Wg Jiang

The role of growth differentiation factor-9 (GDF-9) and its analog, GDF-9b/BMP-15, in human breast cancer.

BackgroundThere has been a recent surge of interest in the role of growth differentiation factors and other bone morphogenic proteins in the development and spread of cancer. In this study we have provided evidence that highlights the significance of growth and differentiation factor-9a (GDF-9a) and GDF-9b (bone morphogenic protein-15, BMP-15) in breast cancer development and progression.MethodsPrimary breast cancer samples (n = 109) and matched background tissues from same patients (n = 33) were processed for frozen section and RNA extraction. Frozen sections from matched tissues were immunostained with GDF-9a and GDF-9b antibodies. Staining intensity was analyzed by computer image analysis. RNA was reverse transcribed and quantified before analysis by quantitative polymerase chain reaction (Q-PCR). Results were expressed as number of transcripts (standardized by β-actin). The data were compared with the clinical outcome of the disease. The biological effects of the molecule were studied using in vitro assays after forced expression in breast cancer cells.ResultsHighly aggressive breast cancer cells did not express GDF-9a. On forced expression of GDF-9a, breast cancer cells became less invasive. These laboratory findings were analyzed against the clinical information. Primary breast cancer samples with good predicted prognosis had a significantly higher level of GDF-9a than in samples with poor predicted prognosis (P = .004). Patients who remained disease-free at the end of a 10-year follow-up had significantly higher levels of both GDF-9a and GDF-9b in their tissue than those with poor clinical outcome (P = .001 and .014, respectively).ConclusionGrowth differentiation factor-9 family expressed in breast cancer has an inhibitory effect on the progression of human breast cancer.

The Role of Claudin- 5 and the Paracellular Barrier Function in Endothelial Cells during Invasion of Breast Cancer Cells.

Material and Methods: Human endothelial (HECV) and breast cancer cells (MDA-MB-231) were used to assess the effect of over-expression (HECV CL-5 , MDA CL-5 ) or knockdown (HECV D CL-5 , MDA D CL-5 ) of Claudin-5. Cell adhesion and growth were evaluated using in vitro assays. An in vivo tumour model using athymic nude mice was used to assess growth of tumours. Transendothelial and transepithelial resistance (TER) was employed to measure changes in TJ function. Cellular motility was analyzed using Cytodex-2 bead motility assay. ECIS (electrical cell impedance sensing) was used to assess the attachment and migration of the cells.Results: There were no significant differences in in vitro growth of cells that over-expressed or had knockdown of Claudin-5 (both HECV and MDA-MB-231 cells). Expression of CL5 in both HECV and MDA-MB-231 did not have a significant impact on in vivo growth. There were however, significant differences in adhesion between control cells (HEVC pEF6 ) and HECV CL-5 , HECV D CL-5 (changes in adhesion: HEVC pEF6 vs. HECV CL- , P>0.05; HECV D CL-5 , P=0.046). There was a distinct modulation of TJ function as assessed using TER. HECV D CL-5 cells exhibited increased resistance compared to HEVC pEF6 and HECV CL-5 (change in TER: HEVC pEF6 : -151.33±6.11 vs. HECV CL-5 -187±1.73, HECV D CL-5 -89.33±2.08, P CL-5 and MDA D CL-5 cells. HGF, a powerful motogen and modulator of TJ function, caused a significant increase in TER in HECV CL-5 and HECV D CL-5 , but resulted in a significant decrease in TER in MDA-MB-231 cells. Motility studies revealed that HECV CL-5 and MDA CL-5 after treatment with HGF cells were significantly more motile than cells without treatment (HECV CL-5 vs. HECV CL-5+HGF , P=0.015; MDA CL-5 vs. MDA CL-5+HGF , P=0.033). There was no difference in motility in HECV D CL-5 cells, however, MDA D CL-5 cells were significantly less motile after treatment with HGF (P=0.017). Interestingly, ECIS results showed that the ROCK inhibitor Y27632 inhibited the attachment and migration of HECV CL-5 cells but was no longer able to inhibit attachment and migration of HECV D CL-5 cells.Discussion: These results demonstrate for the first time that Claudin-5 can be modulated to effect changes in cell motility, attachment and TJ function in human endothelial and breast cancer cells. Moreover, there appears to be a link between Claudin-5 expression and cell motility, particularly with respect to the ROCK signalling cascade. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6170.

P1-01-08: Expression of Interleukin-15 (IL-15) and the IL-15 Receptor in Human Breast Cancer.

Introduction: Interleukin-15 (IL15) is a cytokine that influences activation and proliferation of T-lymphocytes. IL-15 is produced in the body leukocytes such as phagocytes and in many ways has similar immunoregulatory functions to IL-2 including stimulation of NK cells and CD8(+) T cells. It has been suggested that IL-15 may increase the immunity to cancer cells and cancer cell9s response to therapeutic agents. IL-15 has also been shown to be able to inhibit tumour growth in vivo. However, the expression profile of IL-15 and IL-15 receptor (IL-15) in solid tumours including human breast cancer is not clear. The present study investigated the expression profile of both IL-15 and IL-15R in human breast cancer and deduced a clinical and pathological relevance with breast cancer. Methods: Immunohistochemical methods were used to detect IL-15 and IL-15RA in mammary tissues. IL-15 and IL-15RA transcripts were analysed using real-time quantitative PCR method. Levels of IL-15 and IL-15RA were compared in normal and tumour tissues as well as against tumour staging, nodal status, disease progression and clinical outcome after a 10-year followup. Results: Both IL-15 and IL-15RA were detectable in mammary tissues and were seen in both epithelial cells and infiltrating cells. Node positive tumours had low levels of IL-15 compared with node negative tumours (21.7±10 vs 103±46, respectively). Late stage tumour also had lower levels of IL-15 (95±43, 31±15, 3.7±2 and 1.3±1.15 for stage I, II, III and IV tumours respectively. p=0.036 and p=0.032, stage-II and Stage-III vs stage-I). Patients with metastatic disease (10.3±3) and patients died of breast cancer related conditions (8.5±6) had markedly low levels of IL-15 when compared with those who were disease free (54.7±25). The disease free survival time for patients with low levels IL-15 was 126 (114-138, 95%CI) months, compared with 139 (131-148) months for those with high levels of IL-15. Despite the reduced expression of IL-15 in aggressive tumours, expression of IL-15 receptor, IL-15RA, did not display a significant change and failed to showed a link with nodal status, tumour staging and clinical outcome. Conclusions: Interleukin-15, an immuoregulatory cytokine, has an aberrant expression in human breast cancer. Low levels of IL-15, but not IL-15 receptor, is associated with the aggressiveness and disease progression of breast cancer. Together with reported effect of IL-15 on NK cells and other anti-tumour lymphocytes, IL-15 appears to be a useful therapeutic option. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-08.

nWASP, the Neural Wiskott-Aldrich Syndrome Protein, Plays a Pivotal Role in the Control of Endothelial Migration and Angiogenesis.

Introduction. nWASP, the neural Wiskott-Aldrich Syndrome Protein, is a member of the Wiskott-Aldrich syndrome (WAS) protein family which are known regulators of actin polymerization. Via interactions with actin, ERM (ezrin/moesin/radixin) family proteins, and other cytoskeletal associated proteins, nWASP is a key cell migration mediator. It has been shown that nWASP is aberrantly expressed in human breast cancer and that a reduction of nWASP is linked to a poor clinical outcome. Over-expression of nWASP in breast cancer cells also reduces their aggressiveness in vitro. In the present study, we investigated the potential influence of nWASP on the biological functions of endothelial cells and the angiogenic aspect of endothelial cells.Materials and methods. A panel of human cell lines of variety origins (n=43) were screened for the presence/absence of nWASP expression. A human vascular endothelial cell line, HECV, was found to be negative for nWASP expression and was chosen as a cell model in the present study. An expression construct for human nWASP was prepared using a mammalian expression vector, pcDNA3/V5/GFP/TOPO vector. HECV cells were transfected with the expression construct, or control vectors. The impact of nWASP over-expression in the endothelial cell line on the adhesiveness, migration and in vitro angiogenesis was assessed using a panel of in vitro models.Results. HECV cells, following transfection with the nWASP construct, successfully expressed nWASP, a subline subsequently established from which was so named as HECV nWASPexp7 . Using an electric cell impedance sensing (ECIS) method, it was shown that HECV nWASPexp7 cells showed a significantly reduced adhesiveness to matrix surface (333.4±1.4 ohm), p wt 419.7±1.6 ohm) and transfection control (HECV pControl 387.9±1.46 ohm). In a similar fashion, HECV nWASPexp7 cells also displayed a marked reduction in cell migration when compared with control cells. Using an in vitro microtubule formation assay, it was shown that both HECV wt and HECV pControl cells responded well in their microtubule formation to an angiogenic factor, hepatocyte growth factor (HGF). However, HECV nWASPexp7 cells were less responsive to HGF when assessed for microtubules. We have further shown that in response to an nWASP inhibitor, Wiskostatin, HECV nWASPexp7 cells markedly increased their tubule formation, a clear contrast to the wild type and control cells.Conclusion. It is concluded that nWASP is a key regulator of the migratory property of vascular endothelial cells and that this effect on cell migration is reflected by changes in the angiogenic activities of the cells. Collectively, nWASP is a potential intrinsic angiogenic regulator in endothelial cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2156.

The Role of ALCAM, Activated Leukocyte Cell Adhesion Molecule, in the Aggressive Nature of Breast Cancer Cells, a Potential Connection to Bone Metastasis.

Introduction. ALCAM, activated leukocyte cell adhesion molecule, has been previously reported to be connected to the progression of certain solid tumours. In breast cancer, ALCAM has been shown to be expressed at a reduced level in aggressive tumours (King et al 2004) and is a prominent feature for tumours subsequently developed bone metastasis (Davies et al 2008). In the present study, we investigate the molecular impact of ALCAM on the biological behaviours of breast cancer cells with a particular reference to condition that is linked to bone biological environment.Methods. A mammalian ALCAM expression construct was prepared from normal mammary cDNA bank using a pEF6.V5/His vector. Anti-ALCAM transgenes were prepared based on human ALCAM mRNA structure and cloned into a mammalian expression vector. Suitable cells were transfected with either the expression construct or anti-ALCAM transgene, to create sublines that had differential expression of ALCAM. The growth, migration and invasion of the aforementioned cells, together with their parent and control cell lines were evaluated using a panel of in vitro cell models, in the presence or absence of matrix proteins prepared from human bones. Statistical analysis was Student t test or Mann-Whitney test, where appropriate.Results. Of the 12 human breast cancer lines tested, ZR-75-1 was found to be strongly positive for ALCAM expression, whereas MDA-MB-231 was negative. ZR-75-1 was therefore transfected with the anti-ALCAM transgene. After selection, a subline, ZR-751ΔALCAM was created in which ALCAM expression was knocked out. Likewise, MDA-MB-231 was transfected with ALCAM expression construct, followed by creation of new subline, MDA-MB-231ALCAMexp, a sublined that over-expressed ALCAM. MDA-MB-231ALCAMexp cells showed a marginally slower rate of growth compared with control cells. However, in the presence of bone matrix proteins, MDA-MB-231ALCAMexp showed a significantly reduced rate of growth (growth index 0.62±0.11), p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6174.

The Role of Dragon (Repulsive Guidance Molecule-B, RGM-B) in Human Breast Cancer.

Introduction : Repulsive guidance molecules (RGMs) are involved in embryonic development and iron homeostasis. RGM-A mediates repulsive axonal guidance and neural tube closure, and RGM-C is mutated in juvenile hemochromatosis. RGM-B, also known as Dragon, is a myelin-derived inhibitor of axon growth in the central nervous system. The RGM family was also identified as co-receptors of bone morphogenetic proteins (BMPs), a group of proteins that are involved in development of bones, the differentiation and progression of cancer. However, the role of RGMs played in breast cancer remains unclear. In the present study, we examined the pattern of expression of the RGM family in human breast cancer cells and investigated the impact of Dragon (RGM-B) on BMP-induced cell function in breast cancer cells. Material and Methods: Conventional RT-PCR was performed to screen the expression of RGMs in human breast cancer samples and a range of breast cancer cell lines. Dragon/RGM-B ribozyme transgenes were generated and in order to knock down the Dragon transcript. Subsequently, MDA-231 RGMB-Knock-down variants were created by way of the transgenes. A series of cell function assays were employed to investigate any biological effects upon RGM-B knockdown on the breast cancer cells as well as cell9s response to recombinant BMP proteins. Results : RGM-A and RGM-C transcripts were barely detectable in breast cancer cell lines (MDA-MB-231 and MCF-7) and tissues. However RGM-B transcripts were expressed in both cell lines. Using anti-RGM-B transgenes, MDA-231 RGMB-Knock-down variant cell lines, in which RGM-B transcripts were knocked down, were created. Compared with wild type and control transfection, MDA-231 RGMB-Knock-down variants displayed a significant increase in both the adhesiveness and cells growth (p Conclusion: The present study is the first to examine the role of RGM-B in breast cancer and has demonstrated that knock-down of RGMB could enhance breast cancer cells9 ability to grow and attach, indicating that RGM-B may act as an inhibitor in breast cancer. This property is unique to RGMB, as this ability is not associated with any other member in the RGM family. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6158.

Hammerhead Ribozyme Suppresses the Expression of MLN64 and Affects the Function of Breast Cancer Cells In Vitro.

Background: The MLN64 gene, which is localized in q12-q21 of the human chromosome 17, encodes a novel transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The function of MLN64, initially reported in the metastatic lymph nodes, remains unclear, but has been indicated to probably regulate cholesterol transport. MLN64 is highly expressed in certain breast carcinomas, often co-amplified with erbB-2, and thus may contribute to the development and progression of breast tumours. The present study investigated the molecular and cellular impact of MLN64 on the functions of breast cancer cells. Methods: An anti-human MLN64 hammerhead ribozyme transgene was constructed using the mammalian expression vector pEF6/V5-His TOPO, and transfected into breast cancer cells MDA MB 231 and MCF-7 by electroporation. Expression of MLN64 mRNA and protein was determined using RT-PCR and Western blotting respectively. Cell growth, invasiveness, adhesion, motility and migration assays were used to assess, in vitro, the impact of MLN64 knockdown on the cells. In addition, expression of MLN64 was evaluated in a cohort of breast tumours, at protein and message levels. Result: Loss of MLN64 in MCF-7 cells resulted in a significant reduction of cellular growth (growth index for MCF-7 Δ MLN64 being 0.87±0.07, P WT 1.13±0.06) and transfection control (MCF-7 pEF 1.27±0.05). Interestingly, the same change in cell growth was not seen with MDA-MB-231 cells, in that MDA-MB-231 Δ MLN64 didn9t exhibit a significant effect on the cell growth comparing with corresponding WT and PEF controls. In matrix adhesion assay, MDA-MB-231 Δ MLN64 cells showed a significant increase in adhesion (86±14), compared with MDA-MB-231 WT and MDA-MB-231 pEF (61 ±20 and 45±27, respectively, P Conclusion: In this first report on the functions of MLN64 in breast cancer cell lines, we have shown that MLN64 levels are correlated with the growth and adhesion of breast cancer cells in vitro. It is suggested that MLN64 has a direct influence on the aggressiveness of breast cancer cells and may play a role in progression and metastasis of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6163.

Aberrant Expression of ARP2/3 in Breast Cancer and the Association with Disease Progression.

Background: Increased motility is an important characteristics of neoplastic cells, a cell function mediated through actin polymerization. During this process, aside from creation of new branches and lengthening of pre-existing actin molecule, Actin-Related Proteins (ARP)- 2 and ARP-3 work as a complex promotes polymerization through production of new nuclei for actin polymerization. This regulation is orchestrated by other intracellular regulators including and WAVE and WASP proteins, which have been shown to be aberrant in breast cancer (1,2). In this study we determined the differential expression of ARP-2 and ARP-3 and correlated the expression with various prognostic factors.Methods: Expression of ARP-2 and ARP-3 was examined in a cohort of mammary tissues (n=33 normal breast tissue and n=127 primary breast tumor tissue samples). Transcript levels of ARP2 and ARP3 were then determined using quantitative real time PCR (Q-PCR) and protein levels were assessed using immunohistochemical (IHC) staining. Results were analyzed by Mann-Whitney U test.Results: Cytoplasmic staining for both ARP-2 and ARP-3 was noted along with strong epithelial staining as compared to stromal cells. Quantitative real time PCR (Q-PCR) data analysis showed lower expression of both ARP -2 & -3 in tumour tissue as compared to normal but without statistical significance. ARP-2 expression was significantly reduced in tumour samples from patients with poor prognosis (p=0.037) and patients who died of breast cancer (p=0.0265). Primary breast tumor tissue samples from patients classified as TNM stage 3 and 4 showed statistically significant lower expression of ARP-3 as compared to normal tissue (p= 0.019 and 0.020, respectively). ARP-3 expression was also significantly lower in patients who developed local recurrence of breast cancer (p=0.027). Using a Spearman correlation analysis, ARP-3 transcripts were find to be significantly correlated with the WAVE-2 transcript (r=0.42, p References 1. Fernando HS et al. Expression of the WASP verprolin-homologues (WAVE members) in human breast cancer. Oncology. 2007;73:376-3832. Martin TA, et al. N-WASP is a putative tumour suppressor in breast cancer cells, in vitro and in vivo, and is associated with clinical outcome in patients with breast cancer. Clin Exp Metastasis. 2008;25:97-108 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6169.

Abstract P6-02-02: mRNA expression of death associated protein 3 (DAP3) and human breast cancer: Clinical correlations and in vitro evidence

BACKGROUND: To our knowledge, this study is the first to focus on the potential role for death-associated protein 3 (DAP3) in human breast cancer. MATERIAL AND METHODS: mRNA expression of DAP3 in breast cancer tissues (n = 127) and normal background tissues (n = 33) were determined using quantitative polymerase chain reaction (qPCR) and were correlated with clinico-pathological data accumulated over a 10-year follow-up period. Furthermore the effects of DAP3 knock down in breast cancer cell lines (MCF-7 and MDA-MB-231) were investigated. The cells were subjected to conventional growth, adhesion and invasion assays. In addition to the above, electric cell-substrate impedance sensing (ECIS) assay and annexin V/propidium iodide binding apoptosis assay were performed. For the apoptosis assay, the cells were subjected to 48 to 72 hours of serum hunger (depending on cell line) before being analyzed using a flow cytometer. RESULTS: The expression of DAP3 mRNA was demonstrated to decrease with increasing Nottingham Prognostic Index (NPI2 vs. NPI3, p = 0.036), TNM stage (TNM1 vs. 3, p = 0.07), and tumour grade (grade 1 vs. 3, p = 0.08). Lower DAP3 expression levels were significantly associated with local recurrence (p = 0.013), distant metastasis (p = 0.0057) and mortality (p = 0.019). Kaplan-Meier plot analysis suggests that patient with higher levels of DAP3 expression had better overall survival compared to patients with lower levels of DAP3 expression (p = 0.075). DAP3 knock down strains in both cell lines demonstrated increased growth and migration compared to controls during ECIS. In addition, DAP3 knock down strains of MCF-7 appeared more resistant to serum hunger when compared to controls during the apoptosis assay. CONCLUSIONS: This study demonstrates an inverse association between DAP3 mRNA levels and tumour stage and clinical stage in breast cancer. This is in keeping with the role of DAP3 as a pro-apoptotic protein. The in vitro evidence lends further credence to this hypothesis. The role of DAP3 may require further investigation to better understand the role of apoptosis in breast carcinogenesis, and may potentially serve as a prognostic marker for human breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-02-02.

Abstract P6-04-14: mRNA expressions of lamin B1 and lamin B receptor: Clinical correlations with human breast cancer

BACKGROUND: Lamin B (LMNB) and lamin B receptor (LBR) are key components of the nuclear envelope, with roles in chromosomal stability, DNA repair, and cell differentiation. Lamins provide structural support to the nuclear envelope, while LBR is believed to anchor lamin B to the envelope. In addition, LBR also has a number of downstream mediators which affect cell cycle and cell differentiation. Classically, they have been implicated in a spectrum of largely congenital ailments including certain forms of muscle dystrophy and progeria. These conditions are collectively referred to as “laminopathies”. More recently, defects in the expression of lamins have been implicated in neoplasias of the colon, prostate, liver and ovaries. In this study, we have endeavoured to elucidate the relationships between the mRNA expressions of LMNB1 and LBR and the clinicopathological parameters of human breast cancer. METHODS: Breast cancer tissues (n = 115) and associated non-cancerous tissue (ANCT) (n = 30) underwent reverse transcription and quantitative PCR. Transcript levels were correlated with clinicopathological data. RESULTS: LMNB1 mRNA expression was higher in ANCT as compared to cancerous tissue (ANCT vs. cancerous tissue: 0.12 vs. 0.00; p = <0.0001). This difference remained highly significant in all patient categories by tumour grade and clinical stage. In addition, the expression of LMNB1 declined with worsening clinical outcome. This association was statistically significant when comparing patient with disease-free survival with disease related mortalities (Disease-free vs. mortalities: 0.0011 vs. 0.000; p = 0.0177). LBR mRNA expression was found to be directly associated with tumour grade (grade 1 vs.3: 0.00 vs. 0.00; p = 0.0479) and the Nottingham Prognostic Index (NPI1 vs. 3: 0.00 vs. 0.00; p = 0.0551). CONCLUSIONS: To our knowledge, this is the first study to suggest such a role for LMNB1 and LBR in human breast cancer. The contrasting relationships of LMNB1 and LBR expressions with the clinicopathological parameters of human breast cancer may suggest that disruptions in the physiologically normal interactions between these molecules in the nuclear envelope may serve as pathway leading towards the pathogenesis of human breast cancer. Potentially this may be a significant pathway requiring further investigation to be better understood, in order to identify further potential areas for therapeutic intervention. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-14.