Temporally resolved proteomics identifies nidogen-2 as a cotarget in pancreatic cancer that modulates fibrosis and therapy response.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC (Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+) and poorly metastatic KPflC (Pdx1-Cre, LSL-KrasG12D/+, Trp53fl/+) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

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.

Oral Squamous Cell Carcinoma in Young Patients Show Higher Rates of EGFR Amplification: Implications for Novel Personalized Therapy.

There is an increasing worldwide incidence of patients under 50 years of age presenting with oral squamous cell carcinoma (OSCC). The molecular mechanisms driving disease in this emerging cohort remain unclear, limiting impactful treatment options for these patients. To identify common clinically actionable targets in this cohort, we used whole genome and transcriptomic sequencing of OSCC patient samples from 26 individuals under 50 years of age. These molecular profiles were compared with those of OSCC patients over 50 years of age (n=11) available from TCGA. We show for the first time that a molecular signature comprising of EGFR amplification and increased EGFR RNA abundance is specific to the young subset of OSCC patients. Furthermore, through functional assays using patient tumor-derived cell lines, we reveal that this EGFR amplification results in increased activity of the EGFR pathway. Using a panel of clinically relevant EGFR inhibitors we determine that an EGFR-amplified patient-derived cell line is responsive to EGFR inhibition, suggesting EGFR amplification represents a valid therapeutic target in this subset of OSCC patients. In particular, we demonstrate sensitivity to the second-generation EGFR tyrosine kinase inhibitor afatinib, which offers a new and promising therapeutic avenue versus current EGFR-targeting approaches. We propose that testing for EGFR amplification could easily be integrated into current diagnostic workflows and such measures could lead to more personalized treatment approaches and improved outcomes for this younger cohort of OSCC patients.

TLR2 activation promotes tumour growth and associates with patient survival and chemotherapy response in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, and is plagued by a paucity of targeted treatment options and tumour resistance to chemotherapeutics. The causal link between chronic inflammation and PDAC suggests that molecular regulators of the immune system promote disease pathogenesis and/or therapeutic resistance, yet their identity is unclear. Here, we couple endoscopic ultrasound-guided fine-needle aspiration, which captures tumour biopsies from all stages, with whole transcriptome profiling of PDAC patient primary tumours to reveal enrichment of the innate immune Toll-like receptor 2 (TLR2) molecular pathway. Augmented TLR2 expression associated with a 4-gene "TLR2 activation" signature, and was prognostic for survival and predictive for gemcitabine-based chemoresistance. Furthermore, antibody-mediated anti-TLR2 therapy suppressed the growth of human PDAC tumour xenografts, independent of a functional immune system. Our results support TLR2-based therapeutic targeting for precision medicine in PDAC, with further clinical utility that TLR2 activation is prognostic and predictive for chemoresponsiveness.

Substrate-biased activity-based probes identify proteases that cleave receptor CDCP1.

CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

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 neighbors 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.

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.

Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer.

BACKGROUND: The ERG oncogene, a member of the ETS family of transcription factor encoding genes, is a genetic driver of prostate cancer. It is activated through a fusion with the androgen-responsive TMPRSS2 promoter in 50% of cases. There is therefore significant interest in developing novel therapeutic agents that target ERG. We have taken an antisense approach and designed morpholino-based oligonucleotides that target ERG by inducing skipping of its constitutive exon 4. METHODS: We designed antisense morpholino oligonucleotides (splice-switching oligonucleotides, SSOs) that target both the 5' and 3' splice sites of ERG's exon 4. We tested their efficacy in terms of inducing exon 4 skipping in two ERG-positive cell lines, VCaP prostate cancer cells and MG63 osteosarcoma cells. We measured their effect on cell proliferation, migration and apoptosis. We also tested their effect on xenograft tumour growth in mice and on ERG protein expression in a human prostate cancer radical prostatectomy sample ex vivo. RESULTS: In VCaP cells, both SSOs were effective at inducing exon 4 skipping, which resulted in a reduction of overall ERG protein levels up to 96 h following a single transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and significantly increased apoptosis. We observed a concomitant reduction in protein levels for cyclin D1, c-Myc and the Wnt signalling pathway member ß-catenin as well as a marker of activated Wnt signalling, p-LRP6. We tested the 3' splice site SSO in MG63 xenografts in mice and observed a reduction in tumour growth. We also demonstrated that the 3' splice site SSO caused a reduction in ERG expression in a patient-derived prostate tumour tissue cultured ex vivo. CONCLUSIONS: We have successfully designed and tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for oncogene targeting in vivo. As such, this study encourages further in vivo therapeutic studies using SSOs targeting the ERG oncogene.

Rho-ROCK Signaling in Normal Physiology and as a Key Player in Shaping the Tumor Microenvironment.

