Drive Breast Cancer Progression Research Articles

Abstract 5103: Crosstalk between osteoprotegerin (OPG), fatty acid synthase (FASN), and cyclooxygenase-2 (COX-2) in breast cancer: implications in carcinogenesis

Abstract The cross-talk between malignant and nonmalignant cells in the tumor microenvironment, as maneuvered by cytokines/chemokines, drives breast cancer progression. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to human normal mammary epithelial HMEC cells (1). OPG was involved in modulating aneuploidy, cell proliferation, and angiogenesis in breast cancer (1). Mass spectrometry analysis performed in this study revealed OPG interacts with fatty acid synthase (FASN), which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. Further, electron microscopy, immunofluorescence, and fluorescence quantitation assay highlighted the presence of a large number of lipid bodies (lipid droplets) in SUM149PT and SUM1315MO2 cells in comparison to HMEC. We recently showed upregulation of COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells (2). Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and FASN. Immunofluorescence staining exposed the co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be cross-talk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 decreased the number of lipid body/cell, downregulated phosphorylation of ERK, GSK3β, and induced apoptosis by caspase-3 and caspapse-9 activation. Collectively, the novel biological cross-talk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic to target highly invasive breast cancer.

Crosstalk between osteoprotegerin (OPG), fatty acid synthase (FASN) and, cycloxygenase-2 (COX-2) in breast cancer: implications in carcinogenesis.

The crosstalk between malignant and nonmalignant cells in the tumor microenvironment, as maneuvered by cytokines/chemokines, drives breast cancer progression. In our previous study, we discovered Osteoprotegerin (OPG) as one of the cytokines heavily secreted by breast cancer cells. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to normal human mammary epithelial HMEC cells. OPG was involved in modulating aneuploidy, cell proliferation, and angiogenesis in breast cancer. Mass spectrometry analysis performed in this study revealed OPG interacts with fatty acid synthase (FASN), which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. Further, electron microscopy, immunofluorescence, and fluorescence quantitation assays highlighted the presence of a large number of lipid bodies (lipid droplets) in SUM149PT and SUM1315MO2 cells in comparison to HMEC. We recently showed upregulation of the COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells. Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and fatty acid synthase (FASN). FASN is a multifunctional enzyme involved in lipid biosynthesis. Immunofluorescence staining revealed the co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be crosstalk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Interestingly, knocking down OPG by CRISPR/Cas9 gene editing in breast cancer cells decreased FASN expression at the protein level. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 and COX-2 inhibitor celecoxib individually decreased the number of lipid bodies/cell, downregulated phosphorylation of ERK, GSK3β, and induced apoptosis by caspase-3/7 and caspase-9 activation. But a more efficient and effective decrease in lipid bodies/cell and survival kinase signaling was observed upon combining the drug treatments for the aggressive cancer cells. Collectively, the novel biological crosstalk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic target to treat highly invasive breast cancer.

Abstract P2-05-14: The long noncoding RNA BHLHE40-AS1 as a functional biomarker of invasive breast cancer

Abstract Increased emphasis on breast cancer screening has led to a dramatic surge in diagnosis of pre-cancerous ductal carcinoma in situ (DCIS) over the past 30 years. Unfortunately, diagnosis of late stage invasive and metastatic disease has not proportionally declined, suggesting significant over diagnosis and over treatment of innocuous DCIS. Due to a lack of biomarkers, the heterogeneity of DCIS lesions, and an insufficient understanding of mechanisms of invasive progression, there is currently no clinically relevant method of predicting which DCIS lesions will advance to invasive disease. As a result, all DCIS patients undergo an aggressive treatment regimen to prevent disease progression. Therefore, there is a critical need to determine the underlying mechanisms driving breast cancer progression to better inform patient treatment options and nominate novel therapeutic entry points for treatment of invasive disease. Recently, long noncoding RNAs (lncRNAs) have gained attention as critical regulators of epigenetic states and gene expression. Although the study of lncRNAs is in its infancy, they are being uncovered as pivotal regulators of development and tumorigenesis, thus they are a rich source of potential drivers of breast cancer progression. We propose that lncRNAs functionally drive breast cancer invasion and their expression can discriminate between innocuous and potentially invasive DCIS. Using biopsies from women that exhibit tandem DCIS and invasive breast cancer lesions, we have identified a cohort of long noncoding RNAs (lncRNAs) that are enriched in the invasive biopsy. From this cohort we have identified the lncRNA BHLHE40-AS1 as increasing in a step-wise manner in a breast cancer progression series that escalates from normal, non-transformed cells to highly invasive disease. Preliminary evidence suggests that BHLHE40-AS1 expression regulates invasive potential in vitro. Using a GeneChip® Human Transcriptome 2.0 Array (HTA) we have identified several targets previously associated with driving breast cancer invasion as being potentially regulated by BHLHE40-AS1. Future directions will focus on determining the effect of BHLHE40-AS1 expression on tumor cell invasion in an orthotopic xenograft model, mechanistically elaborating its function, and determining the utility of BHLHE40-AS1 as a clinically relevant biomarker of invasive breast cancer. Citation Format: DeVaux RKS, Davis S, Sheftel J, Coarfa C, Behbod F, Herschkowitz JI. The long noncoding RNA BHLHE40-AS1 as a functional biomarker of invasive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-14.

