Primary Breast Cell Research Articles

Oxidative Stress Mediates the Antiproliferative Effects of Nelfinavir in Breast Cancer Cells

The discovery of the anti-proliferative activity of nelfinavir in HIV-free models has encouraged its investigation as anticancer drug. Although the molecular mechanism by which nelfinavir exerts antitumor activity is still unknown, its effects have been related to Akt inhibition. Here we tested the effects of nelfinavir on cell proliferation, viability and death in two human breast cancer cell lines and in human normal primary breast cells. To identify the mechanism of action of nelfinavir in breast cancer, we evaluated the involvement of the Akt pathway as well as the effects of nelfinavir on reactive oxygen species (ROS) production and ROS-related enzymes activities. Nelfinavir reduced breast cancer cell viability by inducing apoptosis and necrosis, without affecting primary normal breast cells. The antitumor activity of nelfinavir was related to alterations of the cell redox state, coupled with an increase of intracellular ROS production limited to cancer cells. Nelfinavir treated tumor cells also displayed a downregulation of the Akt pathway due to disruption of the Akt-HSP90 complex, and subsequent degradation of Akt. These effects resulted to be ROS dependent, suggesting that ROS production is the primary step of nelfinavir anticancer activity. The analysis of ROS-producers and ROS-detoxifying enzymes revealed that nelfinavir-mediated ROS production was strictly linked to flavoenzymes activation. We demonstrated that ROS enhancement represents the main molecular mechanism required to induce cell death by nelfinavir in breast cancer cells, thus supporting the development of new and more potent oxidizing molecules for breast cancer therapy.

Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) Drives Proliferation of IPH-926 lobular Carcinoma Cells.

BackgroundMost breast cancers depend on estrogenic growth stimulation. Functional genetic screenings in in vitro cell models have identified genes, which override growth suppression induced by anti-estrogenic drugs like tamoxifen. Using that approach, we have previously identified Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) as a mediator of cell proliferation and tamoxifen-resistance. Here, we show high level of expression and function of BCAR4 in human breast cancer.Methods BCAR4 mRNA expression was evaluated by (q)RT-PCR in a panel of human normal tissues, primary breast cancers and cell lines. A new antibody raised against C78-I97 of the putative BCAR4 protein and used for western blot and immunoprecipitation assays. Furthermore, siRNA-mediated gene silencing was implemented to study the function of BCAR4 and its downstream targets ERBB2/3.ResultsExcept for placenta, all human normal tissues tested were BCAR4-negative. In primary breast cancers, BCAR4 expression was comparatively rare (10%), but associated with enhanced proliferation. Relative high BCAR4 mRNA expression was identified in IPH-926, a cell line derived from an endocrine-resistant lobular breast cancer. Moderate BCAR4 expression was evident in MDA-MB-134 and MDA-MB-453 breast cancer cells. BCAR4 protein was detected in breast cancer cells with ectopic (ZR-75-1-BCAR4) and endogenous (IPH-926, MDA-MB-453) BCAR4 mRNA expression. Knockdown of BCAR4 inhibited cell proliferation. A similar effect was observed upon knockdown of ERBB2/3 and exposure to lapatinib, implying that BCAR4 acts in an ERBB2/3-dependent manner.Conclusion BCAR4 encodes a functional protein, which drives proliferation of endocrine-resistant breast cancer cells. Lapatinib, a clinically approved EGFR/ERBB2 inhibitor, counteracts BCAR4-driven tumor cell growth, a clinical relevant observation.

Cell membrane softening in human breast and cervical cancer cells

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias.

Clinically relevant human culture models are essential for developing effective therapies and exploring the biology and etiology of human cancers. Current breast tumour models, such as those from oncogenically transformed primary breast cells, produce predominantly basal-like properties, whereas the more common phenotype expressed by the vast majority of breast tumours are luminal. Reasons for this puzzling, yet important phenomenon, are not understood. We show here that luminal epithelial cells are significantly more resistant to viral transduction than their myoepithelial counterparts. We suggest that this is a significant barrier to generating luminal cell lines and experimental tumours in vivo and to accurate interpretation of results. We show that the resistance is due to lower affinity of luminal cells for virus attachment, which can be overcome by pretreating cells—or virus—with neuraminidase. We present an analytical method for quantifying transductional differences between cell types and an optimized protocol for transducing unsorted primary human breast cells in context.

Epigenetic and transcriptional determinants of the human breast.

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.

English

Numerous studies on Ultrasonographic (US) findings of Thyroid malignancy have been conducted (1-6). The main pathologic types of thyroid carcinoma are papillary, follicular, medullary, and anaplastic. Papillary and follicular thyroid carcinomas both have an excellent prognosis, with a 20-year survival of 90%-95% and 75%, respectively (7-9). Medullary thyroid carcinoma is more aggressive, with a 10-year survival of 42%-90% (10-11). Anaplastic thyroid carcinoma has an extremely poor prognosis, with a 5-year survival of 5% (10-11). Risk factors for thyroid carcinoma include age of less than 20 years or more than 60 years, a history of neck irradiation, and a family history of thyroid cancer (11.).Thyroid lymphoma, usually of the non-Hodgkin type, is uncommon. It may occur as part of generalized lymphoma or as a primary tumor, usually in the setting of Hashimoto thyroiditis. Metastases to the thyroid are rare and usually originate from primary lung, breast, and renal cell carcinomas. Metastatic disease should be suspected when a solid thyroid nodule is found in a patient with a known non-thyroid malignancy. AIMS & OBJECTIVE: In this study we planned to differentiate different type of thyroid malignancies based on Ultrasonography from one another due their different prognosis. MATERIAL & METHOD: We took 120 patients showing palpable neck masses out of which 40 patients were found to have malignant neoplasm of Thyroid gland. Further differentiation of different type of Thyroid neoplasm was done by Ultrasonography. RESULT: Out of 40 malignant neoplasm, 24 patients had papillary carcinoma and 4 patients had medullary carcinoma Thyroid. Rest 16 patients were kept in other type of thyroid malignancies. CONCLUSION: We concluded our study with the fact that just keeping the patient under category of thyroid neoplasm is just not sufficient, rather we should be able to differentiate each type of thyroid malignancy from one another due to different prognosis of each type.

Development of a screen to identify selective small molecules active against patient-derived metastatic and chemoresistant breast cancer cells

IntroductionHigh failure rates of new investigational drugs have impaired the development of breast cancer therapies. One challenge is that excellent activity in preclinical models, such as established cancer cell lines, does not always translate into improved clinical outcomes for patients. New preclinical models, which better replicate clinically-relevant attributes of cancer, such as chemoresistance, metastasis and cellular heterogeneity, may identify novel anti-cancer mechanisms and increase the success of drug development.MethodsMetastatic breast cancer cells were obtained from pleural effusions of consented patients whose disease had progressed. Normal primary human breast cells were collected from a reduction mammoplasty and immortalized with human telomerase. The patient-derived cells were characterized to determine their cellular heterogeneity and proliferation rate by flow cytometry, while dose response curves were performed for chemotherapies to assess resistance. A screen was developed to measure the differential activity of small molecules on the growth and survival of patient-derived normal breast and metastatic, chemoresistant tumor cells to identify selective anti-cancer compounds. Several hits were identified and validated in dose response assays. One compound, C-6, was further characterized for its effect on cell cycle and cell death in cancer cells.ResultsPatient-derived cells were found to be more heterogeneous, with reduced proliferation rates and enhanced resistance to chemotherapy compared to established cell lines. A screen was subsequently developed that utilized both tumor and normal patient-derived cells. Several compounds were identified, which selectively targeted tumor cells, but not normal cells. Compound C-6 was found to inhibit proliferation and induce cell death in tumor cells via a caspase-independent mechanism.ConclusionsShort-term culture of patient-derived cells retained more clinically relevant features of breast cancer compared to established cell lines. The low proliferation rate and chemoresistance make patient-derived cells an excellent tool in preclinical drug development.

