Mercedes Ingelmo-Torres

Caveolin-1 is essential for liver regeneration

Liver regeneration is an orchestrated cellular response that coordinates cell activation, lipid metabolism, and cell division. We found that caveolin-1 gene–disrupted mice (cav1–/– mice) exhibited impaired liver regeneration and low survival after a partial hepatectomy. Hepatocytes showed dramatically reduced lipid droplet accumulation and did not advance through the cell division cycle. Treatment of cav1–/– mice with glucose (which is a predominant energy substrate when compared to lipids) drastically increased survival and reestablished progression of the cell cycle. Thus, caveolin-1 plays a crucial role in the mechanisms that coordinate lipid metabolism with the proliferative response occurring in the liver after cellular injury.

Identification and Characterization of Associated with Lipid Droplet Protein 1: A Novel Membrane-Associated Protein That Resides on Hepatic Lipid Droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix‐assisted laser desorption ionization‐time of flight and LC‐MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation‐related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I [ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD‐associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.

Annexin A6-Induced Alterations in Cholesterol Transport and Caveolin Export from the Golgi Complex

Annexin A6 (AnxA6) belongs to a family of Ca2+‐dependent membrane‐binding proteins and is involved in the regulation of endocytic and exocytic pathways. We previously demonstrated that AnxA6 regulates receptor‐mediated endocytosis and lysosomal targeting of low‐density lipoproteins and translocates to cholesterol‐enriched late endosomes (LE). As cholesterol modulates the membrane binding and the cellular location of AnxA6, but also affects the intracellular distribution of caveolin, we investigated the localization and trafficking of caveolin in AnxA6‐expressing cells. Here, we show that cells expressing high levels of AnxA6 are characterized by an accumulation of caveolin‐1 (cav‐1) in the Golgi complex. This is associated with a sequestration of cholesterol in the LE and lower levels of cholesterol in the Golgi and the plasma membrane, both likely contributing to retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. Further strengthening these findings, knock down of AnxA6 and the ectopic expression of the Niemann–Pick C1 protein in AnxA6‐overexpressing cells restore the cellular distribution of cav‐1 and cholesterol, respectively. In summary, this study demonstrates that elevated expression levels of AnxA6 perturb the intracellular distribution of cholesterol, which indirectly inhibits the exit of caveolin from the Golgi complex.

Gene Expression Signature in Urine for Diagnosing and Assessing Aggressiveness of Bladder Urothelial Carcinoma

Purpose: To develop an accurate and noninvasive method for bladder cancer diagnosis and prediction of disease aggressiveness based on the gene expression patterns of urine samples. Experimental Design: Gene expression patterns of 341 urine samples from bladder urothelial cell carcinoma (UCC) patients and 235 controls were analyzed via TaqMan Arrays. In a first phase of the study, three consecutive gene selection steps were done to identify a gene set expression signature to detect and stratify UCC in urine. Subsequently, those genes more informative for UCC diagnosis and prediction of tumor aggressiveness were combined to obtain a classification system of bladder cancer samples. In a second phase, the obtained gene set signature was evaluated in a routine clinical scenario analyzing only voided urine samples. Results: We have identified a 12+2 gene expression signature for UCC diagnosis and prediction of tumor aggressiveness on urine samples. Overall, this gene set panel had 98% sensitivity (SN) and 99% specificity (SP) in discriminating between UCC and control samples and 79% SN and 92% SP in predicting tumor aggressiveness. The translation of the model to the clinically applicable format corroborates that the 12+2 gene set panel described maintains a high accuracy for UCC diagnosis (SN = 89% and SP = 95%) and tumor aggressiveness prediction (SN = 79% and SP = 91%) in voided urine samples. Conclusions: The 12+2 gene expression signature described in urine is able to identify patients suffering from UCC and predict tumor aggressiveness. We show that a panel of molecular markers may improve the schedule for diagnosis and follow-up in UCC patients. Clin Cancer Res; 16(9); 2624–33. ©2010 AACR.

Hydrophobic and Basic Domains Target Proteins to Lipid Droplets

In recent years, progress in the study of the lateral organization of the plasma membrane has led to the proposal that mammalian cells use two different organelles to store lipids: intracellular lipid droplets (LDs) and plasma membrane caveolae. Experimental evidence suggests that caveolin (CAV) may act as a sensitive lipid‐organizing molecule that physically connects these two lipid‐storing organelles. Here, we determine the sequences necessary for efficient sorting of CAV to LDs. We show that targeting is a process cooperatively mediated by two motifs. CAVs central hydrophobic domain (Hyd) anchors CAV to the endoplasmic reticulum (ER). Next, positively charged sequences (Pos‐Seqs) mediate sorting of CAVs into LDs. Our findings were confirmed by identifying an equivalent, non‐conserved but functionally interchangeable Pos‐Seq in ALDI, a bona fide LD‐resident protein. Using this information, we were able to retarget a cytosolic protein and convert it to an LD‐resident protein. Further studies suggest three requirements for targeting via this mechanism: the positive charge of the Pos‐Seq, physical proximity between Pos‐Seq and Hyd and a precise spatial orientation between both motifs. The study uncovers remarkable similarities with the signals that target proteins to the membrane of mitochondria and peroxisomes

Triton X-100 promotes a cholesterol-dependent condensation of the plasma membrane.

The molecular components of membrane rafts are frequently defined by their biochemical partitioning into detergent-resistant membranes. In the present study, we used a combination of epifluorescence and two-photon microscopy to visualize and quantify whether this insolubility in detergent reflects a pre-existing organization of the PM (plasma membrane). We found that the treatment of cells with cold TX (Triton X-100) promotes a profound remodelling of the PM, including a rapid rearrangement of the glycosphingolipid GM1 and cholesterol into newly formed structures, only partial solubilization of fluid domains and the formation of condensed domains that cover 51% of the remaining membrane. TX does not appear to induce the coalescence of pre-existing domains; instead, the domains that remain after TX treatment seem to be newly formed with a higher degree of condensation than those observed in native membranes. However, when cholesterol was complexed physically by treatment with a second detergent, such as saponin, cholesterol did not separate into the newly formed structures, condensation of the domains was unaltered, and the relative area corresponding to ordered domains increased to occupy 62% of the remaining membrane. Our results suggest that detergent can be used to enrich ordered domains for biochemical analysis, but that TX treatment alone substantially alters the lateral organization of the PM.

Validation Study of a Noninvasive Urine Test for Diagnosis and Prognosis Assessment of Bladder Cancer: Evidence for Improved Models

PURPOSE We validated the performance of our previously reported test for bladder cancer based on urine gene expression patterns using an independent cohort. We also ascertained whether alternative models could achieve better accuracy. MATERIALS AND METHODS Gene expression patterns of the previously reported 48 genes, including the 12 + 2 genes of the signature, were analyzed by TaqMan® arrays in an independent set of 207 urine samples. We pooled all samples analyzed to date to obtain a larger training set of 404 and used it to search for putative improved new models. RESULTS Our 12 + 2 gene expression signature had overall 80% sensitivity with 86% specificity (AUC 0.914) to discriminate between bladder cancer and control samples. It had 75% sensitivity and 75% specificity (AUC 0.83) to predict tumor aggressiveness in the validation set of urine samples. After grouping all samples 3 new signatures for diagnosis containing 2, 5 and 10 genes, respectively, and 1 containing 6 genes for prognosis were designed. Diagnostic performance of the 2, 5, 10 and 12-gene signatures was maintained or improved in the enlarged sample set (AUC 0.913, 0.941, 0.949 and 0.944, respectively). Performance to predict aggressiveness was also improved in the 14 and 6-gene signatures (AUC 0.855 and 0.906, respectively). CONCLUSIONS This validation study confirms the accuracy of the 12 + 2 gene signature as a noninvasive tool for assessing bladder cancer. We present improved models with fewer genes that must be validated in future studies.

Molecular characterization of upper urinary tract tumours.

To assess gene‐expression patterns of BIRC5, FGFR3, IGF2, KRT20, UPK2, EBF1, CDH1, FXYD3, HTERT, TP53, AGR2, HER2 and VEGF, widely known markers of bladder urothelial carcinoma (UC) in upper tract UC, and to determine their value as prognostic factors of tumour progression and cancer‐specific survival.

Gene expression test for the non-invasive diagnosis of bladder cancer: A prospective, blinded, international and multicenter validation study

OBJECTIVE This study aimed to validate, in a prospective, blinded, international and multicenter cohort, our previously reported four non-invasive tests for bladder cancer (BC) diagnosis based on the gene expression patterns of urine. METHODS Consecutive voided urine samples from BC patients and controls were prospectively collected in five European centres (n=789). Finally, 525 samples were successfully analysed. Gene expression values were quantified using TaqMan Arrays and previously reported diagnostic algorithms were applied to gene expression data. Results from the most accurate gene signature for BC diagnosis were associated with clinical parameters using analysis of variance test. RESULTS High diagnostic accuracy for the four gene signatures was found in the independent validation set (area under curve [AUC]=0.903-0.918), with the signature composed of two genes (GS_D2) having the best performance (sensitivity: 81.48%; specificity: 91.26%; AUC: 0.918). The diagnostic accuracy of GS_D2 was not affected by the number of tumours (p=0.58) but was statistically associated with tumour size (p=0.008). Also, GS_D2 diagnostic accuracy increases with increasing BC tumour risk. We found no differences in the performance of the GS_D2 test among the populations and centres in detecting tumours (p=0.7) and controls (p=0.2). CONCLUSIONS Our GS_D2 test is non-invasive, non-observer dependent and non-labour-intensive, and has demonstrated diagnostic accuracy in an independent, international and multicenter study, equal or superior to the current gold standard (cystoscopy combined with cytology). Additionally, it has higher sensitivity than cytology while maintaining its specificity. Consequently, it meets the requirements for consideration as a molecular test applicable to clinical practice in the management of BC.

Prognostic value of microRNA expression pattern in upper tract urothelial carcinoma.

To examine the microRNA (miRNA) expression pattern in tumour samples from patients with progressing and non‐progressing upper tract urothelial carcinoma (UTUC) in order to identify putative miRNAs that may be used as prognostic markers.