Paola Mazzarelli

Mutations of the D310 mitochondrial mononucleotide repeat in primary tumors and cytological specimens

A mononucleotide repeat (D310) in mitochondrial DNA has been recently identified as a mutational hot spot in primary tumors. We analyzed 56 tumors for insertion/deletion mutations in the D310 repeat. A total of 13 mutations were detected. The highest frequency of mutations was found for cervical cancer, followed by bladder tumors, breast cancer and endometrial neoplasia. No alterations were observed in four patients suspected of malignancy but without evidence of malignant tumor. We detected identical changes in four of four urine sediments from patients with bladder cancer and in three of three fine needle aspirates of patients with breast cancer. Our results indicate that D310 abnormalities are detectable in cytology specimens from patients with cancer and support the notion that D310 analysis may represent a new molecular tool for cancer detection.

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.

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.