Pro-tumorigenic role of lnc-ZNF30-3 as a sponge counteracting miR-145-5p in prostate cancer.

BACKGROUND: Prostate cancer remains one of the deadliest neoplasms in developed countries. Identification of new molecular markers that predict the onset and progression of the disease could improve its clinical management. Low miR-145-5p expression is consistently found in primary tumors and metastases, but the regulatory mechanisms governing its functions remain largely unknown. METHODS: Bioinformatics analysis was conducted to identify [1] a set of novel potential competing endogenous lncRNAs for sponging of miRNA-145-5p in prostate cancer and [2] miR-145-5p and other EMT-related miRNAs response elements in lnc-ZNF30-3. Quantification of miR-145-5p, lnc-ZNF30-3, and TWIST1 expression levels in tumor tissues in RNA sequencing datasets of our and TCGA PRAD cohorts revealed a correlation with clinical outcome of prostate cancer patients. Biochemical and cell biology approaches, such as RNA pull-down, western blot, immunostaining, and wound healing assays were used for evaluation of the impact of TWIST1/miR-145/ lnc-ZNF30-3 interactions in prostate cancer cells altered in miRNA and lncRNA expression. RESULTS: We identified a few potential lncRNA sponges of miR-145-5p, including lnc-ZNF30-3. It contains five response elements for miR-145-5p, but also other miRNAs targeting EMT transcription factors. Lnc-ZNF30-3 is significantly upregulated in prostate cancer cell lines and tumor tissues, and its high expression is correlated with poor patient prognosis. We demonstrated that lnc-ZNF30-3 is associated with AGO2 and specifically interacts with the miR-145-5p seed region. Knockdown of lnc-ZNF30-3 results in decreased migration of prostate cancer cells and downregulation of EMT drivers such as TWIST1 and ZEB1 at both the RNA and protein levels. These phenotypic and molecular features of lnc-ZNF30-3-depleted cells are partially rescued by miR-145-5p inhibition. CONCLUSIONS: Collectively, our results point to lnc-ZNF30-3 as a novel competing endogenous lncRNA for miR-145-5p and other miRNAs that target TWIST1 as well as other EMT transcription factors. Prostate cancer patients with high lncRNA expression in primary tumors show lower survival rate suggesting that lnc-ZNF30-3 may contribute to prostate cancer progression and metastasis.

CPEB1 orchestrates a fine-tuning of miR-145-5p tumor-suppressive activity on TWIST1 translation in prostate cancer cells.

TWIST1 is a basic helix-loop-helix transcription factor, and one of the master Epithelial-to-Mesenchymal Transition (EMT) regulators. We show that tumor suppressor miR-145-5p controls TWIST1 expression in an immortalized prostate epithelial cell line and in a tumorigenic prostate cancer-derived cell line. Indeed, shRNA-mediated miR-145-5p silencing enhanced TWIST1 expression and induced EMT-associated malignant properties in these cells. However, we discovered that the translational inhibitory effect of miR-145-5p on TWIST1 is lost in 22Rv1, another prostate cancer cell line that intrinsically expresses high levels of the CPEB1 cytoplasmic polyadenylation element binding protein. This translational regulator typically reduces TWIST1 translation efficiency by shortening the TWIST1 mRNA polyA tail. However, our results indicate that the presence of CPEB1 also interferes with the binding of miR-145-5p to the TWIST1 mRNA 3'UTR. Mechanistically, CPEB1 binding to its first cognate site either directly hampers the access to the miR-145-5p response element or redirects the cleavage/polyadenylation machinery to an intermediate polyadenylation site, resulting in the elimination of the miR-145-5p binding site. Taken together, our data support the notion that the tumor suppressive activity of miR-145-5p on TWIST1 translation, consequently on EMT, self-renewal, and migration, depends on the CPEB1 expression status of the cancer cell. A preliminary prospective study using clinical samples suggests that reconsidering the relative status of miR-145-5p/TWIST1 and CPEB1 in the tumors of prostate cancer patients may bear prognostic value.

A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase.

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.

The WEE1 regulators CPEB1 and miR-15b switch from inhibitor to activators at G2/M.

MicroRNAs (miRNAs) in the AGO-containing RISC complex control messenger RNA (mRNA) translation by binding to mRNA 3' untranslated region (3'UTR). The relationship between miRNAs and other regulatory factors that also bind to mRNA 3'UTR, such as CPEB1 (cytoplasmic polyadenylation element-binding protein), remains elusive. We found that both CPEB1 and miR-15b control the expression of WEE1, a key mammalian cell cycle regulator. Together, they repress WEE1 protein expression during G1 and S-phase. Interestingly, the 2 factors lose their inhibitory activity at the G2/M transition, at the time of the cell cycle when WEE1 expression is maximal, and, moreover, rather activate WEE1 translation in a synergistic manner. Our data show that translational regulation by RISC and CPEB1 is essential in cell cycle control and, most importantly, is coordinated, and can be switched from inhibition to activation during the cell cycle.

CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27(Kip1) mRNA translation.

The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3' untranslated regions (3'UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27(Kip1) by specifically targeting its 3'UTR, and competes with miR-221/222 binding at an overlapping site in the 3'UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27(Kip1) 3'UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27(Kip1) mRNA. This leads to higher levels of p27(Kip1) in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma.

Role of 3'-untranslated region translational control in cancer development, diagnostics and treatment.

The messenger RNA 3'-untranslated region (3'UTR) plays an important role in regulation of gene expression on the posttranscriptional level. The 3'UTR controls gene expression via orchestrated interaction between the structural components of mRNAs (cis-element) and the specific trans-acting factors (RNA binding proteins and non-coding RNAs). The crosstalk of these factors is based on the binding sequences and/or direct protein-protein interaction, or just functional interaction. Much new evidence that has accumulated supports the idea that several RNA binding factors can bind to common mRNA targets: to the non-overlapping binding sites or to common sites in a competitive fashion. Various factors capable of binding to the same RNA can cooperate or be antagonistic in their actions. The outcome of the collective function of all factors bound to the same mRNA 3'UTR depends on many circumstances, such as their expression levels, affinity to the binding sites, and localization in the cell, which can be controlled by various physiological conditions. Moreover, the functional and/or physical interactions of the factors binding to 3'UTR can change the character of their actions. These interactions vary during the cell cycle and in response to changing physiological conditions. Abnormal functioning of the factors can lead to disease. In this review we will discuss how alterations of these factors or their interaction can affect cancer development and promote or enhance the malignant phenotype of cancer cells. Understanding these alterations and their impact on 3'UTR-directed posttranscriptional gene regulation will uncover promising new targets for therapeutic intervention and diagnostics. We will also discuss emerging new tools in cancer diagnostics and therapy based on 3'UTR binding factors and approaches to improve them.

Control of cellular senescence by CPEB.

Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16(INK4A); however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence.

Translational control of the embryonic cell cycle.

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells.