Alternative splicing in the hippo pathway-implications for disease and potential therapeutic targets

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Alternative splicing is a well-studied gene regulatory mechanism that produces biological diversity by allowing the production of multiple protein isoforms from a single gene. An involvement of alternative splicing in the key biological signalling Hippo pathway is emerging and offers new therapeutic avenues. This review discusses examples of alternative splicing in the Hippo pathway, how deregulation of these processes may contribute to disease and whether these processes offer new potential therapeutic targets

Autoregulation of the human splice factor kinase CLK1 through exon skipping and intron retention

© 2018 Elsevier B.V. Alternative splicing is a key process required for the regulation of gene expression in normal development and physiology. It is regulated by splice factors whose activities are in turn regulated by splice factor kinases and phosphatases. The CDC-like protein kinases are a widespread family of splice factor kinases involved in normal physiology and in several diseases including cancer. In humans they include the CLK1, CLK2, CLK3 and CLK4 genes. The expression of CLK1 is regulated through alternative splicing producing both full-length catalytically active and truncated catalytically inactive isoforms, CLKT1 (arising from exon 4 skipping) and CLKT2 (arising from intron 4 retention). We examined CLK1 alternative splicing in a range of cancer cell lines, and report widespread and highly variable rates of exon 4 skipping and intron 4 retention. We also examined the effect of severe environmental stress including heat shock, osmotic shock, and exposure to the alkaloid drug harmine on CLK1 alternative splicing in DU145 prostate cancer cells. All treatments rapidly reduced exon 4 skipping and intron 4 retention, shifting the balance towards full-length CLK1 expression. We also found that the inhibition of CLK1 with the benzothiazole TG003 reduced exon 4 skipping and intron 4 retention suggesting an autoregulatory mechanism. CLK1 inhibition with TG003 also resulted in modified alternative splicing of five cancer-associated genes

SPHINX-based combination therapy as a potential novel treatment strategy for acute myeloid leukaemia

Introduction: Dysregulated alternative splicing is a prominent feature of cancer. The inhibition and knockdown of the SR splice factor kinase SRPK1 reduces tumour growth in vivo. As a result several SPRK1 inhibitors are in development including SPHINX, a 3-(trifluoromethyl)anilide scaffold. The objective of this study was to treat two leukaemic cell lines with SPHINX in combination with the established cancer drugs azacitidine and imatinib. Materials and Methods: We selected two representative cell lines; Kasumi-1, acute myeloid leukaemia, and K562, BCR-ABL positive chronic myeloid leukaemia. Cells were treated with SPHINX concentrations up to 10μM, and in combination with azacitidine (up to 1.5 μg/ml, Kasumi-1 cells) and imatinib (up to 20 μg/ml, K562 cells). Cell viability was determined by counting the proportion of live cells and those undergoing apoptosis through the detection of activated caspase 3/7. SRPK1 was knocked down with siRNA to confirm SPHINX results. Results: The effects of SPHINX were first confirmed by observing reduced levels of phosphorylated SR proteins. SPHINX significantly reduced cell viability and increased apoptosis in Kasumi-1 cells, but less prominently in K562 cells. Knockdown of SRPK1 by RNA interference similarly reduced cell viability. Combining SPHINX with azacitidine augmented the effect of azacitidine in Kasumi-1 cells. In conclusion, SPHINX reduces cell viability and increases apoptosis in the acute myeloid leukaemia cell line Kasumi-1, but less convincingly in the chronic myeloid leukaemia cell line K562. Conclusion: We suggest that specific types of leukaemia may present an opportunity for the development of SRPK1-targeted therapies to be used in combination with established chemotherapeutic drugs

The oncogenic transcription factor ERG represses the transcription of the tumour suppressor gene PTEN in prostate cancer cells

© 2017, Spandidos Publications. All rights reserved. The oncogene ETS-related gene (ERG) encodes a transcription factor with roles in the regulation of haematopoiesis, angiogenesis, vasculogenesis, inflammation, migration and invasion. The ERG oncogene is activated in >50% of prostate cancer cases, generally through a gene fusion with the androgen-responsive promoter of transmembrane protease serine 2. Phosphatase and tensin homologue (PTEN) is an important tumour suppressor gene that is often inactivated in cancer. ERG overexpression combined with PTEN inactivation or loss is often associated with aggressive prostate cancer. The present study aimed to determine whether or not ERG regulates PTEN transcription directly. ERG was demonstrated to bind to the PTEN promoter and repress its transcription. ERG overexpression reduced endogenous PTEN expression, whereas ERG knockdown increased PTEN expression. The ability of ERG to repress PTEN may contribute to a more cancer-permissive environment

WT1 activates transcription of the splice factor kinase SRPK1 gene in PC3 and K562 cancer cells in the absence of corepressor BASP1

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF165b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth

CDC2-like (CLK) protein kinase inhibition as a novel targeted therapeutic strategy in prostate cancer

Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer

EPHA2-dependent outcompetition of KRASG12D mutant cells by wild-type neigbors in the adult pancreas

As we age, our tissues are repeatedly challenged by mutational insult, yet cancer occurrence is a relatively rare event. Cells carrying cancer-causing genetic mutations compete with normal neighbors for space and survival in tissues. However, the mechanisms underlying mutant-normal competition in adult tissues and the relevance of this process to cancer remain incompletely understood. Here, we investigate how the adult pancreas maintains tissue health in vivo following sporadic expression of oncogenic Kras (KrasG12D), the key driver mutation in human pancreatic cancer. We find that when present in tissues in low numbers, KrasG12D mutant cells are outcompeted and cleared from exocrine and endocrine compartments in vivo. Using quantitative 3D tissue imaging, we show that before being cleared, KrasG12D cells lose cell volume, pack into round clusters, and E-cadherin-based cell-cell adhesions decrease at boundaries with normal neighbors. We identify EphA2 receptor as an essential signal in the clearance of KrasG12D cells from exocrine and endocrine tissues in vivo. In the absence of functional EphA2, KrasG12D cells do not alter cell volume or shape, E-cadherin-based cell-cell adhesions increase and KrasG12D cells are retained in tissues. The retention of KRasG12D cells leads to the early appearance of premalignant pancreatic intraepithelial neoplasia (PanINs) in tissues. Our data show that adult pancreas tissues remodel to clear KrasG12D cells and maintain tissue health. This study provides evidence to support a conserved functional role of EphA2 in Ras-driven cell competition in epithelial tissues and suggests that EphA2 is a novel tumor suppressor in pancreatic cancer

EphA2 Drives the Segregation of Ras-Transformed Epithelial Cells from Normal Neighbors

© 2016 Elsevier Ltd In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells) are detected by normal neighbors and as a result are often extruded from the tissue [1–6]. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton [2, 6]. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. Here we identify differential EphA2 signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. We also show that Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells invivo, suggesting that our invitro findings are conserved in evolution. We propose that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium

Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells

© 2018 The Author(s). Background: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. Methods: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. Results: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by >25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. Conclusions: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy