Stages Of Breast Cancer Development Research Articles

Host epithelial geometry regulates breast cancer cell invasiveness

Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment.

On the Role of the Microenvironment in Mammary Gland Development and Cancer

Knowledge of the specific microenvironmental cues involved at the earliest stages of breast cancer development is currently limited.

MiRNAs in breast cancer tumorigenesis (Review)

miRNAs are small, endogenous, non-coding RNAs that negatively regulate protein-coding mRNAs at the post-transcriptional level. It is estimated that in humans thousands of miRNAs are expressed and more than 700 miRNAs have been described to date. About 50% of annotated human miRNAs are detected in regions of fragile sites, which are associated with cancer. The available evidence has shown that miRNAs widely participate in the development or progression of many types of cancers, including breast cancer. The role of miRNAs in breast cancer has been widely investigated; here, we will focus on what is known about the working mechanism of miRNAs in different stages of breast cancer development.

2D Difference Gel Electrophoresis Analysis of Different Time Points during the Course of Neoplastic Transformation of Human Mammary Epithelial Cells

Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesis over an 80 day period, protein extracts were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). Nine proteins were found to be significantly different in abundance: keratin 1, keratin 7, heat shock protein 4A-like, t-complex protein 1, stathmin, gelsolin, FK506 binding protein 5, ribosomal protein P0, and maspin. Keratin 7 and maspin displayed a linear down-regulation over 80 days. All of these proteins have been shown to be involved in the maintenance of a metastatic state including cytoskeletal modifications and motility. We conclude that, following neoplastic induction, THMECs display an early and progressive increase in metastatic potential. Further investigations into the function and regulatory mechanisms of these proteins will provide an unparalleled understanding of the initial states through which a breast cancer cell transitions following acquisition of the genetic abnormalities required for oncogenesis.

Abstract P1-06-01: Omega-3 Fatty Acids for Chemoprevention: NF-Kb as a Molecular Target in Both Pre and Post Menopausal High-Risk Breast Cancer Models

Abstract Genetic heterogeneity of human breast cancer has complicated treatment, prevention and therapeutic regimens in both clinical and personal care settings. With the recent advances in genetic analyses, human breast tumors are now segregated into four principle molecular subtypes: luminal A, luminal B, basal and HER2 overexpressors, each differing in their driver mutations, estrogen receptor status and dependence on specific cell signaling pathways to maintain growth and tumorigenicity. Omega-3 fatty acids have been shown in previous studies to be effective in modulating tumor growth in preclinical models of breast cancer, but epidemiological studies have been less clear regarding their efficacy as chemopreventive agents. Like many targeted therapies, we hypothesize that omega-3 fatty acids will be most effective against specific subtypes of disease, and it will be critical to identify these subtypes if these dietary agents are to be exploited most effectively. In this current study, the genetic diversity of breast cancer was represented by appropriate cell lines of matching molecular backgrounds and each class of breast cancer were subjected to exposures of omega-3 fatty ethyl esters derived from Lovaza®, an anti-hypertriglyceride medication that is currently under clinical evaluation as a chemopreventive for breast cancer. Previous studies have suggested that a target for omega-3 fatty acid modulation is the transcription factor NF-kB, a central key mediator of inflammation and cancer cell survival. Coined the single most important molecular machinery required for cancer initiation and promotion, NF-kB has been identified to play critical roles in all stages of breast cancer development. The omega-3 ethyl esters demonstrated suppression of NF-kB transcriptional activity, nuclear localization and overall function with high efficacy in the cell lines reflective of the luminal A, luminal B and basal subtypes, correlating with inhibition of proliferation and overall survival. Interestingly, among the panel of breast cancer subtypes, the aggressive HER2 overexpressing cell lines were resistant to the inhibitory effects of the omega-3 ethyl esters. One of the proposed mechanisms by which NF-kB is regulated is co-localization with IkB. siRNA knockdown assays suggest IkB involvement in the repression of NF-kB function by the omega-3 ethyl esters in the responding cell lines. Results from an ongoing clinical trial with omega-3 ethyl esters will help confirm if specific subtypes of breast cancers will be more effectively prevented using these agents, and whether NF-kB is the target by which they are mediating their anti-cancer effects. These studies are some of the first to use molecular profiles to identify potential responders and non-responders for dietary intervention and may provide better direction for future clinical studies evaluating the efficacy of diet and lifestyle in the preventive setting. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-06-01.

