transforming-growth-factor-beta and Carcinoma--Intraductal--Noninfiltrating

Normal myoepithelial cells (MECs) play an important tumour-suppressor role in the breast but display an altered phenotype in ductal carcinoma in situ (DCIS), gaining tumour-promoter functions. Matrix metalloproteinase-8 (MMP-8) is expressed by normal MECs but is lost in DCIS. This study investigated the function of MMP-8 in MECs and the impact of its loss in DCIS.. Primary normal and DCIS-associated MECs, and normal (N-1089) and DCIS-modified myoepithelial (β6-1089) cell lines, were used to assess MMP-8 expression and function. β6-1089 lacking MMP-8 were transfected with MMP-8 WT and catalytically inactive MMP-8 EA, and MMP-8 in N-1089 MEC was knocked down with siRNA. The effect on adhesion and migration to extracellular matrix (ECM), localisation of α6β4 integrin to hemidesmosomes (HD), TGF-β signalling and gelatinase activity was measured. The effect of altering MEC MMP-8 expression on tumour cell invasion was investigated in 2D and 3D organotypic models.. Assessment of primary cells and MEC lines confirmed expression of MMP-8 in normal MEC and its loss in DCIS-MEC. Over-expression of MMP-8 WT but not MMP-8 EA in β6-1089 cells increased adhesion to ECM proteins and reduced migration. Conversely, knock-down of MMP-8 in N-1089 reduced adhesion and increased migration. Expression of MMP-8 WT in β6-1089 led to greater localisation of α6β4 to HD and reduced retraction fibre formation, this being reversed by MMP-8 knock-down in N-1089. Over-expression of MMP-8 WT reduced TGF-β signalling and gelatinolytic activity. MMP-8 knock-down enhanced TGF-β signalling and gelatinolytic activity, which was reversed by blocking MMP-9 by knock-down or an inhibitor. MMP-8 WT but not MMP-8 EA over-expression in β6-1089 reduced breast cancer cell invasion in 2D and 3D invasion assays, while MMP-8 knock-down in N-1089 enhanced cancer cell invasion. Staining of breast cancer cases for MMP-8 revealed a statistically significant loss of MMP-8 expression in DCIS with invasion versus pure DCIS (p = 0.001).. These data indicate MMP-8 is a vital component of the myoepithelial tumour-suppressor function. It restores MEC interaction with the matrix, opposes TGF-β signalling and MMP-9 proteolysis, which contributes to inhibition of tumour cell invasion. Assessment of MMP-8 expression may help to determine risk of DCIS progression.

Topics: Biomarkers, Tumor; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Adhesion; Cell Line, Transformed; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Epithelial Cells; Female; Gene Expression; Gene Knockdown Techniques; Humans; Immunohistochemistry; Integrin alpha6beta4; Matrix Metalloproteinase 8; Matrix Metalloproteinase 9; Paracrine Communication; Protein Transport; Proteolysis; Signal Transduction; Transforming Growth Factor beta

Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.

Topics: Biomechanical Phenomena; Biophysical Phenomena; Birefringence; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Transformation, Neoplastic; Collagen; Disease Progression; Extracellular Matrix; Female; Humans; Macrophages; Microscopy, Atomic Force; Microscopy, Fluorescence, Multiphoton; Neoplasm Invasiveness; Signal Transduction; Transforming Growth Factor beta

This study investigated the functional and clinical significance of integrin αvβ6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS).. Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvβ6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvβ6 expression, were used to measure the effect of αvβ6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model. Involvement of TGFβ signaling was established using mink lung epithelial cell (MLEC) assay and antibody inhibition, and expression and activation of matrix metalloproteinase (MMP)-9 established by Real Time-PCR and zymography.. Expression of αvβ6 is significantly associated with progression to invasive cancer (P < 0.006) and with recurrence over a median follow-up of 114 months in a series of matched DCIS cases treated with local excision. We show that expression of αvβ6 drives myoepithelial cells to promote tumor cell invasion in vitro and enhances mammary tumor growth in vivo. The tumor-promoting effect of αvβ6-positive myoepithelial cells is dependent on TGFβ-driven upregulation of MMP9 and can be abrogated by inhibiting this pathway.. These findings indicate that altered myoepithelial cells in DCIS predict disease progression and recurrence and show that upregulation of αvβ6 on myoepithelial cells generates a tumor promoter function through TGFβ upregulation of MMP-9. These data suggest that expression of αvβ6 may be used to stratify patients with DCIS.

