A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes

Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems

A feedback loop between plakophilin 4 and YAP signaling regulates keratinocyte differentiation

The Hippo signaling pathway is an important regulator of organ growth and differentiation, and its deregulation contributes to the development of cancer. The activity of its downstream targets YAP/TAZ depends on adherens junctions. Plakophilin 4 (PKP4) is a cell-type specific adherens junction protein expressed in the proliferating cells of the epidermis. Here, we show that PKP4 diminishes proliferation as well as differentiation. Depletion of PKP4 increased proliferation but at the same time induced premature epidermal differentiation. PKP4 interacted with several Hippo pathway components, including the transcriptional co-activators YAP/TAZ, and promoted nuclear YAP localization and target gene expression. In differentiated keratinocytes, PKP4 recruited LATS and YAP to cell junctions where YAP is transcriptionally inactive. YAP depletion, on the other hand, reduced PKP4 levels and keratinocyte adhesion indicative of a feedback mechanism controlling adhesion, proliferation, and differentiation by balancing YAP functions

The dark side of the epitranscriptome : chemical modifications in long non-coding RNAs

The broad application of next-generation sequencing technologies in conjunction with improved bioinformatics has helped to illuminate the complexity of the transcriptome, both in terms of quantity and variety. In humans, 70–90% of the genome is transcribed, but only ~2% carries the blueprint for proteins. Hence, there is a huge class of non-translated transcripts, called long non-coding RNAs (lncRNAs), which have received much attention in the past decade. Several studies have shown that lncRNAs are involved in a plethora of cellular signaling pathways and actively regulate gene expression via a broad selection of molecular mechanisms. Only recently, sequencing-based, transcriptome-wide studies have characterized different types of post-transcriptional chemical modifications of RNAs. These modifications have been shown to affect the fate of RNA and further expand the variety of the transcriptome. However, our understanding of their biological function, especially in the context of lncRNAs, is still in its infancy. In this review, we will focus on three epitranscriptomic marks, namely pseudouridine (Ψ), N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We will introduce writers, readers, and erasers of these modifications, and we will present methods for their detection. Finally, we will provide insights into the distribution and function of these chemical modifications in selected, cancer-related lncRNAs

Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells.

This is the peer reviewed version of the following article: Yacqub-Usman, K., Pickard, M. R., & Williams, G. T. (2015). Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells. Prostate, 75(7), 693-705. DOI: 10.1002/pros.22952, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/pros.22952/abstract;jsessionid=B9892067CCD9082275B1EFFFC59926F6.f02t02. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-ArchivingBACKGROUND: New therapies are required for castrate-resistant prostate cancer (CRPC), and growth-arrest specific 5 (GAS5) lncRNA, which riborepresses androgen receptor action, may offer novel opportunities in this regard. This lncRNA promotes the apoptosis of prostate cancer cells and its levels decline as prostate cancer cells acquire castrate-resistance, so that enhancing GAS5 expression may improve the effectiveness of chemotherapies. Since GAS5 is a member of the 5' terminal oligopyrimidine gene family, we have examined mTOR inhibition as a strategy to increase GAS5 expression. Furthermore, we have determined if GAS5 itself mediates the action of mTOR inhibitors, as demonstrated for other chemotherapeutic agents in prostate cancer cells. METHODS: The effects of mTOR inhibitors on GAS5 lncRNA levels and cell growth were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNAs and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. RESULTS: First generation mTORC1, combined mTORC1/mTORC2 and dual PI3K/mTOR inhibitors all increased cellular GAS5 levels and inhibited culture growth in androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU 145) cell lines. The latter exhibited low endogenous GAS5 expression, and GAS5 silencing in LNCaP and 22Rv1 cells decreased the sensitivity to mTOR inhibitors, whereas transfection of GAS5 lncRNA sensitized PC-3 and DU 145 cells to these agents. CONCLUSION: mTOR inhibition enhances GAS5 transcript levels in certain prostate cancer cell lines. This selectivity is likely to be related to endogenous GAS5 expression levels, since GAS5 lncRNA is itself required for mTOR inhibitor action in prostate cancer cells.Prostate Cancer Collaborative, United Kingdom (PCUK

Function and clinical implications of long non-coding RNAs in melanoma

Metastatic melanoma is the most deadly type of skin cancer. Despite the success of immunotherapy and targeted agents, the majority of patients experience disease recurrence upon treatment and die due to their disease. Long non-coding RNAs (lncRNAs) are a new subclass of non-protein coding RNAs involved in (epigenetic) regulation of cell growth, invasion, and other important cellular functions. Consequently, recent research activities focused on the discovery of these lncRNAs in a broad spectrum of human diseases, especially cancer. Additional efforts have been undertaken to dissect the underlying molecular mechanisms employed by lncRNAs. In this review, we will summarize the growing evidence of deregulated lncRNA expression in melanoma, which is linked to tumor growth and progression. Moreover, we will highlight specific molecular pathways and modes of action for some well-studied lncRNAs and discuss their potential clinical implications

RNA-binding proteins as regulators of migration, invasion and metastasis in oral squamous cell carcinoma

Nearly 7.5% of all human protein-coding genes have been assigned to the class of RNA-binding proteins (RBPs), and over the past decade, RBPs have been increasingly recognized as important regulators of molecular and cellular homeostasis. RBPs regulate the post-transcriptional processing of their target RNAs, i.e., alternative splicing, polyadenylation, stability and turnover, localization, or translation as well as editing and chemical modification, thereby tuning gene expression programs of diverse cellular processes such as cell survival and malignant spread. Importantly, metastases are the major cause of cancer-associated deaths in general, and particularly in oral cancers, which account for 2% of the global cancer mortality. However, the roles and architecture of RBPs and RBP-controlled expression networks during the diverse steps of the metastatic cascade are only incompletely understood. In this review, we will offer a brief overview about RBPs and their general contribution to post-transcriptional regulation of gene expression. Subsequently, we will highlight selected examples of RBPs that have been shown to play a role in oral cancer cell migration, invasion, and metastasis. Last but not least, we will present targeting strategies that have been developed to interfere with the function of some of these RBPsPublikationsfond ML

NANOS1 restricts oral cancer cell motility and TGF-ß signaling

Oral squamous cell carcinoma (OSCC) is the most frequent type of cancer of the head and neck area accounting for approx. 377,000 new cancer cases every year. The epithelial-to-mesenchymal transition (EMT) program plays an important role in OSCC progression and metastasis therefore contributing to a poor prognosis in patients with advanced disease. Transforming growth factor beta (TGF-ß) is a powerful inducer of EMT thereby increasing cancer cell aggressiveness. Here, we aimed at identifying RNA-binding proteins (RBPs) that affect TGF-ß-induced EMT. To this end we treated oral cancer cells with TGF-ß and identified a total of 643 significantly deregulated protein-coding genes in response to TGF-ß. Of note, 19 genes encoded RBPs with NANOS1 being the most downregulated RBP. Subsequent cellular studies demonstrated a strong inhibitory effect of NANOS1 on migration and invasion of SAS oral cancer cells. Further mechanistic studies revealed an interaction of NANOS1 with the TGF-ß receptor 1 (TGFBR1) mRNA, leading to increased decay of this transcript and a reduced TGFBR1 protein expression, thereby preventing downstream TGF-ß/SMAD signaling. In summary, we identified NANOS1 as negative regulator of TGF-ß signaling in oral cancer cells