Stages Of Breast Cancer Progression Research Articles

The ShcA PTB Domain Functions as a Biological Sensor of Phosphotyrosine Signaling during Breast Cancer Progression

ShcA (SHC1) is an adapter protein that possesses an SH2 and a PTB phosphotyrosine-binding motif. ShcA generally uses its PTB domain to engage activated receptor tyrosine kinases (RTK), but there has not been a definitive determination of the role of this domain in tumorigenesis. To address this question, we employed a ShcA mutant (R175Q) that no longer binds phosphotyrosine residues via its PTB domain. Here, we report that transgenic expression of this mutant delays onset of mammary tumors in the MMTV-PyMT mouse model of breast cancer. Paradoxically, we observed a robust increase in the growth and angiogenesis of mammary tumors expressing ShcR175Q, which displayed increased secretion of fibronectin and expression of integrin α5/β1, the principal fibronectin receptor. Sustained integrin engagement activated Src, which in turn phosphorylated proangiogenic RTKs, including platelet-derived growth factor receptor, fibroblast growth factor receptor, and Met, leading to increased VEGF secretion from ShcR175Q-expressing breast cancer cells. We defined a ShcR175Q-dependent gene signature that could stratify breast cancer patients with a high microvessel density. This study offers the first in vivo evidence of a critical role for intracellular signaling pathways downstream of the ShcA PTB domain, which both positively and negatively regulate tumorigenesis during various stages of breast cancer progression.

Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts

To learn more about cancer-associated fibroblasts (CAFs), we have isolated fibroblasts from different stages of breast cancer progression and analysed their function and gene expression. These analyses reveal that activation of the YAP transcription factor is a signature feature of CAFs. YAP function is required for CAFs to promote matrix stiffening, cancer cell invasion and angiogenesis. Remodelling of the ECM and promotion of cancer cell invasion requires the actomyosin cytoskeleton. YAP regulates the expression of several cytoskeletal regulators, including ANLN, and DIAPH3, and controls the protein levels of MYL9/MLC2. Matrix stiffening further enhances YAP activation, thus establishing a feed-forward self-reinforcing loop that helps to maintain the CAF phenotype. Actomyosin contractility and Src function are required for YAP activation by stiff matrices. Further, transient ROCK inhibition is able to disrupt the feed-forward loop leading to a long-lasting reversion of the CAF phenotype.

Abstract 5239: Unraveling the complex regulatory relationship between PI3K signaling and metabolic transformation in breast cancer.

Abstract Cancer cells exhibit a metabolic phenotype characterized by high rates of glucose uptake and lactate production, known as the Warburg effect. While the Warburg effect and normal proliferative metabolism appear similar, important molecular differences exist. We hypothesize that molecular and metabolic drivers of the Warburg effect can be modulated to impede cancer proliferation without substantial effects on normal tissue growth. Intracellular networks exhibit a variety of emergent non-linear behaviors and, as a result, the use of experimental intuition alone will not be enough to identify these drivers. Using a combination of experimental and theoretical methods, we developed a model of breast cancer progression that includes metabolism and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, an important regulator of carbon metabolism. A key component of our model is a detailed logic network of molecular interactions associated with PI3K signaling as well as regulatory connections to central carbon metabolism, including the ATP/AMP ratio, GLUT receptor activation, hexokinase activation, and changes in the catalytic activity of pyruvate kinase. To validate our model, a series of phospho Western blot analyses were performed using a normal-like breast cell line and a diverse set of breast cancer cell lines exposed to PI3K pathway inhibitors. From these data, a series of predictive network models were constructed representing distinct stages of breast cancer progression. We also generated detailed metabolic flux maps for each cell line using metabolic flux analysis (MFA), a method that relies on carbon-13 tracers, mass-spectrometry, and measurements of extracellular flux to infer intracellular flux. In agreement with recent studies, we found an increase in the reductive carboxylation of glutamine derived alpha-ketoglutarate in cells constitutively adapted to hypoxia. We also identified a potentially important metabolic vulnerability in aggressive breast cancers. Moreover, we found important PI3K network differences at the RNA and protein levels, some of which were isoform specific. Together our data indicate that very different system-level properties are associated with distinct stages of breast cancer progression and metabolic transformation. Our model is suitable for performing in silico molecular perturbations to predict a normal as well as tumor level response to a targeted therapy or combination of therapies. Our approach also serves as a prototype for the use of systems biology methods in personalized medicine where molecular and metabolic data collected from a patient's biopsied tumor is input into a predictive model designed to develop a strategic treatment plan for the patient. The use of predictive models to integrate data from an individual patient will have a profound impact on cancer care decisions and patient outcomes in the future. Citation Format: Michelle L. Wynn, Megan Egbert, Lauren D. Van Wassenhove, Zhi Fen Wu, Firas Midani, Charles Evans, Charles F. Burant, Santiago Schnell, Sofia D. Merajver. Unraveling the complex regulatory relationship between PI3K signaling and metabolic transformation in 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 5239. doi:10.1158/1538-7445.AM2013-5239

Abstract 5223: Elucidating the complex cross-talk between the MAPK and PIK3 pathways.

Abstract Targeted molecular inhibitors have emerged as a leading anti-cancer strategy; however, despite promising pre-clinical data, many targeted inhibitors induce undesirable off-target effects in the clinic. The large number of off-target effects associated with molecular inhibitors was recently termed the ‘‘whack a mole problem’’ because inhibiting one molecular target often unintentionally activates another molecule. It is increasingly clear that the high incidence of off-target effects associated with targeted inhibitors is related to the complex interactions and emergent behaviors inherent to the highly complex and dysregulated intracellular networks of cancer. Both the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways are known to be dysregulated in cancer. In previous work, we and others have demonstrated that the MAPK pathway promotes motility, invasion, and angiogenic factors while the PI3K pathway plays an important role in controlling anchorage independent growth. In addition, the PI3K pathway plays an essential role in stimulating glucose metabolism and the Warburg effect. We hypothesize that robust interactions exist between these two pathways that influence efficacy and potentially also acquired resistance to targeted therapies. Using a combination of experimental and theoretical techniques, we developed a predictive network model linking growth factor signaling to the MAPK and PI3K pathways as well as to glucose metabolism. Specifically, we constructed a logic-based network of the cross-talk between MAPK and PI3K signaling that relied on a detailed literature survey to identify known molecular interactions as well as proposed interactions and regulatory feedback connections in the literature. We next performed a set of experiments using a normal-like breast epithelial cell line and a series of pathway specific inhibitors with and without growth factor stimulation to validate our model. Finally, we repeated these experiments using a diverse set of breast cancer cell lines and integrated this data to produce a series of cancer networks representative of different stages of breast cancer progression. Our model was able to recapitulate both our own experimental data and published data in the literature using a smaller subset of regulatory feedback mechanisms than we started with. Together, our results suggest that some proposed interactions and feedback mechanisms attributed to MAPK and PI3K cross-talk in the literature may not be valid. Citation Format: Megan E. Egbert, Michelle L. Wynn, Zhi Fen Wu, Santiago Schnell, Sofia D. Merajver. Elucidating the complex cross-talk between the MAPK and PIK3 pathways. [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 5223. doi:10.1158/1538-7445.AM2013-5223

Real-Time Motion Analysis Reveals Cell Directionality as an Indicator of Breast Cancer Progression

Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.

Characterisation of Walker 256 breast carcinoma cells from two tumour cell banks as assessed using two models of secondary brain tumours

BackgroundMetastatic brain tumours are a common end stage of breast cancer progression, with significant associated morbidity and high mortality. Walker 256 is a rat breast carcinoma cell line syngeneic to Wistar rats and commonly used to induce secondary brain tumours. Previously there has been the assumption that the same cancer cell line from different cell banks behave in a similar manner, although recent studies have suggested that cell lines may change their characteristics over time in vitro.MethodsIn this study internal carotid artery injection and direct cerebral inoculation models of secondary brain tumours were used to determine the tumorigenicity of Walker 256 cells obtained from two cell banks, the American Type Culture Collection (ATCC), and the Cell Resource Centre for Medical Research at Tohoku University (CRCTU).ResultsTumour incidence and volume, plus immunoreactivity to albumin, IBA1 and GFAP, were used as indicators of tumorigenicity and tumour interaction with the host brain microenvironment. CRCTU Walker 256 cells showed greater incidence, larger tumour volume, pronounced blood–brain barrier disruption and prominent glial response when compared to ATCC cell line.ConclusionsThese findings indicate that immortalised cancer cell lines obtained from different cell banks may have diverse characteristics and behaviour in vivo.

RhoC Impacts the Metastatic Potential and Abundance of Breast Cancer Stem Cells

Cancer stem cells (CSCs) have been shown to promote tumorigenesis of many tumor types, including breast, although their relevance to cancer metastasis remains unclear. While subpopulations of CSCs required for metastasis have been identified, to date there are no known molecular regulators of breast CSC (BCSC) metastasis. Here we identify RhoC GTPase as an important regulator of BCSC metastasis, and present evidence suggesting that RhoC also modulates the frequency of BCSCs within a population. Using an orthotopic xenograft model of spontaneous metastasis we discover that RhoC is both necessary and sufficient to promote SUM149 and MCF-10A BCSC metastasis–often independent from primary tumor formation–and can even induce metastasis of non-BCSCs within these cell lines. The relationship between RhoC and BCSCs persists in breast cancer patients, as expression of RhoC and the BCSC marker ALDH1 are highly correlated in clinical specimens. These results suggest new avenues to combating the deadliest cells driving the most lethal stage of breast cancer progression.

Vitamin D and the mammary gland: a review on its role in normal development and breast cancer

Breast cancer is a heterogeneous disease associated with diverse biological behaviours and clinical outcome. Although some molecular subgroups of breast cancer have a targeted therapy, the most aggressive tumours still lack a molecular target. Despite vitamin D being classically associated with the physiological role of calcium regulation and phosphate transport in bone metabolism, several studies have demonstrated a wide range of functions for this hormone, which are particularly important in the field of cancer. The mechanisms underlying the protective actions of vitamin D in cancer development are only sparsely understood, but evidence shows that vitamin D participates in cell growth regulation, apoptosis and cell differentiation. In addition, it has been implicated in the suppression of cancer cell invasion, angiogenesis and metastasis. Most of vitamin D biological actions are mediated by the vitamin D receptor and the synthesis and catabolism of this hormone are regulated by the enzymes CYP27B1 and CYP24A1. In the present review we highlight research data concerning the function of this hormone in the mammary gland, with a special focus on breast carcinogenesis. Hence, and although the available data are controversial, we consider not only updated information on the epidemiology of vitamin D in breast cancer and its potential value as a therapeutic agent or prophylactic (with an emphasis on molecular mechanisms and effectors of vitamin D action), but include data on its role in other stages of breast cancer progression as well. Accordingly, we review data on the influence of vitamin D in the development of normal breast and the expression of vitamin D-related proteins (VDR, CYP27B1 and CYP24A21) in benign mammary lesions and ductal carcinomas in situ.

Abstract LB-18: Expression of discoidin domain receptor tyrosine kinases in breast cancer

Abstract A key step in breast cancer progression is the transition from in situ to invasive carcinoma. This process is partly mediated by defective interactions between the epithelial cells and the extracellular matrix, leading to loss of cell-cell contacts and polarity, increased proliferation, and acquisition of invasive activity. Here we report that a unique set of receptor tyrosine kinases (RTKs) known as the discoidin domain receptors (DDRs) may play a key role during the transition of in situ to invasive breast carcinoma. DDRs are the only RTKs that recognize collagen as their ligands and thus mediate collagen-initiated signaling in both physiological and pathological conditions. The expression of DDRs at various stages of breast cancer progression is unknown. We used a high-density tissue microarray containing samples of normal breast, ductal carcinoma in situ (DCIS), and invasive carcinomas of different clinico-pathological characteristics to examine the expression of DDR1 by immunohistochemistry using an antibody to the C-terminus of DDR1. Expression of DDR1 was evaluated taking into consideration the percentage and intensity of staining, and categorized as low or high. DDR1 was highly expressed in the cytoplasm and membrane of normal epithelial cells in ducts and acini, and in DCIS. In contrast, 88 of 134 (66%) invasive carcinomas displayed low or absent DDR1 expression, while 44% exhibited high levels. DDR1 expression was not associated with histologic type, grade, hormonal receptor or HER2-neu status. Interestingly, 15 of 40 (37.5%) patients with high DDR1 expression developed tumor recurrence compared to 9 of 46 (19.6%) patients with low DDR1 (Kaplan Meier log-rank p=0.05). The expression of DDR1 was also evaluated in a mouse model of breast cancer progression using pre-neoplastic and malignant human MCF10A cell variants. DDR1 was highly expressed in the benign hyperplastic lesions generated by MCF10AT1 cells, and in the comedo DCIS tumors generated by MCF10DCIS.COM cells. In contrast, low or no DDR1 expression was observed in tumors generated by the highly malignant MCF10CA1 variant. Expression analyses of DDR1 isoforms in cultured cells by real-time PCR demonstrated that non-malignant MCF10A cells expressed higher levels of DDR1b mRNA while malignant MCF10CA1 cells showed higher levels of DDR1a suggesting that malignant progression is associated with a DDR1 isoform switch. At present the functional roles of the DDR1 isoforms in breast cancer progression remain unknown. Our analyses suggest that loss of or reduced DDR1 expression is associated with the transition from in situ to invasive carcinomas while patients with invasive carcinomas displaying high DDR1 expression have worse disease free survival. These novel studies highlight a complex role of DDR1 in breast cancer progression that deserves further exploration. 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 LB-18. doi:1538-7445.AM2012-LB-18

PP 14 mTOR inhibition decreases the malignant properties of cancer cells at selective stages of breast cancer progression in vitro

PP 14 mTOR inhibition decreases the malignant properties of cancer cells at selective stages of breast cancer progression in vitro

1070 POSTER mTOR Inhibition Arrests Selective Stages of Breast Cancer Progression in Vitro

1070 POSTER mTOR Inhibition Arrests Selective Stages of Breast Cancer Progression in Vitro

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P < 0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Inflammatory mediators in breast cancer: Coordinated expression of TNFα &amp; IL-1β with CCL2 &amp; CCL5 and effects on epithelial-to-mesenchymal transition

BackgroundThe inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1β were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course.MethodsThe expression of CCL2, CCL5, TNFα and IL-1β was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma In Situ (DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments.ResultsCCL2, CCL5, TNFα and IL-1β were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1β in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1β expression. These results suggest progression-related roles for TNFα and IL-1β in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1β compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1β) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1β acts jointly with other pro-malignancy factors to promote disease relapse.ConclusionsOur findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1β may be important for disease course, and that TNFα & IL-1β may promote disease relapse. Further in vitro and in vivo studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.

Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers

Mitochondria are key organelles in mammary cells responsible for several cellular functions including growth, division, and energy metabolism. In this study, mitochondrial proteins were enriched for proteomics analysis with the state-of-the-art two-dimensional differential gel electrophoresis and matrix-assistant laser desorption ionization-time-of-flight mass spectrometry strategy to compare and identify the mitochondrial protein profiling changes between three breast cell lines with different tumorigenicity and metastasis. The proteomics results demonstrate more than 1,500 protein features were resolved from the equal amount pooled from three purified mitochondrial proteins, and 125 differentially expressed spots were identified by their peptide finger print, in which, 33 identified proteins belonged to mitochondrial proteins. Eighteen out of these 33 identified mitochondrial proteins such as SCaMC-1 have not been reported in breast cancer research to our knowledge. Additionally, mitochondrial protein prohibitin has shown to be differentially distributed in mitochondria and in nucleus for normal breast cells and breast cancer cell lines, respectively. To sum up, our approach to identify the mitochondrial proteins in various stages of breast cancer progression and the identified proteins may be further evaluated as potential breast cancer markers in prognosis and therapy.

The molecular pathology of breast cancer progression.

The current model of human breast cancer progression proposes a linear multi-step process which initiates as flat epithelial atypia (FEA), progresses to atypical ductal hyperplasia (ADH), evolves into DCIS and culminates in the potentially lethal stage of invasive ductal carcinoma. For several decades a major challenge to human breast cancer research has been the identification of the molecular alterations associated with the different stages of breast cancer progression. Until recently, progress in attaining this goal has been hampered by technical limitations associated with applying advanced molecular technologies to the microscopic preinvasive stages of breast tumorigenesis. Recent advances in comprehensive, high-throughput genetic, transcriptomic and epigenetic technologies in combination with advanced microdissection and ex vivo isolation techniques have provided for a more complete understanding of the complex molecular genetic and molecular biological inter-relationships of the different stages of human breast cancer evolution. Here we review the molecular biological data suggesting that breast cancer develops and evolves along two distinct molecular genetic pathways. We also briefly review gene expression and epigenetic data that support the view of the tumour microenvironment as an important co-conspirator rather than a passive bystander during human breast tumorigenesis.

Small Players With Big Roles: MicroRNAs as Targets to Inhibit Breast Cancer Progression

As modulators of gene expression, microRNAs (miRNAs) are essential for normal development. Not surprisingly, aberrant expression of miRNAs is associated with many diseases, including cancer. Studies of various breast cancer subtypes have demonstrated that, like gene expression profiles and pathological differences, miRNA profiles can distinguish various tumor subtypes. Over the last few years, roles for miRNAs during many stages of breast cancer progression have been established. This includes potential breast cancer associated polymorphisms in miRNA target sites or miRNAs themselves, miRNAs that can act as tumor suppressors or oncogenes, and miRNAs that can modulate metastatic spread. Recent studies have also suggested key roles for miRNAs in regulating cancer stem cells. Thus, miRNAs have now become important therapeutic targets. This can be achieved by replacing miRNA expression where it has been lost or decreased, or conversely by inhibiting miRNA expression where it has been amplified or overexpressed in cancers. Ultimately, miRNAs should provide both important prognostic biomarkers as well as new targetable molecules for the treatment of breast cancer.

