CITED1 as a marker of favourable outcome in anti-endocrine treated, estrogen-receptor positive, lymph-node negative breast cancer.

OBJECTIVE: To investigate CITED1 as a potential biomarker of anti-endocrine response and breast cancer recurrence, given its previously determined role in mediating estrogen-dependant transcription. The study is a continuation of earlier work establishing the role of CITED1 in mammary gland development. RESULTS: CITED1 mRNA is associated with estrogen-receptor positivity and selectively expressed in the GOBO dataset of cell lines and tumours representing the luminal-molecular subtype. In patients treated with tamoxifen, higher CITED1 correlated with better outcome, suggesting a role in anti-estrogen response. The effect was particularly evident in the subset of estrogen-receptor positive, lymph-node negative (ER+/LN-) patients although noticeable divergence of the groups was apparent only after five years. Tissue microarray (TMA) analysis further validated the association of CITED1 protein, by immunohistochemistry, with favourable outcome in ER+, tamoxifen-treated patients. Although we also found a favourable response to anti-endocrine treatment in a larger TCGA dataset, the tamoxifen-specific effect was not replicated. Finally, MCF7 cells overexpressing CITED1 showed selective amplification of AREG but not TGFα suggesting that maintenance of specific ERα-CITED1 mediated transcription is important for the long-term response to anti-endocrine therapy. These findings together confirm the proposed mechanism of action of CITED1 and support its potential use as a prognostic biomarker.

The effect of statin treatment on intratumoral cholesterol levels and LDL receptor expression: a window-of-opportunity breast cancer trial.

BACKGROUND: Deregulated lipid metabolism is common in cancer cells and the mevalonate pathway, which synthesizes cholesterol, is central in lipid metabolism. This study aimed to assess statin-induced changes of the intratumoral levels of cholesterol and the expression of the low-density lipoprotein receptor (LDLR) to enhance our understanding of the role of the mevalonate pathway in cancer cholesterol metabolism. METHODS: This study is based on a phase II clinical trial designed as a window-of-opportunity trial including 50 breast cancer patients treated with 80 mg of atorvastatin/day for 2 weeks, between the time of diagnosis and breast surgery. Lipids were extracted from frozen tumor tissue sampled pre- and post-atorvastatin treatment. Intratumoral cholesterol levels were measured using a fluorometric quantitation assay. LDLR expression was evaluated by immunohistochemistry on formalin-fixed paraffin-embedded tumor tissue. Paired blood samples pre- and post-atorvastatin were analyzed for circulating low-density lipoprotein (LDL), high-density lipoprotein (HDL), apolipoprotein A1, and apolipoprotein B. In vitro experiments on MCF-7 breast cancer cells treated with atorvastatin were performed for comparison on the cellular level. RESULTS: In the trial, 42 patients completed all study parts. From the paired tumor tissue samples, assessment of the cholesterol levels was achievable for 14 tumors, and for the LDLR expression in 24 tumors. Following atorvastatin treatment, the expression of LDLR was significantly increased (P = 0.004), while the intratumoral levels of total cholesterol remained stable. A positive association between intratumoral cholesterol levels and tumor proliferation measured by Ki-67 expression was found. In agreement with the clinical findings, results from in vitro experiments showed no significant changes of the intracellular cholesterol levels after atorvastatin treatment while increased expression of the LDLR was found, although not reaching statistical significance. CONCLUSIONS: This study shows an upregulation of LDLR and preserved intratumoral cholesterol levels in breast cancer patients treated with statins. Together with previous findings on the anti-proliferative effect of statins in breast cancer, the present data suggest a potential role for LDLR in the statin-induced regulation of breast cancer cell proliferation. TRIAL REGISTRATION: The study has been registered at ClinicalTrials.gov (i.e., ID number: NCT00816244 , NIH), December 30, 2008.

Adipocytes and Obesity-Related Conditions Jointly Promote Breast Cancer Cell Growth and Motility: Associations With CAP1 for Prognosis.