Methyl Sulfone Manifests Anticancer Activity in a Metastatic Murine Breast Cancer Cell Line and in Human Breast Cancer Tissue - Part 2: Human Breast Cancer Tissue

Background: Methyl sulfone is a small molecule that reverses cancerous phenotypes of a melanoma cell line. Here, we sought to determine whether methyl sulfone was effective against human breast cancer tissue. Methods: We studied normal and cancerous breast tissue obtained from 17 patients. Results: Methyl sulfone introduced structural order, with cancer tissue taking on the morphology of normal in vivo breast tissue; this structural order was sustainable over long-term culture. Methyl sulfone promoted proper wound healing, including migration of cells into wounded areas and establishment of stable contact inhibition once wounds were covered. Methyl sulfone decreased expression of two breast stem cell marker proteins, HCAM and OCT3/4, which are associated with aberrantly rapid migration of metastatic cells. Finally, normal and cancerous primary breast cells remained viable and healthy in methyl sulfone culture for at least 90 days. Conclusion: Methyl sulfone reintroduced a normal structural phenotype to human breast cancer tissues.

C2ORF40 suppresses breast cancer cell proliferation and invasion through modulating expression of M phase cell cycle genes

Recently, it has been suggested that C2ORF40 is a candidate tumor suppressor gene in breast cancer. However, the mechanism for reduced expression of C2ORF40 and its functional role in breast cancers remain unclear. Here we show that C2ORF40 is frequently silenced in human primary breast cancers and cell lines through promoter hypermethylation. C2ORF40 mRNA level is significantly associated with patient disease-free survival and distant cancer metastasis. Overexpression of C2ORF40 inhibits breast cancer cell proliferation, migration and invasion. By contrast, silencing C2ORF40 expression promotes these biological phenotypes. Bioinformatics and FACS analysis reveal C2ORF40 functions at G2/M phase by downregulation of mitotic genes expression, including UBE2C. Our results suggest that C2ORF40 acts as a tumor suppressor gene in breast cancer pathogenesis and progression and is a candidate prognostic marker for this disease.

Hornerin, an S100 family protein, is functional in breast cells and aberrantly expressed in breast cancer

BackgroundRecent evidence suggests an emerging role for S100 protein in breast cancer and tumor progression. These ubiquitous proteins are involved in numerous normal and pathological cell functions including inflammatory and immune responses, Ca2+ homeostasis, the dynamics of cytoskeleton constituents, as well as cell proliferation, differentiation, and death. Our previous proteomic analysis demonstrated the presence of hornerin, an S100 family member, in breast tissue and extracellular matrix. Hornerin has been reported in healthy skin as well as psoriatic and regenerating skin after wound healing, suggesting a role in inflammatory/immune response or proliferation. In the present study we investigated hornerin’s potential role in normal breast cells and breast cancer.MethodsThe expression levels and localization of hornerin in human breast tissue, breast tumor biopsies, primary breast cells and breast cancer cell lines, as well as murine mammary tissue were measured via immunohistochemistry, western blot analysis and PCR. Antibodies were developed against the N- and C-terminus of the protein for detection of proteolytic fragments and their specific subcellular localization via fluorescent immunocytochemisty. Lastly, cells were treated with H2O2 to detect changes in hornerin expression during induction of apoptosis/necrosis.ResultsBreast epithelial cells and stromal fibroblasts and macrophages express hornerin and show unique regulation of expression during distinct phases of mammary development. Furthermore, hornerin expression is decreased in invasive ductal carcinomas compared to invasive lobular carcinomas and less aggressive breast carcinoma phenotypes, and cellular expression of hornerin is altered during induction of apoptosis. Finally, we demonstrate the presence of post-translational fragments that display differential subcellular localization.ConclusionsOur data opens new possibilities for hornerin and its proteolytic fragments in the control of mammary cell function and breast cancer.