PTEN mutation spectrum in breast cancers and breast hyperplasia

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently mutated human tumor suppressor genes. The present study aims to investigate the role of PTEN mutation in breast carcinogenesis by analyzing PTEN mutation spectrum and the protein expression in breast cancers, adjacent hyperplastic lesions, benign breast lesions and normal breast tissues. All 9 exons of PTEN gene were amplified by PCR with DNA extracted from 50 of human breast cancers and corresponding adjacent breast hyperplasia tissues, adjacent normal breast tissues, as well as 50 breast benign lesions residing in or around Yunnan, China, respectively. PCR products were then sequenced for mutation screening. And we also proved the effect of mutations on the expression of PTEN protein by immunohistochemistry. PTEN mutations were detected in 11 of 50 (22%) breast cancers and 4 of 50 (8%) adjacent ductal hyperplasia, all of which were atypical ductal hyperplasia and same PTEN mutation were detected in the corresponding cancer tissues. No PTEN mutation was detected in all adjacent normal breast tissues and 50 cases of breast benign lesions. The mutation sites concentrated at exon 3, 4, 5 and 7; no mutation was detected in exon 1, 2, 6, 8, or 9 and splicing sites of all introns. The hottest mutation spots were exon 5 with missense mutations. Immunohistochemical analysis showed that 24 of 50 (48%) breast cancers and 6 of 50 (12%) adjacent breast hyperplasia demonstrated negative immuno-staining of PTEN (loss of PTEN protein expression). All the 4 adjacent breast tissues harbored PTEN mutations and 9 of 11 breast cancers with PTEN mutation were loss of PTEN expression. Statistical analysis revealed that PTEN gene mutations were correlated with the PTEN expression. The incidence of PTEN mutations is relatively high in patients with sporadic breast cancer in the region of Yunnan, China and exists at the early stage of breast cancer development. The PTEN mutations have significant effect on the expression silencing of PTEN protein indicating the important role of PTEN mutation in carcinogenesis of breast cancers.

Regulation of Macrophage Inhibitory Factor (MIF) by Epidermal Growth Factor Receptor (EGFR) in the MCF10AT Model of Breast Cancer Progression

Genetic aberration of EGFR is one of the major molecular characteristics of breast cancer. However, the molecular changes associated with EGFR signaling during different stages of breast cancer development have not been studied. In this study, complementary two-dimensional-DIGE and iTRAQ technologies were used to profile the expression level of proteins in 4 isogenic cell lines in the MCF10AT model of breast cancer progression following a time course of EGF stimulation. A total of 80 proteins (67 from iTRAQ, 15 from DIGE, 2 common in both) were identified to be up- or down-regulated by EGF treatment. Following EGF stimulation, the expression level of MIF, a cytokine that has been implicated in many human cancers, was decreased in MCF10A1 normal breast mammary epithelial cells, increased in MCF10AT1k preneoplastic and MCF10CA1h low grade breast cancer cells, but showed no obvious difference in the MCF10CA1a high grade cancer cells. The increase in MIF expression level following EGF treatment could also be observed in A431 cervical cancer cells. EGF-induced increases of MIF expression levels in CA1h breast cancer cells were abrogated when MEK, but not PIK3CA, was knocked down. In addition, silencing of MIF diminished the proliferation of EGF-stimulated CA1h cells when compared to control cells. Taken together, our data suggested an EGFR --> MEK --> MIF proliferative pathway that has never been reported previously and that this pathway "evolves" during disease progression as modeled by the MCF10AT system. Revelation of the novel relationship between MIF and EGF may contribute to an integrated understanding of the roles of these oncogenic factors during breast cancer development.

