Strategies for surviving down-regulation : effects on tumour cell growth potential and chemosensitivity profile

Survivin is a bifunctional protein that acts as a suppressor of apoptosis and plays a central role in cell division. The protein is strongly expressed in the most common human neoplasms, has prognostic relevance for some of them, and appears to be involved in tumour cell resistance to anticancer agents and ionizing radiation. On the basis of these findings, survivin has been proposed as an attractive target for new anticancer interventions. We generated a hammerhead ribozyme (Rz) targeting the CUA110 triplet in the survivin mRNA and transfected them into the JR8 human melanoma cell line. Cells endogenously expressing Rz were characterized by a lower survivin protein level than parental cells, and showed an increased caspase-9-dependent apoptotic response to treatment with the cytotoxic agents cisplatin and topotecan as well as with ɣ-irradiation. Moreover, an increased anti-tumour activity of oral topotecan was observed in Rz-expressing cells grown as xenograft tumours in athymic nude mice. In addition, we constructed a Moloney-based retroviral vector expressing Rz, 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 characterized by a significant reduction of survivin expression; the cells became polyploid and underwent caspase-9-dependent apoptosis. Survivin inhibition also enhanced their susceptibility to cisplatin-induced apoptosis and prevented tumour formation when cells were xenografted into athymic nude mice. Again we used RNAi to specifically repress survivin in DU145 and PC-3 cell lines. RNAi-mediated survivin knock-down was able to significantly reduce cell proliferation and to enhance the rate of caspase-9-dependent apoptosis. Moreover, sequential treatment with survivin-specific siRNA followed by the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin produced supra-additive anti-proliferative effects in both cell lines. Finally, we investigated the effects of the novel cdk inhibitor NU6140, in term of ability to potentiate the response to paclitaxel in HeLa cells, in relation to its interference with survivin. Sequential administration of cdk inhibitors resulted in escape from the mitotic block imposed by paclitaxel and significantly increased the apoptotic rate, with inhibition of survivin expression/phosphorylation as the potential mechanism. Overall, such results suggest that strategies aimed at interfering with survivin expression/activity can be adopted to improve the chemo/radio-sensitivity profile of treatment-refractory human malignancies

miR-21: an oncomir on strike in prostate cancer

<p>Abstract</p> <p>Background</p> <p>Aberrant 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. <it>PTEN</it>) 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 <it>PTEN </it>gene status.</p> <p>Results</p> <p>We provide evidence that miR-21 knockdown in prostate cancer cells is not sufficient <it>per se </it>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.</p> <p>Conclusions</p> <p>Overall, 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.</p

Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands

Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 æ from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, cochaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary

Ligand selection from the analysis of protein conformational substates: new leads targeting the N-terminal domain of Hsp90

The dynamic properties of proteins underlie every aspect of their functions in the cell. The atomistic description of protein motions and their inclusion in ligand selection processes may provide new opportunities for hit identification and drug discovery. Herein, we present a novel rational strategy that allowed us to computationally select new N-terminal targeted inhibitors of the molecular chaperone Hsp90 starting from the atomistic analysis of the conformational dynamics of the complex between the protein and its natural ligand ATP. First of all, we have identified the relevant representatives of distinct conformational substates of the Hsp90-ATP complex through the application of a novel structural clustering strategy and, for each of them, we have afterwards characterized the nucleotide-protein interactions to build a pharmacophore model recapitulating the binding hotspots conserved in different ensembles of protein conformations. The resulting pharmacophore has been finally used to screen a database of small molecules and allowed us to identify novel drug-like molecules with interesting activities against Hsp90 functions in experimental models of cancer cells. The results and the experimental validation of the selected molecules provide support for the feasibility of including protein flexibility in drug selection strategies through the characterization of relevant substates

Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/MDM2 pathway in HeLa cells

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation