Human Breast Cancer Epithelial Cells Research Articles

A Rac–Pak signaling pathway is essential for ErbB2-mediated transformation of human breast epithelial cancer cells

The activation of receptor tyrosine kinases, particularly ErbB2, plays an important role in the genesis of breast cancer. ErbB2 kinase activity promotes Ras-mediated stimulation of downstream protein kinase cascades, including the Ras/Raf-1/Mek/extracellular-signal regulated kinase (Erk) pathway, leading to tumor cell growth and migration. Signaling through the Ras-Erk pathway can be influenced by p21-activated kinase-1 (Pak1), an effector of the Rho family GTPases Rac and Cdc42. In this study, we asked if ErbB2 expression correlates with Pak1 and Erk activity in human breast cancer specimens, and if Pak1 signaling is required for ErbB2 transformation in a 3D in vitro setting and in xenografts. We found a correlation between ErbB2 expression and activation of Pak in estrogen-receptor positive human breast tumor samples and observed that in 3D cultures, activation of Rac-Pak1 pathway by ErbB2 homodimers induced growth factor independent proliferation and promoted disruption of three-dimensional mammary acinar-like structures through activation of the Erk and Akt pathways. Further, we found that inhibition of Pak1 by small molecules compromised activation of Erk and Akt, resulting in reversion of the malignant phenotype and restoration of normal acinar architecture. Finally, ErbB2-amplified breast cancer cells expressing a specific Pak inhibitor showed delayed tumor formation and down-regulation of Erk and Akt signaling in vivo. These data imply that the Rac-Pak pathway is vital to ErbB2-mediated transformation and that Pak inhibitors represent plausible drug targets in breast cancers in which ErbB2 signaling is activated.

Zeranol enhances leptin-induced proliferation in primary cultured human breast cancer epithelial cells

Breast cancer is the leading type of cancer in women in the United States. One of the known risk factors of breast cancer is obesity. Leptin is a product of the obese (ob) gene and plays an important role in breast cancer development. Its expression is up-regulated in obesity and it promotes breast cancer cell growth. Exposure to environmental estrogenic disruptors has been found to be directly related to the increase in the incidence of breast cancer. Zeranol (Z) is a non-steroidal anabolic growth promoter with potent estrogenic activity that is widely used in the US beef industry. The objective of this study was to determine the mechanisms of Z- and leptin-induced proliferation of primary cultured human breast cancer epithelial cells (HBCECs). A cell proliferation assay was used to determine the extent to which Z is capable of enhancing the mitogenic activity of leptin in HBCECs. RT-PCR was used to explore the possible mechanisms by quantifying the transcription of cyclin D1 and ObR genes. Our results demonstrated that when the HBCECs were pre-treated with 3 nM leptin for 24 h, the sensitivity to Z exposure greatly enhanced the mitogenic action of leptin. The experimental data observed show that there is interaction between leptin and Z in HBCEC growth.

Inositol 1,4,5-trisphosphate-induced Ca2+ signalling is involved in estradiol-induced breast cancer epithelial cell growth

BackgroundCa2+ is a ubiquitous messenger that has been shown to be responsible for controlling numerous cellular processes including cell growth and cell death. Whereas the involvement of IP3-induced Ca2+ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. Our purpose was to characterize the role of IICS in the control of growth of the estrogen-dependent human breast cancer epithelial cell line MCF-7 and its potential regulation by 17β-estradiol (E2).ResultsOur results show that the IP3 receptor (IP3R) inhibitors caffeine, 2-APB and xestospongin C (XeC) inhibited the growth of MCF-7 stimulated by 5% foetal calf serum or 10 nM E2. Furthermore, Ca2+ imaging experiments showed that serum and E2 were able to trigger, in a Ca2+-free medium, an elevation of internal Ca2+ in a 2-APB and XeC-sensitive manner. Moreover, the phospholipase C (PLC) inhibitor U-73122 was able to prevent intracellular Ca2+ elevation in response to serum, whereas the inactive analogue U-73343 was ineffective. Western-blotting experiments revealed that the 3 types of IP3Rs are expressed in MCF-7 cells and that a 48 hours treatment with 10 nM E2 elevated IP3R3 protein expression level in an ICI-182,780 (a specific estrogen receptor antagonist)-dependent manner. Furthermore, IP3R3 silencing by the use of specific small interfering RNA was responsible for a drastic modification of the temporal feature of IICS, independently of a modification of the sensitivity of the Ca2+ release process and acted to counteract the proliferative effect of 10 nM E2.ConclusionsAltogether, our results are in favour of a role of IICS in MCF-7 cell growth, and we hypothesize that the regulation of IP3R3 expression by E2 is involved in this effect.

Induction of apoptosis by (-)-gossypol-enriched cottonseed oil in human breast cancer cells

Induction of apoptosis is one of the mechanisms of chemotherapeutic agents against breast cancer. In addition, recent studies have shown that diets containing polyphenolic components possess anticancer activities either in vitro or in vivo by inhibiting cell proliferation and inducing apoptosis. The aim of our study was to explore the effects of (-)-gossypol-enriched cottonseed oil [(-)-GPCSO], a polyphenolic compound, on the proliferation of the breast cancer cell line MCF-7 as well as primary cultured human breast cancer epithelial cells (PCHBCEC). We investigated whether the mechanism of the effects of (-)-GPCSO was mediated via the induction of cell apoptosis and the regulation of Bcl-2 gene expression at both the mRNA and protein levels. Our results showed that (-)-GPCSO inhibited the proliferation of MCF-7 and PCHBCEC in a dose-dependent manner. (-)-GPCSO (0.1 and 0.2%) induced DNA fragmentation in both MCF-7 cells and PCHBCEC. (-)-GPCSO suppressed the expression of Bcl-2 at both the mRNA and protein levels in MCF-7 cells and PCHBCEC in a dose-dependent fashion. Our results suggest that the growth inhibitory effect of (-)-GPCSO on MCF-7 and PCHBCEC is due, at least partially, to the induction of cell apoptosis, which is mediated by down-regulation of Bcl-2 expression at both the mRNA and protein levels. It might be possible for (-)-GPCSO to be developed as a novel chemotherapeutic agent for breast cancer patients.

Effect of radiation treatment on newly established human breast cancer cell lines MACL-1 and MGSO-3

The characterization of new cell lines is an important tool to understand the biological processes involved in cancer treatments. In the present study, we used two newly established epithelial human breast cancer cell lines from primary sites MACL-1 and MGSO-3 and compared their susceptibility to the treatment with ionizing radiation (IR) with the commercial cell line MDA-MB-231. In the doses used (10 or 20 Gy), IR induced a reduction in cell viability and cell death, measured as DNA fragmentation, at 48 and 72 h after treatment. In addition, 48 h after treatment with IR, we observed an enhancement in the percentage of apoptotic cells. The broad-range caspases inhibitor zVAD-FMK inhibited cytotoxicity induced by IR. After 24 h, treatment with IR activated caspase-9 in MACL-1 and MDA-MB-231 but not in MGSO-3 cells. Thirty hours after treatment with IR (20 Gy), we observed an activation of caspases 8 and 3. These results suggest the involvement of caspases in the cell death induced by IR in two newly established cell lines. These cells may be useful in studies of breast cancer in defining basic mechanisms in molecular and cellular radiobiology and may contribute to the rational design of future models of cancer therapies.

Abstract 5364: Zeranol Down-regulates p53 Expression in Primary Cultured Human Breast Cancer Epithelial Cells through Epigenetic Modification

Abstract Despite great improvements in diagnosis, treatment advances, and healthier life-styles, breast cancer remains the most common cancer among women in the United States. It is estimated that 192,370 new cases arise and 40,170 women will die from breast cancer in 2009. Epidemiological studies have suggested that there are many risk factors associated with breast cancer such as dietary fat and environmental estrogenic endocrine disruptors. Zeranol (Z), as one of the six growth promoters approved by FDA, has been widely used to enhance the growth of beef cattle. Researchers are still in disagreement whether the growth promoters pose any adverse health risk from consuming beef products produced from Z-implanted beef cattle. Our previous results showed that pre-adipocytes isolated from the cattle 60 days post Z-implantation (72 mg/pellet/animal) grow about 12 fold faster than the pre-adipocytes isolated from the control cattle. Treatment with 0.2, 1 and 5% sera (Z-Sera) harvested from the cattle 30 days post implantation in culture medium significantly stimulated MCF-10A, a human normal breast epithelial cells, and MCF-7 cell growth. Proliferation might be caused by Z-Sera down-regulating the expression of a tumor suppressor gene, protein tyrosine phosphatase γ (PTP γ). The current study investigated the effects of Z on primary cultured human normal and cancerous breast epithelial cell growth (PCHNBECs, PCHBCECs), and the regulation of p53 expression, as well as the potential mechanism. Using non-radioactive cell proliferation assay, real time PCR and western blot analysis, we demonstrated that PCHBCECs are more sensitive in response to treatment with Z than PCHNBECs. Treatment with 7.5, 15 and 30 nM Z increased the growth of PCHNBECs at 27, 45, 50%, respectively, as compared to the control group. The same concentrations enhanced the proliferation of PCHBCECs at 25, 84 and 91%. The expression of p53 protein in PCHNBECs did not decrease by the treatment of Z. However, in PCHBCECs, treatment with 7.5, 15 and 30 nM Z significantly down-regulated the expression of p53 protein by 10, 20 and 40%, respectively. Further investigation suggests that the effect of Z on decreasing the expression of p53 in PCHBCECs might be mediated through its epigenetic modification. Our results showed that Z treatment up-regulates the expression of DNMT1 mRNA by 36, 58 and 160%, and protein by 14, 25 and 42% in PCHBCECs after 24 hour exposure. Our data suggest that the stimulatory effect of Z on PCHBCECs growth might be via down-regulation of p53 expression as a result of epigenetic modification mediated through Z enhancing the expression of DNMT1 mRNA and protein levels. These results imply the potential adverse health risks of the consumption of beef products with Z or its bio-active metabolites. Further investigation on this critical issue is in-progress in our laboratory (Supported by NIH grant R01 ES015212-01). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5364.

Abstract 3269: Calpain knockdown in human breast carcinoma cells reduced xenograft tumor growth rates and inhibited in vitro cell migration and proliferation

Abstract Ubiquitously expressed µ- and m-calpain are calcium dependent intracellular cysteine proteases that have been implicated in the regulation of cell proliferation, survival, apoptosis, migration, and invasion. Stable expression and proteolytic activity of the µ- and m-calpain catalytic subunits (encoded by capn1 and capn2 genes, respectively) requires a common regulatory subunit encoded by the capn4/capns1 gene; thus both µ- and m-calpain activities can be blocked by genetic knockout or knockdown of capn4 expression. Calpain is also regulated by an endogenously expressed inhibitor peptide, calpastain. We screened a panel of human breast cancer and normal epithelial cell lines for expression of calpain and calpastatin. µ- and m-calpain and calpastatin were abundantly expressed in MDA-MB-231, SKBR3, T47D, and MCF-7 breast cancer lines, as well as the normal human breast epithelial cell line MCF-10A. A stably expressed lentivirally encoded shRNA directed against the capn4 transcript was used to effectively knockdown µ- and m-calpain expression and activity in MDA-MB-231 breast cancer cells. This correlated with attenuated proliferation, migration and invasion, but resistance to anoikis. Biochemical analysis also suggests a role for calpain in the regulation of focal adhesion complex proteins such FAK, PTP-1B, talin and F-actin. Finally, knockdown of calpain in MDA-MB-231 cells significantly reduced their ability to form tumors when orthotopically injected into the mammary fat pads of nude mice. These observations provide direct genetic evidence that calpain plays a pro-tumorigenic role in the MDA-MB-231 breast cancer cell model system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3269.