Silencing of TMS1/ASC Promotes Resistance to Anoikis in Breast Epithelial Cells

Ductal carcinoma in situ (DCIS) is characterized by ductal epithelial cells that have filled the luminal space of the breast duct and survive despite loss of extracellular matrix contact. In normal epithelial cells, the loss of such contact triggers a form of apoptosis known as detachment-induced apoptosis or "anoikis." TMS1/ASC is a bipartite adaptor molecule that participates in inflammatory and apoptotic signaling pathways. Epigenetic silencing of TMS1 has been observed in a significant proportion of human breast and other cancers, but the mechanism by which TMS1 silencing contributes to carcinogenesis is unknown. Here, we examined the role of TMS1 in anoikis. We found that TMS1 expression is induced in response to loss of substratum interactions in breast epithelial cells. siRNA-mediated knockdown of TMS1 leads to anoikis resistance, due in part to the persistent activation of extracellular signal-regulated kinase and an impaired ability to up-regulate the BH3-only protein Bim. We further show that the detachment-induced cleavage of procaspase-8, a newly described mediator of cellular adhesion, is significantly inhibited in the absence of TMS1. These data show a novel upstream role for TMS1 in the promotion of anoikis, and suggest that silencing of TMS1 may contribute to the pathogenesis of breast cancer by allowing epithelial cells to bypass cell death in the early stages of breast cancer development. This conclusion is supported by in vivo data showing that TMS1 is selectively down-regulated in the aberrant epithelial cells filling the lumen of the breast duct in a subset of primary DCIS lesions.

The role of bevacizumab in breast cancer

Neoangiogenesis and expression of the main angiogenic factor Vascular endothelial growth factor (VEGF) are critical steps already present at an early stage of breast cancer development. As tumor growth progresses other pro-angiogenic factors, including bFGF, TGFa and IL-8, are overexpressed and act in addition to VEGF. VEGF expression has been associated to poor prognosis and therapy outcome in breast cancer. Moreover, several studies have documented that HER-2 overexpression induces VEGF expression/secretion and neovascularization and that VEGF expression increases when tumors become resistant to treatment. On these bases the anti-VEGF drug Bevacizumab has been evaluated in the clinical setting in breast cancer patients. Recently, phase III studies have demonstrated that Bevacizumab in combination with taxanes produces a high rate of responses and increases the Progression Free survival of breast cancer patients. A large array of studies is currently ongoing with Bevacizumab in combination with chemotherapy and hormone-therapy in the metastatic, neoadjuvant and adjuvant setting.

Proteomics Analysis of Conditioned Media from Three Breast Cancer Cell Lines: A Mine for Biomarkers and Therapeutic Targets

A "bottom-up" proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 microg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers.

Development of Vaccines for High-Risk Ductal Carcinoma In situ of the Breast

Certain ductal carcinoma in situ (DCIS) lesions overexpress the HER-2/neu receptor at this early stage of breast cancer development. Recently, we showed that a HER-2-targeted dendritic cell vaccine could be used to eliminate HER-2-overexpressing cells in patients that harbor these high-risk DCIS lesions. Our findings suggest that vaccinating such patients might diminish the risk of recurrence, protect against the development of invasive breast cancer, and minimize morbidity associated with current treatments. We discuss several implications of this work for developing effective cancer vaccines.

Rapamycin Inhibits Growth of Premalignant and Malignant Mammary Lesions in a Mouse Model of Ductal Carcinoma In situ

Rapamycin has been shown to have antitumor effects in various tumor models. To study the effect of rapamycin at different stages of breast cancer development, we used two unique mouse models of breast cancer with activated phosphatidylinositol 3-kinase (PI3K) pathway. Met-1 tumors are highly invasive and metastatic, and mammary intraepithelial neoplasia-outgrowths (MIN-O), a model for human ductal carcinoma in situ, are transplantable premalignant mammary lesions that develop invasive carcinoma with predictable latencies. Both of these models were derived from mammary lesions in Tg(MMTV-PyV-mT) mice. Met-1 tumors were used to study the effect of rapamycin treatment on invasive disease. Transplanted MIN-O model was used to study the effect of rapamycin on premalignant mammary lesions. Animals were in vivo micro-positron emission tomography imaged to follow the lesion growth and transformation to tumor during the treatment. Cell proliferation, angiogenesis, and apoptosis was assayed by immunohistochemistry. Rapamycin inhibited in vitro tumor cell proliferation and in vivo Met-1 tumor growth. The growth inhibition was correlated with dephosphorylation of mammalian target of rapamycin (mTOR) targets. Rapamycin treatment significantly reduced the growth of the premalignant MIN-O lesion, as well as tumor incidence and tumor burden. Growth inhibition was associated with reduced cell proliferation and angiogenesis and increased apoptosis. In PyV-mT mouse mammary models, rapamycin inhibits the growth of premalignant lesions and invasive tumors. Although the inhibitory effect of rapamycin was striking, rapamycin treatment did not completely obliterate the lesions.

