Clinical Breast Cancer Samples Research Articles

Abstract 1485: Identification of p38γ as a metastatic oncogene: Mechanism of action and clinical implications

Abstract Understanding the molecular alterations that confer metastatic properties to otherwise benign cells is essential to controlling breast cancer and significantly improving patient survival. p38γ is a member of the p38 MAPK family and is normally expressed predominantly in muscle tissue. Developmentally, the primary role of p38γ is to promote myoblast differentiation into myotubes. Viewing the function of p38γ through the perspective of its developmental role led us to hypothesize that p38γ is involved in enabling mesenchymal-like behavior in breast cancer cells by controlling their motility properties. To elucidate the role of p38γ in breast cancer progression we used RNAi to knock down (KD) p38γ in three aggressive breast cancer cell lines. We then subjected the KD cells to a panel of in vitro assays targeting the hallmarks of cancer progression. We investigated in detail the relationship between cytoskeletal defects and motility in p38γ KD cells using an innovative fusion of mathematical modeling and experimental cell biology. To evaluate the in vivo relevance of our in vitro observations, stable p38γ KD cells were orthotopically xenografted into athymic nude mice. Finally, we analyzed p38γ and RhoC expression in over 300 clinical breast cancer samples. We found that p38γ phosphorylation is elevated in all three breast cancer cell lines compared to two control cell lines. p38γ KD also significantly impairs cell motility and invasion while increasing proliferation, and also drastically alters actin cytoskeletal architecture. By developing an in silico mechanical model of cell motility we show that the modified cytoskeletal architecture of p38γ KD cells is a driving force behind the observed altered motility. Our analysis also uncovered a novel leading edge behavior used by both p38γ KD and control cells to accommodate varying cytoskeletal architectures. Biochemically, p38γ concurrently alters RhoC expression, and rescuing RhoC expression restores aggressiveness to p38γ KD cells. When xenografted, p38γ KD cells are significantly less metastatic than scrambled control cells despite forming larger primary tumors. p38γ and RhoC expression correlate across clinical breast cancer specimens and may be predictive of disease severity. We demonstrate for the first time that p38γ is a metastatic oncogene that specifically acts as a switch between primary tumor proliferation and metastatic cell motility and thus presumably between ductal carcinoma in situ and invasive breast cancer. p38γ elicits its effects, at least in part, through cytoskeletal remodeling and by modulating expression of another known metastatic oncogene, RhoC. These findings have important therapeutic implications as p38γ and RhoC expression were found to be concurrently altered in patient tissue. 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 1485. doi:10.1158/1538-7445.AM2011-1485

MicroRNA-26b is underexpressed in human breast cancer and induces cell apoptosis by targeting SLC7A11

MicroRNAs are widely dysregulated in various cancers and integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. Here, we show that miR-26b, which is down-regulated in human breast cancer specimens and cell lines, impairs viability and triggers apoptosis of human breast cancer MCF7 cells. SLC7A11 is identified as a direct target of miR-26b and its expression is remarkably increased in both breast cancer cell lines and clinical samples. Furthermore, SLC7A11 silence mimics miR-26b-aroused viability impairment and apoptosis in MCF7 cells. Our studies reveal a protective role of miR-26b in the molecular etiology of human breast cancer by promoting apoptosis.