Abstract P2-05-07: RAGE-ligand signaling drives breast cancer metastasis through affecting cells of the tumor and microenvironment

Abstract Breast cancer is most common malignant state in women, with 20% of these patients developing metastasis during the course of their disease. Further understanding is needed of the process and mechanisms of metastasis. Our lab and others have been shown that Receptor of Advanced-Glycation End-products (RAGE) plays a role in tumorigenesis and metastasis. RAGE is highly expressed in various cancers including breast cancer and its protein levels correlate with poor patient outcome in breast cancer and other cancers. Activation of RAGE results in increased proliferation, migration and invasion of cancer cells. Further studies in mice have shown it may be a therapeutic target to reduce tumor growth and the resulting metastasis. Further understanding is needed of the role of RAGE in driving metastasis through affecting cells of both the tumor and tumor stroma to design novel therapeutics. Using the breast cancer cell model (MDA-MB-231) and its organotropic sister cells lines selected in vivo for increased metastasis to lung (4175) and bone (1833), we tested the role of RAGE in driving tumor metastasis in vitro and in vivo with xenograft mouse models. To test the role of RAGE in the tumor microenvironment we used the AT-3 syngeneic breast cancer cell model in C57BL6 wild-type and RAGE knockout mice. We demonstrated that the highly metastatic variant of 231 cells (4175 and 1833) have increased expression level of RAGE compared to MDA-MB-231 parental cells. Ectopic over-expression of RAGE in parental 231 cells led to increased migratory and invasive properties compared to vector control cells, without affecting cell proliferation or viability. RAGE knockdown by shRNA in 4175 and 231 parental cells showed decreased cell invasion in transwell assays compared to control scramble shRNA. To validate our data in vivo, we performed mammary fat pad injection of 4175 cells (RAGE and scr shRNA) in NOD SCID gamma mice. Tumor growth and weight was impaired in RAGE gene knockdown 4175 cells compared to scramble (scr) controls. Analysis of lung and liver tissue retrieved from mice revealed RAGE knockdown in 4175 cells prevented metastasis compared to 4175 scr control cells. To test the role of RAGE on non-tumor cells of the breast stroma we next performed syngeneic studies with AT-3 cells (MMTV-PyMT spontaneous BC cell model), by injection into the mammary fat pad of wild-type and RAGE knockout C57BL6 immunocompetent mice. RAGE knockout mice (RAGE -/-) displayed striking impairment of tumor cell growth compared to wild-type (RAGE +/+) mice. We are currently testing whether novel RAGE inhibitors impact breast cancer progression and metastasis. These data highlight RAGE drives breast cancer progression and metastasis through affecting both tumor cell intrinsic and non-tumor cell microenvironment effects. Future studies will demonstrate the potential of RAGE inhibition as a novel therapeutic approach for preventing and treating metastatic disease in breast and other cancers. Citation Format: Kwak T, Drews-Elger K, Ergonul A, Zhao D, Besser A, Slingerland JM, Lippman ME, Hudson BI. RAGE-ligand signaling drives breast cancer metastasis through affecting cells of the tumor and microenvironment. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-07.

