Malignant Progression Of Breast Cancer Research Articles

136. Inhibition of Breast Cancer Malignant Progression by Neutralizing Hypoxia-Induced Cell Motility

136. Inhibition of Breast Cancer Malignant Progression by Neutralizing Hypoxia-Induced Cell Motility

Sphingosine‐1‐phosphate (S1P) Signaling for Breast Cell Invasion

Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. Sphingosine 1‐phosphate (S1P), acting on the G‐protein‐coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase‐9 (MMP‐9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP‐9 by S1P. In addition, activation of extracellular‐signal‐regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine‐protein kinase (Akt) are involved in the process of S1P‐mediated induction of MMP‐9 expression and invasion. Activation of the S1P receptor S1P3 and Gαq are required for S1P‐induced invasive/migratory responses, suggesting that the enhancement of S1P‐mediated invasiveness is triggered by the specific coupling of S1P3 to the eterotrimeric Gαq subunit. Activation of phospholipase C‐β4 and intracellular Ca2+ release are required for S1P‐induced MMP‐9 upregulation. Taken together, this study demonstrated that S1P regulates MMP‐9 induction and invasiveness through coupling of S1P3 and Gαq in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.

The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

The polypeptide hormone prolactin (PRL) stimulates breast epithelial cell growth, differentiation, and motility through its cognate receptor, PRLr. PRLr is expressed in most breast cancers; however, its exact role remains elusive. Our laboratory previously described a novel mode of PRLr signaling in which Stat5a-mediated transcription is regulated through ligand-induced phosphorylation of the PRLr transactivation domain (TAD). Herein, we used a PRLr transactivation-deficient mutant (PRLrYDmut) to identify novel TAD-specific target genes. Microarray analysis identified 120 PRL-induced genes up-regulated by wild type but not PRLrYDmut. Compared with control, PRLr expression significantly induced expression of approximately 4700 PRL-induced genes, whereas PRLrYDmut ablated induction of all but 19 of these genes. Ingenuity pathway analysis found that the PRLr TAD most profoundly affected networks involving cancer and proliferation. In support of this, PRLrYDmut expression reduced anchorage-dependent and anchorage-independent growth. In addition, pathway analysis identified a link between the PRLr TAD and the estrogen and progesterone receptors (ERα/PR). Although neither ERα nor PR was identified as a PRL target gene, a TAD mutation significantly impaired ERα/PR expression and estrogen responsiveness. TMA analysis revealed a marked increase in nuclear, but not cytoplasmic, PRLr TAD phosphorylation as a function of neoplastic progression. We propose that PRLr TAD phosphorylation contributes to breast cancer pathogenesis, in part through regulation of ERα and PR, and has potential utility as a biomarker in this disease.

Expression and significance of phosphorylated Girdin in breast cancer

To detect the expression of phosphorylated girdin (p-girdin) in breast cancer and its association with pathological characteristics and molecular subtypes of breast cancer. Immunohistochemical SP staining was used to investigate the expression of p-girdin in 27 cases of lobular hyperplasia of mammary gland, 61 cases of ductal carcinoma in situ (DCIS), and 94 cases of non-special type invasive carcinoma (IDC-NOS) of breast. p-girdin was located in the cell cytoplasm and (or) nuclei in breast cancer. There was statistically a very significant difference among lobular hyperplasia, DCIS and IDC-NOS (χ2=26.724, P<0.001). Its cytoplasmic and nuclear reactivity were 25.9% (7/27), 39.3% (24/61), and 66.0% (62/94), respectively. The expression of p-girdin was positively associated with pathologic stage (r=0.204, P=0.049), lymph node metastasis (r=0.212, P=0.041) and HER2/neu (r=0.248, P=0.016). But no significant association was identified between p-girdin expression and histological grade (r=-0.015, P=0.918), age of patients (r=-0.011, P=0.918), tumor size (r=0.075, P=0.471), ER(r=0.071, P=0.498), PR (r=-0.050, P=0.634). Mann-Whitney test showed that p-girdin expression in luminal A, luminal B, triple negative and HER-2(+) type was significantly different (χ2=14.017, P=0.003). Among the four types, its positive rate was 55.8% (24/43), 95.8% (23/24), 66.7% (10/15), and 41.7% (5/12), respectively. p-Girdin expression is closely correlated with the malignant progression of breast cancer. Its expression may have clinical value as a new target for the treatment of breast cancer.

