The human rs1050286 polymorphism alters LOX-1 expression through modifying miR-24 binding.

The up-regulation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, plays a fundamental role in the pathogenesis of atherosclerosis. Moreover, OLR1 polymorphisms were associated with increased susceptibility to acute myocardial infarction (AMI) and coronary artery diseases (CAD). In these pathologies, the identification of therapeutic approaches that can inhibit or reduce LOX-1 overexpression is crucial. Predictive analysis showed a putative hsa-miR-24 binding site in the 3'UTR of OLR1, 'naturally' mutated by the presence of the rs1050286 single nucleotide polymorphism (SNP). Luciferase assays revealed that miR-24 targets OLR1 3'UTR-G, but not 3'UTR-A (P < 0.0005). The functional relevance of miR-24 in regulating the expression of OLR1 was established by overexpressing miR-24 in human cell lines heterozygous (A/G, HeLa) and homozygous (A/A, HepG2) for rs1050286 SNP. Accordingly, HeLa (A/G), but not HepG2 (A/A), showed a significant down-regulation of OLR1 both at RNA and protein level. Our results indicate that rs1050286 SNP significantly affects miR-24 binding affinity to the 3'UTR of OLR1, causing a more efficient post-transcriptional gene repression in the presence of the G allele. On this basis, we considered that OLR1 rs1050286 SNP may contribute to modify OLR1 susceptibility to AMI and CAD, so ORL1 SNPs screening could help to stratify patients risk.

Urine LOX-1 and Volatilome as Promising Tools towards the Early Detection of Renal Cancer.

Renal cell carcinoma (RCC) represents around 3% of all cancers, within which clear cell RCC (ccRCC) are the most common type (70-75%). The RCC disease regularly progresses asymptomatically and upon presentation is recurrently metastatic, therefore, an early method of detection is necessary. The identification of one or more specific biomarkers measurable in biofluids (i.e., urine) by combined approaches could surely be appropriate for this kind of cancer, especially due to easy obtainability by noninvasive method. OLR1 is a metabolic gene that encodes for the Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), implicated in inflammation, atherosclerosis, ROS, and metabolic disorder-associated carcinogenesis. Specifically, LOX-1 is clearly involved in tumor insurgence and progression of different human cancers. This work reports for the first time the presence of LOX-1 protein in ccRCC urine and its peculiar distribution in tumoral tissues. The urine samples headspace has also been analyzed for the presence of the volatile compounds (VOCs) by SPME-GC/MS and gas sensor array. In particular, it was found by GC/MS analysis that 2-Cyclohexen-1-one,3-methyl-6-(1-methylethyl)- correlates with LOX-1 concentration in urine. The combined approach of VOCs analysis and protein quantification could lead to promising results in terms of diagnostic and prognostic potential for ccRCC tumors.

Pro-oncogenic action of LOX-1 and its splice variant LOX-1Δ4 in breast cancer phenotypes.

The identification of new predictive biomarkers and therapeutic target for tailored therapy in breast cancer onset and progression is an interesting challenge. OLR-1 gene encodes the cell membrane receptor LOX-1 (lectin-like oxidized low-density lipoprotein receptor). We have recently identified a novel alternative OLR-1 isoform, LOX-1Δ4, whose expression and functions are still not clarified. In the present paper, we demonstrated that LOX-1 is overexpressed in 70% of human breast cancer (n = 47) and positively correlated to the tumor stage and grade (p < 0.01). Observations on LOX-1 and its splice variant Δ4 pointed out a different expression pattern correlated to breast cancer phenotypes. Overexpressing LOX-1 and LOX-1Δ4 in vitro, we obtained a strong enhancement of proliferative rate and a downregulation of cell death-related proteins. In addition, we observed a strong modulation of histone H4 acetylation and Ku70, the limiting factor of DNA double-strand breaks repair machinery implied in apoptosis inhibition and drug resistance acquisition. Moreover, LOX-1Δ4 overexpression is able to increase proliferation in a non-tumorigenic epithelial cell line, MCF12-F, acting as an oncogene. Altogether, these results suggest that LOX-1 may acts as a molecular link among metabolism, inflammation and cancer, indicating its potential role as biomarker and new molecular target, representing an attractive and concrete opportunity to improve current strategies for breast cancer tailored therapy.