Sympathetic innervation, norepinephrine content, and norepinephrine turnover in orthotopic and spontaneous models of breast cancer

Activation of the sympathetic nervous system (SNS) drives breast cancer progression in preclinical breast cancer models, but it has yet to be established if neoplastic and stromal cells residing in the tumor are directly targeted by locally released norepinephrine (NE). In murine orthotopic and spontaneous mammary tumors, tyrosine hydroxylase (TH)+ sympathetic nerves were limited to the periphery of the tumor. No TH+ staining was detected deeper within these tumors, even in regions with a high density of blood vessels. NE concentration was much lower in tumors compared to the more densely innervated spleen, reflecting the relative paucity of tumor TH+ innervation. Tumor and spleen NE concentration decreased with increased tissue mass. In mice treated with the neurotoxin 6-hydroxydopamine (6-OHDA) to selectively destroy sympathetic nerves, tumor NE concentration was reduced approximately 50%, suggesting that the majority of tumor NE is derived from local sympathetic nerves. To evaluate NE utilization, NE turnover in orthotopic 4T1 mammary tumors was compared to spleen under baseline and stress conditions. In non-stressed mice, NE turnover was equivalent between tumor and spleen. In mice exposed to a stressor, tumor NE turnover was increased compared to spleen NE turnover, and compared to non-stressed tumor NE turnover. Together, these results demonstrate that NE in mammary tumors is derived from local sympathetic nerves that synthesize and metabolize NE. However, differences between spleen and tumor NE turnover with stressor exposure suggest that sympathetic NE release is regulated differently within the tumor microenvironment compared to the spleen. Local mammary tumor sympathetic innervation, despite its limited distribution, is responsive to stressor exposure and therefore can contribute to stress-induced tumor progression.

A44: Detection of HER2 mRNA splice variants in exosomes of breast cancer cells

Background HER2 is a known driver of breast cancer progression in 15–30% of primary breast carcinomas and an important target for therapy. More recently, it was established that transcript splice variants of HER2, including delta16-HER2, herstatin, p100 participate in malignant progression and drug resistance. Exosomes are small vesicles of endocytic origin, which are released by many different cell types and can be used as potential novel biomarkers, since they contain DNA and RNA derived from cancer cells. The aim of this study is to explore the use of exosomes for detecting HER2 splice variants and compare levels HER2 splice variants in cells and their exosomes. Material and methods Breast cancer cell lines MDA-MB-361, MDA-MB- 231, MCF7, MCF7-Adr and BT549 were grown as monolayer cultures as recommended by American Type Culture Collection. The exosomes were isolated by an ultracentrifugation protocol. Total RNA was isolated and cDNA was generated to be used for classic PCR and SYBR Green I quantitative ( q ) PCR. Results We determined HER2 splice variants levels in MDA-MB-361, MDA-MB-231 breast cancer cell lines and in exosomes derived from MDA-MB-36. MDA-MB-361 cells expressed high levels of full length HER2 mRNA, delta16-HER2 mRNA, whereas no expression was detected in other cell lines. Exosomes derived from MDA-MB-231 cells line were negative for Herstatin, delta16-HER2 and p100 but positive for mRNA of full length HER2. Conclusion The absence of HER2 mRNA and splice variants in MCF7, MCF7-Adr and BT549 cells lines corresponded to their absence in exosomes. Although the other cell lines remain to be examined, we speculate that exosomes will reflect the HER2 splice variants content present in the breast cancer cells.