Abstract 1562: PYK2 and FAK in breast cancer

Abstract Proliferation and invasion of malignant tumor cells are mediated by multiple events, including activation of focal adhesion signalings and cytoskeleton remodeling. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are cytoplasmic tyrosine kinases identified as key mediators in regulation of cytoskeleton proteins in focal adhesion. Pyk2 were found to be overexpressed in Glioblastoma and played a central role in brain tumor invasion. Studies also showed that FAK increased expression and activation in various human cancers. Recently, studies with patients’ tumor samples have demonstrated that Pyk2 and FAK are co-overexpressed in over 70% human breast cancers from the very early disease stage of in situ carcinoma to the advanced metastatic stage. Pyk2 was also involved in the cytoskeleton changes initiated by heregulin through the ErbB-2 receptor in breast cancer cell lines. To further investigate role of Pyk2 and FAK in breast cancer progression and metastasis, we studied protein expression and activation of Pyk2 and FAK, as well as their substrates and interacting proteins in human breast cancer and normal breast epithelial cell lines. Our results showed that activation of both Pyk2 and FAK are marked increased in breast cancer cell lines in comparison with normal breast epithelial cells. In addition, serum stimulation was able to further activate Pyk2 and FAK and to increase tyrosine phosphorylation of their substrates and interacting proteins, including paxillin, Src, and p130CAS. The activated Pyk2 and FAK formed protein complex with paxillin, Src, and p130CAS. Our results and other published studies support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of Pyk2 and FAK is a potential therapeutic approach for invasive breast cancer. Lead compounds of Pyk2 and FAK inhibitors have been identified by Southern Research drug discovery team and will be further evaluated in vitro and in vivo as potential breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1562.

Characterization of human breast cancer epithelial cells (HBCEC) derived from long term cultured biopsies

IntroductionFor a more individualized therapeutic approach we explored a protease-free method to culture primary cells from breast cancer biopsies.Methods and ResultsTumor tissue from breast cancer patients after surgery was cultured ex vivo without enzymatic digestion for more than one year and revealed the continuous outgrowth of adherent and proliferating primary cell populations. Immunofluorescence staining of these human breast cancer-derived epithelial cells (HBCEC) and quantification by flow cytometry revealed nearly exclusively cytokeratin-expressing cells. Analysis of surface markers during long term tumor culture of primary HBCEC (more than 476d) demonstrated a prominent expression of CD24, CD44 and MUC1 (CD227). According to aging markers, expression of senescence-associated β-galactosidase revealed little if any positive staining in a primary tumor-derived HBCEC population after 722d in culture, whereas the majority of normal human mammary epithelial cells (HMEC) demonstrated senescent cells already after a culture period of 32d. In this context, HBCEC populations derived from a tumor culture after 152d and 308d, respectively, exhibited a significant telomerase activity, suggesting continuous proliferative capacity. Treatment with several chemotherapeutic compounds and their combinations revealed distinct cytotoxic effects among HBCEC from different breast cancer patients, indicating an individualized response of these tumor-derived primary cells.ConclusionThe protease-free outgrowth of primary HBCEC offers a patient-specific approach to optimize an individually-designed cancer therapy. Moreover, HBCEC from long term breast tumor tissue cultures resemble tumor cell-like properties by an intact ECM formation and a stable cell surface protein expression providing a reproducible screening platform to identify new biomarkers and to test new therapeutics in individual tumor samples.

