Marco Folini

miR-205 Exerts Tumor-Suppressive Functions in Human Prostate through Down-regulation of Protein Kinase Cε

Limited information is available concerning the expression and role of microRNAs in prostate cancer. In this study, we investigated the involvement of miR-205 in prostate carcinogenesis. Significantly lower miR-205 expression levels were found in cancer than in normal prostate cell lines as well as in tumor compared with matched normal prostate tissues, with a particularly pronounced reduction in carcinomas from patients with local-regionally disseminated disease. Restoring the expression of miR-205 in prostate cancer cells resulted in cell rearrangements consistent with a mesenchymal-to-epithelial transition, such as up-regulation of E-cadherin and reduction of cell locomotion and invasion, and in the down-regulation of several oncogenes known to be involved in disease progression (i.e., interleukin 6, caveolin-1, EZH2). Our evidence suggests that these events are driven by the concurrent repression of specific predicted miR-205 targets, namely N-chimaerin, ErbB3, E2F1, E2F5, ZEB2, and protein kinase Cepsilon. Strikingly, the latter seemed to play a direct role in regulating epithelial-to-mesenchymal transition. In fact, its down-regulation led to a cell phenotype largely reminiscent of that of cells ectopically expressing miR-205. Overall, we showed for the first time that miR-205 exerts a tumor-suppressive effect in human prostate by counteracting epithelial-to-mesenchymal transition and reducing cell migration/invasion, at least in part through the down-regulation of protein kinase Cepsilon.

Targeting survivin in cancer therapy.

Background: Survivin is a structurally unique member of the inhibitor of apoptosis protein (IAP) family that acts as a suppressor of apoptosis and plays a central role in cell division. Owing to its massive upregulation in human tumors and its involvement in cancer progression and treatment resistance, survivin is currently undergoing extensive investigation as a novel therapeutic target. Objective: The purpose of this review is to define the potential of survivin as a therapeutic target for new anticancer interventions. Methods: The literature dealing with the therapeutic targeting of survivin has been carefully reviewed. Results/conclusion: Several preclinical studies have demonstrated that downregulation of survivin expression or function, accomplished by means of various strategies, reduced tumor growth potential, increased the apoptotic rate and sensitized tumor cells to chemotherapeutic drugs and radiation in different human tumor models. Moreover, the first survivin inhibitors are being currently evaluated in clinical settings.

miR-21: an oncomir on strike in prostate cancer.

BackgroundAberrant expression of microRNAs, small non-coding RNA molecules that post-transcriptionally repress gene expression, seems to be causatively linked to the pathogenesis of cancer. In this context, miR-21 was found to be overexpressed in different human cancers (e.g. glioblastoma, breast cancer). In addition, it is thought to be endowed with oncogenic properties due to its ability to negatively modulate the expression of tumor-suppressor genes (e.g. PTEN) and to cause the reversion of malignant phenotype when knocked- down in several tumor models. On the basis of these findings, miR-21 has been proposed as a widely exploitable cancer-related target. However, scanty information is available concerning the relevance of miR-21 for prostate cancer. In the present study, we investigated the role of miR-21 and its potential as a therapeutic target in two prostate cancer cell lines, characterized by different miR-21 expression levels and PTEN gene status.ResultsWe provide evidence that miR-21 knockdown in prostate cancer cells is not sufficient per se i) to affect the proliferative and invasive potential or the chemo- and radiosensitivity profiles or ii) to modulate the expression of the tumor-suppressors PTEN and Pdcd4, which in other tumor types were found to be regulated by miR-21. We also show that miR-21 is not differently expressed in carcinomas and matched normal tissues obtained from 36 untreated prostate cancer patients subjected to radical prostatectomy.ConclusionsOverall, our data suggest that miR-21 is not a central player in the onset of prostate cancer and that its single hitting is not a valuable therapeutic strategy in the disease. This supports the notion that the oncogenic properties of miR-21 could be cell and tissue dependent and that the potential role of a given miRNA as a therapeutic target should be contextualized with respect to the disease.

Ribozyme-mediated attenuation of survivin expression sensitizes human melanoma cells to cisplatin-induced apoptosis

Survivin is a structurally unique member of the inhibitor of apoptosis (IAP) family of proteins that is potentially involved in both control of cell division and inhibition of apoptosis (1). Specifically, its antiapoptotic function is related to the ability to directly or indirectly inhibit caspases (2). The notion that survivin is overexpressed in most of the common human tumors (3, 4) but absent in normal adult tissues with only a few exceptions (5, 6) has led to the proposal of survivin as a promising therapeutic target for novel anticancer therapies (7). Indeed, in October 2001, Mesri et al. (8) reported in the JCI that infection with a replication-deficient adenovirus encoding a Thr34→Ala mutant of survivin caused apoptosis in human tumor cell lines of different histology and reduced tumor growth in xenograft breast cancer models. Moreover, inhibition of survivin expression enhanced taxol-induced cell death in tumor cells. As an alternative strategy for survivin inhibition we developed hammerhead ribozymes targeting the 3′ end of the CUA110 (RZ1) and the GUC294 (RZ7) triplets in the survivin mRNA. In a cell-free system, both ribozymes induced cleavage of a synthetic RNA substrate obtained by cloning a portion of survivin mRNA, with cleavage products being detectable starting from a ribozyme/substrate ratio of 1:0.5. Conversely, the catalytically inactive mutRZ1 (which was produced by introducing a mutation in the catalytic core of the active ribozyme RZ1 and was used as control throughout the study) did not show any cleavage activity. RZ1, RZ7, and mutRZ1 sequences were inserted into the pRC expression vector under the control of the cytomega-lovirus promoter and transfected into the human metastatic melanoma cell line JR8 overexpressing survivin. For the present study we selected three stably transfected clones proven to endogenously express RZ1 (clone RZ1/C), RZ7 (clone RZ7/H), or mutRZ1 (clone mutRZ1/B). RZ1/C and RZ7/H cells were characterized by a markedly lower survivin protein level (68% and 60% lower, respectively) than JR8 parental cells, whereas a negligible reduction (13%) in survivin expression was observed in mutRZ1/B cells (Figure ​(Figure1a).1a). To evaluate the effect of survivin inhibition on the susceptibility of melanoma cells to undergo cisplatin-induced apoptosis, we treated the different clones with 10 μg/ml of the drug for 1 hour and determined the presence of apoptotic nuclei in cells stained with propidium iodide under fluorescence micro-scopy at 72 hours after treatment. A very modest apoptotic response was ob-served in the mutRZ1/B cells, whereas a significant increase in the percentage of apoptotic cells was observed in RZ1/C (P = 0.01) and RZ7/H (P = 0.005) cells (Figure ​(Figure1b).1b). Processing of caspase-3 to its active subunits of approximately 17 and 19 kDa was observed in all three drug-treated clones. However, the caspase-3 catalytic activity as assessed by hydrolysis of the fluorogenic substrate N-Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-AMC) was about threefold higher in RZ1/C and RZ7/H clones than in the mutRZ1/B clone (Figure ​(Figure11c). Figure 1 (a) Survivin protein expression in JR8 parental cells and melanoma cell clones transfected with the active ribozymes RZ1 (RZ1/C clone) and RZ7 (RZ7/H clone) or with the mutant ribozyme mutRZ1 (mutRZ1/B clone). Western blots were probed with a polyclonal ... These results are in agreement with the previous finding of Grossman et al. (9), who observed enhancement of cisplatin-induced apoptosis by expression of the survivin Thr34→Ala mutant in YUSAC2 melanoma cells. Unlike what was reported by these authors, attenuation of survivin expression in our melanoma cell system was not sufficient to appreciably trigger apoptosis in the absence of other stimuli. Other antiapoptotic factors besides survivin, such as bcl-2 and bcl-xL, are strongly expressed in JR8 cells and may contribute to preventing programmed cell death in this tumor model. However, it should be stressed that in JR8 cells survivin expression was attenuated but not completely abrogated. It may be that inhibition below a certain threshold is insufficient to determine a proapoptotic effect. Interestingly, and in accordance with such a hypothesis, when we transduced the human prostate cancer cells DU145 with a Moloney-based retroviral vector carrying the catalytic sequence of the ribozyme RZ7, we were able to select a ribozyme-expressing clone characterized by an almost complete abrogation of survivin expression (99.5% lower compared with control cells, as assessed by Western blotting, and lack of detectable protein expression by confocal microscopy). This ribozyme-expressing clone also showed a markedly higher percentage of apoptotic cells than control culture (20% and 3% of cells on the overall cell population, respectively). In conclusion, the present results obtained with the ribozyme-mediated approach in melanoma cells extend and corroborate earlier evidence indicating that attenuating survivin expression renders these cells more susceptible to cisplatin-induced apoptosis. These data also suggest a possible strategy to enhance the chemosensitivity profile of such a drug-refractory human malignancy.

Photochemical Internalization: A New Tool for Drug Delivery

The utilisation of macromolecules in the therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In many cases the targets of macromolecular therapeutics are intracellular. However, degradation of macromolecules in endocytic vesicles after uptake by endocytosis is a major intracellular barrier for the therapeutic application of macromolecules having intracellular targets of action. Photochemical internalisation (PCI) is a novel technology for the release of endocytosed macromolecules into the cytosol. The technology is based on the activation by light of photosensitizers located in endocytic vesicles to induce the release of macromolecules from the endocytic vesicles. Thereby, endocytosed molecules can be released to reach their target of action before being degraded in lysosomes. PCI has been shown to stimulate intracellular delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), DNA delivered as gene-encoding plasmids or by means of adenovirus or adeno-associated virus, peptide nucleic acids (PNAs) and chemotherapeutic agents such as bleomycin and in some cases doxorubicin. PCI of PNA may be of particular importance due to the low therapeutic efficacy of PNA in the absence of an efficient delivery technology and the 10-100-fold increased efficacy in combination with PCI. The efficacy and specificity of PCI of macromolecular therapeutics has been improved by combining the macromolecules with targeting moieties, such as the epidermal growth factor. In general, PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that it may have a variety of useful applications for site-specific drug delivery as for example in gene therapy, vaccination and cancer treatment.

Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which has been implicated in inhibition of apoptosis and control of mitotic progression. The finding that survivin is overexpressed in most human tumors but absent in normal adult tissues has led to the proposal of survivin as a promising therapeutic target for anticancer therapies. We decided to evaluate the effects of a ribozyme-based strategy for survivin inhibition in androgen-independent human prostate cancer cells. We constructed a Moloney-based retroviral vector expressing a ribozyme targeting the 3′ end of the CUA110 triplet in survivin mRNA, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations obtained by infection with the retroviral vector of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were selected for the study. Ribozyme-expressing prostate cancer cells were characterized by a significant reduction of survivin expression compared to parental cells transduced with a control ribozyme; the cells became polyploid, underwent caspase-9-dependent apoptosis and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of prostate cancer cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted in athymic nude mice. These findings suggest that manipulation of the antiapoptotic survivin pathway may provide a novel approach for the treatment of androgen-independent prostate cancer.

G-Quadruplex Structures in the Human Genome as Novel Therapeutic Targets

G-quadruplexes are secondary structures that may form within guanine-rich nucleic acid sequences. Telomeres have received much attention in this regard since they can fold into several distinct intramolecular G-quadruplexes, leading to the rational design and development of G-quadruplex-stabilizing molecules. These ligands were shown to selectively exert an antiproliferative and chemosensitizing activity in in vitro and in vivo tumor models, without appreciably affecting normal cells. Such findings point to them as possible drug candidates for clinical applications. Other than in telomeres, G-quadruplexes may form at additional locations in the human genome, including gene promoters and untranslated regions. For instance, stabilization of G-quadruplex structures within the promoter of MYC, KIT, or KRAS resulted in the down-regulation of the corresponding oncogene either in gene reporter assays or in selected experimental models. In addition, the alternative splicing of a number of genes may be affected for a therapeutic benefit through the stabilization of G-quadruplexes located within pre-mRNAs. It is now emerging that G-quadruplex structures may act as key regulators of several biological processes. Consequently, they are considered as attractive targets for broad-spectrum anticancer therapies, and much effort is being made to develop a variety of ligands with improved G-quadruplex recognition properties. Quarfloxin, a fluoroquinolone derivative designed to target a G-quadruplex within ribosomal DNA and disrupt protein-DNA interactions, has entered clinical trials for different malignancies. This review will provide some hints on the role of G-quadruplex structures in biological processes and will evaluate their implications as novel therapeutic targets.