40. Cytoplasmic localisation of β catenin is a marker of poor outcome in breast cancer patients

β catenin is involved in cell adhesion via catenin-cadherin complexes and as a transcriptional regulator in the Wnt signaling pathway. Its deregulation is important in the genesis of a number of human malignancies, particularly colorectal cancer. A range of studies have been undertaken in breast cancer but there is a lack of consensus on associations reported between β catenin expression, clinicopathological parameters and disease outcome. We undertook an immunohistochemical study measuring the levels and subcellular localisation of β catenin in 292 invasive ductal breast cancers with known treatment and outcome. Forty percent of tumours were >20mm, 45% were grade >2, 43% were lymph node positive, 68% were ER positive, 57% were PR positive and 18% were HER2 FISH positive. The median age was 54 years and patients were treated with endocrine therapy (49%), chemotherapy (38%) or both (24%). Cases were prospectively followed-up for a median of 64 months and outcome events measured were: recurrence (local or distant) (25%), metastasis (23%) and all deaths were recorded but only breast cancer-related deaths (18%) were considered for survival analyses. β catenin expression was found predominantly in the membrane and cytoplasm of cells, with only 3 cases showing detectable nuclear expression, hence we described a histoscore value for the cytoplasmic and the membrane expression independently, and then derived a third histoscore which represents both categories of expression in one continuous variable (membrane minus cytoplasmic histoscore) as a measure of subcellular localisation, the ‘membrane to cytoplasmic score' (MTC). When this continuous value is positive the tissue shows predominantly membranous expression and when the value is negative the tissue shows mainly cytoplasmic expression. No association with outcome was observed for cytoplasmic or membrane expression alone, however a shift from membrane to cytoplasm expression calculated using the MTC score was associated with worse outcome in univariate analysis (<i>p</i>=0.004), and approached significance in a multivariate analysis model that included lymph node, PR and HER2 status (<i>p</i>=0.054). The MTC score was then used for further statistical analyses due to the importance of both subcellular location and levels of expression of β catenin. An association was identified between a shift to high cytoplasmic expression (low MTC score) and high tumour grade (<i>p</i>=0.004), positive Ki67(<i>p</i>=0.0001), negative ER (<i>p</i>=0.005), positive HER2(<i>p</i>=0.01) status and an active PI3K pathway (<i>p</i>=0.005), measured as PIK3CA mutations (<i>p</i>=0.05) or PTEN loss (<i>p</i>=0.05). A shift to low cytoplasmic expression (high MTC score) was associated with the Luminal A subtype (<i>p</i>=0.004). A shift in subcellular localisation of β catenin is associated with alterations in cell cycle regulatory proteins and signalling transduction pathways, in clinical breast cancer samples. Furthermore, cytoplasmic accumulation of β catenin is associated with a poor prognosis in invasive ductal carcinoma. It is possible that in a larger cohort, independence may be established and further efforts are needed to validate these findings in other breast cancer cohorts.

Prolyl-4-hydroxylase PHD2- and hypoxia-inducible factor 2-dependent regulation of amphiregulin contributes to breast tumorigenesis

Hypoxia-elicited adaptations of tumor cells are essential for tumor growth and cancer progression. Although ample evidence exists for a positive correlation between hypoxia-inducible factors (HIFs) and tumor formation, metastasis and bad prognosis, the function of the HIF-α protein stability regulating prolyl-4-hydroxylase domain enzyme PHD2 in carcinogenesis is less well understood. In this study, we demonstrate that downregulation of PHD2 leads to increased tumor growth in a hormone-dependent mammary carcinoma mouse model. Tissue microarray analysis of PHD2 protein expression in 281 clinical samples of human breast cancer showed significantly shorter survival times of patients with low-level PHD2 tumors over a period of 10 years. An angiogenesis-related antibody array identified, amongst others, amphiregulin to be increased in the absence of PHD2 and normalized after PHD2 reconstitution. Cultivation of endothelial cells in conditioned media derived from PHD2-downregulated cells resulted in enhanced tube formation that was blocked by the addition of neutralizing anti-amphiregulin antibodies. Functionally, amphiregulin was regulated on the transcriptional level specifically by HIF-2 but not HIF-1. Our data suggest that PHD2/HIF-2/amphiregulin signaling has a critical role in the regulation of breast tumor progression and propose PHD2 as a potential tumor suppressor in breast cancer.

Array-Based DNA Methylation Profiling for Breast Cancer Subtype Discrimination

BackgroundAbnormal DNA methylation is well established for breast cancer and contributes to its progression by silencing tumor suppressor genes. DNA methylation profiling platforms might provide an alternative approach to expression microarrays for accurate breast tumor subtyping. We sought to determine whether the distinction of the inflammatory breast cancer (IBC) phenotype from the non-IBC phenotype by transcriptomics could be sustained by methylomics.Methodology/Principal FindingsWe performed methylation profiling on a cohort of IBC (N = 19) and non-IBC (N = 43) samples using the Illumina Infinium Methylation Assay. These results were correlated with gene expression profiles. Methylation values allowed separation of breast tumor samples into high and low methylation groups. This separation was significantly related to DNMT3B mRNA levels. The high methylation group was enriched for breast tumor samples from patients with distant metastasis and poor prognosis, as predicted by the 70-gene prognostic signature. Furthermore, this tumor group tended to be enriched for IBC samples (54% vs. 24%) and samples with a high genomic grade index (67% vs. 38%). A set of 16 CpG loci (14 genes) correctly classified 97% of samples into the low or high methylation group. Differentially methylated genes appeared to be mainly related to focal adhesion, cytokine-cytokine receptor interactions, Wnt signaling pathway, chemokine signaling pathways and metabolic processes. Comparison of IBC with non-IBC led to the identification of only four differentially methylated genes (TJP3, MOGAT2, NTSR2 and AGT). A significant correlation between methylation values and gene expression was shown for 4,981 of 6,605 (75%) genes.Conclusions/SignificanceA subset of clinical samples of breast cancer was characterized by high methylation levels, which coincided with increased DNMT3B expression. Furthermore, an association was observed with molecular signatures indicative of poor patient prognosis. The results of the current study also suggest that aberrant DNA methylation is not the main force driving the molecular biology of IBC.

Critical roles of LGN/GPSM2 phosphorylation by PBK/TOPK in cell division of breast cancer cells

To investigate the molecular mechanism of mammary carcinogenesis and identify novel molecular targets for breast cancer therapy, we analyzed genome-wide gene expression profiles of 81 clinical breast cancer samples. Here, we report the critical role of LGN/GPSM2 (Leu-Gly-Asn repeat-enriched protein/G-protein signaling modulator 2) in the growth of breast cancer cells. Semiquantitative RT-PCR and Northern blot analyses confirmed upregulation of LGN/GPSM2 in a large proportion of breast cancers. Immunocytochemical staining identified LGN/GPSM2 at the spindle in cells at metaphase, and at midzone and midbody in cytokinetic cells. Western blot analysis indicated the highest expression and the phosphorylated form of LGN/GPSM2 protein in G2/M phase. Treatment with small-interfering RNAs (siRNAs) targeting LGN/GPSM2 caused incompletion of cell division and resulted in significant growth suppression of breast cancer cells. We found that the 450th threonine (Thr450) of LGN/GPSM2 was phosphorylated by the serine/threonine kinase PBK/TOPK during mitosis. Overexpression of LGN/GPSM2-T450A in which Thr450 was substituted with alanine induced growth suppression and aberrant chromosomal segregation. These findings imply an important role of LGN/GPSM2 in cell division of breast cancer cells and suggest that the PBK/TOPK-LGN/GPSM2 pathway might be a promising molecular target for treatment of breast cancer.

An estrogen receptor α suppressor, microRNA‐22, is downregulated in estrogen receptor α‐positive human breast cancer cell lines and clinical samples

Previous studies have suggested that microRNAs (miRNAs) may play important roles in tumorigenesis, but little is known about the functions of most miRNAs in cancer development. In the present study, we set up a cell-based screen using a luciferase reporter plasmid carrying the whole approximately 4.7 kb 3'-UTR of estrogen receptor alpha (ERalpha) mRNA cotransfected with a synthetic miRNA expression library to identify potential ERalpha-targeting miRNAs. Among all the miRNAs, miR-22 was found to repress robustly the luciferase signal in both HEK-293T and ERalpha-positive MCF-7 cells. Mutation of the target site was found to abrogate this repression effect of miR-22, whereas antagonism of endogenous miR-22 in MDA-MB-231 cells resulted in elevated reporter signals. We assessed the miR-22 expression patterns in five breast cancer cell lines and 23 clinical biopsies and revealed that there is a significant inverse association between the miR-22 levels and ERalpha protein expression. To evaluate the potential of miR-22 as a potential therapeutic intervention, we found that reduction of endogenous ERalpha protein levels and suppression of cancer cell growth could be achieved in MCF-7 cells by miR-22 overexpression in a way that can be recapitulated by the introduction of specific small interfering RNA against ERalpha. The phenomena can be rescued by the reintroduction of ERalpha. Taken together, our data indicate that miR-22 was frequently downregulated in ERalpha-positive human breast cancer cell lines and clinical samples. Direct involvement in the regulation of ERalpha may be one of the mechanisms through which miR-22 could play a pivotal role in the pathogenesis of breast cancer.