Separation of malignant human breast cancer epithelial cells from healthy epithelial cells using an advanced dielectrophoresis-activated cell sorter (DACS)

In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 V(pp). Finally, under the applied voltage of 48 MHz-8 V(pp) and a flow rate of 290 microm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications.

Expression of TRPC6 channels in human epithelial breast cancer cells

BackgroundTRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.MethodsMolecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).ResultsA non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.ConclusionOur results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

Imatinib Mesylate Inhibits Proliferation and Exerts an Antifibrotic Effect in Human Breast Stroma Fibroblasts

Tumor stroma plays an important role in cancer development. In a variety of tumors, such as breast carcinomas, a desmoplastic response, characterized by stromal fibroblast and collagen accumulation, is observed having synergistic effects on tumor progression. However, the effect of known anticancer drugs on stromal cells has not been thoroughly investigated. Imatinib mesylate is a selective inhibitor of several protein tyrosine kinases, including the receptor of platelet-derived growth factor, an important mediator of desmoplasia. Recently, we have shown that imatinib inhibits the growth and invasiveness of human epithelial breast cancer cells. Here, we studied the effect of imatinib on the proliferation and collagen accumulation in breast stromal fibroblasts. We have shown that it blocks the activation of the extracellular signal-regulated kinase and Akt signaling pathways and up-regulates cyclin-dependent kinase inhibitor p21(WAF1), leading to the inhibition of fibroblast proliferation, by arresting them at the G(0)/G(1) phase of the cell cycle. Imatinib inhibits more potently the platelet-derived growth factor-mediated stimulation of breast fibroblast proliferation. By using specific inhibitors, we have found that this is due to the inhibition of the Akt pathway. In addition, imatinib inhibits fibroblast-mediated collagen accumulation. Conventional and quantitative PCR analysis, as well as gelatin zymography, indicates that this is due to the down-regulation of mRNA synthesis of collagen I and collagen III-the main collagen types in breast stroma-and not to the up-regulation or activation of collagenases matrix metalloproteinase 2 and matrix metalloproteinase 9. These data indicate that imatinib has an antifibrotic effect on human breast stromal fibroblasts that may inhibit desmoplastic reaction and thus tumor progression.

Inhibition of cyclin D1 gene transcription by Brg-1

The evolutionarily conserved SWI-SNF chromatin remodeling complex regulates cellular proliferation. A catalytic subunit, BRG-1, is frequently down regulated, silenced or mutated in malignant cells, however, the mechanism by which BRG-1 may function as a tumor suppressor or block breast cancer cellular proliferation is not understood. The cyclin D1 gene is a collaborative oncogene overexpressed in greater than 50% of human breast cancers. Herein, BRG-1 inhibited DNA synthesis and cyclin D1 expression in human MCF-7 breast cancer epithelial cells. The cyclin D1 promoter AP-1 and CRE sites were required for repression by BRG-1 in promoter assays. BRG-1 deficient cells abolished and siRNA to BRG-1 reduced, formation of the BRG-1 chromatin complex. The endogenous cyclin D1 promoter AP-1 site bound BRG-1. Estradiol treatment of MCF7 cells induced recruitment of BRG-1 to the endogenous hpS2 gene promoter. Estradiol, which induced cyclin D1 abundance, was associated with a reduction in recruitment of the co-repressors HP1α/HDAC1 to the endogenous cyclin D1 promoter AP-1/BRG-1 binding sites. These studies suggest the endogenous cyclin D1 promoter BRG-1 binding site functions as a molecular scaffold in the context of local chromatin upon which coactivators and corepressors are recruited to regulate cyclin D1.

In vitroexpansion of human breast cancer epithelial and mesenchymal stromal cells: optimization of a coculture model for personalized therapy approaches

Molecularly targeted, customized therapies are designed based on the molecular portraits of cancer tissue. The efficacy of targeted therapy in individual patients depends on the contribution of single individual cancer cells within the context of their microenvironment. We have developed an in vitro model of human mammary epithelial-stromal cocultures to answer specific clinical questions related to breast cancer, to provide a tool with which to identify a signature in each breast tumor, and to identify the metabolic molecular targets of therapy in an attempt to optimize the efficacy of targeted therapy in each patient. Fifty-five human breast cancer samples were obtained through surgery. Epithelial and stromal cells were isolated from tissue specimens by differential centrifugation, and cryopreserved. Western blot analysis and RT-PCR were used to identify the tissue-specific expression patterns of cancer cells. Dose-response curves were constructed for the aromatase inhibitor formestane and for herceptin, and a 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay was done for combined treatment. We collected and cryopreserved, for future use, viable living cells from 55 breast tumor specimens from which we derived short-term cocultures. The presence of cytokeratins and vimentin was evaluated in 20 samples, and pHER2/neu and aromatase were evaluated in 4 cocultures. Formestane and herceptin had a cumulative growth-inhibitory effect on cocultures expressing epidermal growth factor receptors and aromatase. The in vitro model of human mammary epithelial-stromal cocultures reported herein can be used to examine, and to store, a patient's tumor-derived, living cells that retain the characteristics of the mother-tissue and respond, in vitro, to therapy.

Altered chemotherapeutic response of primary human breast cancer epithelial cells (HBCEC) and breast cancer cell lines

11506 Background: A novel technique to obtain individual primary cultures of human breast cancer biopsies was filed for patent (PCT/DE 2006/000608). The different individualized HBCEC (human breast cancer epithelial cell) cultures will be characterized and chemotherapeutic effects will be compared to established breast cancer cell lines. Methods: Primary HBCEC from 20 different breast cancer patients were characterized for epithelial cell and tumor markers by immunofluorescence and PCR. Following treatment with 1μM epirubicin for 1h up to 72h differences in protein expression patterns were compared to the similarly treated MCF-7 cell line by 2D gel electrophoresis. Differentially expressed protein spots were identified by mass spectrometry and confirmed by appropriate Western blot analysis. Results: Characterization of primary HBCEC revealed continuous mitosis and cell cycle progression for more than one year in culture with no significant contamination by fibroblasts or other cell types. Whereby HBCEC underwent cell death within 72h of epirubicin treatment analysis by 2D gel and subsequent protein identification by MALDI-TOF/TOF mass spectrometry exhibited a variety of differences compared to MCF-7 cells including HSP27 and prohibitin. Appropriate Western blots confirmed these differences and revealed altered expression levels for HSP27 and prohibitin in the course of epirubicin exposure in HBCEC and MCF-7 cells, respectively, suggesting altered signalling pathways in either primary breast cancer cells or the tumor cell line. Conclusions: Individualized primary HBCEC from various patients could provide a cellular platform beyond breast cancer cell lines, which eventually meet the requirements for an appropriate breast cancer testing system including the characterization of biomarkers and the identification of potential molecular targets. No significant financial relationships to disclose.

S-phase reduction in T47D human breast cancer epithelial cells induced by an S100P antisense-retroviral construct

