Dino Amadori

In vitro and in vivo evaluation of NCX 4040 cytotoxic activity in human colon cancer cell lines

BackgroundNitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity.MethodsIn vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks.ResultsIn the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO2 group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all.ConclusionsNCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.

Switching to Anastrozole Versus Continued Tamoxifen Treatment of Early Breast Cancer: Preliminary Results of the Italian Tamoxifen Anastrozole Trial

PURPOSE Tamoxifen, which is actually the gold standard adjuvant treatment in estrogen receptor-positive early breast cancer, is associated with an increased risk of endometrial cancer and other life-threatening events. Moreover, many women relapse during or after tamoxifen therapy because of the development of resistance. Therefore new approaches are required. PATIENTS AND METHODS We conducted a prospective randomized trial to test the efficacy of switching postmenopausal patients who were already receiving tamoxifen to the aromatase inhibitor anastrozole. After 2 to 3 years of tamoxifen treatment, patients were randomly assigned either to receive anastrozole 1 mg/d or to continue receiving tamoxifen 20 mg/d, for a total duration of treatment of 5 years. Disease-free survival was the primary end point. Event-free survival, overall survival, and safety were secondary end points. RESULTS Four hundred forty-eight patients were enrolled. All women had node-positive, estrogen receptor-positive tumors. At a median follow-up time of 36 months, 45 events had been reported in the tamoxifen group compared with 17 events in the anastrozole group (P = .0002). Disease-free and local recurrence-free survival were also significantly longer in the anastrozole group (hazard ratio [HR] = 0.35; 95% CI, 0.18 to 0.68; P = .001 and HR = 0.15; 95% CI, 0.03 to 0.65; P = .003, respectively). Overall, more adverse events were recorded in the anastrozole group compared with the tamoxifen group (203 v 150, respectively; P = .04). However, more events were life threatening or required hospitalization in the tamoxifen group than in the anastrozole group (33 of 150 events v 28 of 203 events, P = .04). CONCLUSION Switching to anastrozole after the first 2 to 3 years of treatment is well tolerated and significantly improves event-free and recurrence-free survival in postmenopausal patients with early breast cancer.

Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization

Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis-suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin–dependent epithelial traits and Sec23a-mediated tumor cell secretome.

A New Palliative Prognostic Score: A First Step for the Staging of Terminally Ill Cancer Patients

In recent years, extensive research has been performed to identify prognostic factors that predict survival in terminally ill cancer patients. This study describes the construction of a simple prognostic score based on factors identified in a prospective multicenter study of 519 patients with a median survival of 32 days. An exponential multiple regression model was adopted to evaluate the joint effect of some clinico-biological variables on survival. From an initial model containing 36 variables, a final parsimonious model was obtained by means of a backward selection procedure. The Palliative Prognostic Score (PaP Score) is based on the final model and includes the following variables: Clinical Prediction of Survival (CPS), Karnofsky Performance Status (KPS), anorexia, dyspnea, total white blood count (WBC) and lymphocyte percentage. A numerical score was given to each variable, based on the relative weight of the independent prognostic significance shown by each single category in the multivariate analysis. The sum of the single scores gives the overall PaP Score for each patient and was used to subdivide the study population into three groups, each with a different probability of survival at 30 days: (1) group A: probability of survival at 30 days > 70%, with patient score < or = 5.5; (2) group B: probability of survival at 30 days 30-70%, with patient score 5.6-11.0; and (3) group C: probability of survival at 30 days < 30%, with patient score > 11.0. Using this method, 178/519 (34.3%) patients were classified in risk group A, 205 (39.5%) patients were in risk group B, and 136 (26.2%) patients were in risk group C. The patients classified in the three risk groups had a very different survival experience (logrank = 294.8, P < 0.001), with a median survival of 64 days for group A, 32 days for group B, and 11 days for group C. The PaP Score based on simple clinical and biohumoral variables proved to be statistically significant in a multivariate analysis. The score is valid in this population (training set). An independent validation on another patient series (testing set) is required and is the object of a companion paper.

Successful Validation of the Palliative Prognostic Score in Terminally Ill Cancer Patients

The aim of this work was to validate a previously constructed prognostic score for terminally ill cancer patients in order to determine its value in clinical practice. The Palliative Prognostic Score (PaP Score) was tested on a population of 451 evaluable patients consecutively entered in the hospice programs of 14 Italian Palliative Care Centers. The score subdivided patients into three specific risk classes based on the following six predictive factors of death: dyspnea, anorexia, Karnofsky Performance Status (KPS), Clinical Prediction of Survival (CPS), total white blood count (WBC), and lymphocyte percentage. The performance of the PaP Score index in the training and testing sets was evaluated by comparing mortality rates in the 3 prognostic risk categories. The score was able to subdivide the validation-independent case series into three risk groups. Median survival was 76 days in group A (with a 86.6% probability of 30-day survival), 32 days in group B (with a 51.6% probability of 30-day survival), and 14 days in group C (with a 16.9% probability of 30-day survival). Survival medians were remarkably similar to those of the training set (64 days in group A, 32 days in group B, and 11 days in group C). In the complex process of staging terminally ill patients, the PaP Score is a simple instrument which permits a more accurate quantification of expected survival. It has been validated on an independent case series and is thus suitable for use in clinical practice.

