An androgen receptor mutation in the MDA-MB-453 cell line model of molecular apocrine breast cancer compromises receptor activity

Recent evidence indicates that the estrogen receptor-a-negative, androgen receptor (AR)- positive molecular apocrine subtype of breast cancer is driven by AR signaling. The MDA-MB-453 cell line is the prototypical model of this breast cancer subtype; its proliferation is stimulated by androgens such as 5a-dihydrotestosterone (DHT) but inhibited by the progestin medroxyprogesterone acetate (MPA) via AR-mediated mechanisms. We report here that the AR gene in MDAMB- 453 cells contains a G-T transversion in exon 7, resulting in a receptor variant with a glutamine to histidine substitution at amino acid 865 (Q865H) in the ligand binding domain. Compared with wild-type AR, the Q865H variant exhibited reduced sensitivity to DHT and MPA in transactivation assays in MDA-MB-453 and PC-3 cells but did not respond to non-androgenic ligands or receptor antagonists. Ligand binding, molecular modeling, mammalian two-hybrid and immunoblot assays revealed effects of the Q865H mutation on ligand dissociation, AR intramolecular interactions, and receptor stability. Microarray expression profiling demonstrated that DHT and MPA regulate distinct transcriptional programs in MDA-MB-453 cells. Gene Set Enrichment Analysis revealed that DHT- but not MPA-regulated genes were associated with estrogen-responsive transcriptomes from MCF-7 cells and the Wnt signaling pathway. These findings suggest that the divergent proliferative responses of MDA-MB-453 cells to DHT and MPA result from the different genetic programs elicited by these two ligands through the AR-Q865H variant. This work highlights the necessity to characterize additional models of molecular apocrine breast cancer to determine the precise role of AR signaling in this breast cancer subtype. Endocrine-Related Cancer (2012) 19 599–613

Quantifying alterations in cell migration : tracking fluorescently-tagged migrating cells by FACs and live-imaging

Cell migration is fundamental to many different physiological processes including embryonic development, inflammation and wound healing. Given the range and importance cell migration plays a number of assays have been developed to measure different aspects of cell migration. Here we describe two different methods to analyze cell migration. The first method analyzes the migration of fluorescently tagged cells using Boyden chambers and FACs and the second looks at migration properties using time-lapse microscopy.

A biological staging model for operable non-small cell lung cancer

Background Currently the best prognostic index for operable non-small cell lung cancer (NSCLC) is the TNM staging system. Molecular biology holds the promise of predicting outcome for the individual patient and identifying novel therapeutic targets. Angiogenesis, matrix metalloproteinases (MMP)-2 and -9, and the erb/HER type I tyrosine kinase receptors are all implicated in the pathogenesis of NSCLC. Methods A retrospective analysis of 167 patients with resected stage I-IIIa NSCLC and >60 days postoperative survival with a minimum follow up of 2 years was undertaken. Immunohistochemical analysis was performed on paraffin embedded sections for the microvessel marker CD34, MMP-2 and MMP-9, EGFR, and c-erbB-2 to evaluate the relationships between and impact on survival of these molecular markers. Results Tumour cell MMP-9 (HR 1.91 (1.23-2.97)), a high microvessel count (HR 1.97 (1.28-3.03)), and stage (stage II HR 1.44 (0.87-2.40), stage IIIa HR 2.21 (1.31-3.74)) were independent prognostic factors. Patients with a high microvessel count and tumour cell MMP-9 expression had a worse outcome than cases with only one (HR 1.68 (1.04-2.73)) or neither (HR 4.43 (2.29-8.57)) of these markers. EGFR expression correlated with tumour cell MMP-9 expression (p<0.001). Immunoreactivity for both of these factors within the same tumour was associated with a poor prognosis (HR 2.22 (1.45-3.41)). Conclusion Angiogenesis, EGFR, and MMP-9 expression provide prognostic information independent of TNM stage, allowing a more accurate outcome prediction for the individual patient. The development of novel anti-angiogenic agents, EGFR targeted therapies, and MMP inhibitors suggests that target specific adjuvant treatments may become a therapeutic option in patients with resected NSCLC.

Design, construction, and analysis of cell line arrays and tissue microarrays for gene expression analysis

Cell line array (CMA) and tissue microarray (TMA) technologies are high-throughput methods for analysing both the abundance and distribution of gene expression in a panel of cell lines or multiple tissue specimens in an efficient and cost-effective manner. The process is based on Kononen's method of extracting a cylindrical core of paraffin-embedded donor tissue and inserting it into a recipient paraffin block. Donor tissue from surgically resected paraffin-embedded tissue blocks, frozen needle biopsies or cell line pellets can all be arrayed in the recipient block. The representative area of interest is identified and circled on a haematoxylin and eosin (H&E)-stained section of the donor block. Using a predesigned map showing a precise spacing pattern, a high density array of up to 1,000 cores of cell pellets and/or donor tissue can be embedded into the recipient block using a tissue arrayer from Beecher Instruments. Depending on the depth of the cell line/tissue removed from the donor block 100-300 consecutive sections can be cut from each CMA/TMA block. Sections can be stained for in situ detection of protein, DNA or RNA targets using immunohistochemistry (IHC), fluorescent in situ hybridisation (FISH) or mRNA in situ hybridisation (RNA-ISH), respectively. This chapter provides detailed methods for CMA/TMA design, construction and analysis with in-depth notes on all technical aspects including tips to deal with common pitfalls the user may encounter. © Springer Science+Business Media, LLC 2011.

Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas

Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO 2 and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IκB kinase β, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO 2 measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO 2, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO 2 (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy. © 2007 Elsevier Inc. All rights reserved.

Multiple types of data are required to identify the mechanisms influencing the spatial expansion of melanoma cell colonies

Background The expansion of cell colonies is driven by a delicate balance of several mechanisms including cell motility, cell-to-cell adhesion and cell proliferation. New approaches that can be used to independently identify and quantify the role of each mechanism will help us understand how each mechanism contributes to the expansion process. Standard mathematical modelling approaches to describe such cell colony expansion typically neglect cell-to-cell adhesion, despite the fact that cell-to-cell adhesion is thought to play an important role. Results We use a combined experimental and mathematical modelling approach to determine the cell diffusivity, D, cell-to-cell adhesion strength, q, and cell proliferation rate, ?, in an expanding colony of MM127 melanoma cells. Using a circular barrier assay, we extract several types of experimental data and use a mathematical model to independently estimate D, q and ?. In our first set of experiments, we suppress cell proliferation and analyse three different types of data to estimate D and q. We find that standard types of data, such as the area enclosed by the leading edge of the expanding colony and more detailed cell density profiles throughout the expanding colony, does not provide sufficient information to uniquely identify D and q. We find that additional data relating to the degree of cell-to-cell clustering is required to provide independent estimates of q, and in turn D. In our second set of experiments, where proliferation is not suppressed, we use data describing temporal changes in cell density to determine the cell proliferation rate. In summary, we find that our experiments are best described using the range D = 161 - 243 ?m2 hour-1, q = 0.3 - 0.5 (low to moderate strength) and ? = 0.0305 - 0.0398 hour-1, and with these parameters we can accurately predict the temporal variations in the spatial extent and cell density profile throughout the expanding melanoma cell colony. Conclusions Our systematic approach to identify the cell diffusivity, cell-to-cell adhesion strength and cell proliferation rate highlights the importance of integrating multiple types of data to accurately quantify the factors influencing the spatial expansion of melanoma cell colonies.

Gene Silencing in Arabidopsis Spreads from the Root to the Shoot, through a Gating Barrier, by Template-Dependent, Nonvascular, Cell-to-Cell Movement

Upward long-distance mobile silencing has been shown to be phloem mediated in several different solanaceous species. We show that the Arabidopsis (Arabidopsis thaliana) seedling grafting system and a counterpart inducible system generate upwardly spreading long-distance silencing that travels not in the phloem but by template-dependent reiterated short-distance cell-to-cell spread through the cells of the central stele. Examining the movement of the silencing front revealed a largely unrecognized zone of tissue, below the apical meristem, that is resistant to the silencing signal and that may provide a gating or protective barrier against small RNA signals. Using a range of auxin and actin transport inhibitors revealed that, in this zone, alteration of vesicular transport together with cytoskeleton dynamics prevented or retarded the spread of the silencing signal. This suggests that small RNAs are transported from cell to cell via plasmodesmata rather than diffusing from their source in the phloem.

Targeting amino acid transport in metastatic castration-resistant prostate cancer : effects on cell cycle, cell growth, and tumor development

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin

Background Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified. Methods We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR. Results We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P < 0.05) in the PC3 prostate cancer cell line, however, ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines. Conclusions This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific. The expression of GOAT in prostate cancer supports the hypothesis that the ghrelin axis has autocrine/paracrine roles. We propose that the RWPE-1 prostate cell line and the PC3 prostate cancer cell line may be useful for investigating GOAT regulation and function.

Effect of cell wall properties of plant tissue on porosity and shrinkage of dried apple

In this study cell wall properties; moisture distribution, stiffness, thickness and cell dimension have been taken into consideration. Cell wall stiffness dependent on complex combination of plant cell microstructures, composition and water holding capacity of the cell. In this work, some preliminary steps taken by investing cell wall properties of apple in order to predict change of porosity and shrinkage during drying. Two different types of apple cell wall characteristic were investigated to correlate with porosity and shrinkage after convective drying. A scanning electron microscope (SEM), 2N Intron, a pyncometer and image J software were used in order to measure and analyze cell characteristics, water dynamics, porosity and shrinkage. Cell stiffness of red delicious apple was found higher than granny smith apples. A significant relationship has found between cell wall characteristics and both heat and mass transfer. Consequently, evolution of porosity and shrinkage noticeably influenced during convective drying by the nature of cell wall. This study has brought better understanding of porosity and shrinkage of dried food stuff in microscopic (cell) level and would provide better insight to attain energy effective drying process and quality food stuff.

CD151 is associated with prostate cancer cell invasion and lymphangiogenesis in vivo

CD151, a member of the tetraspanin family, is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. CD151 was found in our laboratory to have a prognostic value in prostate cancer and is a promoter of prostate cancer migration and invasion. These roles involve association with integrins on both cell-cell and cell-stroma levels. Furthermore, CD151 plays a role in endothelial cell motility. CD151 expression was examined in three commonly used prostate cancer cell lines. We investigated CD151 expression, angiogenesis (microvessel density; MVD) and lymphangiogenesis (lymphatic vessel density; LVD) in an orthotopic xenograft model of prostate cancer in matched tumours from primary and secondary sites. CD151 was found to be heterogeneously expressed across different prostate cancer cell lines and the levels of CD151 expression were significantly higher in the highly tumorigenic, androgen-insensitive cells PC-3 and DU-145 compared to the androgen-sensitive cell line LNCaP (P<0.05). The majority of in vivo xenografts developed pelvic lymph node metastases. Importantly, primary tumours that developed metastasis had significantly higher CD151 expression and MVD compared to those which did not develop metastasis (P<0.05). We identified, for the first time, that CD151 expression is associated with LVD in prostate cancer. These findings underscore the potential role of CD151 and angiogenesis in the metastatic potential of prostate cancer. CD151 has a prognostic value in this mouse model of prostate cancer and may play a role in lymphangiogenesis. CD151 is likely an important regulator of cancer cell communication with the surrounding microenvironment.

Comparing methods for modelling spreading cell fronts

Spreading cell fronts play an essential role in many physiological processes. Classically, models of this process are based on the Fisher-Kolmogorov equation; however, such continuum representations are not always suitable as they do not explicitly represent behaviour at the level of individual cells. Additionally, many models examine only the large time asymptotic behaviour, where a travelling wave front with a constant speed has been established. Many experiments, such as a scratch assay, never display this asymptotic behaviour, and in these cases the transient behaviour must be taken into account. We examine the transient and asymptotic behaviour of moving cell fronts using techniques that go beyond the continuum approximation via a volume-excluding birth-migration process on a regular one-dimensional lattice. We approximate the averaged discrete results using three methods: (i) mean-field, (ii) pair-wise, and (iii) one-hole approximations. We discuss the performace of these methods, in comparison to the averaged discrete results, for a range of parameter space, examining both the transient and asymptotic behaviours. The one-hole approximation, based on techniques from statistical physics, is not capable of predicting transient behaviour but provides excellent agreement with the asymptotic behaviour of the averaged discrete results, provided that cells are proliferating fast enough relative to their rate of migration. The mean-field and pair-wise approximations give indistinguishable asymptotic results, which agree with the averaged discrete results when cells are migrating much more rapidly than they are proliferating. The pair-wise approximation performs better in the transient region than does the mean-field, despite having the same asymptotic behaviour. Our results show that each approximation only works in specific situations, thus we must be careful to use a suitable approximation for a given system, otherwise inaccurate predictions could be made.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.