Hypoxia inducible factor 1α gene (HIF-1α) splice variants: potential prognostic biomarkers in breast cancer

BackgroundHypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known.MethodsUsing real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival.ResultsHIF-1α isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1αTAG) and HIF-1α736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1αTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1αTAG (P = 0.048), and total HIF-1α (P = 0.048) mRNA levels. HIF-1αTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1αTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01).ConclusionsOur results show for the first time that mRNA expression of a HIF-1αTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45

Proteome-Wide Profiling of the MCF10AT Breast Cancer Progression Model

BackgroundMapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer.Methodology/Principal FindingsWe conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the “Warburg effect”). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions.Conclusion/SignificanceThe information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.

Abstract 1480: Characterization of C35, a novel protein binding partner of p73, in gynaecological cancers

Abstract The p53 family protein p73 encodes pro-apoptotic (TAp73) and anti-apoptotic (DNp73) isoforms. The functional role of the two isoforms in cancer development is still unclear. In this study, by yeast-2-hybrid screening using DNp73 as a bait, we identified C35 (C17orf37) as a novel protein interacting partner of p73. C35 was mapped to chromosome 17q12. It was overexpressed in breast cancer and prostate cancer. Its expression was positively correlated with the grade and stage of breast cancer progression. A recent study reported that C35 promotes cell migration and invasion in prostate cancer cells. However, the significance of C35 in cancer development remains to be elucidated. In this study, we focused on the functional role of C35 in gynaecological cancers. The binding of C35 and DNp73 was confirmed by co-immunoprecipitation. Quantitative real-time PCR revealed that mRNA expression of C35 in ovarian and cervical cancer cell lines was higher than that in normal cell lines. In addition, mRNA expression of C35 was significantly higher in ovarian cancer clinical samples than that in normal samples (p&amp;lt;0.0001, t-test). To investigate the function of C35, SiHa and OV2008 cells stably expressing C35 were established. The protein expression of C35 was confirmed by western blot analysis. In studying the effect of C35 on cell transforming ability, soft agar assay was performed. The result showed that the number of colonies formed by the C35 stable clones was significantly higher than those formed by the parental cells and control vector stably transfected cells. In addition, the size of the colonies formed by the C35 stable clones was larger. Wound healing assay and transwell migration assay were performed to investigate the effect of C35 on cell migration rate. We found that the migration rate of the C35 stable clones was higher than that of the parental cells and vector transfected cells. XTT assay revealed that the proliferation rate of the C35 stable clones was higher than that of the parental cells and vector controls. In summary, our findings suggested that C35 is overexpressed in ovarian cancer. C35 likely exerts its oncogenic roles to enhance transformation, cell migration and cell proliferation in gynaecological cancers. Further study of the significance of the DNp73-C35 interaction may give insight into their oncogenic properties. 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 1480.

Abstract LB-215: Hypoxia inducible factor-1 alpha (HIF-1α) splice variants: Potential prognostic biomarkers in breast cancer

Abstract Background: Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. HIF-1 protein is composed of two HIF-1α and HIF-1ß/ARNT subunits. Prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variants expression on breast cancer prognosis in term of metastasis risk is not well known. Methods: With real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1α and four variants in breast specimens of a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1α splice variant levels were compared to clinicopathological parameters including tumor microvessel density and metastasis-free survival. Results: HIF-1α isoforms containing three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1 TAG) and HIF-1 736mRNAs were found expressed at higher levels in estrogen receptor negative carcinomas compared to normal/benign tissues (p=0.009 and p=0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1 TAG mRNA levels (p=0.037). Significant statistical association was found between tumor grade and HIF-1 TAG (p=0.048), and total HIF-1 (p=0.048) mRNA levels. HIF-1 TAGmRNA levels also correlated inversely with both estrogen and progesterone receptor status (p=0.005 and p=0.033 respectively). Univariate analysis showed that high HIF-1 TAG mRNA levels correlated with shortened metastasis free survival (p=0.01). Conclusion: Our results show for the first time that mRNA expression of HIF-1 TAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 LB-215.