Redox-Active Polymer Microcapsules for the Delivery of a Survivin-Specific siRNA in Prostate Cancer Cells

In this report, we describe the delivery of small interfering RNA (siRNA) using LbL-assembled microcapsules. The microcapsules are based on negatively charged poly(methacrylic acid) nanometer thin films containing cross-linking disulfide bonds. One system is polycation-free and another contains polylysine for siRNA complexation in the microcapsule void. When microcapsules containing a siRNA targeting survivin were delivered to PC-3 prostate cancer cells, a significant inhibition of the expression of the antiapoptotic protein was observed. However, down-regulation of survivin was also observed in PC-3 cells exposed to microcapsules embedded with a scrambled siRNA as well as in cells treated with empty microcapsules. These findings indicate a capsule-dependent off-target effect, which is supported by a reduction in the expression of other survivin-unrelated proteins. The microcapsules and their polymeric constituents do not affect cell proliferation, as determined by a metabolic assay, even after 4 days of exposure. In addition, in PC-3 cells exposed to microcapsules, we observed a marked accumulation of LC3b, a marker related to autophagy (i.e., self-digestion), a degradation pathway involved in the maintenance of cell homeostasis in response to different stresses. This evidence suggests that empty microcapsules can induce a perturbation of the intracellular environment, which causes the activation of a cell safeguard mechanism that may limit the therapeutic effect of the microcapsules in tumor cells.

Inhibition of telomerase activity by a cell‐penetrating peptide nucleic acid construct in human melanoma cells

We investigated the effect of two peptide nucleic acids (PNAs), which are complementary to the RNA component of human telomerase, on the catalytic activity of the enzyme. PNAs induced a dose‐dependent reduction of telomerase activity in cell extracts from human melanoma cell lines and surgical specimens. To down‐regulate telomerase in intact cells, we generated a chimeric molecule synthesized by coupling the 13‐mer PNA to the Antennapedia peptide. The PNA construct induced a dose‐ and time‐dependent inhibition of telomerase activity. However, a 20‐day exposure to the PNA construct only caused a slight increase in melanoma cell doubling time and failed to induce any telomere shortening.

MicroRNAs as new therapeutic targets and tools in cancer

Introduction: MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that negatively regulate gene expression at the post-transcriptional level. Several studies have provided evidence that abnormal expression of selected miRNAs is associated with the pathogenesis of cancer. As they can act as either oncogenes or tumor suppressors, miRNAs have been proposed as potential new therapeutic targets or tools for cancer therapy. Areas covered: This paper reviews a significant body of the experimental data collected to date which indicate that specific miRNA inhibition or replacement can successfully modify the proliferative and invasive properties of tumor cells. It discusses recent evidence that has also revealed a direct involvement of miRNAs in drug resistance, underlying an entirely new mechanism by which tumor cells may be refractory to the treatment with cytotoxic agents. Based on these findings, in the therapeutic setting, interference with cancer-specific miRNAs could be exploited not only to produce a direct anticancer effect but also to improve the response of tumor cells to conventional treatments. Expert opinion: Overall, manipulation of miRNA functions, either by mimicking or inhibiting them, is emerging as a highly promising therapeutic strategy. However, before miRNA-based therapeutics enters the clinical armamentarium, important issues concerning specific delivery to cells/tissues of interest, safety as well as pharmacokinetic profiles needs to be addressed.