Marzia Pennati

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.

Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer

Abstract. Stable transfection of human ovarian carcinoma cells with survivin cDNA caused a four- to sixfold increase in cell resistance to taxotere and taxol (two-sided Students t test, p <0.05), with a concomitant reduction in the apoptotic response to taxol, but did not affect cell sensitivity to cisplatin or oxaliplatin. Such findings were indirectly supported by similar observations obtained with clinical tumours. In fact, high levels of survivin protein expression (>30% positive cells), detected by immunohistochemistry in 90/124 (73%) advanced ovarian carcinomas, were significantly associated with clinical resistance to a taxol/platinum-based regimen but unrelated to tumour shrinkage following cisplatin-including combinations (non-taxol based). In the 95 patients receiving a taxol/platinum-based regimen, survivin overexpression correlated with a lower clinical or pathologic complete remission rate than absent/low protein expression (43 vs 75%, p = 0.0058 by logistic regression adjusted for tumour stage, histological grade and p53 expression). Conversely, in the 29 cases treated with cisplatin-containing regimens (not taxol based), survivin expression was unrelated to tumour response. Cellular studies and clinical data suggest a direct link between survivin expression and tumour cell susceptibility to taxol.

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.

Pazopanib in advanced and platinum-resistant urothelial cancer: an open-label, single group, phase 2 trial

BACKGROUND The development of new drugs for patients with refractory urothelial cancer is still an unmet medical need. Preclinical evidence lends support to a rationale for targeting of the VEGF or platelet-derived growth-factor axis. We therefore investigated the activity and safety of pazopanib, a multitarget drug with antiangiogenic activity, in patients with urothelial cancer. METHODS In an open-label, single-group, phase 2 study, patients (aged ≥18 years) with relapsed or refractory urothelial cancer were given pazopanib 800 mg per day, orally. They were treated until disease progression or prohibitive toxicity occurred. The primary endpoint was the proportion of patients who achieved a confirmed objective response, defined as complete or partial response, after independent review, and was analysed by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT01031875. FINDINGS The trial has been completed. 21 (51%) of 41 patients enrolled were given pazopanib as third-line or further-line treatment. 26 (63%) patients had an Eastern Cooperative Oncology Group performance status of 1 or 2. Seven patients had a confirmed objective response (17·1%, 95% CI 7·2-32·1), all of which were partial responses. The most frequent treatment-related grade 3 adverse events were hypertension (three [7%]), fatigue (two [5%]), and gastrointestinal and vaginal fistulisations (two each [5%]). One patient died as a result of duodenal fistulisation that was related to tissue response of bulky tumour masses. INTERPRETATION Pazopanib has single-agent activity in patients with heavily pretreated metastatic urothelial cancer, and warrants further study in this setting. Particular attention should be paid to patients with bulky tumour masses adjacent to viscera because fistulisation is probably related to the response to pazopanib and is the most frequent serious adverse event. FUNDING Fondazione IRCCS Istituto Nazionale dei Tumori provided the grant. GlaxoSmithKline provided the study drug and provided funding for the independent radiological review.

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.

Silencing of survivin gene by small interfering RNAs produces supra-additive growth suppression in combination with 17-allylamino-17-demethoxygeldanamycin in human prostate cancer cells

Survivin is an antiapoptotic gene, which is overexpressed in most human tumors and involved in mitotic checkpoint control. Recent evidence points to an essential role for heat shock protein 90 (Hsp90) in survivin function regulation. Although the survivin-Hsp90 association may promote tumor cell proliferation, it may also suggest new opportunities for the design of novel anticancer approaches. We evaluated the effect of small interfering RNA (siRNA)–mediated inhibition of survivin on the proliferative potential of prostate cancer cells and their sensitivity to the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Human androgen-independent prostate cancer cell lines (DU145 and PC-3) were transfected with four 21-mer double-stranded siRNAs (100 nmol/L) directed against different portions of survivin mRNA. After transfection, cells were collected and analyzed for survivin mRNA and protein expression, cell proliferation rate, ability to undergo apoptosis, and sensitivity to 17-AAG. Transfection of prostate cancer cells with siRNAs induced a variable extent of inhibition of survivin mRNA expression (39–60% compared with controls), which was paralleled by a 38% to 75% reduction in survivin protein abundance. The three siRNAs able to induce the greatest inhibition of survivin expression also significantly reduced cell proliferation and enhanced the rate of apoptosis, with a concomitant increase in caspase-9 activity. Sequential treatment with siRNA and 17-AAG induced supra-additive antiproliferative effects in all cell lines, with an enhanced caspase-9-dependent apoptotic response. These findings suggest that combined strategies aimed at interfering with the survivin-Hsp90 connection may provide novel approaches for treatment of androgen-independent prostate cancer. [Mol Cancer Ther 2006;5(1):179–86]

Potentiation of paclitaxel-induced apoptosis by the novel cyclin-dependent kinase inhibitor NU6140: a possible role for survivin down-regulation

Cyclin-dependent kinases (CDK) play a crucial role in the control of the cell cycle. Aberrations in the control of cell cycle progression occur in the majority of human malignancies; hence, CDKs are promising targets for anticancer therapy. Here, we define the cellular effects of the novel CDK inhibitor NU6140, alone or in association with paclitaxel, with respect to inhibition of cell proliferation and cell cycle progression and induction of apoptosis in HeLa cervical carcinoma cells and in comparison with purvalanol A. Both CDK inhibitors induced a concentration-dependent cell cycle arrest at the G2-M phase and an increase in the apoptotic rate, with a concomitant down-regulation of the antiapoptotic protein survivin, a member of the inhibitors of apoptosis protein family. Notably, the addition of NU6140 to paclitaxel-treated cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with the paclitaxel-purvalanol A combination (86 ± 11% and 37 ± 8%, respectively). Similarly, the extent of caspase-9 and caspase-3 activation in paclitaxel-NU6140–treated cells was ∼4-fold higher than after the paclitaxel-purvalanol A combination. Moreover, an almost complete abrogation of the expression of the active, Thr34-phosphorylated form of survivin was observed in cells exposed to the paclitaxel-NU6140 combination. A synergistic effect of the paclitaxel-NU6140 combination, as a consequence of survivin inhibition and increased activation of caspase-9 and caspase-3, was also observed in OAW42/e ovarian cancer line but not in the derived OAW42/Surv subline ectopically expressing survivin. Results from this study indicate that NU6140 significantly potentiates the apoptotic effect of paclitaxel, with inhibition of survivin expression/phosphorylation as the potential mechanism.

Novel 1H-Pyrrolo[2,3-b]pyridine Derivative Nortopsentin Analogues: Synthesis and Antitumor Activity in Peritoneal Mesothelioma Experimental Models

In this study, we describe the synthesis of new nortopsentin analogues, 1H-pyrrolo[2,3-b]pyridine derivatives and their biological effects in experimental models of diffuse malignant peritoneal mesothelioma (DMPM), a rare and rapidly fatal disease, poorly responsive to conventional therapies. The three most active compounds, 1f (3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine), 3f (3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine), and 1l (3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-b] pyridine), which were shown to act as cyclin-dependent kinase 1 inhibitors, consistently reduced DMPM cell proliferation and induced a caspase-dependent apoptotic response, with a concomitant reduction of the expression of the active Thr(34)-phosphorylated form of the antiapoptotic protein survivin. Moreover, the combined treatment of DMPM cells with 3f derivative and paclitaxel produced a synergistic cytotoxic effect, which was paralleled by an enhanced apoptotic response. In the mouse model, i.p. administration of 1f, 3f, and 1l derivatives was effective, resulting in a significant tumor volume inhibition of DMPM xenografts (range, 58-75%) at well-tolerated doses, and two complete responses were observed in each treatment group.