miR-205 regulates basement membrane deposition in human prostate: implications for cancer development.

The basement membrane (BM) is a layer of specialized extracellular matrix that surrounds normal prostate glands and preserves tissue integrity. Lack or discontinuity of the BM is a prerequisite for tumor cell invasion into interstitial spaces, thus favoring metastasis. Therefore, BM maintenance represents a barrier against cancer development and progression. In the study, we show that miR-205 participates in a network involving ΔNp63α, which is essential for maintenance of the BM in prostate epithelium. At the molecular level, ΔNp63α is able to enhance miR-205 transcription by binding to its promoter, whereas the microRNA can post-transcriptionally limit the amount of ΔNp63α protein, mostly by affecting ΔNp63α proteasomal degradation rather than through a canonical miRNA/target interaction. Functionally, miR-205 is able to control the deposition of laminin-332 and its receptor integrin-ß4. Hence, pathological loss of miR-205, as widely observed in prostate cancer, may favor tumorigenesis by creating discontinuities in the BM. Here we demonstrate that therapeutic replacement of miR-205 in prostate cancer (PCa) cells can restore BM deposition and 3D organization into normal-like acinar structures, thus hampering cancer progression.

Apollon gene silencing induces apoptosis in breast cancer cells through p53 stabilisation and caspase-3 activation.

We analysed the effects of small interfering RNA (siRNA)-mediated silencing of Apollon, a member of the inhibitors of apoptosis protein family, on the proliferative potential and ability of human breast cancer cell lines to undergo apoptosis. In wild-type p53 ZR75.1 cells, Apollon knockdown resulted in a marked, time-dependent decline of cell growth and an increased rate of apoptosis, which was associated with p53 stabilisation and activation of the mitochondrial-dependent apoptotic pathway. Pre-incubation of cells with a p53-specific siRNA resulted in a partial rescue of cell growth inhibition, as well as in a marked reduction of the apoptotic response, indicating p53 as a major player in cell growth impairment consequent on Apollon silencing. Apollon knockdown induced consistently less pronounced anti-proliferative and pro-apoptotic effects in mutant p53 MDA-MB-231 cells than in ZR75.1 cells. Furthermore, the activation of caspase-3 seemed to be essential for the induction of apoptosis after Apollon knockdown, as the Apollon-specific siRNA had no effect on the viability of caspase-3-deficient, wild-type p53 MCF-7 cells or the ZR75.1 cells after RNA interference-mediated caspase-3 silencing. Our results indicate that p53 stabilisation and caspase-3 activation concur to determine the apoptotic response mediated by Apollon knockdown in breast cancer cells, and suggest Apollon to be a potential new therapeutic target for this malignancy.

Photochemically enhanced delivery of a cell-penetrating peptide nucleic acid conjugate targeting human telomerase reverse transcriptase: effects on telomere status and proliferative potential of human prostate cancer cells.

OBJECTIVES: Peptide nucleic acids (PNAs) are DNA mimics that have been demonstrated to be efficient antisense/antigene tools in cell-free systems. However, their potential as in vivo regulators of gene expression has been hampered by their poor uptake by living cells, and strategies need to be developed for their intracellular delivery. This study has aimed to demonstrate the possibility (i) of efficiently delivering a PNA, which targets mRNA of the catalytic component of human telomerase reverse transcriptase (hTERT), into DU145 prostate cancer cells through a combined approach based on conjugation of the PNA to Tat internalizing peptide (hTERT-PNA-Tat) and subsequent photochemical internalization, and (ii) to interfere with telomerase function. MATERIALS AND METHODS: Treated cells were analysed for telomerase activity, hTERT expression, growth rate, ability to undergo apoptosis and telomere status. RESULTS: After exposure to light, DU145 cells treated with hTERT-PNA-Tat and the photosensitiser TPPS2a showed dose-dependent inhibition of telomerase activity, which was accompanied by marked reduction of hTERT protein expression. A dose-dependent decline in DU145 cell population growth and induction of caspase-dependent apoptosis were also observed from 48 h after treatment. Such an antiproliferative effect was associated with the presence of telomeric dysfunction, as revealed by cytogenetic analysis, in the absence of telomere shrinkage, and with induction of DNA damage response as suggested by the increased expression of gamma-H2AX. CONCLUSIONS: Our results (i) indicate photochemical internalization as an efficient approach for intracellular delivery of chimaeric PNAs, and (ii) corroborate earlier evidence suggesting pro-survival and anti-apoptotic roles of hTERT, which are independent of its ability to maintain telomere length.

Oligomer-mediated modulation of hTERT alternative splicing induces telomerase inhibition and cell growth decline in human prostate cancer cells.

The expression of telomerase in human cells is strictly controlled by multiple mechanisms including transcription and alternative splicing of telomerase reverse transcriptase (hTERT). In this study, we demonstrated the possibility of modulating the hTERT splicing pattern in DU145 human prostate carcinoma cells through the use of 2'-O-methyl-RNA phosphorothioate oligonucleotides targeting the splicing site located between intron 5 and exon 6 in the hTERT pre-mRNA. An 18-h oligonucleotide exposure induced a decrease in the full-length hTERT transcript and a concomitant increase in the alternatively spliced transcripts, which resulted in significant inhibition of telomerase catalytic activity. Moreover, exposure to the R7 oligomer (which induced the most pronounced modulation of the hTERT splicing pattern and the greatest telomerase inhibition) caused a marked reduction in DU145 cell growth and the induction of apoptosis starting 2 days after treatment. Such data support the concept that down-regulation of hTERT expression can cause short-term effects on tumour cell growth, which are telomere-shortening independent.