Antonio Addario

The miR-15a – miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities

MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.

Role of microRNAs in drug-resistant ovarian cancer cells

OBJECTIVES Chemotherapy is the preferred therapeutic approach for the therapy of advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This work was aimed to identify selected microRNAs involved in the development of chemoresistance in ovarian cancer. METHODS High-throughput analysis of the miRNA profile in a panel of paclitaxel- (A2780TAX, A2780TC1 and A2780TC3) and cisplatin-resistant (A2780CIS) cells was assessed using a microarray platform and subsequent validation with qPCR and Northern blots. Downstream target validation was performed for miR-130a and the target M-CSF.] RESULTS Six miRNAs (let-7e, miR-30c, miR-125b, miR-130a and miR-335) were always diversely expressed in all the resistant cell lines. Let-7e was upregulated in A2780TAX cells, while it was downregulated in the other resistant cell lines. The opposite phenomenon was obtained for miR-125b, which was downregulated in A2780TAX and upregulated in the other cell lines. The miR-30c, miR-130a and miR-335 were downregulated in all the resistant cell lines, thereby suggesting a direct involvement in the development of chemoresistance. Finally downstream target validation was proven for the miR-130a, whose downregulation was linked to the translational activation of the M-CSF gene, a known resistance factor for ovarian cancer. CONCLUSIONS Our results indicate that ovarian cancer drug resistance is associated with a distinct miRNA fingerprint, and miRNA microarrays could represent a prognostic tool to monitor the chemotherapy outcome.

Control of tumor and microenvironment cross-talk by miR-15a and miR-16 in prostate cancer

The interaction between cancer cells and microenvironment has a critical role in tumor development and progression. Although microRNAs regulate all the major biological mechanisms, their influence on tumor microenvironment is largely unexplored. Here, we investigate the role of microRNAs in the tumor-supportive capacity of stromal cells. We demonstrated that miR-15 and miR-16 are downregulated in fibroblasts surrounding the prostate tumors of the majority of 23 patients analyzed. Such downregulation of miR-15 and miR-16 in cancer-associated fibroblasts (CAFs) promoted tumor growth and progression through the reduced post-transcriptional repression of Fgf-2 and its receptor Fgfr1, which act on both stromal and tumor cells to enhance cancer cell survival, proliferation and migration. Moreover, reconstitution of miR-15 and miR-16 impaired considerably the tumor-supportive capability of stromal cells in vitro and in vivo. Our data suggest a molecular circuitry in which miR-15 and miR-16 and their correlated targets cooperate to promote tumor expansion and invasiveness through the concurrent activity on stromal and cancer cells, thus providing further support to the development of therapies aimed at reconstituting miR-15 and miR-16 in advanced prostate cancer.

BTG2 loss and miR-21 upregulation contribute to prostate cell transformation by inducing luminal markers expression and epithelial-mesenchymal transition

Prostate cancer is one of the leading causes of cancer-related death in men. Despite significant advances in prostate cancer diagnosis and management, the molecular events involved in the transformation of normal prostate cells into cancer cells have not been fully understood. It is generally accepted that prostate cancer derives from the basal compartment while expressing luminal markers. We investigated whether downregulation of the basal protein B-cell translocation gene 2 (BTG2) is implicated in prostate cancer transformation and progression. Here we show that BTG2 loss can shift normal prostate basal cells towards luminal markers expression, a phenotype also accompanied by the appearance of epithelial–mesenchymal transition (EMT) traits. We also show that the overexpression of microRNA (miR)-21 suppresses BTG2 levels and promotes the acquisition of luminal markers and EMT in prostate cells. Furthermore, by using an innovative lentiviral vector able to compete with endogenous mRNA through the overexpression of the 3′-untranslated region of BTG2, we demonstrate that in prostate tumor cells, the levels of luminal and EMT markers can be reduced by derepression of BTG2 from microRNA-mediated control. Finally, we show that the loss of BTG2 expression confers to non-tumorigenic prostate cells ability to grow in an orthotopic murine model, thus demonstrating the central role of BTG2 downregulaton in prostate cancer biology.

C-Met/miR-130b axis as novel mechanism and biomarker for castration resistance state acquisition

Although a significant subset of prostate tumors remain indolent during the entire life, the advanced forms are still one of the leading cause of cancer-related death. There are not reliable markers distinguishing indolent from aggressive forms. Here we highlighted a new molecular circuitry involving microRNA and coding genes promoting cancer progression and castration resistance. Our preclinical and clinical data demonstrated that c-Met activation increases miR-130b levels, inhibits androgen receptor expression, promotes cancer spreading and resistance to hormone ablation therapy. The relevance of these findings was confirmed on patients’ samples and by in silico analysis on an independent patient cohort from Taylor’s platform. Data suggest c-Met/miR-130b axis as a new prognostic marker for patients’ risk assessment and as an indicator of therapy resistance. Our results propose new biomarkers for therapy decision-making in all phases of the pathology. Data may help identify high-risk patients to be treated with adjuvant therapy together with alternative cure for castration-resistant forms while facilitating the identification of possible patients candidates for anti-Met therapy. In addition, we demonstrated that it is possible to evaluate Met/miR-130b axis expression in exosomes isolated from peripheral blood of surgery candidates and advanced patients offering a new non-invasive tool for active surveillance and therapy monitoring.

Blocking the APRIL circuit enhances acute myeloid leukemia cell chemosensitivity

The findings of this study suggest that APRIL acts in an autocrine fashion to protect acute myeloid leukemia cells from drug-induced death. Resistance to chemotherapy-induced cell death represents a major obstacle in the treatment of acute myeloid leukemia. APRIL (A Proliferation Inducing Ligand) is a member of the tumor necrosis factor superfamily that plays a key role in normal B-cell development, while promoting survival and proliferation of malignant B cells. We investigated APRIL expression and activity in acute myeloid leukemia. We found that APRIL mRNA and protein, including the secreted form, are expressed in leukemic cells of patients with M0, M2 and M4 acute myeloid leukemia subtypes but not in normal hematopoietic progenitors. Retrovirus-mediated APRIL expression in normal hematopoietic progenitors confers resistance to chemotherapeutic drugs-induced apoptosis. Conversely, blocking APRIL function by recombinant soluble APRIL receptors increased chemotherapeutic drugs-induced cell adeath in acute myeloid leukemia cells. These results indicate that APRIL acts in an autocrine fashion to protect acute myeloid leukemia cells from drug-induced death and foresee a therapeutic potential of APRIL antagonists in the treatment of acute myeloid leukemia.

Abstract LB-040: Establishment of a predictive patient-derived xenograft model for renal cell carcinoma

Renal Cell Carcinoma (RCC) is classically considered a difficult malignancy to diagnose and treat. Although the surgery is a resolving approach for many local tumors, the metastatic disease generally is characterized by poor outcomes. The lack of valid preclinical cancer models has hampered the discovery of valuable diagnostic, prognostic and predictive biomarkers to develop effective therapeutic options. In order to create preclinical models, we established an orthotopic patient derived xenograft (PDX) murine model using an enriched stem cell like-heterogeneous bulk obtained from surgery patients’ specimens. To predict effective conventional and innovative therapeutic treatment, the stem cell like-heterogeneous bulks were analyzed before injection for key total and phosphorylated protein expression by Reverse Phase Protein Array (RPPA). METHODS: Using specific enzymatic dissociation and culture conditions, we first isolated enriched stem cell like-heterogeneous bulks from fresh surgery specimens derived from 30 patients who underwent nephrectomy. After one week, bulks were orthotopically injected in immunocompromised mice (NSG mice). Cells were infected with lentiviral vector (Tween-Luc) and in vivo monitored by IVIS imaging system. The percentage of engrafting bulks, sizes and distal spreading capacity were evaluated. Several xenografts were further dissociated, serially inoculated and propagated for up to eighth generations. Bulks were analyzed before injection by RPPA. This technique performs the detection of total and phosphoproteins allowing the analysis of hundreds of proteins. This RPPA platform includes specific antibodies for key proteins belonging or involved in targeted therapy signaling cascade. RESULTS: Approximately 67% of the implanted samples engrafted. We observed a correlation between the engraftment success and the aggressiveness of the parental tumor. Moreover, the PDXs obtained from more aggressive tumors showed increased size. Hematoxylin and Eosin staining showed that PDX displayed similar histological architectures to parental tumors. Furthermore, immunohistochemical analysis demonstrated that PDX retained CD10 and PAX 8 expression, two typical RCC biomarkers, at similar level than parental tumors. Finally, we obtained maps highlighting the specific activated proteins which are key candidates of targeted therapy pathways by RPPA. CONCLUSIONS: Our established in vitro and PDX models recapitulate tumor histology and molecular characteristic creating new source for ameliorating diagnosis, prognosis and therapy sensitiveness prediction. Furthermore, we were able to propagate PDXs from the same patient for up to eight generations. Since in average tumor uptake is 4-5 weeks, it would be theoretically possible to test in parallel therapeutics in murine models while the corresponding metastatic patient is under therapy. The ability to generate large PDX animal cohorts is very promising and could be exploited to obtain valuable preclinical platforms for innovative drug testing. Moreover, the possibility to map a wide variety of intracellular pathways and biological functions in enriched stem cell like-heterogeneous bulks enable us to explore protein-protein interaction networks with potential impact on drug activity and to identify deregulated circuits related to pharmacological inhibition. Citation Format: Simona Di Martino, Gabriele De Luca, Ludovica Grassi, Giulia Federici, Laura De Salvo, Anna Laura Di Pace, Antonio Addario, Giovanni Muto, Manuela Costantini, Mauro Biffoni, Michele Signore, Steno Sentinelli, Michele Milella, Michele Gallucci, Desiree Bonci, Ruggero De Maria. Establishment of a predictive patient-derived xenograft model for renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-040.