Sabina Pucci

Modulation of different clusterin isoforms in human colon tumorigenesis

Clusterin is a ubiquitous secretory heterodimeric disulfide-linked glycoprotein, which is implicated in several physiological processes, including immune regulation, cell adhesion and morphological transformation, lipid transportation, tissue remodelling, membrane recycling and cell–cell interactions. A large number of studies have focused their interest on clusterin gene products as mediators of cell cycle progression and cell death induction, although data on the different isoforms and their role in the different cell processes are still obscure. Recently, an increased clusterin expression in breast cancer has been reported. In order to elucidate the role of clusterin in tumor progression and whether one of its isoforms is preferentially expressed in tumorigenesis, we examined its presence throughout the different steps of human colon carcinoma, one of the best-characterized models of human tumor progression. The immunohistochemical observation of 30 bioptic and surgical colon specimens demonstrated a cell compartment clusterin translocation from the nucleus to the cytoplasm directly related to tumor progression. In fact, a nuclear localization found in healthy colonic mucosa is consistent with the involvement of the proapoptotic nuclear form in the regulation of cell cycle progression and in cell death induction. The progression towards high-grade and metastatic carcinoma leads to cytoplasmic clusterin distribution. Protein extracts from freshly isolated cells of the same patients confirm in high-grade carcinomas with metastatic nodes the complete loss of the proapoptotic nuclear form and a cytoplasmic overexpression of the highly glycosylated form. Data obtained from in vitro experiments confirm that this form is released in the extracellular space and corresponded to the fully glycosylated one. These data suggest that the controversial data on clusterin function in tumors may be related to the pattern shift of its isoform production. As the secreted form of clusterin is correlated to cell matrix formation, cell membrane remodeling and cell–cell adhesion, the overexpression of this form in highly aggressive tumors and metastatic nodes could be a potential new prognostic and predictive marker for colon carcinoma aggressiveness.

Clusterin in stool: a new biomarker for colon cancer screening?

OBJECTIVES:The identification of useful markers for early diagnosis of human colon cancer is a major goal still in progress. Clusterin is a pleiotropic protein with a broad range of functions. It has recently drawn much attention because of its association with cancer promotion and metastasis. It is involved in prosurvival and apoptosis processes that are carried out by two different isoforms. Secreted clusterin isoform (sCLU) is cytoprotective and its prosurvival function is the basis of the current phase I/II clinical trials against prostate, lung, and breast cancers. We have already shown that in colorectal cancer (CRC) there is an increased expression of sCLU. In this report, we investigated whether sCLU is released in the blood and stool of colon cancer patients in order to study sCLU as a potential diagnostic molecular marker for colon cancer screening.METHODS:The quantitative expression of sCLU was determined by dot blot immunodosage in the serum and stool of CRC patients (n=63) and age-matched controls without clinical history of neoplasia, CRC, or systemic or bowel inflammatory disease (n=50). Unpaired t-tests and Mann–Whitney U-tests were used for continuous variables. The diagnostic performance of clusterin was appraised by means of receiver operating characteristic (ROC) curves.RESULTS:We found a significant increase of sCLU in the serum and stool of CRC patients (P=0.0002 and P<0.000, respectively) as compared with controls. ROC curves provided cutoff points showing a trade-off between sensitivity and specificity. With a cutoff point of 88.5 μg/ml, sCLU in blood showed a 55.6% sensitivity and 100% specificity, and with a cutoff point of 34.6 μg/g, the stool test reached 66.7% sensitivity and 84% specificity in discriminating between nonneoplastic and colorectal neoplastic lesions. Human cancer xenografts in nude mice indicated a positive correlation between increasing serum clusterin level and tumor size.CONCLUSIONS:This study highlights the potential of clusterin detection in stool to be a valuable tool to improve the effectiveness and efficiency of large-scale clinical cancer screening.

Interleukin-6 affects cell death escaping mechanisms acting on Bax-Ku70-Clusterin interactions in human colon cancer progression.

Activation of pro-survival pathways and apoptotic cell death escape are considered hallmarks of oncogenic cell transformation. Tissue microenvironment strongly influences tumorigenesis, redirecting some pathways versus a persisting pro-survival state. Here, we report evidence on the role of interleukin 6 (IL-6) in affecting pro-survival pathways in colon cancer progression, modulating the expression and the molecular interactions among the pro-apoptotic factor Bax, the DNA repair proteins Ku70/86 and Clusterin isoforms.