Topics: Animals; Antigens, Neoplasm; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Case-Control Studies; Cell Line; Cell Line, Tumor; Disease Progression; Epithelial Cells; Female; Follow-Up Studies; Humans; Immunohistochemistry; Integrins; Matrix Metalloproteinase 9; Mice; Mink; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Prognosis; Transforming Growth Factor beta; Tumor Burden; Tumor Microenvironment

Ductal carcinoma in situ (DCIS) is an early stage noninvasive breast cancer that originates in the epithelial lining of the milk ducts, but it can evolve into comedo DCIS and ultimately, into the most common type of breast cancer, invasive ductal carcinoma. Understanding the progression and how to effectively intervene in it presents a major scientific challenge. The extracellular matrix (ECM) surrounding a duct contains several types of cells and several types of growth factors that are known to individually affect tumor growth, but at present the complex biochemical and mechanical interactions of these stromal cells and growth factors with tumor cells is poorly understood. Here we develop a mathematical model that incorporates the cross-talk between stromal and tumor cells, which can predict how perturbations of the local biochemical and mechanical state influence tumor evolution. We focus on the EGF and TGF-β signaling pathways and show how up- or down-regulation of components in these pathways affects cell growth and proliferation. We then study a hybrid model for the interaction of cells with the tumor microenvironment (TME), in which epithelial cells (ECs) are modeled individually while the ECM is treated as a continuum, and show how these interactions affect the early development of tumors. Finally, we incorporate breakdown of the epithelium into the model and predict the early stages of tumor invasion into the stroma. Our results shed light on the interactions between growth factors, mechanical properties of the ECM, and feedback signaling loops between stromal and tumor cells, and suggest how epigenetic changes in transformed cells affect tumor progression.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Epidermal Growth Factor; Female; Humans; Mathematical Concepts; Models, Biological; Neoplasm Invasiveness; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Tumor Microenvironment

A gene expression signature indicative of activated wound responses is common to more than 90% of non-neoplastic tissues adjacent to breast cancer, but these tissues also exhibit substantial heterogeneity. We hypothesized that gene expression subtypes of breast cancer microenvironment can be defined and that these microenvironment subtypes have clinical relevance.. Gene expression was evaluated in 72 patient-derived breast tissue samples adjacent to invasive breast cancer or ductal carcinoma in situ. Unsupervised clustering identified two distinct gene expression subgroups that differed in expression of genes involved in activation of fibrosis, cellular movement, cell adhesion and cell-cell contact. We evaluated the prognostic relevance of extratumoral subtype (comparing the Active group, defined by high expression of fibrosis and cellular movement genes, to the Inactive group, defined by high expression of claudins and other cellular adhesion and cell-cell contact genes) using clinical data. To establish the biological characteristics of these subtypes, gene expression profiles were compared against published and novel tumor and tumor stroma-derived signatures (Twist-related protein 1 (TWIST1) overexpression, transforming growth factor beta (TGF-β)-induced fibroblast activation, breast fibrosis, claudin-low tumor subtype and estrogen response). Histological and immunohistochemical analyses of tissues representing each microenvironment subtype were performed to evaluate protein expression and compositional differences between microenvironment subtypes.. Extratumoral Active versus Inactive subtypes were not significantly associated with overall survival among all patients (hazard ratio (HR) = 1.4, 95% CI 0.6 to 2.8, P = 0.337), but there was a strong association with overall survival among estrogen receptor (ER) positive patients (HR = 2.5, 95% CI 0.9 to 6.7, P = 0.062) and hormone-treated patients (HR = 2.6, 95% CI 1.0 to 7.0, P = 0.045). The Active subtype of breast microenvironment is correlated with TWIST-overexpression signatures and shares features of claudin-low breast cancers. The Active subtype was also associated with expression of TGF-β induced fibroblast activation signatures, but there was no significant association between Active/Inactive microenvironment and desmoid type fibrosis or estrogen response gene expression signatures. Consistent with the RNA expression profiles, Active cancer-adjacent tissues exhibited higher density of TWIST nuclear staining, predominantly in epithelium, and no evidence of increased fibrosis.. These results document the presence of two distinct subtypes of microenvironment, with Active versus Inactive cancer-adjacent extratumoral microenvironment influencing the aggressiveness and outcome of ER-positive human breast cancers.