MAD2 depletion triggers premature cellular senescence in human primary fibroblasts by activating a P53 pathway preventing aneuploid cells propagation

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that ensures faithful chromosome segregation during mitosis and its failure can result in aneuploidy. Previously, it was suggested that reduction of the MAD2 gene, encoding a major component of the SAC, induced aneuploidy in human tumor cells. However, tumor cell lines contain multiple mutations that might affect or exacerbate the cellular response to Mad2 depletion. Thus, the scenario resulting by Mad2 depletion in primary human cells could be different and more complex that the one depicted so far. We used primary human fibroblasts (IMR90) and epithelial breast cells (MCF10A) to gain further insight on the effects of genomic instability caused by transient Mad2 depletion. To this aim we depleted Mad2 by RNAi to a level shown by Mad2 haplo-insufficient cells and found that induced aneuploidy caused premature cellular senescence in IMR90 cells. IMR90 cells showed typical features of senescent cells, like senescence-associated (SA) beta galactosidase expression, including up-regulation of p53 and p14ARF proteins and of p21(waf1) as well, but not of p16(INK4A) cyclin-dependent kinase (Cdk) inhibitor. In contrast, after MAD2 post-transcriptional silencing MCF10A cells in which the INK4A/ARF locus is deleted, showed both aneuploidy and a small increase of p53 and p21(waf1) proteins, but not premature cellular senescence. Finally, our results provides an explanation of how a p53 controlled pathway, involving initially p21(waf1) and then p14ARF, could minimize the occurrence of genomic alterations derived from chromosome instability induced by low amounts of MAD2 protein.

Cyclosporine A inhibits breast cancer cell growth by downregulating the expression of pyruvate kinase subtype M2

The high proliferative rate of tumor cells leads to metabolic needs distinct from those of their normal counterparts. An embryonic- and tumor-specific isoform of the enzyme pyruvate kinase M2 (PKM2) is overexpressed in cancer cells to increase the use of glycolytic intermediates for macromolecular biosynthesis and tumor growth. We report that Cyclosporin A (CsA) can regulate the expression and activity of PKM2 in breast cancer cell lines MCF-7, MDA-MB-435 and MDA-MB-231. PKM2 was found to be highly expressed in the three breast cancer cell lines compared to normal primary breast cells. Treatment with CsA inhibited the viability of breast cancer cells in a time- and dose-dependent manner. CsA significantly downregulated the expression of PKM2 in breast cancer cells and decreased adenosine triphosphate (ATP) synthesis, which induced cancer cells to undergo necrosis. Furthermore, the growth suppression effect of CsA was impaired in MCF-7 cells when they were transfected with the PKM2 overexpression plasmid, suggesting that CsA was an effective inhibitor of PKM2-dependent proliferation of breast cancer cells. These results may provide new insights into the mechanism of CsA in cancer therapy.

Abstract 2189: Novel transcriptional regulation of Notch1 by EZH2 in mammary stem cells

Abstract Background: EZH2 has oncogenic functions in the breast and other organs, and has been recently shown to regulate the differentiation of tissue specific stem cells. However, the underlying mechanisms are largely unknown. We hypothesized that EZH2 may regulate mammary stem cells by transcriptional mechanisms of key signaling pathways. Methods: We developed EZH2 KD in SUM149 breast cancer cells, and an inducible and transient cell culture model of EZH2 overexpression, (EZH2-OV), and its deletion mutants in MCF10A and in primary breast cells. Mammary stem cells were identified using ALDH1+ and CD44+/CD24- by FACS. ChIP assays were used to determine EZH2 recruitment to specific areas in NOTCH1 promoter. Ectopic expression of EZH2 and deletion mutants leads to identify the EZH2 domain responsible for the transcription up regulation of NOTCH1. Results: We identified that EZH2 KD in SUM149 significantly decreased NOTCH1 signaling in the stem cell population. We observed preferential expression of EZH2 in the stem cell population of normal primary breast cells compared to non-stem cells. In MCF10A cells, EZ-OV was predominantly increased in the ALDH-1+ population. We found that EZH2-OV increased the stem cell population of MCF10A cells. Consistently, EZH2-OV in MCF10A and primary breast cells increased Notch1 mRNA specifically in the ALDH1+ population. EZH2-OV increased Notch1 protein levels and promoter activity in MCF10A and primary cells. Mechanistically, Notch1 inhibition by GSI reversed the effect of EZH2-OV in the stem cell population of MCF10A cells. ChIP assays revealed that EZH2 is recruited to specific regions of the NOTCH1 promoter depending on EZH2 specific context. Moreover, ChIP assays with EZH2 deletion mutants identify that the Nter- EZH2 HII domain is responsible for the NOTCH1 promoter transcriptional up regulation and that this function is independent of the SET domain (methytransferase activity) and as shown by comparing the EZH2 tri-methylation mark (3H3K27) in the NOTCH1 promoter. Conclusion: Collectively, these results indicate that EZH2 regulates mammary stem cells and requires, at least in part the activation of Notch1 signaling by recruitment of EZH2 to the NOTCH1 promoter through the Nter domain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2189. doi:1538-7445.AM2012-2189

Coherent angular motion in the establishment of multicellular architecture of glandular tissues

Glandular tissues form ducts (tubes) and acini (spheres) in multicellular organisms. This process is best demonstrated in the organization of the ductal tree of the mammary gland and in 3D models of morphogenesis in culture. Here, we asked a fundamental question: How do single adult epithelial cells generate polarized acini when placed in a surrogate basement membrane 3D gel? Using human breast epithelial cells from either reduction mammoplasty or nonmalignant breast cell lines, we observed a unique cellular movement where single cells undergo multiple rotations and then maintain it cohesively as they divide to assemble into acini. This coherent angular motion (CAMo) was observed in both primary cells and breast cell lines. If CAMo was disrupted, the final geometry was not a sphere. The malignant counterparts of the human breast cell lines in 3D were randomly motile, did not display CAMo, and did not form spheres. Upon "phenotypic reversion" of malignant cells, both CAMo and spherical architecture were restored. We show that cell-cell adhesion and tissue polarity are essential for the formation of acini and link the functional relevance of CAMo to the establishment of spherical architecture rather than to multicellular aggregation or growth. We propose that CAMo is an integral step in the formation of the tissue architecture and that its disruption is involved in malignant transformation.

Generation of tumor-initiating cells by exogenous delivery of OCT4transcription factor

IntroductionTumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations.MethodsOCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments.ResultsOTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A. In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew.ConclusionsTransduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors.

RAB family gene expression in breast cancer cells under influence of paclitaxel

AbstractThe aim of this study was to investigate the role of paclitaxel on RAB family of genes in primary breast cancer cell lines. The cancer breast cells obtained from 40 women during mastectomy were used to address this issue. The group included patients with intraductal breast cancer - lesions in I or II advancement level by TNM classification and G1-G2 by Bloom classification. (tumor dimensions up to 2.0 cm without metastases to lymph nodes). Cytostatic drugs before surgery were not administered to these patients. The cultures were conducted in 25 cm^2^ plastic containers at RPMI medium with addiction of 10% fetal bovine serum (FBS) at the standard conditions. After reaching concentration levels of 10 000/ml of the cells, the cultures were treated with 60 ng/ml and 300 ng/ml doses of paclitaxel. The concentrations were calculated in relation to therapeutic doses of paclitaxel, applied in polytherapy in patients with breast cancer. The cell cultures untreated for cytostatic were used as a control group. Analysis was conducted for RAB family of genes: RAB3D, RAB5B, RAB5C, RAB7, RAB7L1, RAB9P1, RAB10. RAB11A, RAB311B, RAB13, RAB18, RAB22A, RAB23, RAB26, RAB27A, RAB27B, RAB28, RAB30, RAB31, RAB33A, RAB3D6, RAB 38, RABL2B Total RNA was extracted from the harvest control group and the treated cells, and this was followed by cDNA synthesis, which was used for hybridization assays using arrays. A lower dose of paclitaxel (60 ng/ml) treatment resulted in an increase (2-4 fold- statistically significant), whereas a higher dose (300 ng/ml) caused a decrease (2-fold - statistically insignificant) in expression of examined oncogenes, compared to that of the control group.In summary, this data indicates that 60 ng/ml paclitaxel dose induced the RAB gene expression in an up-regulated pathway. A higher concentration of cytostatic (300 ng/ml) is a toxic dose for primary breast cells in vitro.

HER2 Overexpression Elicits a Proinflammatory IL-6 Autocrine Signaling Loop That Is Critical for Tumorigenesis

HER2 overexpression occurs in approximately 25% of breast cancers, where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis, although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of interleukin-6 (IL-6) expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2-amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL-6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers.

Abstract 3345: Enhancer of zeste homolog 2 (EZH2) promotes expansion of breast tumor initiation cells through activation of Notch1-jagged signaling

Abstract EZH2 is an oncogene which has been recently shown to regulate the differentiation of tissue specific stem cells. However the role of EZH2 on mammary stem cells is unknown. Notch1 signaling has been implicated in cell-fate determination of human mammary stem cells. We hypothesized that EZH2 may contribute to initiation and expansion of mammary stem cells through activation of Notch1 signaling. Methods: We developed two cell culture models: an inducible EZH2 up regulation system in MCF10A benign mammary cells and a lentivirus-mediated shRNA interference targeting EZH2 in SUM149 breast cancer cells. Mammary stem cells were identified using ALDH1+ and CD44+/CD24- by FACS. The effect of EZH2 on the tumorigenic ability of ALDH-1 positive and negative SUM149 cells was studied in a xenograft mouse model. Results: We found preferential expression of EZH2 in the stem cell population of normal primary breast cells compared to non stem cells. In MCF10A cells, EZH2 overexpression, (EZ-OV), was predominantly increased in the ALDH-1+ population. When we tested the role of EZH2 in the stem cell population we found that EZH2-OV increased the stem cell population of MCF10A cells while EZH2-KD decreased it in SUM149 cells. Microarray analysis identified Notch1-Jagged1 signaling as the most significantly downregulated targets of EZH2 in ALDH+ vs. ALDH- SUM149 cells. Consistently, EZH2-OV in MCF10A and primary breast cells increased Notch1 mRNA specifically in the ALDH1+ population. EZH2-OV increased Notch1 protein level and promoter activity in MCF10A cells. Mechanistically, Notch1 inhibition by GSI reversed the effect of EZH2-OV in the stem cell population of MCF10A cells. Our in vivo studies demonstrated that EZH2 KD in SUM149 cells decreased the growth of ALDH-1+ cells in xenograft experiments. Consistently, EZH2 KD tumors exhibited decreased Notch1 and Jagged1 protein levels. Conclusion: Collectively, these results indicate that EZH2 regulates mammary stem cells and requires, at least in part the activation of Notch1 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3345. doi:10.1158/1538-7445.AM2011-3345

Abstract 1203: Therapeutic opportunities for anti-Met antibody (MetMAb) in breast cancer