The transcription factor Fra-2 promotes mammary tumour progression by changing the adhesive properties of breast cancer cells

The transcription factor Fra-2 (Fos-related antigen-2) has been implicated in invasion of breast cancer cells, but there is only sparse information about its role in clinical tumours. In the present study, we analysed Fra-2 mRNA expression in a cohort of 167 patients, and found significant correlations between high Fra-2 expression and nodal involvement or reduced disease-free survival. To get more information about the underlying mechanisms, we generated stably transfected MDA-MB231 breast cancer cells with increased Fra-2 expression. Compared with the controls, these clones did not differ in proliferation and motility, but had higher invasive potential. By global gene expression analysis and subsequent validation of selected genes, we identified a number of proteins involved in cell–cell or cell–matrix interactions that were up- or down-regulated in Fra-2 overexpressing cells, e.g. connexin 43, ICAM-1, L1-CAM, integrin beta 2, integrin beta 4, and integrin alpha 6. The association of Fra-2 overexpression and high ICAM-1 or L1-CAM levels could also be demonstrated in our clinical cohort of mammary tumours. In both MDA-MB231 and MCF7 cells, we found an increased attachment of Fra-2 transfectants to components of the extracellular matrix. In addition, we could show a striking increase in the number of rolling cells in flow-through assays using E-selectin coated capillaries, which might indicate a higher capacity of extravasation. In conclusion, our data obtained on breast cancer cell lines and clinical tissue samples suggest that overexpression of Fra-2 promotes breast cancer progression and metastasis by deregulation of genes involved in cell–cell and cell–ECM contacts.

Protéomique et cancer du sein : à la recherche de nouveaux biomarqueurs diagnostiques et théragnostiques

A major challenge of breast cancer research is the identification of accurate biomarkers that improve screening, early diagnosis, prediction of aggressiveness and prediction of therapeutic response or toxicity as well as the identification of novel molecular therapeutic targets. Following the sequencing of the human genome and pioneer developments of transcriptomics, analysis of the proteome (the repertoire of proteins expressed in a biological samples) was recently introduced as a promising tool in the study of biology of human cancers. In spite of their current limitations, proteomics, either array-based (such as tissue microarrays, antibody arrays or reverse phase protein microarrays) or mass spectrometry-based, make possible to envision major improvements in the molecular knowledge of breast cancer and for the identification of tissue-based or circulating diagnostic and theragnostic biomarkers relevant to this disease. The current techniques applied to clinical samples of breast cancer and the most important results obtained are summarized in this review.

Abnormalities of 53BP1 in Basal-Like Breast Cancer.

Abstract Background: Abnormalities in the DNA repair mechanism have been proposed to play a central role in the development of breast cancers. However the specific mechanism underlying DNA repair defects in sporadic breast cancer are mostly unknown. As 53BP1 plays a critical role in DNA repair response and has been identified as a potential tumor suppressor, its expression in basal-like breast cancer cell lines and clinical samples was investigated.Experimental Design: mRNA expression of 53BP1 was investigated in publicly available gene expression data sets. Protein expression of 53BP1 was evaluated in a large set of clinically annotated breast cancer specimens by immunohistochemistry. Analysis of 53BP1 and related proteins was performed in a set of human and mouse breast cancer cell lines.Results: Gene expression analysis demonstrated that a subset of basal-like breast cancer had decreased expression of 53BP1. Analysis of over 400 breast cancers in a tissue microarray showed that loss of 53BP1 staining was almost exclusively found in tumors that lacked ER, PR and HER2 expression; this "triple-negative" phenotype is characteristic of BLC. Approximately 42% of "triple-negative" breast cancers have abnormally low levels of 53BP1, while &amp;lt;2% of tumor that express either ER or HER2 lack 53BP1 expression. Tumors that have low 53BP1 expression are associated with worse distant metastasis free survival when compared to tumors with normal 53BP1 expression. A set of breast cancer cell lines with abnormal 53BP1 expression was also identified. 53BP1 abnormalities were present in some tumors and cell lines with known BRCA1 mutations.Discussion: These data suggest that a subset of basal-like breast cancers lack normal 53BP1 expression and imply that these tumors may have profound defects in DNA repair/check point function that could be exploited therapeutically. As 53BP1 abnormalities were found in some tumors with known BRCA1 mutations, this suggests that loss of 53BP1 may cooperate with abnormalities in the BRCA1-related DNA repair pathways in the pathogenesis of basal-like breast cancers. Data regarding the molecular mechanisms underlying 53BP1 abnormalities in breast cancer cell lines and samples will be presented. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1122.

MTA2 Enhances Breast Cancer Metastasis by Regulating the Rho Signaling Pathway.