The Rho-ROCK signaling network has a range of specialized functions of key biological importance, including control of essential developmental processes such as morphogenesis and physiological processes including homeostasis, immunity, and wound healing. Deregulation of Rho-ROCK signaling actively contributes to multiple pathological conditions, and plays a major role in cancer development and progression. This dynamic network is critical in modulating the intricate communication between tumor cells, surrounding diverse stromal cells and the matrix, shaping the ever-changing microenvironment of aggressive tumors. In this chapter, we overview the complex regulation of the Rho-ROCK signaling axis, its role in health and disease, and analyze progress made with key approaches targeting the Rho-ROCK pathway for therapeutic benefit. Finally, we conclude by outlining likely future trends and key questions in the field of Rho-ROCK research, in particular surrounding Rho-ROCK signaling within the tumor microenvironment.

The Evolutionarily Conserved Cassette Exon 7b Drives ERG's Oncogenic Properties.

The oncogene ERG encodes an ETS family transcription factor and is implicated in blood, vascular, and bone development and in prostate, blood, and bone cancer. The ERG gene is alternatively spliced; of particular interest is its cassette exon 7b which adds 24 amino acids, in frame, to the transcriptional activation domain. Higher exon 7b inclusion rates are associated with increased cell proliferation and advanced prostate cancer. The 24 amino acids encoded by exon 7b show evolutionary conservation from humans to echinoderms, highlighting their functional importance. Throughout evolution, these 24 amino acids are encoded by a distinct short exon. Splice-switching oligonucleotides based on morpholino chemistry were designed to induce skipping of ERG exon 7b in MG63 osteosarcoma and VCaP prostate cancer cells. Induction of exon 7b skipping reduced cell proliferation and invasion, increased apoptosis in vitro, and reduced xenograft growth in vivo. We also show that ERG's exon 7b is required for the induction of tissue nonspecific alkaline phosphatase. Together, these findings show that the evolutionarily conserved cassette exon 7b is central to ERG's oncogenic properties.

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

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.

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

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.

Alternative Splicing in the Hippo Pathway-Implications for Disease and Potential Therapeutic Targets.

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.

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

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.

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

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 in vivo, suggesting that our in vitro 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.

YAP is essential for tissue tension to ensure vertebrate 3D body shape.

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

Essential techniques for introducing medaka to a zebrafish laboratory--towards the combined use of medaka and zebrafish for further genetic dissection of the function of the vertebrate genome.

The medaka, Oryzias latipes, a small egg-laying freshwater fish, is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens have been carried out. Despite a number of large-scale screens in zebrafish, a substantial number of mutants with new distinct phenotypes were identified in similar large-scale screens in the medaka. This observed difference in phenotype is due to the two species having a unique combination of genetic, biological and evolutional properties. The two genetic models share a whole-genome duplication event over that of tetrapods; however, each has independently specialized or lost the function of one of the two paralogues. The two fish species complement each other as genetic systems as straightforward comparison of phenotypes, ease of side-by-side analysis using the same techniques and simple and inexpensive husbandry of mutants make these small teleosts quite powerful in combination. Furthermore, both have draft genome sequences and bioinformatic tools available that facilitate further genetic dissection including whole-genome approaches. Together with the gene-driven approach to generate gene knockout mutants of the fish models, the two fish models complement the mouse in genetically dissecting vertebrate genome functions. The external embryogenesis and transparent embryos of the fish allow systematic isolation of embryonic lethal mutations, the most difficult targets in mammalian mutant screens. This chapter will describe how to work with both medaka and zebrafish almost as one species in a lab, focusing on medaka and highlighting the differences between the medaka and zebrafish systems.

Insufficiency of BUBR1, a mitotic spindle checkpoint regulator, causes impaired ciliogenesis in vertebrates.

Budding uninhibited by benzimidazole-related 1 (BUBR1) is a central molecule of the spindle assembly checkpoint. Germline mutations in the budding uninhibited by benzimidazoles 1 homolog beta gene encoding BUBR1 cause premature chromatid separation (mosaic variegated aneuploidy) [PCS (MVA)] syndrome, which is characterized by constitutional aneuploidy and a high risk of childhood cancer. Patients with the syndrome often develop Dandy-Walker complex and polycystic kidneys; implying a critical role of BUBR1 in morphogenesis. However, little is known about the function of BUBR1 other than mitotic control. Here, we report that BUBR1 is essential for the primary cilium formation, and that the PCS (MVA) syndrome is thus a novel ciliopathy. Morpholino knockdown of bubr1 in medaka fish also caused ciliary dysfunction characterized by defects in cerebellar development and perturbed left-right asymmetry of the embryo. Biochemical analyses demonstrated that BUBR1 is required for ubiquitin-mediated proteasomal degradation of cell division cycle protein 20 in the G0 phase and maintains anaphase-promoting complex/cyclosome-CDC20 homolog 1 activity that regulates the optimal level of dishevelled for ciliogenesis.