S100P is expressed in several malignant neoplasms. It was previously demonstrated that S100P is involved in the very early stages of breast carcinogenesis. In the present study we used a retrovirus-mediated transfer of antisense-S100P in order to check whether the decrease in expression of this protein could lead to alterations in the cell cycle of epithelial cells of human breast cancer. The T47D breast carcinoma cell line, a human breast epithelial cell that expresses high levels of S100P, was a tool used in this study to investigate the alteration in cell cycle induced by a retrovirus-mediated transfer of antisense-S100P. First we used the real-time PCR technique to quantify the gene expression. The results showed a reduction of 63% of expression within the T47D-S100P-A/S infected population compared with control T47D-LXSN clones. To determine the impact of the S100P antisense technique on protein expression in T47D cells, we performed immunofluorescence staining and analyzed the resulting images using a confocal microscope. The images showed much less pronounced antibody marking of the S100P protein in the T47D-S100P-A/S compared with control cells. To evaluate whether the antisense approach caused any alteration in the cell cycle, we concluded the study with flow cytometric analysis of the cell distribution. Our findings indicated that, in our model, S100P-antisense cells showed a 23% reduction of cells at the S-phase. Using transduction techniques with an S100P antisense-retroviral construct we were able to demonstrate a significant reduction in S-phase of the T47D cell cycle. To the best of our knowledge, this is the first time that an antisense approach has been used against S100P mRNA in breast cancer epithelial cells. The results showed here seem to further classify S100P as a protein that might be involved in the cell cycle imbalance observed during breast carcinogenesis.

The Alexander Disease–Causing Glial Fibrillary Acidic Protein Mutant, R416W, Accumulates into Rosenthal Fibers by a Pathway That Involves Filament Aggregation and the Association of αB-Crystallin and HSP27

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

Messenger RNA Expression of Leptin and Leptin Receptors and their Prognostic Value in 322 Human Primary Breast Cancers

Leptin and obesity are clearly related, and obesity is associated with an increased risk of breast cancer. We therefore measured the expression of leptin and its two main receptor isoforms, OBR-L and OBR-S, in 322 breast cancers. We analyzed their relations with the classical prognostic factors and with survival to establish their links with breast cancer. The expression of leptin and its receptors was quantified by real-time reverse transcription-PCR, using TaqMan fluorogenic probes and an ABI PRISM 7700 sequence detector system (Applied Biosystems, Courtaboeuf, France). TATA box binding protein was used to normalize expression. The human breast cancer cell, SK-BR-3, expressing the three targets, was chosen as the calibrator sample (i.e., target expression = 1). All the tumors expressed both receptors, and 318 of 322 expressed leptin. These three variables correlated positively with each other and with estradiol and progesterone receptors, whereas they correlated negatively with histoprognostic grading and tumor diameter. OBR-L/OBR-S expression was inversely correlated with progesterone receptors. Patients with elevated OBR-S expression had longer relapse-free survival (P = 0.008), whereas high OBR-L/OBR-S was associated with a shorter relapse-free survival (P = 0.05). In Cox multivariate analyses, OBR-S maintained its prognostic value (P = 0.02; relative risk, 0.51). This study shows that (a) almost all of the breast cancers coexpress leptin and its two main isoforms of receptors, suggesting that the human epithelial breast cancer cells respond to leptin acting via an autocrine pathway; (b) high expression levels of leptin and leptin receptors are biological markers of a more differentiated phenotype; and that (c) OBR-S is an independent prognostic factor.

Innate Immunity and Tumor Growth

BIOMEDICINE The Nod-like family of receptors of the intraepithelial cell are considered important sensors of pathogenic bacteria. Nod1 is activated by bacterial peptidoglycan and is associated with apoptotic pathways in the cell. Using retroviral mutagenesis in a human breast cancer epithelial cell

Nod1-dependent control of tumor growth

Nod1, a cytosolic protein that senses meso-diaminopimelic acid-containing ligands derived from peptidoglycan, plays a role in host responses to invasive bacteria. Here we describe a function for Nod1, whereby it controls tumor formation. Cell lines derived from the human breast cancer epithelial cell line MCF-7 were used in a severe combined immune deficiency (SCID) mouse xenograft model to characterize a pathway linking Nod1 to the growth of estrogen-sensitive tumors. In MCF-7 cells, the absence of Nod1 correlates with tumor growth, an increased sensitivity to estrogen-induced cell proliferation, and a failure to undergo Nod1-dependent apoptosis. Conversely, overexpression of Nod1 in MCF-7 cells results in inhibition of estrogen-dependent tumor growth and reduction of estrogen-induced proliferative responses in vitro.