Abstract Background: Metastasis is the ultimate cause of death for patients succumbing to breast cancer. The metastasis tumor associated (MTA) gene family has been linked with metastasis in breast cancer. Previously, we found that MTA2 overexpression enhanced the metastatic ability of the estrogen receptor-alpha (ERα)-negative cell line MDA-MB-231. This was accompanied by an increase in cell rounding, indicating a change in cytoskeletal organization. Here we explored the molecular pathways contributing to the MTA2-enhanced metastatic phenotype.Materials and Methods: MDA-MB-231 cells were engineered to overexpress MTA2. Changes in protein expression were determined by immunoblot assay. Differential gene expression was determined using Affymetrix microarrays and analyzed using dChip, BRB Array Tools, and R. Pathway analysis was carried out using the Database for Visualization, Annotation, and Integrated Discovery (DAVID). Validation was done by comparing to publicly available datasets. MTA2-overexpressing or control cells were transfected with a Rho GTP dissociation inhibitor alpha (Rho GDIα) or vector control expression constructs. Anchorage-independent growth was measured using soft-agar assays. Analysis of the impact of MTA2 and Rho GDIα combined expression was carried out using publically available datasets.Results: Analysis of enriched ontologies from significantly different genes between MTA2-overexpressing cells and clinical breast cancer samples of the basal subtype previously published by Richardson et al (1) indicated that the focal adhesion pathway was enhanced in MTA2-overexpressing samples. We found that a negative regulator of the focal adhesion pathway, Rho GDIα, was decreased in MTA2-overexpressing cells at the protein level. When a yellow fluorescent protein-tagged Rho GDIα expression vector was re-expressed in MTA2-overexpressing cells, there was a concomitant decrease in the expression of MTA2 and increased expression of endogenous Rho GDIα. When tested in soft agar assays, re-expression of Rho GDIα significantly reduced the colony-forming ability of MTA2-overexpressing cells. We examined the publically available dataset published by Wang et al. (2), and found that ERα-negative patients with high MTA2 expression and low Rho GDIa levels had earlier recurrence compared with all other patients (P=0.02).Conclusion: MTA2 overexpression was associated with an aggressive phenotype in MDA-MB-231 cells. These data indicate that MTA2 overexpression may be associated with increased signaling through the Rho pathway. We found that MTA2 and Rho GDIα form a regulatory loop which reinforces either MTA2 high or MTA2 low expression levels. Furthermore, we found that combined high MTA2 and low Rho GDIα expression had a negative impact on patient outcomes in the ERα-negative population. We are currently exploring the use of Rho pathway inhibitors for their potential to block this phenotype, and to explore the MTA2/Rho GDIα regulatory loop.1) Richardson AL, Wang ZC, De Nicolo A, Lu X et al. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 2006 Feb;9(2):121-32.2) Wang Y, Klijn JG, Zhang Y, Sieuwerts AM et al. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 2005 Feb 19-25;365(9460):671-9. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6143.

Oncogenic function of microtubule end‐binding protein 1 in breast cancer

Microtubule end-binding protein 1 (EB1) is an evolutionarily conserved protein that regulates microtubule dynamics and participates in diverse cell activities. Here, we demonstrate that EB1 expression is up-regulated in human breast cancer specimens and cell lines. The level of EB1 correlates with clinicopathological parameters indicating the malignancy of breast cancer, including higher histological grade, higher pathological tumour node metastasis (pTNM) stage, and higher incidence of lymph node metastasis. Knockdown of EB1 expression remarkably inhibits cancer cell proliferation, and conversely, elevation of its expression promotes cell proliferation. Our data further show that EB1 promotes colony formation and enhances tumour growth in nude mice. In addition, EB1 stimulates Aurora-B activity in breast cancer cells, and EB1 expression correlates with increased Aurora-B activity in clinical samples of breast cancer. These findings thus suggest an oncogenic role for EB1 in breast cancer.