Abstract P4-04-15: Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression

Abstract Background: Increasing evidence has demonstrated that stromal cells play a pivotal role to promote breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). Upon interaction with tumor cells, CAFs promote tumor progression by providing paracrine oncogenic signals mediated by activation of various pathways including developmental pathways, integrin signaling, and the MAPK pathway in tumor cells. CAFs have also been shown to promote the survival of CTCs and help them in metastasis at distant sites. Using breast cancer patient tumor datasets, we have previously identified a microRNA signature reflective of hyperactive MAPK signaling and that is significantly associated with reduced recurrence-free and overall survival. We have established 3 primary breast CAF lines, one from a Luminal A breast cancer, one from an ER-/Her2 amplified cancer, and one from a triple negative cancer, along with several primary tumor-derived dissociated tumor (DT) culture models that are tumorigenic in vivo and vary in metastatic ability. The CAFs express several hMAPK-microRNAs preferentially compared to the DTs. In addition to the paracrine interaction of stromal and tumor cells mediated by chemokines or hormones, miRNA cross talk between stromal and tumor cells can also occur. Results: To further investigate the connection between our miRNA signature and stroma, we analyzed the TCGA and METABRIC breast cancer datasets and found that the hMAPK-miRNA identifies tumors that with signficant stromal cell infiltrate. To investigate the role of specific expression of hMAPK-miRNAs in the CAFs, CM was isolated from CAFs from "aggressive" tumors, from the "indolent" tumor, and from normal human mammary fibroblasts (HMFs) and analyzed for exosome and microRNA secretion. CAFs from the aggressive tumors secrete more exosomes and more hMAPK-microRNAs into the CM than do HMFs or CAFs from the indolent tumor. Importantly, conditioned media (CM) from the "aggressive" CAFs activate MAPK and repress ER protein, mRNA and ER 3’UTR-reporter activity in ER+ MCF-7 breast cancer cells, while HMFs and "indolent" CAFs did not. Exosomes from the "aggressive" CAFs were responsible for the ER repression. To determine if the secreted miRNA differences exhibited by the CAFs could be seen in patients, serum from breast cancer patients with metastatic breast cancer and patients without metastases was analyzed for microRNA expression. Differentially expressed circulating hMAPK-miRNAs were identified in serum from metastatic breast cancer patients compared with patients without metastatses. Further analyses of the serum for CTCs and CAFs show that serum samples from metastatic patients had a significantly higher number of CTCs with CAFs compared to serum from patients without metastases. Conclusions: Collectively these data suggest that different CAF populations have distinct abilities to influence the phenotype and behavior of associated cancer cells and that CAF secreted hMAPK-miRNAs may play important roles in breast cancer progression. They further suggest that these CAF secreted miRNAs can be found in patient serum along with circulating CAFs. Citation Format: Sanket H Shah, Phil Miller, Zheng Ao, Emilio Issa, Katherine Drews Elger, Ram Datar, Marc Lippman, Dorraya El-Ashry. Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-15.

Cancer-associated fibroblasts: A multifaceted driver of breast cancer progression

Cancerous tissue is a complex mix of tumor cells, stromal cells and extracellular matrix (ECM), all of which make up a disordered and aggressive niche in comparison with organized and homeostatic normal tissue. It is well accepted that the tumor microenvironment plays an indispensable role in cancer development, and thus can be recognized as an additional cancer hallmark alongside those that are well established. In breast cancer, cancer associated fibroblasts (CAFs) are the predominant cellular components and play a centric role in the tumor microenvironment since they not only promote cancer initiation, growth, invasion, metastasis and therapeutic resistance but are also involved in microenvironmental events including angiogenesis/lymphangiogenesis, ECM remodeling, cancer-associated inflammation and metabolism reprogramming, all of which are known to have pre-malignancy potency. At the molecular level, there is a sophisticated network underlying the interactions between CAFs and epithelial cells as well as other stromal components. Accordingly, targeting CAFs provides a novel strategy in cancer therapy. Herein, we summarize the current understanding of the role of CAFs in breast cancer.

The DEK oncogene promotes cellular proliferation through paracrine Wnt signaling in Ron receptor-positive breast cancers.

Disease progression and recurrence are major barriers to surviving breast cancer. Understanding the etiology of recurrent or metastatic breast cancer and underlying mechanisms is critical for the development of new treatments and improved survival. Here, we report that two commonly over-expressed breast cancer oncogenes, Ron and DEK, cooperate to promote advanced disease through multi-pronged effects on β-catenin signaling. The Ron receptor is commonly activated in breast cancers, and Ron over-expression in human disease stimulates β-catenin nuclear translocation and is an independent predictor of metastatic dissemination. Dek is a chromatin-associated oncogene whose expression has been linked to cancer through multiple mechanisms, including β-catenin activity. We demonstrate here that Dek is a downstream target of Ron receptor activation in murine and human models. The absence of Dek in the MMTV-Ron mouse model led to a significant delay in tumor development, characterized by decreased cell proliferation, diminished metastasis, and fewer cells expressing cancer stem cell markers. Dek complementation of cell lines established from this model was sufficient to promote cellular growth and invasion in vitro and in vivo. Mechanistically, Dek expression stimulated the production and secretion of Wnt ligands to sustain an autocrine/paracrine canonical β-catenin signaling loop. Finally, we show that Dek over-expression promotes tumorigenic phenotypes in immortalized human mammary epithelial MCF10A cells and, in the context of Ron receptor activation, correlates with disease recurrence and metastasis in patients. Overall, our studies demonstrate that DEK over-expression, due in part to Ron receptor activation, drives breast cancer progression through the induction of Wnt/β-catenin signaling.

Expression of integrin α3β1 and cyclooxygenase-2 (COX2) are positively correlated in human breast cancer.

BackgroundExpression of integrin α3β1 is associated with tumor progression, metastasis, and poor prognosis in several cancers, including breast cancer. Moreover, preclinical studies have revealed important pro-tumorigenic and pro-metastatic functions for this integrin, including tumor growth, survival, invasion, and paracrine induction of angiogenesis. Our previously published work in a preclinical breast cancer model showed that integrin α3β1 promotes expression of cyclooxygenase-2 (COX2/PTGS2), a known driver of breast cancer progression. However, the clinical significance of this regulation was unknown. The objective of the current study was to assess the clinical relevance of the relationship between integrin α3β1 and COX2 by testing for their correlated expression among various forms of human breast cancer.MethodsImmunohistochemistry was performed to assess co-expression of α3 and COX2 in specimens of human invasive ductal carcinoma (IDC), either on a commercial tissue microarray (n = 59 samples) or obtained from Albany Medical Center archives (n = 68 samples). Immunostaining intensity for the integrin α3 subunit or COX2 was scored, and Spearman’s rank correlation coefficient analysis was performed to assess their co-expression across and within different tumor subtypes or clinicopathologic criteria.ResultsAlthough expression of integrin α3 or COX2 varied among clinical IDC samples, a statistically significant, positive correlation was detected between α3 and COX2 in both tissue microarrays (rs = 0.49, p < 0.001, n = 59) and archived samples (rs = 0.59, p < 0.0001, n = 68). In both sample sets, this correlation was independent of hormone receptor status, histological grade, or disease stage.ConclusionsCOX2 and α3 are correlated in IDC independently of hormone receptor status or other clinicopathologic features, supporting the hypothesis that integrin α3β1 is a determinant of COX2 expression in human breast cancer. These results support the clinical relevance of α3β1-dependent COX2 gene expression that we reported previously in breast cancer cells. The findings also suggest that COX2-positive breast carcinomas of various subtypes might be vulnerable to therapeutic strategies that target α3β1, and that α3 expression might serve as an independent prognostic biomarker.

Identification of mTORC2 as a Necessary Component of HRG/ErbB2-Dependent Cellular Transformation

Overexpression of the receptor tyrosine kinase HER2/ErbB2 (ERBB2) has been linked to a poor prognosis for patients with breast cancer; thus, its activity is a central target for cancer therapy. Likewise, overexpression of heregulin (HRG/NRG1), a growth factor responsible for ErbB2 activation, has also been shown to be a driver of breast cancer progression. Although ErbB2 inhibitors offer a major advancement in the treatment of ErbB2-dependent breast cancers, patients are highly susceptible to developing clinical resistance to these drugs. Therefore, a detailed understanding of the molecular mechanism that underlies HRG/ErbB2-induced tumorigenesis is essential for the development of effective therapeutic strategies for this subset of patients with breast cancer. Here, it was demonstrated that HRG promoted anchorage-independent breast cancer cell growth more potently than EGF, and that the HRG-dependent activation of phosphoinositide 3-kinase and mTORC1 are necessary events for cell transformation. Functional evaluation of two distinct mTOR (MTOR) inhibitors, rapamycin and INK-128, on HRG-dependent signaling activities, uncovered a necessary role for mTORC2 in the regulation of the AKT/TSC2/mTORC1 axis by affecting the phosphorylation of AKT at the PDK1(PDPK1)-dependent site (T308) as well as at the mTORC2-dependent site (S473). The elimination of Rictor (RICTOR), a critical component of mTORC2, is detrimental to both the activation of mTORC1 and HRG-mediated cellular transformation. Similar results were obtained in multiple breast cancer model systems, highlighting an important role for mTORC2 in HRG/ErbB2-dependent breast cancer. These findings suggest the potential benefits of targeting mTORC2 in HRG/ErbB2-induced breast cancer.

Gamma secretase inhibitors of Notch signaling

The numerous processes involved in the etiology of breast cancer such as cell survival, metabolism, proliferation, differentiation, and angiogenesis are currently being elucidated. However, underlying mechanisms that drive breast cancer progression and drug resistance are still poorly understood. As we discuss here in detail, the Notch signaling pathway is an important regulatory component of normal breast development, cell fate of normal breast stem cells, and proliferation and survival of breast cancer initiating cells. Notch exerts a wide range of critical effects through a canonical pathway where it is expressed as a type I membrane precursor heterodimer followed by at least two subsequent cleavages induced by ligand engagement to ultimately release an intracellular form to function as a transcriptional activator. Notch and its ligands are overexpressed in breast cancer, and one method of effectively blocking Notch activity is preventing its cleavage at the cell surface with γ-secretase inhibitors. In the context of Notch signaling, the application of clinically relevant anti-Notch drugs in treatment regimens may contribute to novel therapeutic interventions and promote more effective clinical response in women with breast cancer.

Can pharmacological receptor tyrosine kinase inhibitors sensitize poor outcome breast tumors to immune-based therapies?

Receptor tyrosine kinases (RTKs) drive breast cancer progression, particularly in human epidermal growth factor receptor 2 and basal tumors, the two worst prognosis subtypes. Tumor cells recruit host stromal components, including immune cells, which strongly influence disease progression. This has been studied in human breast cancer and translated to murine models of breast cancer. Stromal immune components including cytotoxic T lymphocytes (CTLs) and natural killer cells, destroy cancer cells through a process termed immune surveillance. Unfortunately, clinically detectable tumors escape these immune protective effects through their ability to limit the infiltration, activation, and/or survival of CTLs in breast tumors. The immunosuppressed state of established tumors limits the success rate of immune-based therapies, and possibly other therapeutic modalities that depend on host immunity. Published studies demonstrate that RTKs facilitate breast cancer progression, in part, by establishing immune suppression. This raises the intriguing possibility that pharmacological RTK inhibitors may be exploited to sensitize breast cancer patients to immune-based therapies.

Abstract 952: The SCF F box protein, Skp2, is a key component of an E3 ubiqutin ligase that governs estrogen receptor ≤ stability

Abstract Breast carcinoma is the most common cancer among women in developed countries, and about 70% of these tumors express estrogen receptor ≤ (ERα). ERα is a transcription factor and master regulator of estrogen stimulated proliferation and its expression indicates potential for response to estrogenic stimulation. A majority of ERα positive breast cancers initially respond well to selective estrogen receptor modulators (such as tamoxifen) or to aromatase inhibitors. Understanding the regulation of ERα levels and its role in transcription of estrogen driven genes is thus highy germane to breast cancer therapy. There is evidence that for many transcription factors, activation is linked to factor degradation. Moreover, estrogen stimulation is known to activate ERα proteolysis. Given the importance of estrogen as a driver of breast cancer progression, we have investigated mechanisms governing ERα proteolysis. S phase kinase-associated protein 2 (Skp2) is an F box component of a multi-protein SCF ubiquitin ligase that acts on multiple substrates. Levels of Skp2 have been reported to be inversely related with the levels of ERα in breast cancer. Here we report that SCFSkp2, comprised of Skp2, in association with Skp1, Cul1 and Rbx1, is an E3 ubiquitin ligase for ERα. Ectopic expression of dominant negative Cul1 increases ERα levels by impeding ERα degradation in breast cancer cells. Knockdown of Skp2 impairs estrogen-triggered ERα proteolysis, while ectopic Skp2 expression decreased ERα stability. We show that Skp2, Skp1, Rbx1 and Cul1 co-precipitate with cellular ERα and the formation of this ERα/SCFSkp2 complex is cell cycle regulated and parallels CDK2 activation. We also show ER is an in vitro substrate that is ubiquitylated and degraded by SCFSkp2. The involvement of CDK2-dependent ERα phosphorylation in estrogen activated ERα/SCFSkp2 binding, ERα proteolysis and the functional consequences of this on ERα transcriptional activity are under investigation. These data suggest that Skp2 plays an important role in the regulation of ERα stability, and potentially in the biologic action of this key steroid hormone receptor in breast cancer. 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 952. doi:1538-7445.AM2012-952