Topics: Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Claudins; Female; Fibroblasts; Fibrosis; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Nuclear Proteins; Receptors, Estrogen; Transforming Growth Factor beta; Tumor Microenvironment; Twist-Related Protein 1

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating "synthetic" cancer associated fibroblasts (CAFs). More specifically, these "synthetic" CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti-cancer agents, to therapeutically restore the expression of stromal Cav-1 in cancer associated fibroblasts. We discuss this possibility, in light of the launch of a new clinical trial that uses chloroquine to treat DCIS patients: PINC (Preventing Invasive Breast Neoplasia with Cholorquine) [See http://clinicaltrials.gov/show/NCT01023477].

Topics: Actins; Autophagy; Biomarkers, Tumor; Breast Neoplasms; Calcium-Binding Proteins; Calponins; Carcinoma, Intraductal, Noninfiltrating; Caveolin 1; Cell Line, Tumor; Chloroquine; Coculture Techniques; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Microfilament Proteins; Phenotype; Prognosis; Smad2 Protein; Transforming Growth Factor beta; Vimentin

Invasive lobular carcinoma (ILC) and lobular carcinoma in situ characteristically show loss of E-cadherin expression and so immunohistochemistry for E-cadherin is being increasingly used as a tool to differentiate between lobular and ductal lesions in challenging situations. However, misinterpretation of "aberrant" positive staining may lead some to exclude a diagnosis of lobular carcinoma. E-cadherin and beta-catenin immunohistochemistry was analyzed in 25 ILCs. E-cadherin "positive" ILCs were subjected to molecular analysis including comparative genomic hybridization. Different morphologic components of case 25, showing heterogenous E-cadherin expression, were analyzed by E-cadherin gene sequencing, methylation, and DASL gene expression profiling. Four ILCs were positive for E-cadherin, but each also had neoplastic cells with aberrant staining. Two of these ILCs were positive for beta-catenin, again with some aberrantly stained neoplastic cells, and 2 were negative. The solid component of case 25 was positive for E-cadherin whereas the classic and alveolar areas were negative. All components harbored an in-frame deletion in exon 7 (867del24) of the E-cadherin gene and loss of the wild type allele. Comparative genomic hybridization demonstrated evidence of clonal evolution from E-cadherin-positive to E-cadherin-negative components. E-cadherin down-regulation seems to be through transcriptional repression via activation of transforming growth factor-beta/SMAD2 rather than methylation. Positive staining for E-cadherin should not preclude a diagnosis of lobular in favor of ductal carcinoma. Molecular evidence suggests that even when E-cadherin is expressed, the cadherin-catenin complex maybe nonfunctional. Misclassification of tumors may lead to mismanagement of patients in clinical practice, particularly in the context of in situ disease at margins.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cadherins; Carcinoma in Situ; Carcinoma, Ductal; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Diagnosis, Differential; DNA Methylation; DNA, Neoplasm; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Loss of Heterozygosity; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; RNA, Messenger; RNA, Neoplasm; Sequence Analysis, DNA; Smad2 Protein; Transforming Growth Factor beta

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.

Topics: Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Adhesion; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The activity of T regulatory cells (Tregs) is known to be closely associated with the expression of forkhead/winged helix transcription factor, FOXP3. To determine, whether accumulation and activation of intratumoral Tregs help in the progression of breast carcinoma, we have analyzed the intratumoral expression of FOXP3 in invasive breast carcinoma and compared it with its level in ductal carcinoma in situ (DCIS) and adjacent normal tissue with the main aim of using this factor as marker of tumor progression. Intratumoral FOXP3 levels were correlated with the levels of transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF, an invasogenic and angiogenic growth factor) and intratumoral microvessel density (IMD, a prognostic marker for angiogenesis). We also analyzed whether FOXP3 gene expression correlated with other clinicopathological variables like age, tumor stage, histological grade and lymph node metastasis. Infiltrating cancers had higher FOXP3 transcription (7.43+/-3.44) than did ductal carcinoma in situ (4.27+/-1.97, p<0.05) and normal tissues (3.51+/-1.22, p<0.001). Intratumoral FOXP3 expression was significantly higher in patients with stage III disease (TNM classification) compared to patients who had stage II disease (p=0.037). In infiltrating carcinoma, a significant positive correlation between FOXP3 expression and TGF-beta1 expression was noted (p<0.001). Furthermore, a positive correlation between FOXP3 expression with VEGF expression and IMD values was also detected, however, statistically that was non-significant. A linear association of intratumoral FOXP3 expression with invasion, size and vascularity suggests a utility of FOXP3, an indicator of Treg activity as a marker of tumor progression and metastasis in breast carcinoma.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Disease Progression; Female; Forkhead Transcription Factors; Gene Expression; Humans; Mastectomy; Middle Aged; Neoplasm Staging; Pilot Projects; T-Lymphocyte Subsets; T-Lymphocytes; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

A comparative study of the products of the cell cycle control genes p53 (mutated form), p21, Rb (nonphosphorylated and phosphorylated form) and TGFbeta was performed by immunohistochemistry and Western blot, in benign breast disorders and breast cancer (in situ and infiltrating tumors). For the five proteins studied, the relative numbers of positively stained cells were higher in in situ carcinoma than in benign breast diseases. In infiltrating breast tumors, the relative numbers of positively stained cells were even higher than in in situ tumors except for the percentage of pRb immunostained cells, which decreased slightly in infiltrative tumors. For the other four proteins, the percentages of positively stained cases were similar to those found in in situ tumors. In the three groups of patients, TGFbeta immunoreaction appeared in the cytoplasm while immunoreactions to p53, p21, Rb, and pRb were found always in the nucleus except for p21 in in situ tumors, which showed cytoplasmic immunoreaction. Present results suggest that accumulation of mutated p53, cytoplasmic p21, and pRb in breast gland epithelium might be a crucial point in the development of in situ adenocarcinoma. In the infiltrating tumors, the expression of p21 in the nuclei and the decrease in pRb expression suggest an insufficient attempt to hinder cell proliferation.

Topics: Adult; Aged; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Immunohistochemistry; Middle Aged; Retinoblastoma Protein; Transforming Growth Factor beta; Tumor Suppressor Protein p53

DCs are the most potent antigen-presenting cells that play a major role in initiating the antitumor immune response. Although the clinical significance of TIDCs has been investigated in a variety of human cancers, few studies have focused on the in situ maturation status of DCs. We have analyzed the maturation-specific significance of TIDCs in the prognosis of patients with breast carcinoma. We evaluated 130 breast carcinomas for the presence of TIDCs using immunohistochemistry with an anti-CD1a antibody for immature DCs and an anti-CD83 antibody for mature DCs. Intratumoral expression of immunosuppressive cytokines was also examined. All samples contained CD1a(+) TIDCs, and 82 (63.1%) samples contained CD83(+) TIDCs. The number of CD83(+) TIDCs was inversely correlated with lymph node metastasis and with tissue expression of VEGF and TGF-beta, whereas the number of CD1a(+) TIDCs was not. Kaplan-Meier analysis (log rank statistics) revealed a significant association of increasing number of CD83(+) TIDCs with longer relapse-free (p = 0.002) and overall (p < 0.001) survival. Furthermore, among patients with lymph node metastasis, the survival rate of those with larger numbers of CD83(+) TIDCs was significantly better than that of patients with fewer CD83(+) TIDCs. Multivariate analysis revealed that CD83(+) TIDCs had independent prognostic relevance in breast carcinomas. The infiltration of tumors by mature DCs expressing CD83 may be of great importance in initiating the primary antitumor immune response and is confirmed as an independent, immunologic prognostic parameter for survival in patients with breast cancer.

Topics: Adenocarcinoma, Scirrhous; Adult; Aged; Antigens, CD; Antigens, CD1; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; CD83 Antigen; Dendritic Cells; Endothelial Growth Factors; Female; Humans; Immunoglobulins; Intercellular Signaling Peptides and Proteins; Life Tables; Lymphatic Metastasis; Lymphokines; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Prognosis; Receptors, Estrogen; Receptors, Progesterone; S100 Proteins; Survival Analysis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Several cytokines including members of the transforming growth factor-beta (TGF-beta) and tumor necrosis factor (TNF) families have been implicated in the homing mechanism of breast cancer metastasis. We hypothesize that primary breast tumor tissues differentially express modulators of bone cell function and that this expression pattern contributes to their aggressive and metastatic potential and to their capacity to establish and grow in bone. We, therefore, examined the gene expression pattern of the TGF-beta family members (inhibin/activin betaA subunit (activin betaA), inhibin alpha subunit, and bone morphogenetic protein-2 (BMP-2)), the TNF family members (receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG)), and osteopontin (OPN) in normal, non-invasive, invasive, and metastatic human breast cancer specimens. The mRNA transcript levels of these genes were quantified by reverse transcription (RT) and fluorescent-based kinetic PCR in 18 normal breast tissues, five ductal carcinoma in situ (DCIS). 24 primary breast tumor tissue, and five distant metastases. The mRNA transcript level of each gene was normalized to the amount of beta-actin present in the samples. We observed differential gene expression of the selected TGF-beta family members as well as OPN in breast cancer progression. The average gene expression of the putative tumor suppressor, inhibin alpha, did not significantly change in any of the tumor tissues examined compared to normal breast tissue. The mRNA level of BMP-2, a protein with anti-proliferative effects in breast cancer cell lines and involved in bone formation, significantly decreased in non-invasive, invasive, and liver metastatic breast tumor tissue compared to normal breast tissue. The gene expression of activin betaA, a protein involved in cell proliferation and osteoclast induction, increased in invasive and bone metastatic tumor tissue compared to normal breast tissue. The mRNA level of OPN, a bone matrix protein associated with enhanced malignancy, increased in non-invasive, invasive, and liver and bone metastatic breast tumor tissue compared to normal breast tissue. In contrast, the average gene expressions of the TNF family members, RANKL and OPG, proteins involved in the regulation of osteoclastogenesis, were only slightly if at all changed in the different stage breast tumor tissues. These results suggest that differential gene expression of bone-related proteins, especially OPN, activin betaA, and BMP-

Topics: Adult; Aged; Aged, 80 and over; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Breast; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Inhibin-beta Subunits; Liver Neoplasms; Middle Aged; Neoplasm Metastasis; Osteopontin; Ovarian Neoplasms; Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta

Localization of growth factors such as transforming growth factor alpha (TGF-alpha) and beta1 (TGF-beta1), insulin-like growth factor 1 (IGF-1), and epidermal growth factor receptor (EGFR) in breast cancer tissue is controversial. We immunohistochemically investigated expression patterns of these growth factors and EGFR along with estrogen receptor (ER) status in 36 breast carcinomas (21 invasive ductal, 11 invasive lobular, 4 noninvasive ductal) and compared the results with those found in 10 fibroadenomas. Twenty-four of 36 carcinomas and all of the 10 fibroadenomas showed positivity for ER. TGF-alpha was immunoreactive in all of the carcinomas and fibroadenomas. TGF-beta1 was negative in all of the invasive ductal carcinomas and positive in all of the fibroadenomas and in five lobular carcinomas. EGFR was regularly expressed preferentially in the myoepithelial cells of mammary ducts in the fibroadenomas and in nontumorous glands. Six of the 36 carcinomas were positive for EGFR. Those tumors were negative for ER (P < .001). There was IGF-1 expression in all of the cases of carcinoma and fibroadenoma. We conclude that TGF-alpha is expressed abundantly in invasive and intraductal breast carcinomas and in fibroadenomas. EGFR expression significantly correlates with negative ER status in breast carcinoma. In breast carcinoma, IGF-1 is broadly expressed by the tumor as well as by stromal cells and might act as a growth stimulator in endocrine, paracrine, and autocrine manners.

Topics: Adenocarcinoma; Adult; Aged; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; ErbB Receptors; Female; Fibroadenoma; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Middle Aged; Transforming Growth Factor alpha; Transforming Growth Factor beta

Transforming growth factor alpha (TGF alpha) and Transforming growth factor beta-1 (TGF-beta 1) are growth regulatory for breast cancer cell lines in vitro and several studies have suggested that levels of the receptor for TGF alpha, the epidermal growth factor (EGFR) in tumour biopsies predict relapse and survival. We have examined the prognostic significance of TGF alpha, TGF-beta 1 and EGFR mRNA expression in a series of patients with primary breast cancer with a median follow up period of 60 months. In 167 patients the expression of TGF-beta 1 was inversely correlated with node status (P = 0.065) but not ER status, tumour size or menopausal status. Patients with high levels of TGF-beta 1 had a longer disease free interval with a significantly longer probability of survival at 80 months although the overall relapse free survival was not increased. EGFR mRNA expression was measured in 106 patients and was inversely correlated with ER status (P = 0.018). EGFR levels did not predict for early relapse or survival. TGF alpha mRNA levels were measured in 104 patients, no correlation was seen tumour size, node status, Er status, or clinical outcome.

Topics: Adenocarcinoma, Mucinous; Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma; Carcinoma, Intraductal, Noninfiltrating; ErbB Receptors; Female; Follow-Up Studies; Humans; Immunoblotting; Middle Aged; Prognosis; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factors

TGF alpha and beta expression was examined using rabbit polyclonal antibodies and immunohistochemistry on a series of 195 breast carcinomas. TGF alpha immunoreactivity was observed in all but nine of the tumours, with over 50 per cent staining strongly. The polyclonal TGF alpha antibody (CIM1), when compared with a commercially available mouse monoclonal TGF alpha antibody used on the same sections, gave a good correlation (r = 0.52, P < 0.001). Both TGF alpha antibodies produced a granular cytoplasmic staining pattern, that with CIM1 being coarser, suggestive of binding to an aggregated protein or organelle. Eighty-one per cent of tumours stained with the TGF beta antibody, 35 per cent strongly. There was significant co-expression of TGF alpha and TGF beta (P < 0.001). However, they were not found to be useful prognostic indicators, lacking any significant correlation with histological classification, tumour size, nodal status, oestrogen receptor status, S-phase fraction, or overall survival over a 9-12 year period. The expression of these growth factors in most breast carcinomas suggests that they have important biological roles, but the exact nature of these roles remains unclear at the moment.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Immunoenzyme Techniques; Middle Aged; Prognosis; Transforming Growth Factor alpha; Transforming Growth Factor beta

The level of expression and localization of transforming growth factor-beta 1 (TGF-beta 1) were analyzed by immunocytochemistry using antibodies that distinguished the sites of intracellular synthesis and extracellular secretion of TGF-beta 1 in 28 cases of infiltrating duct carcinoma of breast, 12 of which had lymph node metastases. Twenty-seven of 28 primary tumors and all 12 lymph node metastases showed extracellular deposition of TGF-beta 1. The extracellular TGF-beta 1 staining was either confined to or more strongly expressed at the advancing edges of the tumor than in the center of the primary tumor. By contrast, 19 of 28 primary tumors and 11 of 12 metastases contained intracellular TGF-beta 1, and no variation in the intensity was seen. The metastatic tumors were significantly more intensely stained for both intra- and extracellular TGF-beta 1 than the primary tumor tissues. The preferential expression of secreted TGF-beta 1 at the advancing tumor edges and in lymph node metastases suggests a role for TGF-beta 1 in the malignant progression of breast carcinoma.

Topics: Axilla; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Immunoenzyme Techniques; Lymph Nodes; Lymphatic Metastasis; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) is a multi-functional regulatory protein which can affect growth, immune responses, angiogenesis and the formation of extracellular matrix. Its role in breast carcinomas has been investigated using an antiserum to TGF-beta 1 and immunohistochemistry. 27 ductal carcinomas in situ and 54 invasive carcinomas were examined, employing formalin-fixed, paraffin-embedded material. There was no reactivity in 55.5% of in situ carcinomas in comparison with the invasive tumour where only a third were negative. Prominent reactivity was seen in 11% of in situ tumours, and 20% of invasive carcinomas. There was no correlation between detection of transforming growth factor beta 1, and histological grade, oestrogen receptor status, epidermal growth factor receptor status and Ki-67 labelling for the invasive carcinomas. There was a significant relationship between prominent reactivity and node status, all carcinomas with this degree of staining having metastasised. This, along with the differences between in situ and invasive carcinomas, suggests that TGF-beta 1 may be a determining factor for invasion and metastasis.

Topics: Breast Neoplasms; Carcinoma in Situ; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Neoplasm Invasiveness; Transforming Growth Factor beta

We have investigated the ability of tamoxifen to regulate members of the transforming growth factor beta (TGF-beta) family in human breast cancers in vivo. Using immunohistochemical techniques, we find that 3 months of tamoxifen treatment causes a consistent induction of extracellular TGF-beta 1 in breast cancer biopsies, compared with matched pretreatment samples from the same patient. The induced TGF-beta is localized between and around stromal fibroblasts and appears to be derived from these cells. Lower levels of TGF-beta 1,-beta 2, and -beta 3 seen in epithelial cells were not altered by tamoxifen treatment. The increased stromal staining of TGF-beta 1 occurred in estrogen receptor-negative as well as estrogen receptor-positive tumors. These results provide in vivo evidence for a novel, estrogen receptor-independent mechanism of action for tamoxifen, involving the stromal induction of a potent growth inhibitor for epithelial cells.

Topics: Biomarkers, Tumor; Biopsy; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Immunohistochemistry; Neoplasm Invasiveness; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor beta