MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index. (Cancer Res 2005; 65(16): 7065-70)

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.

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Alteration of the FHIT (fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis. FHIT gene transfer into lung cancer cell line H460 lacking Fhit protein expression resulted in reversion of tumorigenicity. To gain insight into the biological function of FHIT, we compared the H460 cell line with its Fhit transfectants (H460͞FHIT). A significant inhibition of cell growth was observed in H460͞FHIT cells. The analysis of apoptosis by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed a high rate of apoptosis-induced DNA strand breaks in stable clones. In situ results were confirmed by FACScan analysis that showed an apoptotic rate of 44-47% compared with a 15% level in the control H460 cells. Analysis of cell cycle-phase distribution indicated a significant G 0 ͞G 1 arrest and the presence of a sub-G 1 peak in the stable clones. No significant changes in Bcl2, BclX, and Bax protein expression level were observed in the transfected clones as compared with the control H460 cells whereas a 2-fold increase in Bak protein levels was noticed. An increased level of p21 waf protein paralleled by an up-regulation of p21 waf transcripts also was found in Fhit-expressing clones compared with the H460 cell line. No differences in p53 levels were observed in the same cells, suggesting a p53-independent effect. These data suggest that the observed growth-inhibitory effect in FHIT-reexpressing cells could be related to apoptosis and cell cycle arrest and link the tumor-suppressor activity of FHIT to its proapoptotic function.The FHIT (fragile histidine triad) gene (1), at 3p14.2, is a frequent target of deletions associated with abnormal RNA and protein expression in primary tumors and cell lines of lung, head and neck, kidney, cervix, and breast cancer (2-6). Stable FHIT-transduced clones expressing exogenous wild-type Fhit, isolated after transfection of various epithelial cell lines carrying inactivated endogenous Fhit, show reduced colonyformation efficiency in vitro and inhibition of tumor development in nude mice, indicating that FHIT acts as a tumorsuppressor gene (7). The Fhit protein is a diadenosine triphosphate (Ap 3 A) hydrolase belonging to the histidine triad superfamily (HIT) of nucleotide-binding proteins (8). Our observation that the His(96)Asn mutant, lacking hydrolytic activity, still inhibits tumor formation in vivo (7) suggests that the tumor-suppressing function of Fhit is not related to catalysis of nucleotide substrates. However, the biological mechanism of FHIT activity and the cellular pathways associated with its tumor-suppressor function are not known. Crystallographic studies suggested that Ap 3 A nucleotide binding is crucial for Fhit biological activity and that enzymesubstrate complexes may be a signaling form (9). Interestingly, it has been reported that apoptosis in human cultured cells is associated with a decrease of free Ap 3 A levels (10).To study a possible involvement of FHIT in cell growth control and apoptosis, we...