GPER and ER: Estrogen Receptors with Distinct Biological Roles in Breast Cancer

Comparative clinical studies indicate that blockade of estrogen biosynthesis by the use of aromatase inhibitors may have benefit over estrogen receptor (ER) antagonism as a strategy for treating breast cancer. One plausible explanation for this idea is that more than one type of estrogen receptor may promote the biological effects of estrogen. Recent findings that G-protein-coupled receptor-30, (GPR30/GPER) promotes specific estrogen binding and manifests plasma membrane-initiated signaling events suggests that this previously unappreciated receptor may have importance in breast cancer, a concept further supported by the observation that GPER expression is linked to disease progression in cancers that arise from breast, ovary and endometrium. In breast cancer cells, estrogen action via GPER coordinates two cellular activities that have important implications for disease progression, integrin α5β1-dependent fibrillogenesis and release of plasma membrane tethered HB-EGF. GPER has further potential significance in breast cancer in that it acts independently of nuclear steroid hormone receptors, ERα and ERβ, and ER antagonists function as GPER agonists. In support of the idea that GPER may have independent influence from ER in breast cancer, these receptors have been reported to exhibit distinct patterns of association with clinicopathological parameters of disease progression. This review article evaluates this concept and outlines a model by which GPER and ER may act independently to drive breast cancer progression. Keywords: Estrogen, G-protein-coupled estrogen receptor (GPER), seven transmembrane receptors, estrogen receptors (ERs), nuclear steroid hormone receptors, tamoxifen, faslodex, epidermal growth factor receptors (EGFRs), aromatase inhibitors, breast cancer

Abstract 2337: Defining significance of the novel tGLI1 transcription factor in cancer growth and progression

Abstract The goal of this study is to gain a greater understanding of the molecular pathways that drive breast cancer progression and metastasis, the leading cause of breast cancer mortality. In achieving this goal, our research has been concentrated on GLI1 (glioma-associated oncogene homolog 1) zinc-finger transcription factor, the terminal effector of the Hedgehog pathway that is important for many physiological processes, constitutively activated in breast cancer and associated with poor survival of breast cancer patients. While no mutations in the human GLI1 gene have been reported in any cell or tumor type, we recently discovered a novel alternatively spliced truncated variant of GLI1, tGLI1, with an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene (Can Res 17:6790-8, 2009). In the current study, we further discovered that the tGLI1 transcript and protein are highly expressed in the majority of cell lines and specimens of breast cancer we have examined to date, but is undetectable in normal cells or tissues. tGLI1 has retained the functional domains of GLI1 and a similar ability as GLI1 to undergo nuclear import and to activate GLI1-targeted genes. To gain insights into the role of tGLI1 in breast cancer progression, we used MDA-MB-231 cell line (known to be highly invasive and metastatic) to create three isogenic cell lines that stably express control vector, wild-type GLI1 and tGLI1. Functional characterization of these cell lines uncovered that tGLI1, but not wild-type GLI1, has rendered MDA-MB-231 cells significantly more invasive and migratory. This finding is also observed in other breast cancer cells we examined. Colony formation soft agar assays further indicate that tGLI1-expressing MDA-MB-231 cells had a higher propensity to undergo anchorage-independent growth than control cells and those with wild-type GLI1. To identify the molecular mechanisms that underlie tGLI1-associated phenotypes, we investigated the effects of tGLI1 (and wild-type GLI1) on several genes known to regulate cancer cell invasion, migration and growth. The results of these studies showed that tGLI1, but not GLI1, transcriptionally upregulates CD24 (an invasion/metastasis-associated gene) and downregulates PUMA (a proapoptotic gene) in breast cancer cells. In light of these exciting observations, ongoing efforts in our laboratory are to investigate the involvement of CD24 and PUMA (and other genes) in tGLI1-mediated breast cancer growth and progression. Animal studies are also in progress to examine the in vivo impact of tGLI1 on breast tumor invasion and metastasis. In summary, we report in this study that the newly discovered tGLI1 transcription factor is expressed tumor-specifically in breast cancer and associated with increased invasiveness, motility and anchorage-independent growth of breast cancer, and thus could be an important regulator of breast cancer growth and progression. 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 2337. doi:10.1158/1538-7445.AM2011-2337

P95HER2 and Breast Cancer

A subtype of HER2-positive tumors with distinct biological and clinical features expresses a series of carboxy-terminal fragments collectively known as p95HER2. One of these fragments, named 100- to 115-kDa p95HER2 or 611-CTF, is hyperactive because of its ability to form homodimers maintained by intermolecular disulfide bonds. Despite lacking the majority of the extracellular domain, this HER2 fragment drives breast cancer progression in vivo. The recent availability of specific anti-p95 antibodies has confirmed previous results indicating that the expression of p95HER2 is predictive of poor prognosis and correlates with resistance to the treatment with trastuzumab, a therapeutic antibody directed against the extracellular domain of HER2.

High Expression of Sphingosine 1-Phosphate Receptors, S1P 1 and S1P 3, Sphingosine Kinase 1, and Extracellular Signal-Regulated Kinase-1/2 Is Associated with Development of Tamoxifen Resistance in Estrogen Receptor-Positive Breast Cancer Patients

Various studies in cell lines have previously demonstrated that sphingosine kinase 1 (SK1) and extracellular signal-regulated kinase 1/2 (ERK-1/2) interact in an estrogen receptor (ER)-dependent manner to influence both breast cancer cell growth and migration. A cohort of 304 ER-positive breast cancer patients was used to investigate the prognostic significance of sphingosine 1-phosphate (S1P) receptors 1, 2, and 3 (ie, S1P1, S1P2, and S1P3), SK1, and ERK-1/2 expression levels. Expression levels of both SK1 and ERK-1/2 were already available for the cohort, and S1P1, S1P2, and S1P3 levels were established by immunohistochemical analysis. High membrane S1P1 expression was associated with shorter time to recurrence (P=0.008). High cytoplasmic S1P1 and S1P3 expression levels were also associated with shorter disease-specific survival times (P=0.036 and P=0.019, respectively). Those patients with tumors that expressed high levels of both cytoplasmic SK1 and ERK-1/2 had significantly shorter recurrence times than those that expressed low levels of cytoplasmic SK1 and cytoplasmic ERK-1/2 (P=0.00008), with a difference in recurrence time of 10.5 years. Similarly, high cytoplasmic S1P1 and cytoplasmic ERK-1/2 expression levels (P=0.004) and high cytoplasmic S1P3 expression and cytoplasmic ERK-1/2 expression levels (P=0.004) were associated with shorter recurrence times. These results support a model in which the interaction between SK1, S1P1, and/or S1P3 and ERK-1/2 might drive breast cancer progression, and these findings, therefore, warrant further investigation.

Devious signals from NFκB driving breast cancer progression

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Clinical Remarks on Repeated Haemoptyses in Non-Tuberculous Subjects, with Illustrative Cases

<h3>Abstract</h3> The mechanical properties of the extracellular matrix within tumours control multiple cellular functions that drive cancer invasion and metastasis. However, the mechanisms controlling microenvironmental force sensation and transmission, and how these regulate transcriptional reprogramming and invasion, are unclear. Our aim was to understand how mechanical inputs are transmitted bidirectionally and translated into biochemical and transcriptional outputs to drive breast cancer progression. We reveal that adhesion receptor and growth factor receptor crosstalk regulates a bidirectional feedback mechanism co-ordinating force-dependent transcriptional regulation and invasion. Integrin αVβ6 drives invasion in a range of carcinomas and is a potential therapeutic target. αVβ6 exhibits unique biophysical properties that promote force-generation and increase matrix rigidity. We employed an inter-disciplinary approach incorporating proteomics, biophysical techniques and multi-modal live-cell imaging to dissect the role of αVβ6-EGFR crosstalk on transmission of mechanical signals bidirectionally between the extracellular matrix and nucleus. We show that αVβ6 expression correlates with poor prognosis in triple-negative breast cancer (TNBC) and drives invasion of TNBC cells. Moreover, our data show that a complex regulatory mechanism exists involving crosstalk between αVβ6 integrin and EGFR that impacts matrix stiffness, force transmission to the nucleus, transcriptional reprogramming and microenvironment rigidity. αVβ6 engagement triggers EGFR &amp; MAPK signalling and αVβ6-EGFR crosstalk regulates mutual receptor trafficking mechanisms. Consequently, EGF stimulation suppresses αVβ6-mediated force-application on the matrix and nuclear shuttling of force-dependent transcriptional co-activators YAP/TAZ. Finally, we show that crosstalk between αVβ6 &amp; EGFR regulates TNBC invasion. We propose a model whereby αVβ6-EGFR crosstalk regulates matrix stiffening, but also the transmission of extracellular forces into the cell in order to co-ordinate transcriptional reprogramming and invasion. To exploit adhesion receptors and receptor tyrosine kinases therapeutically, it will be essential to understand the integration of their signalling functions and how crosstalk mechanisms influence invasion and the response of tumours to molecular therapeutics.