Abstract The hepatocyte growth factor (HGF)/Met pathway drives cell proliferation, survival, migration and invasion, which are hallmarks of cancer. Animal models and microarray data suggest that elevated expression of Met is a poor prognostic factor in breast tumors. Our evaluation of primary breast tumors and cell lines has confirmed that expression of Met is found predominantly in the basal sub-type, which includes estrogen receptor (ER), progesterone receptor (PR) and HER2 triple negative (3N) tumors as well as some HER2+ basal tumors. In a panel of 3N basal breast tumors, 96% (25/26) of samples express Met by enzyme-linked immunosorbant assay (ELISA), with 42% (11/26) positive by immuno-histochemistry (IHC). All of the samples were positive for HGF as determined by ECLA, with 32% (8/25) of these primary tumors showing robust staining by IHC. To further explore the biological response of basal breast cancers to HGF, a panel of basal breast cancer cell lines were evaluated in cell proliferation, migration and invasion assays. While most lines tested showed a modest response to HGF in proliferation assays, the migratory and invasive response to HGF was more robust. Assessment of signaling pathways and gene signatures was also performed to better understand the nature of Met signaling leading to biological outcomes. HGF responsive lines were evaluated for growth in transgenic SCID mice expressing human HGF. MetMAb, a potent HGF-blocking Met monovalent monoclonal antibody, acts to inhibit HGF-driven effects in basal breast cancer cell lines in vitro and xenograft growth in vivo. Taken together, these data suggest that Met can drive invasive growth of basal breast tumors, and that treatment with MetMAb, alone or in combination with standard of care, may be therapeutically beneficial in these tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1203. doi:10.1158/1538-7445.AM2011-1203

Abstract 3147: HER2 gene amplification and CDC25A overexpression in human breast cancer

Abstract HER2 gene amplification/overexpression has been identified in 10-34% of invasive breast cancers and correlates with reduced disease-free and overall survival. The HER2-targeted therapy with trastuzumab decreases the risk of relapse and prolongs survival. Resistance to trastuzumab is a significant barrier to the effective HER2-positive breast cancer treatment: response rate ranges from 20-35%, underlining that HER2-positive breast cancer is hardly a homogeneous disease and, more importantly, that other HER2-related pathways need to be unveiled. Recent studies provided evidence that HER2 overexpression increases skin tumorigenesis through CDC25A maintenance: HER2 suppresses DNA damage-induced checkpoint activation through lack of CDC25A degradation. CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. CDC25A misregulation contributes to genomic instability and cancer development. Oncogene activation during early stages of tumor development causes DNA replication stress resulting in the DNA damage response (DDR) induction and the selection of cells defecting in their DDR could lead to malignant progression. Accordingly, CDC25A overexpression in primary human breast cells promotes the DDR alteration, increasing the chances of tumorigenic conversion by enhancing genomic instability at common fragile sites. The aim of our work was to verify a relation between HER2 and CDC25A in human breast cancer. To this end HER2 amplification and CDC25A overexpression were determined in 220 breast cancer patients. We found a statistically significant correlation between HER2 gene status and CDC25A expression (p=0,000): 64,4% of HER2 amplified tumours show CDC25A overexpression and 75,5% of tumours with no HER2 amplification do not overexpress CDC25A. Furthermore HER2 status and CDC25A expression significantly correlate with prognosis (p=0,003 and p=0,047 respectively). A mortality risk stratification was identified when four different combinations of HER2 status and CDC25A expression were evaluated for their relative effect on survival: the group with the highest mortality was the one in which breast carcinomas showed both HER2 amplification and CDC25A overexpression (p=0,019). Our in vitro studies demonstrate that HER2 can stabilize CDC25A protein levels in breast cancer cell lines, through PI3K-AKT pathway: decreased HER2-mediated signaling by HER2 inhibitors resulted in CDC25A decreased expression; remarkably CDC25A inactivation by specific inhibitor can restore response to IR-induced DNA damage in HER2-positive, both trastuzumab sensitive and resistant, breast cancer cell lines. These findings suggest a CDC25A role for a more specific characterization of HER2-positive breast cancers, warranting further study of its potential as therapeutic target in combination with HER2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2011-3147