Breast Tumor-derived Cell Lines Research Articles

The correlation between PLC-β2 and miR-146a in breast ductal carcinoma in situ (DCIS) defines its malignant potential

Ductal carcinoma in situ (DCIS), which represents the most frequently diagnosed tumor in women in industrialized countries, may be a crucial step in the progression of breast lesions to invasive ductal carcinoma (IDC) (1). Among the signaling molecules deregulated in breast tumors, the beta2 isoform of the phosphoinositide-dependent phospholipase C (PLC-b2) strongly correlates with malignancy of invasive tumors and breast tumor-derived cells (2, 3). In breast tumor-derived cell lines cultured under hypoxia, PLC-b2 regulates the levels of cancer stem cell and epithelial-to-mesenchymal transition (EMT) markers (4), suggesting its involvement in breast cancer progression. By using archival FFPE breast tumor samples, we demonstrated that PLC-β2 is up-regulated in DCIS, in which it inversely correlates to the levels of miR-146a, known to act as a tumor suppressor in breast cancer (5). By using the MCF10DCIS cell line, a well-established model of DCIS-derived cells, we demonstrated that the de-regulation of miR-146a is sufficient to modulate the expression of PLC-β2, in turn able to affect the epithelial-to-mesenchymal shift as well as the number of cells expressing CD133. These data indicate that miR-146a and PLC-β2 are members of an intracellular network able to ensure the maintenance of the non-invasive phenotype of DCIS and suggest that alterations in their levels can determine the appearance of an invasive phenotype. The potential prognostic relevance of PLC-β2/miR-146a relationship was investigated in primary DCIS from patients who developed an invasive ductal carcinoma in the contralateral breast. The PLC-β2/miR-146a correlation was found negative in primary DCIS from patients who did not recur and strongly positive in DCIS from patients who developed a contralateral IDC. We propose that the assessment of the correlation between the levels of PLC-β2 and miR-146a in primary DCIS at diagnosis could be beneficial to identify patients with either low or high propensity to develop invasive recurrence. Since a major problem in the management of patients with DCIS is the lack of reliable prognostic markers, our results might be of value in selecting the most appropriate therapies for individual women with non-invasive breast neoplasia.

Abstract 5604: Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer.

Abstract Many tumor cells are dependent on glutamine (Gln) and Gln-derived metabolites to meet bioenergetic and biosynthetic demands. A key cellular reaction in the utilization of Gln is its deamidation by the enzyme glutaminase to yield glutamate (Glu). We have developed a series of potent and selective small molecule glutaminase inhibitors for evaluation as novel cancer therapeutics. These inhibitors exhibit 5-20 nM potency against the broadly-expressed form of glutaminase (GLS) with minimal activity against the liver form of the enzyme (GLS2). Expression analysis (see below) has identified triple-negative breast cancer (TNBC) as a potential clinical target population for GLS inhibitors. TNBC is a poor prognosis breast cancer subtype lacking estrogen receptor (ER), progesterone receptor (PR) and the growth factor receptor Her2. TNBC is insensitive to approved targeted therapies (ER antagonists and anti-Her2 agents) and is currently treated with conventional cytotoxic drugs. Therefore, TNBC represents a critical unmet medical need for which new targeted therapeutics are urgently needed. Analysis of a primary breast tumor mRNA expression dataset (The Cancer Genome Atlas; n=756) revealed that low ER, PR, and Her2 expression is associated with high GLS expression and low expression of both GLS2 and glutamine synthetase, an enzyme that opposes the action of glutaminase by synthesizing Gln from Glu. This expression pattern, together with published work on the Gln dependence of breast tumor cell lines [Kung et al. (2011) PLOS Genet 7:e1002229] suggests that TNBC may be particularly dependent on GLS. To test this hypothesis, we evaluated the anti-tumor activity of our GLS inhibitors on a panel of breast tumor-derived cell lines (n>25) that included a mixture of TNBC and ER-positive subtypes. The TNBC subtype displayed the greatest sensitivity to GLS inhibitor treatment (IC50s ranging from 5-100 nM) and this sensitivity correlated with a dependence on extracellular Gln for cell growth. In the TNBC cell line MDA-MB-231 the antiproliferative effect of the GLS inhibitor was associated with a dose-dependent accumulation of Gln and depletion of Glu. Additionally, GLS inhibition showed additive in vitro activity in combination with paclitaxel, a standard-of-care treatment in TNBC. In a mouse orthotopic tumor xenograft model with MDA-MB-231 cells implanted in the mammary fat pad, oral delivery of a GLS inhibitor caused an accumulation of tumor Gln, a reduction in tumor Glu, and enhanced the anti-tumor efficacy of the paclitaxel. Experiments aimed at expanding this observation with other TNBC xenograft models are in progress. Overall, these results demonstrate that a selective inhibitor of GLS, either as a single agent or in combination with standard-of-care chemotherapeutics, may be effective as a targeted therapeutic in TNBC. Citation Format: Susan Demo, Tania Chernov-Rogan, Matthew Gross, Julie Janes, Raja Kawas, Evan Lewis, Francesco Parlati, Hector Rodriguez, Mirna Rodriguez, Jinfu Yang, Frances Zhao, Adam Richardson, Mark K. Bennett. Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5604. doi:10.1158/1538-7445.AM2013-5604

Cell surface profiling with peptide libraries yields ligand arrays that classify breast tumor subtypes

Cancer heterogeneity renders risk stratification and therapy decisions challenging. Thus, genomic and proteomic methodologies have been used in an effort to identify biomarkers that can differentiate tumor subtypes to improve therapeutic outcome. Here, we report a generally applicable strategy to generate tumor type-specific peptide ligand arrays. Peptides that specifically recognize breast tumor-derived cell lines (MDA-MB-231, MCF-7, and T47-D) were identified using cell-displayed peptide libraries carrying an intrinsic fluorescent marker allowing for sorting and characterization with quantitative flow cytometry. Tumor cell specificity was achieved by depleting libraries of ligands binding to normal mammary epithelial cells (HMEC and MCF-10A). Although integrin binding RGD motifs were favored by some cell lines, screening with RGD competitors yielded several novel consensus motifs exhibiting improved tumor specificity. The resultant peptide array contained multiple consensus motifs exhibiting strong similarity to breast tumor-associated proteins. Profiling a panel of breast cancer cell lines with the peptide array revealed receptor expression patterns distinctive for luminal or basal tumor subtypes. In addition, peptide displaying bacteria and peptide functionalized microparticles enabled fluorescent labeling of tumor cells and frozen tumor tissue sections. Our results indicate that cell surface profiling using highly specific breast tumor cell binding ligands may provide an efficient route for tumor subtype classification, biomarker identification, and for the development of targeted diagnostics and therapeutics.

Oncostatin M (OSM) Cytostasis of Breast Tumor Cells: Characterization of an OSM Receptor β–Specific Kernel

The interleukin-6 cytokine oncostatin M (OSM) induces potent growth-inhibitory and morphogenic responses in several different tumor cell types, highlighting the importance of OSM signaling mechanisms as targets for therapeutic intervention. The specific molecular pathways involved are not well understood, as OSM can signal through two separate heterodimeric receptor complexes, glycoprotein 130 (gp130)/leukemia inhibitory factor receptor (LIFR) alpha and gp130/OSM receptor beta (OSMRbeta). In this investigation, we used a LIFR antagonist to help resolve signaling responses and identify patterns of gene expression elicited by the different receptor complexes. OSM-induced biological effects on breast tumor-derived cell lines were specifically mediated through the gp130/OSMRbeta complex. Each cytokine tested exhibited differential signaling capability and manifested both shared and unique patterns of gene activation, emphasizing compositional differences in activator protein-1 transcription factor activity and expression. In particular, OSM strongly activated the c-Jun NH(2)-terminal kinase (JNK) serine/threonine kinase and downstream components, including activating transcription factor (ATF)/cyclic AMP-responsive element binding protein family member, ATF3. JNK/stress-activated protein kinase kinase inhibition abrogated cell morphogenesis induced by OSM, indicating an important role for this pathway in OSM specificity. These findings identify a core signaling/transcriptional mechanism specific to the OSMRbeta in breast tumor cells.

Detection of Low Molecular Weight Derivatives of Cyclin E1 Is a Function of Cyclin E1 Protein Levels in Breast Cancer

Cyclin E1 regulates the initiation of the S phase program in the mammalian cell division cycle. In normal cells, cyclin E1 protein expression is tightly controlled through a combination of transcriptional and proteolytic regulatory processes. However, in many types of human tumor, cyclin E1 expression is frequently dysregulated, including overexpression, nonperiodic expression relative to cell division, and generation of low molecular weight (LMW) derivatives. LMW derivatives of cyclin E1 have been proposed to be generated by the in vivo proteolytic cleavage of the full-length cyclin E1 protein by a yet to be identified tumor-specific protease. Recently, it was suggested that overexpression of full-length or LMW derivatives of cyclin E1 are independent variables associated with poor outcome in patients with breast cancer. However, we have extensively analyzed cyclin E1 protein expression in primary breast tumors and breast tumor-derived cell lines and found that the ability to detect LMW derivatives of cyclin E1 correlates only with the level of cyclin E1 protein. When cyclin E1 levels on Western blots are normalized, LMW derivatives of cyclin E1 were observed at roughly equal levels in all primary breast tumors, breast tumor-derived cell lines, immortalized nontransformed human mammary epithelial cells, and normal breast tissue. Therefore, the detection of LMW derivatives of cyclin E1 is likely a function of cyclin E1 protein levels, and the activity of the proteolytic machinery responsible for their generation is not a tumor-specific property.

Competition on Nitrocellulose-immobilized Antibody Arrays: From Bacterial Protein Binding Assay to Protein Profiling in Breast Cancer Cells

Large scale comparative evaluation of protein expression requires miniaturized techniques to provide sensitive and accurate measurements of the abundance of molecules present as individual and/or assembled protein complexes in cells. The principle of competition between target molecules for binding to arrayed antibodies has recently been proposed to assess differential expression of numerous proteins with one-color or two-color fluorescence detection methods. To establish the limiting factors and to validate the use of alternative detection for protein profiling, we performed competitive binding assays under different conditions. A model experimental protocol was developed whereby the competitive displacement of multi-subunit bacterial RNA polymerase and/or its subunits was evaluated through binding to subunit-specific immobilized monoclonal antibodies. We show that the difference in physico-chemical properties of unlabeled and labeled molecules significantly affects the performance of one-color detection, whereas epitope inaccessibility in the protein complex can prohibit the assessment of competition by both detection methods. Our data also demonstrate that antibody cross-reactivity, target protein truncation and abundance, as well as the cellular compartment of origin are major factors that affect protein profiling on antibody arrays. The experimental conditions established for prokaryotic proteins were adopted to compare protein profiles in the breast tumor-derived cell lines MDA MB-231 and SKBR3. Competitive displacement was detected and confirmed for a number of proteins using both detection methods; however, we show that overall the two-color method is better suited for accurate expression profile evaluation of a large, complex set of proteins. Antibody array data confirm the functional linkage between the ErbB2 receptor and AP-2 transcription factors in these cell lines and highlight unexpected differences in G1 cyclin expression.

Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked upregulation (10-15 fold) of the telomere-binding protein, TRF2. Upregulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite lifespan and immortal HMEC. TRF2 protein was not upregulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least twofold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells' ability to maintain an indefinite lifespan.

Differential expression of VEGF-A mRNA by 17 -estradiol in breast tumor cells lacking classical ER- may be mediated through a variant form of ER-

Beta-estradiol (17beta-E2) augments VEGF-A expression in various estrogen targeted organs and cells including breast tumor derived cell lines, via an ER-alpha mediated pathway. Ironically, 17beta-E2 is able to regulate some genes via ER-alpha independent pathways. In the present study, we sought to determine whether 17beta-E2 can modulate VEGF-A expression in absence of ER-alpha, and therefore, three different cell lines including ER-alpha+ MCF-7, and ER-alpha SKBR-3 and HMEC were used for this study. The present study demonstrates that 17beta-E2 also induces VEGF-A mRNA expression in ER-negative SKBR-3 breast tumor cells in a manner similar to that observed in ER-positive MCF-7 cells. Blocking the induced-expression by antiestrogen ICI 182,780 indicates the induction pathway is ER dependent. While ER-alpha mRNA is absent in both HMEC and SKBR-3 cells, the impact of estrogen was found only in SKBR-3 cells, suggesting the existence of an analogue to ER-alpha or overlapping signal in these cells. Consistent with this suggestion, the present studies demonstrate the existence of an ER-alpha(var2) protein in MCF-7 and in SKBR-3 cells. This variant is predominantly localized in the nuclei of SKBR-3 cells. Importantly, specific binding of 17beta-E2 by these cells suggest the ER-alpha(var2) may act as active receptor in SKBR-3 cells.

Cyclin-Dependent Kinase 6 Inhibits Proliferation of Human Mammary Epithelial Cells

Many defects in cancer cells are in molecules regulating G(1)-phase cyclin-dependent kinases (cdks), which are responsible for modulating the activities of Rb family growth-suppressing proteins. Models for understanding how such defects affect proliferation assume that cdks are responsible for sequentially phosphorylating, and hence inactivating, the growth-suppressing functions of Rb family proteins, thus promoting cell cycle progression. However, cdks also play a role in formation of growth-suppressing forms of pRb family molecules, including the "hypophosphorylated" species of pRb itself. Here, it is shown that normal human mammary epithelial cells have a high amount of cdk6 protein and activity, but all breast tumor-derived cell lines analyzed had reduced levels, with several having little or no cdk6. Immunohistochemical studies showed reduced levels of cdk6 in breast tumor cells as compared with normal breast tissue in vivo. Cdk6 levels in two breast tumor cell lines were restored to those characteristic of normal human mammary epithelial cells by DNA transfection. The cells had a reduced growth rate compared with parental tumor cells; cells that lost ectopic expression of cdk6 reverted to the faster growth rate of parental cells. Cell lines with restored cdk6 levels accumulated higher amounts of the Rb family protein p130 as well as E2F4, a suppressing member of the E2F family of transcription factors, in their nuclei. The results suggest that cdk6 restrains rather than stimulates breast epithelial cell proliferation and that its loss or down-regulation could play a role in breast tumor development.

Reovirus oncolysis of human breast cancer.

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.

Prolactin Negatively Regulates Caveolin-1 Gene Expression in the Mammary Gland during Lactation, via a Ras-dependent Mechanism

Caveolin-1 is a 22-kDa integral membrane protein that has been suggested to function as a negative regulator of mitogen-stimulated proliferation in a variety of cell types, including mammary epithelial cells. Because much of our insight into caveolin-1 function has come from the study of human breast tumor-derived cell lines in culture, the normal physiological regulators of caveolin-1 expression in the mammary gland remain unknown. Here, we examine caveolin-1 expression in mice at different stages of mammary gland development. We show that caveolin-1 expression is significantly down-regulated during late pregnancy and lactation. Upon weaning, mammary gland expression of caveolin-1 rapidly returns to non-pregnant "steady-state" levels. Injection of virgin mice with a battery of hormones normally up-regulated during lactation demonstrates that prolactin is the main mediator of caveolin-1 down-regulation. Virtually identical results were obtained with human mammary epithelial cells (hTERT-HME1) in culture. In addition, we demonstrate that prolactin-mediated down-regulation of caveolin-1 expression occurs at the level of transcriptional control and via a Ras-dependent mechanism. Interestingly, in the mammary gland, both mammary epithelial cells and the surrounding mammary adipocytes show prolactin-mediated down-regulation of caveolin-1. This hormone-dependent regulation of caveolin-1 expression is specific to the mammary fat pad. Finally, we employed HC11 cells, a well-established model of mammary epithelial cell differentiation, to study the possible functional effects of caveolin-1 expression. In the presence of lactogenic hormones, recombinant expression of caveolin-1 in HC11 cells dramatically suppresses the induction of the promoter activity and the synthesis of beta-casein, an established reporter of lactogenic differentiation and milk production. These findings may explain why caveolin-1 levels are normally down-regulated during lactation. This report is the first demonstration that caveolin-1 levels are down-regulated during a normal physiological event in vivo, i.e. lactation, because previous reports have only documented that down-regulation of caveolin-1 occurs during cell transformation and tumorigenesis.

Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer

Amplification of the human epidermal growth factor receptor 2 protein (HER2) in primary breast carcinomas has been shown to correlate with poor clinical prognosis for certain patients. Trastuzumab (Herceptin ®, Genentech, Inc., South San Francisco, California) is a highly purified recombinant DNA-derived humanized monoclonal immunoglobulin G1 kappa antibody that binds with high affinity and specificity to the extracellular domain of the HER2 receptor. In vitro and in vivo preclinical studies have shown that administration of trastuzumab alone or in combination with paclitaxel or carboplatin significantly inhibits the growth of breast tumor-derived cell lines that overexpress the HER2 gene product. At therapeutic doses in breast cancer patients, the mean half-life of trastuzumab is 5.8 days. Trastuzumab serum concentrations reach steady state with mean trough and peak concentrations of 79 μg/mL and 123 μg/mL, respectively. In a 222-patient, singlearm clinical study, treatment with a loading dose of trastuzumab 4 mg/kg administered IV followed by weekly IV doses of 2 mg/kg produced an overall response rate of 14% (2% complete remission and 12% partial remission). The beneficial effects were greatest in patients with the greatest degree (3+) of HER2 protein overexpression. In another clinical study, 469 women with metastatic breast carcinoma were randomized to a paclitaxel or anthracycline-pluscyclophosphamide regimen with or without trastuzumab. The overall response rate was significantly greater in the trastuzumabplus-chemotherapy group than in the chemotherapy-alone cohort. The magnitude of observed effects was greatest with paclitaxel plus trastuzumab. The most common adverse effects attributed to trastuzumab in clinical studies were fever and chills, pain, asthenia, nausea, vomiting, increased cough, diarrhea, headache, dyspnea, infection, rhinitis, and insomnia. Trastuzumab in combination with chemotherapy can lead to cardiotoxicity, leukopenia, anemia, diarrhea, abdominal pain, and infection. Trastuzumab has been approved by the US Food and Drug Administration as a single agent for the treatment of patients who have metastatic breast cancer involving overexpression of the HER2 protein and who have received 1 or more chemotherapy regimens; in combination with paclitaxel, it has been approved for the treatment of such patients who have not received chemotherapy.

P185HER2 Monoclonal Antibody Has Antiproliferative Effects In Vitro and Sensitizes Human Breast Tumor Cells to Tumor Necrosis Factor

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.

P185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.