Carnitine palmitoyltransferase I in human carcinomas: A novel role in histone deacetylation?

Carnitine palmitoyl transferase I (CPT1) catalyzes the transport of long-chain fatty acids into mitochondria for β-oxidation. A link between CPT1 and apoptosis has been suggested on the basis of several experimental data. Nevertheless, results are contradictory about the effective role of CPT1 in cell survival control and cancer development. Conversely, Fatty acid synthase (FAS) enzyme, required for the synthesis of fatty acids, is found over-expressed in tumors and inhibition of FAS triggers apoptosis in human cancer cells. We have studied the tumor-specific modulation of CPT1 and FAS in human colorectal cancer (n=11) and breast carcinomas (n=24). CPT1 was significantly decreased in the cytoplasm of tumoral samples (p≤0.04), whereas FAS was increased (p≤0.04). A striking CPT1 nuclear localization was evident in the tumors (p≤0.04). In the nuclear environment the protein would modulate the levels of acetyl/acyl-CoA implicated in the regulation of gene transcription. At this purpose, we performed in vitro experiments using epithelial neoplastic (MCF-7, Caco-2, HepG2 cells) and non neoplastic cell lines (MCF-12F) confirming a nuclear localization of CPT1 protein exclusively in neoplastic cells. Moreover histone deacetylase (HDAC) activity showed significantly higher levels in nuclear extracts from neoplastic than from control cells. HDAC1 and CPT1 proteins co-immunoprecipitated in nuclear extracts from MCF-7 cells. The treatment with HDAC inhibitors such as trichostatin A and butyrate significantly decreased nuclear expression of CPT1 and its bond to HDAC1. We also identified the existence of CPT1A mRNA transcript variant 2 in MCF-7, beside to the classic isoform 1. The peculiar localization of CPT1 in the nuclei of human carcinomas and the disclosed functional link between nuclear CPT1 and HDAC1 propose a new role of CPT1 in the histonic acetylation level of tumors.

CLU and colon cancer. The dual face of CLU: from normal to malignant phenotype

The transition from normal to malignant phenotype implies the activation of some pathways that underlie the aberrant clone expansion. In some way, the conventional function of proteins involved in DNA repair, cell death/growth induction, vascularization, and metabolism is inhibited or shifted toward other pathways by soluble mediators that orchestrate such change depending on the microenvironment conditions. The adenoma-carcinoma sequence of the colon represents one of the most well studied and characterized models of human tumor progression. In this section, we focus our attention on defined pathways that underlie the initiation, promotion, and progression of colon cancer, conferring aggressiveness to the neoplastic cells. Clusterin (CLU) is a pleiotropic protein with a broad range of functions. It has recently drawn much attention because of its association with cancer promotion and metastasis. It is involved in prosurvival and apoptosis processes that are carried out by two different forms. sCLU is cytoprotective and its prosurvival function is the basis of the current Phase I/II clinical trials. In colorectal cancer an increase of sCLU expression occurs, whereas the nuclear proapoptotic form is downregulated. Several controversial data have been published on colon cancer discussing its role as tumor suppressor or prosurvival factor in colon cancer. Here, we report the dynamic interaction of the different forms of CLU with their partners DNA-repair protein Ku70 and proapoptotic factor Bax during colon cancer progression, which seems to be a crucial point for the neoplastic cell fate. We also highlight that the appearance and the progressive increase of the sCLU in colorectal tumors correlate to a significant increase of CLU in serum and stool of patients. On the basis of results obtained by CLU immuno-dosage in blood and stool of colon cancer patients, we report that sCLU could represent a diagnostic molecular marker for colon cancer screening.

Modulation of Clusterin Isoforms Is Associated With All-Trans Retinoic Acid–Induced Proliferative Arrest and Apoptosis of Intimal Smooth Muscle Cells

Objectives— Clusterin is a heterodimeric glycoprotein which is implicated in several biological processes. The nuclear (n-CLU) and cytoplasmic secreted (s-CLU) isoforms have recently been described, but their role is still unclear. The aim of this study is to investigate the expression of clusterin and its isoforms during proliferative arrest and apoptosis of vascular smooth muscle cells (SMCs). Methods and Results— Clusterin expression was evaluated by immunohistochemistry and Western blotting in human arteries and rat aortas. In human diffuse myointimal thickening, clusterin was detected in cell cytoplasm and extracellular space, whereas it was practically absent in the media. In rat aortas 15 days after ballooning, intimal cells (IT cells) overexpressed s-CLU and n-CLU, the latter mainly in the inner neointima; clusterin expression decreased at 60 days. In vitro, IT cells maintained high clusterin expression and its antisense markedly reduced proliferation and increased apoptosis. Western blotting showed that all-trans retinoic acid-induced proliferative arrest and increased &agr;-smooth muscle actin expression did associate to s-CLU and B-myb reduction, whereas bax-related apoptosis was associated to a shift from the s-CLU to n-CLU isoform. Conclusions— Clusterin overexpression characterized neointimal SMCs; s-CLU expression decreased in IT cells during all-trans retinoic acid–induced proliferative arrest and redifferentiation, whereas n-CLU overexpression was characteristic of apoptosis.

The lectin-like oxidized LDL receptor-1: a new potential molecular target in colorectal cancer

The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy.

MicroRNA Dysregulation in Colon Cancer Microenvironment Interactions: The Importance of Small Things in Metastases

The influence of the microenvironment through the various steps of cancer progression is signed by different cytokines and growth factors, that could directly affect cell proliferation and survival, either in cancer and stromal cells. In colon cancer progression, the cooperation between hypoxia, IL-6 and VEGF-A165 could regulate the DNA repair capacity of the cell, whose impairment is the first step of colon cancer development. This cooperation redirects the activity of proteins involved in the metabolic shift and cell death, affecting the cell fate. The pathways triggered by micro environmental factors could modulate cancer-related gene transcription, affecting also small non coding mRNA, microRNAs. MicroRNAs have emerged as key post-transcriptional regulators of gene expression, directly involved in human cancers. The present review will focus first on the intertwined connection between cancer microenvironment and aberrant expression of microRNAs which contribute to carcinogenesis. In particular, the epigenetic mechanisms triggered by tissue microenvironment will be discussed, in view of the recent identification of miRNAs able to directly or indirectly modulate the epigenetic machinery (epi-miRNAs) and that are involved in the epithelial to mesenchimal transition and metastases development.

Exon-Skipping Strategy by Ratio Modulation between Cytoprotective versus Pro-Apoptotic Clusterin Forms Increased Sensitivity of LNCaP to Cell Death

Background In prostate cancer the secreted form of clusterin (sCLU) has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties. Methodology In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress. Results and Conclusions We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line “LNCaP” after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an “on demand alternative splicing” strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

Modulation of Ku70/80, Clusterin/ApoJ Isoforms and Bax Expression in Indocyanine-Green-Mediated Photo-Oxidative Cell Damage

Purpose: In order to characterize the biological effects and molecular mechanism underlying indocyanine-green (ICG)-mediated photo-oxidative cell damage, human cultured retinal pigmented epithelium (RPE) cells preloaded with ICG were exposed to 810-nm laser irradiation. Cell viability and death induction were examined, as well as the modulation of proteins involved in cell death and DNA repair. Methods: ARPE-19 cells preloaded with 100 µM ICG were irradiated using continuous and micropulsed 810-nm laser for the dye photoactivation, and cell viability and apoptosis were evaluated. The expression and subcellular localization of Bax, Ku70, Ku80 and clusterin/ApoJ were analyzed by immunocytochemistry and Western blot. Results: ICG photoactivation induced apoptosis in RPE cells. The micropulsed laser irradiation induced a higher percentage of cell killing as compared to continuous wave. Cell killing was inhibited by sodium azide, suggesting the involvement of reactive oxygen species in the laser-induced cell damage. Bax was strongly induced after 4 and up to 24 h of treatment. The nuclear proapoptotic isoform of clusterin/ApoJ was selectively upregulated after 24 h of treatment. The DNA repair machinery was upregulated after 4 and up to 24 h. Conclusion: These data elucidate some molecular mechanisms involved in cell death induced by ICG photosensitization. The increase and relocalization of Bax into the mitochondria and the upregulation and translocation of the proapoptotic isoform of clusterin/ApoJ in the nucleus demonstrated the involvement of these proteins in the photo-oxidative cell death pathway. These data point out new molecular targets and suggest potential applications in the therapy of the retinal diseases that could benefit by selective RPE treatment.