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<0.01).Conclusions: Breast cancer shows aberrant expression of ARP-2 and ARP-3, a pattern linked to the prognosis of the patients. ARP-3 and WAVE-2 may have an intimate interplay in this association which warrants further investigation.References1. 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.
BACKGROUND: The Na+/H+ exchange regulatory factor 1 (NHERF1) is an adapter protein that, when present in the cytoplasmic region, regulates trafficking and signaling of several G protein-coupled receptors (GPCRs). However, it has been recently suggested that, when located in the nucleus of cells, NHERF1 may have an oncogenic role in cancer. Recent work has shown that NHERF1 may be involved in the progression of certain solid tumours including breast cancer. The aim of the current study were: first to evaluate the spatial and cellular location of the NHEREF1 protein in mammary tissues, mammary epithelial and cancer cells, second to determine the expression pattern of the NHERF1 transcript in human breast cancer and deduce a possible association with clinical and pathological factors and, finally, to investigate the cellular impact of NHERF1 expression on breast cancer cells.MATERIALS AND METHODS: Expression of NHERF1 was examined in a cohort of breast tissue samples. The protein levels and distributions were assessed using immunohistochemical staining (IHC) and imaging analysis tools. The distribution of the NHERF1 protein in nucleus and cytoplasm was calculated using the protein staining intensity ratio between the two compartments. The transcript level was determined using quantitative real time-PCR. Constructed ribozyme transgenes were used to knock-down NHERF1 in MCF-7 cells, and the effect this had on in vitro cell growth was examined using in vitro methods.RESULTS: NHERF1 protein staining was seen in both normal epithelial cells and cancer cells in tissues. However, the staining pattern in cancer cells and normal epithelial cells was different. The protein was seen at a higher level in the nucleaus of cancer cells, as shown by a higher nuclear/cytoplasmic ratio of NHERF1 staining in breast cancer cells when compared with that in normal mammary epithelial cells (p=0.038). NHERF1 expression was increased in high grade tumours compared with low grade tumours (p=0.0016, grade-3 vs grade-1). Quantitative analysis of the NHERF1 transcript revealed a higher level expression in samples from patients with poor prognosis and that this was linked to the long term survival: mean survival for patients with high NHERF1 was 102 (55-148.8, 95%CI) months compared with 136 (126.6-145.9) months for patients with low NHERF1 transcripts. Using human breast cancer cell line, MCF7, we created NHERF1 knockdown subline. Loss of NHERF1 in the MCF-7 subline resulted in an increase in the growth rate, in vitro.CONCLUSION: This study shows that NHERF1 influences the growth of breast cancer cells. However, the effect of NHERF1 is dependent upon the levels of expression of NHERF1 in breast cancer tissues and most importantly determined by the cellular location of this protein in cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3155.
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-7ΔMLN64 being 0.87±0.07, P<0.01 vs wild type control (MCF-7WT 1.13±0.06) and transfection control (MCF-7pEF 1.27±0.05). Interestingly, the same change in cell growth was not seen with MDA-MB-231 cells, in that MDA-MB-231ΔMLN64 didn't exhibit a significant effect on the cell growth comparing with corresponding WT and PEF controls. In matrix adhesion assay, MDA-MB-231ΔMLN64 cells showed a significant increase in adhesion (86±14), compared with MDA-MB-231WT and MDA-MB-231pEF (61 ±20 and 45±27, respectively, P<0.01). In MCF-7 cell line, no significant differences were observed between the knockdown mutants and the wild types and pEF type. Breast cancer tissues showed a higher levels of MLN64 transcript than normal tissues (median (IQR) 1.08 (0.19-4.7) copies vs 0.5 (0.18-1.3) copies. Furthermore, ER positive tumours had significantly higher levels of MLN64 transcript (2.86(0.53-11.9) copies) than ER negative tumours (0.59 (0.15-2.68) copies), p=0.0012.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.
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