Tryptophan metabolism in breast cancers: molecular imaging and immunohistochemistry studies

Tryptophan oxidation via the kynurenine pathway is an important mechanism of tumoral immunoresistance. Increased tryptophan metabolism via the serotonin pathway has been linked to malignant progression in breast cancer. In this study, we combined quantitative positron emission tomography (PET) with tumor immunohistochemistry to analyze tryptophan transport and metabolism in breast cancer. Dynamic α-[(11)C]methyl-l-tryptophan (AMT) PET was performed in nine women with stage II-IV breast cancer. PET tracer kinetic modeling was performed in all tumors. Expression of L-type amino acid transporter 1 (LAT1), indoleamine 2,3-dioxygenase (IDO; the initial and rate-limiting enzyme of the kynurenine pathway) and tryptophan hydroxylase 1 (TPH1; the initial enzyme of the serotonin pathway) was assessed by immunostaining of resected tumor specimens. Tumor AMT uptake peaked at 5-20 min postinjection in seven tumors; the other two cases showed protracted tracer accumulation. Tumor standardized uptake values (SUVs) varied widely (2.6-9.8) and showed a strong positive correlation with volume of distribution values derived from kinetic analysis (P<.01). Invasive ductal carcinomas (n=6) showed particularly high AMT SUVs (range, 4.7-9.8). Moderate to strong immunostaining for LAT1, IDO and TPH1 was detected in most tumor cells. Breast cancers show differential tryptophan kinetics on dynamic PET. SUVs measured 5-20 min postinjection reflect reasonably the tracer's volume of distribution. Further studies are warranted to determine if in vivo AMT accumulation in these tumors is related to tryptophan metabolism via the kynurenine and serotonin pathways.

Thioredoxin stimulates MMP-9 expression, de-regulates the MMP-9/TIMP-1 equilibrium and promotes MMP-9 dependent invasion in human MDA-MB-231 breast cancer cells

Increased expression of thioredoxin (Trx)-1 and matrix metalloproteinase (MMP)-9 associates with malignant breast cancer progression. Here, we describe a functional relationship between Trx-1 and MMP-9 in promoting MDA-MB-231 breast cancer cell invasive behaviour. Trx-1 overexpression stimulated MMP-9 expression, de-regulated the MMP-9/TIMP-1 equilibrium and augmented MMP-9 involvement in a more invasive phenotype. Trx-1 augmented MMP-9 transcription through NF-κB, AP-1 and SP1 elements; stimulated p50/p65 NF-κB activity and recruitment to the MMP-9 promoter; and facilitated MMP-9 promoter-accessibility to NF-κB by preventing HDAC recruitment and maintaining MMP-9 promoter histone acetylation. Our data provide a functional basis for Trx-1 and MMP-9 association in malignant breast cancer and identify Trx-1 and NF-κB as potentially druggable targets for reducing MMP-9 involvement in malignant behaviour.

Sphingosine 1-phosphate regulates matrix metalloproteinase-9 expression and breast cell invasion through S1P3–Gαq coupling

Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. Sphingosine 1-phosphate (S1P), acting on the G-protein-coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S1P on the regulation of invasive/migratory phenotypes of MCF10A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S1P causes induction of matrix metalloproteinase-9 (MMP-9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP-9 by S1P. In addition, activation of extracellular-signal-regulated kinases 1 and 2 (ERK1/2), p38 and alpha serine/threonine-protein kinase (Akt) are involved in the process of S1P-mediated induction of MMP-9 expression and invasion. Activation of the S1P receptor S1P₃ and G(αq) are required for S1P-induced invasive/migratory responses, suggesting that the enhancement of S1P-mediated invasiveness is triggered by the specific coupling of S1P₃ to the heterotrimeric G(αq) subunit. Activation of phospholipase C-β₄ and intracellular Ca²⁺ release are required for S1P-induced MMP-9 upregulation. Taken together, this study demonstrated that S1P regulates MMP-9 induction and invasiveness through coupling of S1P₃ and G(αq) in MCF10A cells, thus providing a molecular basis for the crucial role of S1P in promoting breast cell invasion.