Breast cancer progression in MCF10A series of cell lines is associated with alterations in retinoic acid and retinoid X receptors and with differential response to retinoids.

In most breast carcinomas and in breast cancer cell lines, retinoic acid receptor beta (RARbeta) is lost or down-regulated, whereas retinoic acid receptor alpha and gamma (RARalpha, gamma) and retinoid X receptors (RXRalpha, beta, gamma) are variably expressed. Little is known about alterations of the above receptors in hyperplastic and premalignant stages of breast cancer development. In this study, we employed the MCF10A series of breast epithelial cell lines (the parental and benign MCF10A, premalignant MCF10AT, and malignant MCF10CA1a) to assess whether in the course of their malignant transformation specific alterations in RARalpha, beta, gamma and RXRalpha, beta, gamma expression occur and whether they may affect the sensitivity of cells to retinoids. Malignant properties of the above cell lines were estimated by the nude mice xenograft transplantation assay. Among the above receptors most significant alterations occurred in RARbeta2, which was detected in the normal breast epithelial cells both, at mRNA and protein levels, but expressed in the MCF10A cell lines at mRNA level only. The transformation of benign MCF10A cells into premalignant MCF10AT and malignant MCF10CA1a was also associated with increase in RARalpha, RARgamma, RXRalpha, and RXRbeta proteins expression. All-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA), and 4-(hydroxyphenyl) retinamide (4-HPR) induced RARbeta2 protein expression exclusively in the benign MCF10A cells and the former two retinoids, mRNA expression in MCF10A and MCF10AT cells, but not in malignant, MCF10CA1a cells, suggesting that the loss of inducible RARbeta expression is associated with the progression and malignant transformation of MCF10A cells. Retinoids also variable decreased the RARalpha, RARgamma and RXRalpha protein expression preferentially in the premalignant and malignant, but not in benign MCF10A cells. Among the above retinoids, 4-HPR was most efficacious in inhibiting the growth of the three cell lines and this apparently was not dependent on the levels of the RARbeta2 transcriptional activation. Thus, our data support the hypothesis that breast epithelial cells in the course of their progression and malignant transformation may differentially respond to retinoids and that not only RARbeta, but RARalpha, gamma and/or RXRalpha, beta may also serve as potential targets for retinoids in breast cancer prevention and therapy trials.

S100P calcium-binding protein overexpression is associated with immortalization of human breast epithelial cells in vitro and early stages of breast cancer development in vivo.

The mechanism of cell immortalization of human breast epithelial cells leading to neoplastic transformation is not clear. The isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10F, have provided a valuable tool to identify genes involved in this process. Using the technique of differential display, we have identified seven cDNA bands differentially displayed in the MCF-10F cells when compared with the mortal S130 cells from which MCF-10F was originated. One of these bands was isolated and cloned. Sequence analysis revealed 99% homology to the EF-hand calcium-binding protein S100P (Placental). The clone was overexpressed in the immortal cell line MCF-10F when compared to the mortal counterpart S130 or other primary cultures of human breast epithelial cells. In addition, it was highly expressed in chemically transformed breast epithelial cell lines (BP1E and D3. 1), breast cancer cell line T47D, as well as in three invasive ductal carcinomas when compared to their normal adjacent tissue. The S100P protein was localized by immunohistochemistry, using a monoclonal antibody against the same amino acid sequence of the gene cloned, in ductal hyperplasias, in situ and invasive ductal carcinoma, but not in the normal tissues. We concluded that S100P overexpression is an early event that might play an important role in the immortalization of human breast epithelial cells in vitro and tumor progression in vivo.

Genomic structure of the human M6P/IGF2 receptor.

Genomic structure of the human M6P/IGF2 receptor.

Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor gene correlates with poor differentiation in early breast carcinomas.

Chromosome 6q has been shown to be one of the most frequent sites for allelic loss in human breast cancer. The mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) gene, which maps to chromosome 6q26-27, functions in the activation of TGF-beta1, a potent growth inhibitor for most cell types, the degradation of the mitogen IGF2 and the intracellular trafficking of lysosomal enzymes. Loss of heterozygosity (LOH) at the IGF2R locus with mutations in the remaining allele have been reported in liver cancers and recently in two high-grade cases of ductal carcinoma in situ of the breast. We have sought to confirm that allelic loss of IGF2R is an early event in the aetiology of breast cancer by screening a group of 'early' lesions for LOH at a polymorphic microsatellite marker within the IGF2R gene using polymerase chain reaction (PCR). Several microdissected tumour foci were analysed for each of 40 mammographically detected invasive carcinomas and 22 cases of pure ductal carcinoma in situ (DCIS). None of 25 (62.5%) informative early invasive carcinomas showed any evidence of LOH. This group comprised predominantly of well- to moderately differentiated cases (95%). However, 4 out of 18 informative DCIS cases (22%) showed clear evidence of LOH. Three of these were poorly differentiated (high-grade) lesions. These data suggest that loss of heterozygosity at the IGF2R gene is associated with poor differentiation at this early stage of breast cancer development and progression.

Transforming growth factor-beta in breast cancer: a working hypothesis.

Transforming Growth Factor-beta (TGF beta) is the most potent known inhibitor of the progression of normal mammary epithelial cells through the cell cycle. During the early stages of breast cancer development, the transformed epithelial cells appear to still be sensitive to TGF beta-mediated growth arrest, and TGF beta can act as an anti-tumor promoter. In contrast, advanced breast cancers are mostly refractory to TGF beta-mediated growth inhibition and produce large amounts of TGF beta, which may enhance tumor cell invasion and metastasis by its effects on extracellular matrix. We postulate that this seemingly paradoxical switch in the responsiveness of tumor cells to TGF beta during progression is the consequence of the activation of the latent TGF beta that is produced and deposited into the tumor microenvironment, thereby driving the clonal expansion of TGF beta-resistant tumor cells. While tumor cells themselves may activate TGF beta, recent observations suggest that environmental tumor promoters or carcinogens, such as ionizing radiation, can cause stromal fibroblasts to activate TGF beta by epigenetic mechanisms. As the biological effects of the anti-estrogen tamoxifen may well be mediated by TGF beta, this model has a number of important implications for the clinical uses of tamoxifen in the prevention and treatment of breast cancer. In addition, it suggests a number of novel approaches to the treatment of advanced breast cancer.

E-Cadherin (E-cad) expression in duct carcinoma in situ (DCIS) of the breast

E-cadherin (E-cad) is an epithelial cell-cell adhesion molecule whose loss or reduced expression is associated with a more invasive tumour phenotype. Ninety-six cases of screen detected pure ductal carcinoma in situ (DCIS) were analysed immunocytochemically for expression of E-cad using the HECD-1 mouse monoclonal antibody. The in situ component in each case was classified on the basis of cytonuclear grade, extent of necrosis, Van Nuys classification and a newly devised Cardiff classification. The amount of E-cad expression was assessed semi-quantitatively using an intensity distribution method. There is significantly more expression of E-cad in well-differentiated DCIS when compared with poorly differentiated DCIS and this finding is highly significant (P < 0.001) irrespective of the classification system used. These findings suggest that progressive loss of E-cad expression may occur at an early stage of breast cancer development.

Independent molecular pathways in initiation and loss of hormone responsiveness of breast carcinomas.

These studies were set up to determine whether those oncogenes participating in the initiation of mammary carcinogenesis (for example, ras oncogenes) play a direct role in the outcome of events associated with the late stages of tumor development such as loss of hormone dependency. Mammary carcinomas induced by a single carcinogenic insult in pubescent rats was selected as an in vivo model system with direct relevance to human breast cancer. Acquisition of hormone-independent growth in these carcinogen-induced tumors was found to be independent of the activation of ras oncogenes during the early stages of carcinogenesis. In agreement with these observations, introduction of a human ras oncogene into human MCF-7 breast carcinoma cells did not abrogate their hormonal dependency for growth in vivo. These findings suggest that those events responsible for the critical stages of breast cancer development occur independently and in an uncoordinated manner.