Mammary-Derived Growth Inhibitor Alters Traffic of EGFR and Induces a Novel Form of Cetuximab Resistance

Only few predictive factors for the clinical activity of anti-epidermal growth factor receptor (EGFR) therapy are available. Mammary-derived growth inhibitor (MDGI) is a small cytosolic protein suggested to play a role in the differentiation of epithelial cells. Here, we have investigated the effect of MDGI expression on the EGFR signaling and cetuximab responsiveness of cancer cells. MDGI mRNA expression was investigated in clinical breast and lung cancer samples and in nontransformed and malignant cell lines. The effect of ectopic expression of MDGI on EGFR, ErbB2, and integrin function and traffic was investigated in breast and lung cancer cell lines using multiple methods. The effect of anti-EGFR agents on these cells were tested by cell proliferation measurements and by assessing tumor growth of breast cancer cells in cetuximab treated and control athymic nude mice. Here, we show that although MDGI is absent in cultured cell lines because of epigenetic silencing, MDGI mRNA is expressed in 40% of clinical breast carcinomas and 85% of lung cancers. Ectopic expression of MDGI rendered breast and lung cancer cells resistant to the anti-EGFR antibody cetuximab in vitro and in an orthotopic breast cancer xenograft model in vivo. When expressed in cancer cells, MDGI induces intracellular accumulation of EGFR, but not ErbB2, and the internalized receptor is phosphorylated and not degraded. MDGI-driven inherent desensitization of cancer cells is a novel molecular mechanism for resistance to the anti-EGFR antibody therapy, and MDGI may be a biomarker for responsiveness to anti-EGFR antibody therapy.

Conjugated and non-conjugated androgens differentially modulate specific early gene transcription in breast cancer in a cell-specific manner

The role of androgen in breast cancer development is not fully understood, although androgen receptors (ARs) have been identified in breast cancer clinical samples and cell lines. However the whole spectrum of androgen actions cannot be accounted to the classic AR activation and the possible existence of a cell surface-AR has been suggested. Indeed, androgen, like all steroids, has been reported to trigger membrane-initiated signaling activity and exert specific actions, including ion channels and kinase signaling pathway activation, ultimately affecting gene expression. However, the molecular nature of membrane androgen sites represents another major persisting question. In the present study, we investigated early transcriptional effects of testosterone and the impermeable testosterone–BSA conjugate, in two breast cancer cell lines (T47D and MDA-MB-231), in an attempt to decipher specific genes modified in each case, providing evidences about specific membrane-initiating actions. Our data indicate that the two agents affect the expression of several genes. A group of genes were commonly affected while others were uniquely modified by each agent, including interaction with growth factors and K +-channels. In MDA-MB-231 cells, that are AR negative, the majority of genes affected by testosterone were also affected by testosterone–BSA indicating a membrane-initiated action. Subsequent analysis revealed that the two agents trigger different molecular pathways and cellular/molecular functions, suggestive of a molecular or functional heterogeneity of membrane and intracellular AR. In addition, the reported phenotypic interactions of membrane-acting androgen with growth factor were verified at the transcriptomic level, as well as their ion channel-modifying effects. Finally an interesting interplay between membrane-acting androgen with inflammation-related molecules, with potential clinical implications was revealed.

Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer

IntroductionProgrammed cell death through apoptosis plays an essential role in the hormone-regulated physiological turnover of mammary tissue. Failure of this active gene-dependent process is central both to the development of breast cancer and to the appearance of the therapy-resistant cancer cells that produce clinical relapse. Functional expression cloning in two independent laboratories has identified Finkel–Biskis–Reilly murine sarcoma virus-associated ubiquitously expressed gene (Fau) as a novel apoptosis regulator and candidate tumour suppressor. Fau modifies apoptosis-controller Bcl-G, which is also a key target for candidate oncoprotein maternal embryonic leucine zipper kinase (MELK).MethodsWe have used RNA interference to downregulate Fau and Bcl-G expression, both simultaneously and independently, in breast cancer cells in vitro to determine the importance of their roles in apoptosis. Expression of Fau, Bcl-G and MELK was measured by quantitative RT-PCR in breast cancer tissue and in matched breast epithelial tissue from the same patients. Expression data of these genes obtained using microarrays from a separate group of patients were related to patient survival in Kaplan–Meier analyses.ResultssiRNA-mediated downregulation of either Fau or Bcl-G expression inhibited apoptosis, and the inhibition produced by combining the two siRNAs was consistent with control of Bcl-G by Fau. Fau expression is significantly reduced in breast cancer tissue and this reduction is associated with poor patient survival, as predicted for a candidate breast cancer tumour suppressor. In addition, MELK expression is increased in breast cancer tissue and this increase is also associated with poor patient survival, as predicted for a candidate oncogene. Bcl-G expression is reduced in breast cancer tissue but decreased Bcl-G expression showed no correlation with survival, indicating that the most important factors controlling Bcl-G activity are post-translational modification (by Fau and MELK) rather than the rate of transcription of Bcl-G itself.ConclusionsThe combination of in vitro functional studies with the analysis of gene expression in clinical breast cancer samples indicates that three functionally interconnected genes, Fau, Bcl-G and MELK, are crucially important in breast cancer and identifies them as attractive targets for improvements in breast cancer risk prediction, prognosis and therapy.

Effects of Raf Kinase Inhibitor Protein Expression on Metastasis and Progression of Human Breast Cancer

Raf kinase inhibitor protein (RKIP) has been shown to be a metastasis suppressor in many kinds of malignant tumors. But its function in breast cancer was not yet clarified completely. We detected RKIP expression in clinical samples of primary breast cancer, breast cancer metastases, and in different breast cancer cells. Compared with the normal breast epithelia, benign breast epithelia, or in situ ductal carcinoma, the expression level of RKIP is decreased in invasive carcinoma and significantly reduced or lost in the metastasis lymph node matched to the invasive carcinoma. To explore the potential role of RKIP in breast cancer metastasis, we studied the effect of RKIP on the malignant phenotypes of the breast cancer cells with ectopically overexpression or knockdown of RKIP. Cell proliferation, soft-agar colony formation, in vitro adhesion assay, invasion, and migation assays were done to examine the malignant phenotypes of the transfected cells. Consequently, RKIP has no effect on in vitro proliferation rate or colony-forming ability of MDA-MB-435 cells. In vitro cell invasion and migration assays indicated that the RKIP expression was inversely associated with the invasiveness of MDA-MB-435 cells. Consistent with these results, in the orthotopic murine models, we observed that overexpression of RKIP in breast cancer cells impaired invasiveness and metastasis, whereas down-regulation of RKIP expression promoted invasiveness and metastasis. These results indicate that RKIP is a metastasis suppressor gene of human breast cancer.

Translational disequilibrium as an interference marker to study miRNA and methylation silencing of APRIN, a stem cell regulator in breast cancer microchimerism

11109 Background: Gene silencing is universally observed in cancer and involves promoter DNA methylation. We found that a cohesin-related stem cell regulator, APRIN (Pds5B) was silenced in breast cancer clinical samples. Surprisingly, in 40% of these samples DNA methylation was not involved. Furthermore, in some breast cancer cell lines the APRIN protein was silenced without transcript downregulation or promoter methylation. This “translational disequilibrium” has been frequently reported with other proteins, but without mechanistic explanations. Recent results with RNA interference indicate that gene repression through microRNAs (typically mismatched) is mostly translational without transcript degradation. We propose, therefore, that the puzzling translational disequilibrium phenomenon is a new form of epigenetic silencing by miRNA mechanisms. We aim (i) to verify miRNA epigenetics of APRIN silencing in breast cancer cell lines; (ii) to study clinical breast cancer samples for methylation vs. miRNAs mechanisms in APRIN translational disequilibrium; and (iii) to investigate if miRNA silencing of APRIN affects a fetal embryonic stem cell pool in breast cancer (microchimerism). Methods: (i) We used miRNA mimics and miRNA inhibitors in breast cancer cell lines to verify specific miRNA involvement in APRIN silencing. (ii) We used immunohistochemistry with bisulfite converted DNA for methylation and microdissected RNA for microRNA interference studies from 56 clinical breast cancer samples. (iii) We used Y-chromosome markers on microdissected DNA for fetal microchimerism studies. Results: (i) We found that in breast cancer cell lines with APRIN translational disequilibrium a set of microRNAs correlate with APRIN silencing. (ii) We found miRNA related mechanisms in about 35 percent of breast cancer samples where APRIN was silenced and (iii) APRIN may specifically affect stem cells of fetal origin in the mother's mammary gland and contribute to cancer. Conclusions: The novel miRNA-based mechanism maybe a new epigenetic factor of gene silencing in cancer. We experimentally confirmed a set of APRIN specific miRNAs and established preliminary correlations with fetal microchimerism in breast cancer. No significant financial relationships to disclose.

Correlations of estrogen receptor (ER) related genomic transcription and ER gene expression with increasing AJCC stage of ER-positive breast cancer

1044 Background: Advanced stages of ER-positive breast cancer may have decreased tumor dependence on estrogen, possibly due to biological selection and/or progression. The sensitivity to endocrine therapy (SET) index measures transcriptional activity of the ER genomic pathway (165 ER-related genes). We investigated whether expression of genes for receptors ER (ESR1), progesterone receptor (PGR), or HER-2 (ERBB2), the SET index, or a housekeeper gene (GAPDH) vary by stage of ER-positive breast cancer. Methods: We evaluated gene expression profiles (Affymetrix U133 microarrays, Affymetrix, Santa Clara, CA) from 956 patients’ clinical samples of ER-positive breast cancer, including 290 new samples profiled at MDACC and 666 samples from published datasets. Microarray data were uniformly normalized, log-transformed, and expression levels for single genes and the SET index were compared to pathologic AJCC stage (315 patients were stage I, 362 stage IIA, 151 stage IIB, 29 stage III, 27 stage IV at initial presentation, and 72 stage IV previously treated and/or at relapse) using a median ordered regression analysis (p &lt; 0.05 was significant). Results: SET index significantly decreased with advancing pathologic stage (p &lt; 0.001), and PGR expression levels showed a similar, but lesser, effect (p = 0.014). However, expression levels of ESR1 and ERBB2 did not vary by stage. Overall, GAPDH gene expression increased with stage (p &lt; 0.001), but that effect was only observed in stages III and IV. Expression of these genes was not significantly different between stage IV disease at initial presentation or at relapse. Conclusions: Expression levels of ESR1 and ERBB2 receptor genes did not vary, but ER-related genomic transcription (SET index and, to a lesser extent, PGR) declined significantly with increasing pathologic stage. This suggests that ER-positive breast cancer tends to have less transcriptional dependence on estrogen with increasing pathologic stage. The observed increase in expression of GAPDH in stages III and IV might reflect higher metabolic activity in advanced ER-positive breast cancer and deserves further study. [Table: see text]

Quantum dots-based immunofluorescence technology for the quantitative determination of HER2 expression in breast cancer

HER2 detection is important for breast cancer (BC) treatment and prognosis, but the detection methods currently used have some disadvantages. Quantum dots (QDs)-based probes provide a potentially important new method for HER2 detection in clinical practice. This potential is examined in this paper. A QDs HER2 probe kit and QDs image acquisition and analysis software were developed and applied to 94 clinical samples of BC. Compared to conventional immunohistochemistry techniques, this method provided a superior accurate and sensitive method for the detection of HER2 in clinical breast cancer diagnosis.

Prognostic and predictive value of enzymes of the sphingolipid metabolism in breast cancer.

Abstract Abstract #6014 Backround:&amp;#x2028; Sphingolipids do not only have structural roles for the cell membrane, but also act as effector molecules and second messengers in signal transduction with links to cancer initiation, progression and treatment response. Knowledge of sphingolipid's impact on breast cancer and its prognosis is limited to in vitro data. Clinical data is rare but needed.&amp;#x2028; Material and Methods: We analyzed gene expression of 43 proteins from the sphingolipid pathways in different subtypes of breast cancer using microarray data of 1269 tumor samples (test set n=171; validation sets n=1098). Kaplan Meier analysis of disease free survival was performed to examine the prognostic value of different markers. In addition immunohistochemistry was done to determine whether the genes of the sphingolipid rheostat are expressed in cancer or stroma cells of the tumor.&amp;#x2028; Results: Significant relationships of several genes of the sphingolipid metabolism with clinical parameters were obtained. Sphingosine kinase 1 (SPHK1), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA1) displayed higher expression among ER negative tumors. In contrast, glucosylceramidesynthase (GCS), dihydroceramidesynthases (LASS4, LASS 6) and acid ceramidase (ASAH1) were higher expressed in ER positive samples. Survival analysis revealed a significant worse outcome of patients with high SPHK1 expression. LASS 6 and GCS gene expression had no prognostic impact when the estrogen receptor status was taken into account in multivariate analyses. Immunohistochemistry identified the carcinoma cells as the major source of SPHK1 expression in the tumor.&amp;#x2028; Conclusions: These studies of a large cohort of clinical breast cancer samples reveal that sphingolipids do play a role in breast cancer. Enzymes of the sphingolipid metabolism have impact on the prognosis of breast cancer patients and targeting the sphingolipid rheostat can open new treatment options. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6014.