Optimizing a 3D Culture System to Study the Interaction between Epithelial Breast Cancer and Its Surrounding Fibroblasts

Stromal fibroblasts are suggested to be a key determinant in the malignant progression of breast cancer. To find an in vitro culture model that best mimics the in vivo tumor microenvironment so we can study the effects of stromal fibroblasts on breast cancer progression, we evaluated several three-dimensional (3D) co-culture models in order to identify the most suitable culture model for our study. The purpose of our study is to co-culturing malignant mouse breast cancer 4T1 cells and murine embryonic fibroblasts (MEF) to form spheroids with matrigel. We found the best culture model for forming the 4T1 aggregates/spheroids was, in the absence of fibroblast, by growing 4T1 cells in the culture wells precoated with matrigel and in the overlay medium containing 2% matrigel. We chose this model as our standard 3D culture to co-culture 4T1 and MEF cells at different ratios. We found that the amount of MEF in the 4T1/MEF mixture affects the morphology of 4T1/MEF aggregates/spheroids: the higher the ratio of MEF in the mixture, the more ductal structures formed among the aggregates, and the more polarized-like alveolar structures they tended to become. Fibroblasts produced protection for the breast cancer cells in the 3D culture, as aggregates/spheroids formed by breast-cancer cells alone were more sensitive to cytotoxic chemo-agents than aggregates formed by the breast-cancer/fibroblast mixture. These results indicate that the selection of a suitable 3D culture model for a particular research focus may be critical to collecting clinically relevant information about tumor progression that involves interplay between different cell types. This 3D co-culture model demonstrated that tumor-surrounding fibroblasts play important roles in distributing and connecting epithelial breast cancer cells in a tumor microenvironment, as well as providing protection for breast cancer cells from chemo-agent killing.

Key signalling nodes in mammary gland development and cancer. The Snail1-Twist1 conspiracy in malignant breast cancer progression

Breast cancer is the most common cancer among women, and despite significant advances in diagnosing and treating it, metastatic spread of cancer cells results in a high mortality rate. Epithelial-to-mesenchymal transition (EMT) is an embryonic program in which epithelial cells lose their characteristics and gain mesenchymal features. Therefore, EMT might play a very important role during malignant tumour progression. In this review we summarise recent advances in breast cancer research with a particular focus on the transcription factors Snail1 and Twist1. Besides discussing the role of EMT in normal mammary gland development, we describe regulatory mechanisms involving newly discovered upstream regulators and microRNAs, the association of EMT with breast cancer stem cells, and the involvement of the tumour microenvironment in breast cancer progression.

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance.

The Wilms' tumor suppressor WT1 induces estrogen-independent growth and anti-estrogen insensitivity in ER-positive breast cancer MCF7 cells

A switch from estrogen-dependent to estrogen-independent growth is a critical step in malignant progression of breast cancer and is a major problem in endocrine therapy. However, the molecular mechanisms underlying this switch remain poorly understood. The Wilms' tumor suppressor gene, wt1, encodes a zinc finger protein WT1 that functions as a transcription regulator. High levels of the WT1 expression have been associated with malignancy of breast cancer. The goal of this study was to investigate the function of WT1 in malignant progression of breast cancer. We found that the high passage ER-positive breast cancer MCF7H cells expressed EGFR, HER2 and WT1 at higher levels compared to the low passage MCF7L cells. MCF7H cells responded weakly to estrogen stimulation, grew rapidly in the absence of estrogen and were insensitive to anti-estrogens such as ICI 182,780 and 4-hydroxy-tamoxifen (4OH-TAM). We also established stable cell lines from the low passage MCF7L cells to constitutively express exogenous WT1 and found elevated levels of EGFR and HER2 expression, estrogen-independent growth and anti-estrogen insensitivity in WT1-transfected MCF7L cells. These results suggested WT1 promotes estrogen-independent growth and anti-estrogen resistance in ER-positive breast cancer cells presumably through activation of the signaling pathways mediated by the members of EGFR family.

Protein Acetylation and Histone Deacetylase Expression Associated with Malignant Breast Cancer Progression

Excess histone deacetylase (HDAC) activity can induce hypoacetylation of histone and nonhistone protein substrates, altering gene expression patterns and cell behavior potentially associated with malignant transformation. However, HDAC expression and protein acetylation have not been studied in the context of breast cancer progression. We assessed expression levels of acetylated histone H4 (ac-H4), ac-H4K12, ac-tubulin, HDAC1, HDAC2, and HDAC6 in 22 reduction mammoplasties and in 58 specimens with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Differences among groups were tested for significance using nonparametric tests. From normal epithelium to DCIS, there was a marked reduction in histone acetylation (P < 0.0001). Most cases showed similar levels of acetylation in DCIS and IDC, although some showed further reduction of ac-H4 and ac-H4K12 from DCIS to IDC. Expression of HDAC1, HDAC2, and HDAC6 was also significantly reduced but by a smaller magnitude. Greater reductions of H4 acetylation and HDAC1 levels were observed from normal to DCIS in estrogen receptor-negative compared with estrogen receptor-positive, and in high-grade compared with non-high-grade tumors. Overall, there was a global pattern of hypoacetylation associated with progression from normal to DCIS to IDC. These findings suggest that the reversal of this hypoacetylation in DCIS and IDC could be an early measure of HDAC inhibitor activity.

Cytokeratin and Vimentin Expression in Breast Cancer

The transition from epithelial keratin to mesenchymal vimentin expression marks an important step in the malignant progression of breast cancer. This study analyzed the clinical significance of cytokeratin and vimentin in patients with breast cancer. Expression of cytokeratin and vimentin was evaluated by immunohistochemistry on paraffin-embedded tissue sections of patients with breast cancer. Loss of cytokeratin was seen in 11% of the patients. A clearer trend towards loss of cytokeratin was observed in patients with stage IV disease and PR negativity. Weak cytokeratin expression was present in patients who developed recurrence or metastatic disease. Loss of cytokeratin was associated with reduced overall survival in univariate and multivariate analysis, gain of vimentin expression was seen in 57% of breast carcinoma patients. It was higher in patients with lymph node positivity, advanced stage, HER2 positivity, and disease recurrence or metastasis. Multivariate survival analysis indicated that gain of vimentin expression was associated with reduced relapse-free survival. Loss of cytokeratin and gain of vimentin expression are indicators of biologically aggressive breast carcinoma.

Cytokeratin and vimentin expression in breast cancer

The transition from epithelial keratin to mesenchymal vimentin expression marks an important step in the malignant progression of breast cancer. This study analyzed the clinical significance of cytokeratin and vimentin in patients with breast cancer. Expression of cytokeratin and vimentin was evaluated by immunohistochemistry on paraffin-embedded tissue sections of patients with breast cancer. Loss of cytokeratin was seen in 11% of the patients. A clearer trend towards loss of cytokeratin was observed in patients with stage IV disease and PR negativity. Weak cytokeratin expression was present in patients who developed recurrence or metastatic disease. Loss of cytokeratin was associated with reduced overall survival in univariate and multivariate analysis, gain of vimentin expression was seen in 57% of breast carcinoma patients. It was higher in patients with lymph node positivity, advanced stage, HER2 positivity, and disease recurrence or metastasis. Multivariate survival analysis indicated that gain of vimentin expression was associated with reduced relapse-free survival. Loss of cytokeratin and gain of vimentin expression are indicators of biologically aggressive breast carcinoma.

Integrative bioinformatics analysis of transcriptional regulatory programs in breast cancer cells

BackgroundMicroarray technology has unveiled transcriptomic differences among tumors of various phenotypes, and, especially, brought great progress in molecular understanding of phenotypic diversity of breast tumors. However, compared with the massive knowledge about the transcriptome, we have surprisingly little knowledge about regulatory mechanisms underling transcriptomic diversity.ResultsTo gain insights into the transcriptional programs that drive tumor progression, we integrated regulatory sequence data and expression profiles of breast cancer into a Bayesian Network, and searched for cis-regulatory motifs statistically associated with given histological grades and prognosis. Our analysis found that motifs bound by ELK1, E2F, NRF1 and NFY are potential regulatory motifs that positively correlate with malignant progression of breast cancer.ConclusionThe results suggest that these 4 motifs are principal regulatory motifs driving malignant progression of breast cancer. Our method offers a more concise description about transcriptome diversity among breast tumors with different clinical phenotypes.

Correlation between expression of P38 MAPK-signaling and uPA in breast cancer

To study the expression of phosphorylated p38 mitogen-activated protein kinase (p-p38) and uPA and the correlation of their expression with breast cancer clinicopathological characteristics, and to investigate the role of the p38MAPK-signaling pathway in regulating uPA expression in breast cancer cells. Immunohistochemistry (S-P) was used to test the expression of p-p38 and uPA in 60 specimens of breast cancer tissues. Western blots were adopted to detect expression of the p-p38 and uPA proteins in MDA-MB-231 and MCF-7 breast cancer cells, and uPA expression after treatment with SB203580, a specific inhibitor of p38 MAPK. The positive rate of the p-p38 protein and uPA protein expression in the breast cancer tissues was 56.7% and 60.0%,respectively. The expression of p-p38 was positively related to the expression of uPA (r = 0.316, P 0.05). The expression of p-p38 and uPA in MDA-MB-231 cells was higher than that in MCF-7 cells. SB203580 inhibited the p38 MAPK pathway and reduced uPA protein expression. The p38 MAPK-signaling pathway promotes breast cancer malignant progression by up-regulating uPA expression,and it may be an important process in breast cancer invasion and metastasis.

Identification of estrogenically synthesized LRP16 as an ERα coactivator and its role in ERα-positive breast cancer

10676 Background: Aberrant ERα activity is linked to genesis and malignant progression of breast cancer through direct target gene activation or repression. A complex network of coregulatory proteins is largely believed to determine the transcriptional activity of ERα. LRP16 was identified previously to be an estrogen (E2) responsive gene, but its function involving in conferring estrogen signalling pathway is not clear. Methods: Endogenous LRP16 expression in MCF-7 cells was stably suppressed by retrovirus-mediated small interference RNA (siRNA). The effects of LRP16 expression on E2-stimulated growth and invasive ability of MCF-7 cells were determined in vitro and in vivo assays. The effects of LRP16 expression on ERα transactivation were determined by luciferase assays. The interaction of LRP16 and ERα was examined by GST pull-down and coimmunopricipitation (CoIP) assays. Northern blot and Western blot were used to detect the mRNA and protein levels of ER target genes in LRP16-inhibited MCF-7 cells. The LRP16 expression levels in primary breast cancer were detected by Northern blot. Results: Fristly, LRP16 expression was characterized to be dependent on estrogen activities. Then, LRP16 was identified to be an estrogen-independent ERα cofactor in ER-positive breast cancer cells and demonstrate that LRP16 is an essential coactivator to ERα-mediated transactivation in an estrogen-dependent manner. Suppression of LRP16 expression in ER-positive breast cancer cells specifically inhibits the transcription of ER upregulated genes, results in the increase of E-cadherin expression through ER mediation. In vitro and in vivo data demonstrate that suppression of LRP16 inhibits the ability of estrogen-stimulated proliferation and invasiveness of ER-positive breast cancer cells. The pathological and clinical characteristics of human breast cancer includining ER/PR-positiveness, tumor diameter and the involvement of axillary lymphoid nodes were tightly linked with the LRP16 gene expression level. Conclusions: These results establish a mechanistic link between estrogen receptor status, its coactivator LRP16, and progression of ER-positive breast cancers, and may provide a novel antiestrogenic target for the therapy of ER positive breast cancer. No significant financial relationships to disclose.

Activation of Stat3 in Primary Tumors from High-Risk Breast Cancer Patients Is Associated with Elevated Levels of Activated Src and Survivin Expression

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) protein has been observed in a wide variety of tumors, including breast cancer, and contributes to oncogenesis at least in part by prevention of apoptosis. In a study of 45 patients with high-risk breast cancer enrolled in a phase II neoadjuvant chemotherapy trial with docetaxel and doxorubicin, we evaluated the levels of Stat3 activation and potentially associated molecular biomarkers in invasive breast carcinoma compared with matched nonneoplastic tissues. Using immunohistochemistry and image analysis, we quantified the levels of phospho-Stat3 (pY-Stat3), phospho-Src (pY-Src), epidermal growth factor receptor, HER2/neu, Ki-67, estrogen receptor, Bcl-2, Bcl-xL, Survivin, and apoptosis in formalin-fixed, paraffin-embedded sections from invasive carcinomas and their paired nonneoplastic parenchyma. The levels of molecular biomarkers in nonneoplastic and tumor tissues were analyzed as continuous variables for statistically significant correlations. Levels of activated pY-Stat3 and pY-Src measured by immunohistochemistry were significantly higher in invasive carcinoma than in nonneoplastic tissue (P < 0.001). In tumors, elevated levels of pY-Stat3 correlated with those of pY-Src and Survivin. Levels of pY-Stat3 were higher in partial pathologic responders than in complete pathologic responders. In partial pathologic responders, pY-Stat3 levels correlated with Survivin expression. Our findings suggest important roles for elevated activities of Stat3 and Src, as well as Survivin expression, in malignant progression of breast cancer. Furthermore, elevated Stat3 activity correlates inversely with complete pathologic response. These findings suggest that specific Stat3 or Src inhibitors could offer clinical benefits to patients with breast cancer.

Establishment of three human breast epithelial cell lines derived from carriers of the 999del5 BRCA2 icelandic founder mutation

Germ line mutations in BRCA1 and BRCA2 account for a large proportion of inherited breast and ovarian cancer. Both genes are involved in DNA repair by homologous recombination and are thought to play a vital role in maintaining genomic stability. A major drawback for long-term functional studies of BRCA in general and BRCA2 in particular has been a lack of representative human breast epithelial cell lines. In the present study, we have established three cell lines from two patients harboring the 999del5 germ line founder mutation in the BRCA2 gene. Primary cultures were established from cellular outgrowth of explanted tissue and subsequently transfected with a retroviral construct containing the HPV-16 E6 and E7 oncogenes. Paired cancer-derived and normal-derived cell lines were established from one patient referred to as BRCA2-999del5-2T and BRCA2-999del5-2N, respectively. In addition, one cell line was derived from cancer-associated normal tissue from another patient referred to as BRCA2-999del5-1N. All three cell lines showed characteristics of breast epithelial cells as evidenced by expression of breast epithelial specific cytokeratins. Cytogenetic analysis showed marked chromosomal instability with tetraploidy and frequent telomeric associations. In conclusion, we have established three breast epithelial cell lines from two patients carrying the BRCA2 Icelandic 999del5 founder mutation. These cell lines form the basis for further studies on carcinogenesis and malignant progression of breast cancer on a defined genetic background.

Expression of Heparanase by Primary Breast Tumors Promotes Bone Resorption in the Absence of Detectable Bone Metastases

Heparanase is an enzyme that cleaves heparan sulfate and through this activity promotes tumor growth, angiogenesis, invasion, and metastasis in several tumor types. In human breast cancer patients, heparanase expression is associated with sentinel lymph node metastases. However, the precise role of heparanase in the malignant progression of breast cancer is unknown. To examine this, a variant of MDA-MB-231 cells was transfected with the cDNA for human heparanase (HPSE cells) or with vector alone as a control (NEO cells). Transfection produced a 6-fold increase in heparanase activity in HPSE cells relative to NEO cells. When injected into the mammary fat pads of severe combined immunodeficient mice, the tumors formed by HPSE cells initially grow significantly faster than the tumors formed by NEO cells. The rapid growth is due in part to increased angiogenesis, as microvessel densities are substantially elevated in primary HPSE tumors compared with NEO tumors. Although metastases to bones are not detected, surprisingly vigorous bone resorption is stimulated in animals bearing tumors formed by the HPSE cells. These animals have high serum levels of the C-telopeptide derived from type I collagen as well as significant elevation of the active form of tartrate-resistant acid phosphatase (TRAP)-5b. In contrast, in animals having a high tumor burden of Neo cells, the serum levels of C-telopeptide and TRAP-5b never increase above the levels found before tumor injection. Consistent with these findings, histologic analysis for TRAP-expressing cells reveals extensive osteoclastogenesis in animals harboring HPSE tumors. In vitro osteoclastogenesis assays show that the osteoclastogenic activity of HPSE cell conditioned medium is significantly enhanced beyond that of NEO conditioned medium. This confirms that a soluble factor or factors that stimulate osteoclastogenesis are specifically produced when heparanase expression is elevated. These factors exert a distal effect resulting in resorption of bone and the accompanying enrichment of the bone microenvironment with growth-promoting factors that may nurture the growth of metastatic tumor cells. This novel role for heparanase as a promoter of osteolysis before tumor metastasis suggests that therapies designed to block heparanase function may disrupt the early progression of bone-homing tumors.