Targeting LOX-1 Inhibits Colorectal Cancer Metastasis in an Animal Model.

Recurrence and metastasis are the primary causes of mortality in patients with colorectal cancer (CRC), and therefore effective tools to reduce morbidity and mortality of CRC patients are necessary. LOX-1, the ox-LDL receptor, is strongly involved in inflammation, obesity, and atherosclerosis, and several studies have assessed its role in the carcinogenesis process linking ROS, metabolic disorders and cancer. We have already demonstrated in vitro that LOX-1 expression correlates to the aggressiveness of human colon cancer and its downregulation weakens the tumoral phenotype, indicating its potential function as a biomarker and a target in CRC therapy. Here we further investigate in vivo the role of LOX-1 in colon tumorigenesis by xenografting procedures, injecting nude mice both subcutaneously and intravenously with human high grade metastatic colorectal cancer cells, DLD-1, in which LOX-1 expression has been downregulated by shRNA (LOX-1RNAi cells). Histopathological and immunohistochemical evaluations have been performed on xenograft tumors. The experiments have been complemented by the analysis of the volatile compounds (VOCs) collected from the cages of injected mice and analyzed by gas-chromatography and gas sensors. After intravenous injection of LOX-1RNAi cells, we found that LOX-1 silencing influences both the engraftment of the tumor and the metastasis development, acting by angiogenesis. For the first time, we have observed that LOX-1 inhibition significantly prevents metastasis formation in injected mice and, at the same time, induces a downregulation of VEGF-A165, HIF-1α, and ß-catenin whose expression is involved in cell migration and metastasis, and a variation of histone H4 acetylation pattern suggesting also a role of LOX-1 in regulating gene transcription. The analysis of the volatile compounds (VOCs) collected from the cages of injected mice has evidenced a specific profile in those xenograft mice in which metastasis originates. These findings underline the role of LOX-1 as a potential target for inhibition of tumor progression and metastasis, enhancing current therapeutic strategies against colorectal cancer.

Ku70, Ku80, and sClusterin: A Cluster of Predicting Factors for Response to Neoadjuvant Chemoradiation Therapy in Patients With Locally Advanced Rectal Cancer.

PURPOSE: The identification of predictive biomarkers for neoadjuvant chemoradiation therapy (CRT) is a current clinical need. The heterodimer Ku70/80 plays a critical role in DNA repair and cell death induction after damage. The aberrant expression and localization of these proteins fail to control DNA repair and apoptosis. sClusterin is the Ku70 partner that sterically inhibits Bax-dependent cell death after damage in some pathologic conditions. This study sought to evaluate the molecular relevance of Ku70-Ku80-Clu as a molecular cluster predicting the response to neoadjuvant CRT in patients with locally advanced rectal cancer (LARC). METHODS AND MATERIALS: Patients enrolled in this study underwent preoperative CRT followed by surgical excision. A retrospective study based on individual response, evaluated by computed tomography and diffusion-weighted magnetic resonance imaging, identified responder (56%) and no-responder patients (44%). Ku70/80 and Clu expression were observed in biopsy specimens obtained before and after treatment with neoadjuvant CRT from the same LARC patients. In vitro studies before and after irradiation were also performed on radioresistant (SW480) and radiosensitive (SW620) colorectal cancer cell lines, mimicking sensitive or resistant tumor behavior. RESULTS: We found a conventional nuclear localization of Ku70/80 in pretherapeutic tumor biopsies of responder patients, in agreement with their role in DNA repair and regulating apoptosis. By contrast, in the no-responder population we observed an unconventional overexpression of Ku70 in the cytoplasm (P<.001). In this context we also overexpression of sClu in the cytoplasm, which accorded with its role in stabilizing of Bax-Ku70 complex, inhibiting Bax-dependent apoptosis. Strikingly, Ku80 in these tumor tissues was lost (P<.005). In vitro testing of colon cancer cells finally confirmed the results observed in tumor biopsy specimens, proving that Ku70/80-Clu deregulation is extensively involved in the resistance mechanism. CONCLUSION: These results strongly suggest a potential role of these proteins as a new prognostic tool to predict the response to chemoradiation in LARC.

Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer.

Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for ß-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn't retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors.