB-435 concomitant with its ability to inhibit the invasive process in vitro. Furthermore, treatment of MDA-MB-435 cells with rMaspin results in the selective adhesion of the cell to a fibronectin matrix and conversion from a fibroblastic to a more epithelial-like phenotype. In addition, the ability of rMaspin to inhibit the invasive process can be abrogated with a blocking antibody to the alpha5beta1 integrin, which diminishes the ability of the cells to invade through a fibronectin matrix-containing barrier in vitro. Taken together, these data address the hypothesis that rMaspin reduces the invasive phenotype of MDA-MB-435 cells by altering their integrin profile, particularly alpha5, which in turn converts these cells to a more benign epithelial phenotype, with less invasive ability. These data provide new insights into the biological significance of this tumor suppressor gene found in normal mammary epithelium and may form the basis of novel therapeutic strategies in the management of breast carcinoma.

Glycolysis is increased in cancer cells compared with normal cells. It has been shown that glucose enters cells via a family of five functional glucose transporters (GLUT). However, GLUT expression appears to be altered in human breast cancer, which may serve as a selective advantage and facilitate the metastatic potential of these cells. The relationship of GLUT isoform expression and breast cancer cell invasiveness has not been adequately addressed. Thus, the purpose of this study was to investigate whether an association exists between GLUT expression and human breast cancer cell invasiveness. Invasiveness of the human breast cancer lines MCF-7, MDA-MB-435 and MDA-MB-231 was measured using an in vitro assay and compared with cellular GLUT isoform expression, assessed by Western blot analysis and verified by immunohistochemistry in a poorly differentiated human ductal breast cancer. Cell surface GLUT-1 expression was associated with the invasive ability of MCF-7 (2.0 + 0.02%), MDA-MB-435 (6.4 +/- 0.4%), and MDA-MB-231 (19.3 +/- 2.0%). However, GLUT-2 and GLUT-5 were inversely associated with invasiveness; GLUT-3 expression was variable; and GLUT-4 was undetected. In a poorly differentiated human ductal breast cancer, in situ GLUT-1 staining was intense. GLUT-1 expression was associated with the in vitro invasive ability of human breast cancer cells which was validated in situ. If this relationship is found to exist in a larger number of human breast cancer tissues, it may be possible to develop diagnostic and therapeutic strategies based on targeted GLUT isoform expression.

The expression of intermediate filament proteins is remarkably tissue specific, which suggests that the intermediate filament type(s) present in cells is somehow related to their biological function. However, in some cancers, particularly malignant breast carcinoma, there is a strong indication that vimentin is co-expressed with keratins, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In the present study, we recapitulated the interconverted phenotype by developing stable transfectants of MCF-7 human breast cancer cells, termed MoVi clones, to express both vimentin and keratins. Overexpression of vimentin in these cells led to augmentation of motility and invasiveness in vitra. These activities could be transiently down-regulated by vimentin antisense oligonucleotides in MoVi clones and MDA-MB-231 cells (which constitutively co-express keratins and vimentin). Furthermore, in the MoVi experimental transfectants expressing the highest percentage of vimentin-positive cells, their proliferative capacity, clonogenic potential, and tumorigenicity increased. However, the metastatic ability of the MoVi transfectants remained unchanged compared with MCF-7neo controls. The MDA-MB-231 cells metastasized to axillary lymph nodes in a SCID mouse model. Finally, we explored the possibility that potential changes could occur with respect to cell surface integrins. These studies revealed a decrease in the alpha 2- and alpha 3-containing promiscuous integrins, in addition to beta 1 containing integrins, concomitant with an increase in the alpha 6-containing laminin receptor integrin. Further functional analysis of the alpha 6 observation showed an increase in the baptotactic migration of MoVi transfectants toward a laminin substrate. From these data, it is postulated that the ability to co-express vimentin and keratins confers a selective advantage to breast cancer cells in their interpretation of signaling cues from the extracellular matrix; however the addition of vimentin intermediate filaments alone is not sufficient to confer the metastatic phenotype.

The expression of intermediate filament proteins is remarkably tissue-specific which suggests that the intermediate filament (IF) type(s) present in cells is somehow related to their biological function. However, in some cancers-particularly malignant melanoma and breast carcinoma, there is a strong indication that vimentin and keratin IFs are coexpressed, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In this review, two in vitro models are presented which recapitulate the interconverted phenotype in human melanoma and breast carcinoma, and allow, for the first time, unique observations to be made with respect to the role of IFs in cancer progression. These studies have provided direct evidence linking overexpression of keratin IFs in human melanoma with increased migratory and invasive activity in vitro, which can be down-regulated by substituting dominant-negative keratin mutants. Overexpression of vimentin IFs in the breast carcinoma model leads to augmentation of motility and invasiveness in vitro, which can be transiently down-regulated by treatment with antisense oligonucleotides to vimentin. Additional experimental evidence suggests that the mechanism(s) responsible for the differential expression of metastatic properties associated with the interconverted phenotype rest(s) in the unique interaction, either direct or indirect, of IFs with specific integrins interacting with the extracellular matrix. In this review, we discuss the observations derived from the human melanoma and breast carcinoma models to address the hypothesis that the ability to coexpress vimentin and keratins confers a selective advantage to tumor cells in their interpretation of and response to signaling cues from the extracellular matrix. The ramifications of these observations are discussed with respect to the patholophysiology of the respective in situ tumors.