Modulation of mismatch repair and genomic stability by miR-155

Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10–40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.

MicroRNA-21 induces resistance to 5-fluorouracil by down-regulating human DNA MutS homolog 2 (hMSH2)

The overexpression of microRNA-21 (miR-21) is linked to a number of human tumors including colorectal cancer, where it appears to regulate the expression of tumor suppressor genes including p21, phosphatase and tensin homolog, TGFβ receptor II, and B-cell leukemia/lymphoma 2 -associated X protein. Here we demonstrate that miR-21 targets and down-regulates the core mismatch repair (MMR) recognition protein complex, human mutS homolog 2 (hMSH2) and 6 (hMSH6). Colorectal tumors that express a high level of miR-21 display reduced hMSH2 protein expression. Cells that overproduce miR-21 exhibit significantly reduced 5-fluorouracil (5-FU)-induced G2/M damage arrest and apoptosis that is characteristic of defects in the core MMR component. Moreover, xenograft studies demonstrate that miR-21 overexpression dramatically reduces the therapeutic efficacy of 5-FU. These studies suggest that the down-regulation of the MMR mutator gene associated with miR-21 overexpression may be an important clinical indicator of therapeutic efficacy in colorectal cancer.

Association of a microRNA/TP53 feedback circuitry with pathogenesis and outcome of b-cell chronic lymphocytic leukemia

CONTEXT Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown. OBJECTIVE To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL. DESIGN, SETTING, AND PATIENTS CLL Research Consortium institutions provided blood samples from untreated patients (n = 206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)-associated protein kinase 70 kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in cell lines and primary samples and was validated in a separate cohort of primary CLL samples. MAIN OUTCOME MEASURES Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation. RESULTS In CLLs with 13q deletions the miR-15a/miR-16-1 cluster directly targeted TP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95% confidence interval {CI}, 0.63-0.73]; P = .02; mean for miR-16 vs scrambled control, 0.62 RLU [95% CI, 0.59-0.65]; P = .02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted the ZAP70 kinase (mean luciferase activity for miR-34a vs scrambled control, 0.33 RLU [95% CI, 0.30-0.36]; P = .02; mean for miR-34b vs scrambled control, 0.31 RLU [95% CI, 0.30-0.32]; P = .01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33-0.37]; P = .02). CONCLUSIONS A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70.

miR-126 and miR-126 * repress recruitment of mesenchymal stem cells and inflammatory monocytes to inhibit breast cancer metastasis

The tumour stroma is an active participant during cancer progression. Stromal cells promote tumour progression and metastasis through multiple mechanisms including enhancing tumour invasiveness and angiogenesis, and suppressing immune surveillance. We report here that miR-126/miR-126*, a microRNA pair derived from a single precursor, independently suppress the sequential recruitment of mesenchymal stem cells and inflammatory monocytes into the tumour stroma to inhibit lung metastasis by breast tumour cells in a mouse xenograft model. miR-126/miR-126* directly inhibit stromal cell-derived factor-1 alpha (SDF-1α) expression, and indirectly suppress the expression of chemokine (C–C motif) ligand 2 (Ccl2) by cancer cells in an SDF-1α-dependent manner. miR-126/miR-126* expression is downregulated in cancer cells by promoter methylation of their host gene Egfl7. These findings determine how this microRNA pair alters the composition of the primary tumour microenvironment to favour breast cancer metastasis, and demonstrate a correlation between miR-126/126* downregulation and poor metastasis-free survival of breast cancer patients.

WWOX gene restoration prevents lung cancer growth in vitro and in vivo

The WWOX (WW domain containing oxidoreductase) gene at the common fragile site, FRA16D, is altered in many types of cancer, including lung cancer. We have examined the tumor suppressor function of WWOX in preclinical lung cancer models. The WWOX gene was expressed in lung cancer cell lines through recombinant adenovirus (Ad) infection (Ad-WWOX), and through a drug [ponasterone A, (ponA)]-inducible system. After WWOX restoration in vitro, endogenous Wwox protein-negative cell lines (A549, H460, and H1299) underwent apoptosis through activation of the intrinsic apoptotic caspase cascade in A549 and H460 cells. Ectopic expression of Wwox caused dramatic suppression of tumorigenicity of A549, H460, and H1299 cells in nude mice after Ad-WWOX infection and after ponA induction of Wwox expression in H1299 lung cancer cells. Tumorigenicity and in vitro growth of U2020 (Wwox-positive) lung cancer cells was unaffected by Wwox overexpression. This study confirms that WWOX is a tumor suppressor gene and is highly effective in preventing growth of lung cancer xenografts, whether introduced through viral infection or by induction of a silent WWOX transgene.