The global increase in overweight and obesity rates represent pressing public health concerns associated with severe comorbidities, amongst a rising incidence and impaired outcome of breast cancer. Yet, biological explanations for how obesity affects breast cancer are incompletely mapped. Herein, the joint impact by differentiated 3T3-L1 adipocytes and obesity-related metabolic conditions on breast cancer cells was evaluated in vitro and adipocyte-derived mediators assessed. Adipokine receptor expression was explored among breast cancer cell lines (n = 47) and primary breast tumors (n = 1,881), where associations with survival outcomes were investigated. Adipocytes and metabolic complications jointly stimulated breast cancer cell proliferation and motility, with phenotype-specific differences. Resistin was among the top modulated adipokines secreted by 3T3-L1 adipocytes under obesity-associated metabolic conditions compared with normal physiology. The newly identified resistin receptor, CAP1, was expressed across a large panel of breast cancer cell lines and primary breast tumors. CAP1 was associated with poor tumor characteristics with higher CAP1 expression among estrogen receptor (ER)-negative tumors, relative to ER-positive tumors (P = 0.025), and higher histological grades (P = 0.016). High CAP1 tumor expression was associated with shorter overall survival (adjusted hazard ratio [HRadj] 1.54; 95% confidence interval [CI], 1.11-2.13) and relapse-free survival (HRadj 1.47; 95% CI, 1.10-1.96), compared with low or intermediate CAP1 expression, particularly among ER-positive tumors or lymph node positive tumors. Together, these translational data demonstrate that the adipocyte secretome promote breast cancer cell proliferation and motility and highlight a potential role of CAP1 regarding breast cancer outcome-results that warrant further investigation to elucidate the obesity-breast cancer link in human pathology.

Insensitivity to atorvastatin is associated with increased accumulation of intracellular lipid droplets and fatty acid metabolism in breast cancer cells.

Apart from the relevant lipid-lowering effects, statins have demonstrated significant, although heterogeneous, anti-tumor activities in preventing breast cancer (BC) progression. To characterize the critical pathways behind the diverse responses to therapy, we investigated statin-induced changes in regulation of lipid metabolism and abundance of neutral lipid-containing cytoplasmic lipid droplets (LDs) in BC cells displaying different sensitivity to atorvastatin. Following atorvastatin treatment, accumulated LD levels inversely mirrored the marginal anti-proliferative effects in a dose and time-dependent manner in the less-sensitive BC cells. Transcriptional profiling excluded dysregulation of lipid uptake and efflux as specific mechanisms associated with differences in LD accumulation and anti-proliferative effects of atorvastatin. Notably, significant upregulation of genes involved in unsaturated fatty acid metabolism [stearoyl-CoA desaturase (SCD)] and cholesterol biosynthesis [3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)], were associated with atorvastatin insensitivity. Taken together, the increased ability to store neutral lipids in LDs as consequence of atorvastatin treatment likely confers a proliferative advantage to BC cells and may serve as potential biomarker of statin resistance in BC. Contributions of cholesterol biosynthesis and unsaturated fatty acid metabolism to LD formation should be thoroughly explored for better understanding of the molecular mechanisms underlying statin-induced effects against BC progression.

ImmunoPEGliposome-mediated reduction of blood and brain amyloid levels in a mouse model of Alzheimer's disease is restricted to aged animals.

The accumulation of extracellular amyloid-beta (Aß) and intracellular neurofibrillary tangles (hyper-phosphorylated Tau) in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). Active and passive immunotherapy may limit cerebral Aß deposition and/or accelerate its clearance. With the aid of a newly characterized monoclonal anti-Aß antibody we constructed immunoPEGliposomes with high avidity for capturing Aß in the periphery. The functionality of these vesicles in modulating Aß uptake by both human brain capillary endothelial hCMEC/D3 cells (suppressing uptake) and THP-1 phagocytes (stimulating uptake) was confirmed in vitro. The multivalent immunoliposomes dramatically reduced circulating and brain levels of Aß1-40, and particularly Aß1-42, in "aged" (16 month-old), but not "adult" (10 month-old) APP/PS1 transgenic mice on repeated intraperitoneal administration. Furthermore, the immunoPEGliposome-mediated reduction in amyloidosis correlated with lower levels of glial fibrillary acidic protein (GFAP) and reactive glia (GFAP-positive cells). This treatment also lowered the ratio of phosphorylated Tau to total Tau. The therapeutic efficacy of immunoliposome treatment was superior to free monoclonal antibody administration (at an equivalent antibody dose). The potential mechanisms and significance of age-dependent immunoliposome therapy in AD is discussed.

High expression of cholesterol biosynthesis genes is associated with resistance to statin treatment and inferior survival in breast cancer.

There is sufficient evidence that statins have a protective role against breast cancer proliferation and recurrence, but treatment predictive biomarkers are lacking. Breast cancer cell lines displaying diverse sensitivity to atorvastatin were subjected to global transcriptional profiling and genes significantly altered by statin treatment were identified. Atorvastatin treatment strongly inhibited proliferation in estrogen receptor (ER) negative cell lines and a commensurate response was also evident on the genome-wide transcriptional scale, with ER negative cells displaying a robust deregulation of genes involved in the regulation of cell cycle progression and apoptosis. Interestingly, atorvastatin upregulated genes involved in the cholesterol biosynthesis pathway in all cell lines, irrespective of sensitivity to statin treatment. However, the level of pathway induction; measured as the fold change in transcript levels, was inversely correlated to the effect of statin treatment on cell growth. High expression of cholesterol biosynthesis genes before treatment was associated with resistance to statin therapy in cell lines and clinical biopsies. Furthermore, high expression of cholesterol biosynthesis genes was independently prognostic for a shorter recurrence-free and overall survival, especially among ER positive tumors. Dysregulation of cholesterol biosynthesis is therefore predictive for both sensitivity to anti-cancer statin therapy and prognosis following primary breast cancer diagnosis.

Loss of CITED1, an MITF regulator, drives a phenotype switch in vitro and can predict clinical outcome in primary melanoma tumours.

CITED1 is a non-DNA binding transcriptional co-regulator whose expression can distinguish the 'proliferative' from 'invasive' signature in the phenotype-switching model of melanoma. We have found that, in addition to other 'proliferative' signature genes, CITED1 expression is repressed by TGFß while the 'invasive' signature genes are upregulated. In agreement, CITED1 positively correlates with MITF expression and can discriminate the MITF-high/pigmentation tumour molecular subtype in a large cohort (120) of melanoma cell lines. Interestingly, CITED1 overexpression significantly suppressed MITF promoter activation, mRNA and protein expression levels while MITF was transiently upregulated following siRNA mediated CITED1 silencing. Conversely, MITF siRNA silencing resulted in CITED1 downregulation indicating a reciprocal relationship. Whole genome expression analysis identified a phenotype shift induced by CITED1 silencing and driven mainly by expression of MITF and a cohort of MITF target genes that were significantly altered. Concomitantly, we found changes in the cell-cycle profile that manifest as transient G1 accumulation, increased expression of CDKN1A and a reduction in cell viability. Additionally, we could predict survival outcome by classifying primary melanoma tumours using our in vitro derived 'CITED1-silenced' gene expression signature. We hypothesize that CITED1 acts a regulator of MITF, functioning to maintain MITF levels in a range compatible with tumourigenesis.

Caracterización molecular de cáncer colorrectal hereditario en Perú / Molecular characterization of hereditary colorectal cancer in Peru

Investigar molecularmente la deficiencia en los genes de reparo de DNA (MMR) asociados al síndrome de Lynch. Materiales y métodos: Realizar los análisis de inmunohistoquímica e inestabilidad de microsatélites (MSI) en 5 familias con sospecha de síndrome de Lynch de acuerdo a los criterios clínicos de Amsterdam y/o Bethesda, atendidos en el Hospital Nacional Almanzor Aguinaga Asenjo de la ciudad de Chiclayo (Lambayeque-Perú) durante 2007-2010. Resultados. La falta de expresión de las proteínas MLH1/PMS2 y una alta MSI (MSI-H) fueron observados en un paciente de sexo masculino de 60 años diagnosticado con adenocarcinoma de grado I. Adicionalmente, se realizó el análisis mutacional puntual en el gen BRAF (V600E) a fin de descartar que se trate de un caso esporádico de cáncer colorrectal. La ausencia de mutación en el gen analizado asociado a los resultados moleculares del tumor, sugiere la caracterización de este paciente como sospecha de síndrome de Lynch. Conclusiones: Es el primer estudio molecular reportado en la población peruana y demuestra la importancia del análisis molecular en familias con sospecha de cáncer colorrectal hereditario a fin de ofrecer posibilidades de vigilancia y seguimiento que han demostrado reducir la morbilidad y la mortalidad del cáncer colorrectal así como contribuir a la caracterización a nivel genética y clínica de este tipo de cáncer en la población peruana...

To investigate the molecular deficiency in MMR genes associated to Lynch syndrome. Material and methods: Immunohistochemical and microsatellite instability (MSI) analysis were performed in 5 families with suspected Lynch syndrome according to the clinical criteria, Amsterdam and/or Bethesda that had been treated at the Hospital Nacional Almanzor Aguinaga Asenjo (Lambayeque-Peru) during 2007-2010. Results: The absence of expression of MLH1/PMS2 and high MSI (MSI-H) were observed in a male patient aged 60 with adenocarcinoma grade I. In addition, the point mutational analysis was performed in BRAF (V600E) to rule that it is a sporadic case of colorectal cancer. The absence of mutation in BRAF together with the molecular analysis suggests the suspicion as a Lynch syndrome. Conclusions: It is the first molecular study reported in the Peruvian population and demonstrates the importance of molecular analysis in families with suspected hereditary colorectal cancer in order to provide possibilities of surveillance and monitoring that have been shown to reduce morbidity and mortality of colorectal cancer. The present study contributes to the genetic and clinical characterization of the Lynch syndrome in the Peruvian population...

[Molecular characterization of hereditary colorectal cancer in Peru]. / Caracterización molecular de cáncer colorrectal hereditario en Perú.

OBJECTIVE: To investigate the molecular deficiency in MMR genes associated to Lynch syndrome. MATERIAL AND METHODS: Immunohistochemical and microsatellite instability (MSI) analysis were performed in 5 families with suspected Lynch syndrome according to the clinical criteria, Amsterdam and/or Bethesda that had been treated at the Hospital Nacional Almanzor Aguinaga Asenjo (Lambayeque-Peru) during 2007-2010. RESULTS: The absence of expression of MLH1/PMS2 and high MSI (MSI-H) were observed in a male patient aged 60 with adenocarcinoma grade I. In addition, the point mutational analysis was performed in BRAF (V600E) to rule that it is a sporadic case of colorectal cancer. The absence of mutation in BRAF together with the molecular analysis suggests the suspicion as a Lynch syndrome. CONCLUSIONS: It is the first molecular study reported in the Peruvian population and demonstrates the importance of molecular analysis in families with suspected hereditary colorectal cancer in order to provide possibilities of surveillance and monitoring that have been shown to reduce morbidity and mortality of colorectal cancer in the Peruvian population.

Dynamic investigation of interaction of biocompatible iron oxide nanoparticles with epithelial cells for biomedical applications.

Magnetic nanoparticles have emerged as important players in current research in modern medicine since they can be used in medicine for diagnosis and/or therapeutic treatment of diseases. Among many therapeutic applications of iron-based nanoparticles, drug delivery and photothermal therapy are of particular interest. At cellular level their uptake has been studied and the mechanism by which nanoparticles enter into the cell has important implication not only for their fate but also for their impact on the biological systems. We present here a dynamic investigation of interaction of biocompatible iron oxide nanoparticles coated with L-3,4-dihydroxyphenylalanine and labeled with tetra-methylrhodamine-5/6-isothiocyanate with lung epithelial cells. Our data show that after macropinocytosis-mediated internalization, nanoparticles in form of vesicles approach the nucleus and converge in the more acidic compartments of the cells in a microtubule-dependent manner. During progression the nanoparticles aggregate. Finally, we have demonstrated that a converging laser radiation on the cells, causes the increase in the local temperature and thus damages the cells, suggesting that these nanoparticles may be applied for photothermal therapy studies.

PEGylated nanoparticles bind to and alter amyloid-beta peptide conformation: toward engineering of functional nanomedicines for Alzheimer's disease.

We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aß(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs on the interaction with monomeric and soluble oligomeric forms of Aß(1-42) peptide was demonstrated by capillary electrophoresis, surface plasmon resonance, thioflavin T assay, and confocal microscopy, where the binding affected peptide aggregation kinetics. The capture of peptide by NPs in serum was also evidenced by fluorescence spectroscopy and ELISA. Moreover, in silico and modeling experiments highlighted the mode of PEG interaction with the Aß(1-42) peptide and its conformational changes at the nanoparticle surface. Finally, Aß(1-42) peptide binding to NPs affected neither complement activation in serum nor apolipoprotein-E (Apo-E) adsorption from the serum. These observations have crucial implications in NP safety and clearance kinetics from the blood. Apo-E deposition is of prime importance since it can also interact with the Aß(1-42) peptide and increase the affinity of NPs for the peptide in the blood. Collectively, our results suggest that these engineered long-circulating NPs may have the ability to capture the toxic forms of the Aß(1-42) peptide from the systemic circulation and potentially improve Alzheimer's disease condition through the proposed "sink effect".

Complement system and the brain: selected pathologies and avenues toward engineering of neurological nanomedicines.

Several nanoparticle systems and supramolecular assemblies are under investigation as potential therapeutic entities for Alzheimer's disease and other neurological disorders through both brain-specific targeting and peripheral effects. However, activation of the complement system, a complex innate immune network of over 30 circulating and membrane-bound proteins, remains a serious concern related to the use of these prospective neurological nanomedicines. The role of complement in processes of neurodegeneration in the injured or aged and diseased central nervous system is well known. Nanoparticle-mediated complement activation cannot only induce adverse cardiopulmonary distress in sensitive subjects, but may further aggravate the already-compromised condition of neurological disorders and diseases. This minireview briefly examines the role of complement in neurological diseases and outlines the current status of the development of key neurological nanomedicines with respect to complement activation. Understanding of these topics is crucial for rational design and development of safe neurological nanomedicines.

A biophysical model of intracellular distribution and perinuclear accumulation of particulate matter.

We have measured in human alveolar cells the cytoplasmic distribution of the fluorophore coumarin-6 carried by Solid Lipid Nanoparticles (SLNs) and observed a perinuclear accumulation of the fluorescence that can be described by a single exponential growth along an ideal line joining the plasma membrane to the nuclear border and by a sigmoidal relationship as a function of time. Intracellular distribution was affected by lowering the temperature from 37 to 4° C and by the disruption of cytoskeleton by cytochalasin D, but it was minimally perturbed by the inhibition of ATP dependent molecular motors. A biophysical model was developed for an accumulation of loaded particles against a diffusion gradient based on a mean field interaction energy, whose origin we ascribe to the actin structure of the cytoskeleton. The estimated value for the load diffusion coefficient was four and two orders of magnitude less than that of free coumarin-6 and of SLNs in aqueous solutions, respectively, suggesting that the load moves within the cell cytoplasm in a form still reminiscent of the nanocarrier structure.

Hypoxia-induced modifications in plasma membranes and lipid microdomains in A549 cells and primary human alveolar cells.

We evaluated the response to mild hypoxia exposure of A549 alveolar human cells and of a continuous alveolar cell line from human excised lungs (A30) exposed to 5% O(2) for 5 and 24 h. No signs of increased peroxidation and of early apoptosis were detected. After 24 h of hypoxia total cell proteins/DNA ratio decreased significantly by about 20%. Similarly, we found a decrease in membrane phospholipid and cholesterol content. The membrane fluidity assessed by fluorescence anisotropy measurements was unchanged. We also prepared the detergent resistant membrane fraction (DRM) to analyze the distribution of the two types of lipid microdomains, caveolae and lipid rafts. The DRM content of Cav-1, marker of caveolae, was decreased, while CD55, marker of lipid rafts, increased in both cell lines. Total content of these markers in the membranes was unchanged indicating remodelling of their distribution between detergent-resistant and detergent-soluble fraction of the cellular membrane. The changes in protein markers distribution did not imply changes in the corresponding mRNA, except in the case of Cav-1 for A30 line. In the latter case we found a parallel decrease in Cav-1 and in the corresponding mRNA. We conclude that an exposure to a mild degree of hypoxia triggers a significant remodelling of the lipid microdomains expression, confirming that they are highly dynamic structures providing a prompt signalling platform to changes of the pericellular microenvironment.