Karim Malik

Autolysosomal β-catenin degradation regulates Wnt-autophagy-p62 crosstalk

The Wnt/β‐catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/β‐catenin signalling pathway as a negative regulator of both basal and stress‐induced autophagy. Manipulation of β‐catenin expression levels in vitro and in vivo revealed that β‐catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show that during nutrient deprivation β‐catenin is selectively degraded via the formation of a β‐catenin–LC3 complex, attenuating β‐catenin/TCF‐driven transcription and proliferation to favour adaptation during metabolic stress. Formation of the β‐catenin–LC3 complex is mediated by a W/YXXI/L motif and LC3‐interacting region (LIR) in β‐catenin, which is required for interaction with LC3 and non‐proteasomal degradation of β‐catenin. Thus, Wnt/β‐catenin represses autophagy and p62 expression, while β‐catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction. These findings reveal a regulatory feedback mechanism that place β‐catenin at a key cellular integration point coordinating proliferation with autophagy, with implications for targeting these pathways for cancer therapy.

A Novel Wilms Tumor 1 (WT1) Target Gene Negatively Regulates the WNT Signaling Pathway

Mammalian kidney development requires the functions of the Wilms tumor gene WT1 and the WNT/β-catenin signaling pathway. Recent studies have shown that WT1 negatively regulates WNT/β-catenin signaling, but the molecular mechanisms by which WT1 inhibits WNT/β-catenin signaling are not completely understood. In this study, we identified a gene, CXXC5, which we have renamed WID (WT1-induced Inhibitor of Dishevelled), as a novel WT1 transcriptional target that negatively regulates WNT/β-catenin signaling. WT1 activates WID transcription through the upstream enhancer region. In the developing kidney, Wid and Wt1 are coexpressed in podocytes of maturing nephrons. Structure-function analysis demonstrated that WID interacts with Dishevelled via its C-terminal CXXC zinc finger and Dishevelled binding domains and potently inhibits WNT/β-catenin signaling in vitro and in vivo. WID is evolutionarily conserved, and ablation of wid in zebrafish embryos with antisense morpholino oligonucleotides perturbs embryonic kidney development. Taken together, our results demonstrate that the WT1 negatively regulates WNT/β-catenin pathway via its target gene WID and further suggest a role for WID in nephrogenesis.

Long-range epigenetic silencing of chromosome 5q31 protocadherins is involved in early and late stages of colorectal tumorigenesis through modulation of oncogenic pathways

Loss of tumour suppressor gene function can occur as a result of epigenetic silencing of large chromosomal regions, referred to as long-range epigenetic silencing (LRES), and genome-wide analyses have revealed that LRES is present in many cancer types. Here we utilize Illumina Beadchip methylation array analysis to identify LRES across 800 kb of chromosome 5q31 in colorectal adenomas and carcinomas (n=34) relative to normal colonic epithelial DNA (n=6). This region encompasses 53 individual protocadherin (PCDH) genes divided among three gene clusters. Hypermethylation within these gene clusters is asynchronous; while most PCDH hypermethylation occurs early, and is apparent in adenomas, PCDHGC3 promoter methylation occurs later in the adenoma–carcinoma transition. PCDHGC3 was hypermethylated in 17/28 carcinomas (60.7%) according to methylation array analysis. Quantitative real-time reverse transcription–polymerase chain reaction showed that PCDHGC3 is the highest expressed PCDH in normal colonic epithelium, and that there was a strong reciprocal relationship between PCDHGC3 methylation and expression in carcinomas (R=−0.84). PCDH LRES patterns are reflected in colorectal tumour cell lines; adenoma cell lines are not methylated at PCDHGC3 and show abundant expression at the mRNA and protein level, while the expression is suppressed in hypermethylated carcinoma cell lines (R=−0.73). Short-interfering RNA-mediated reduction of PCDHGC3 led to a decrease of apoptosis in RG/C2 adenoma cells, and overexpression of PCDHGC3 in HCT116 cells resulted in the reduction of colony formation, consistent with tumour suppressor capabilities for PCDHGC3. Further functional analysis showed that PCDHGC3 can suppress Wnt and mammalian target of rapamycin signalling in colorectal cancer cell lines. Taken together, our data suggest that the PCDH LRES is an important tumour suppressor locus in colorectal cancer, and that PCDHGC3 may be a strong marker and driver for the adenoma–carcinoma transition.

Autoregulation of the human WT1 gene promoter

The human Wilms tumour suppressor gene, WT1, encodes a zinc‐finger protein which can function as a transcriptional activator or suppressor. This study reports the analysis of the human WT1 gene promoter, and demonstrates that high levels of WT1 expression lead to autosuppression of the WT1 promoter. Deletion analyses of the promoter region implicate sequences 5′ and 3′ of the transcriptional start site as being crucial in WT1 autosuppression. Loss or alteration of this function of WT1 may be important in tumourigenesis.

Loss of heterozygosity at 7p in Wilms’ tumour development

Chromosome 7p alterations have been implicated in the development of Wilms’ tumour (WT) by previous studies of tumour cytogenetics, and by our analysis of a constitutional translocation (t(1;7)(q42;p15)) in a child with WT and radial aplasia. We therefore used polymorphic microsatellite markers on 7p for a loss of heterozygosity (LOH) study, and found LOH in seven out of 77 informative WTs (9%). The common region of LOH was 7p15–7p22, which contains the region disrupted by the t(1;7) breakpoint. Four WTs with 7p LOH had other genetic changes; a germline WT1 mutation with 11p LOH, LOH at 11p, LOH at 16q, and loss of imprinting of IGF2. Analysis of three tumour-associated lesions from 7p LOH cases revealed a cystic nephroma-like area also having 7p LOH. However, a nephrogenic rest and a contralateral WT from the two other cases showed no 7p LOH. No particular clinical phenotype was associated with the WTs which showed 7p LOH. The frequency and pattern of 7p LOH demonstrated in our studies indicate the presence of a tumour suppressor gene at 7p involved in the development of Wilms’ tumour.

Epigenetic gene deregulation in cancer

A mini-review of the literature concerning epigenetic gene regulation in cancer.

The retinoblastoma protein interacts with Bag-1 in human colonic adenoma and carcinoma derived cell lines

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide range of human tumours, overexpression in colonic carcinomas has been linked to the antiapoptotic function of the protein. In the current study we show that the Retinoblastoma susceptibility protein (Rb) protein interacts with Bag‐1, an apoptotic regulator, in human colonic adenoma‐ and carcinoma‐derived cell lines. Coimmunoprecipitation demonstrated that endogenous Rb and Bag‐1 interact in both adenoma‐ and carcinoma‐derived cell lines. The specificity of the interaction was demonstrated by expression of human Papillomavirus E7 oncoprotein, an inhibitor of Rb protein interactions, which disrupted the Rb/Bag‐1 complex. We report that Bag‐1 is predominantly localised in the nucleus of colorectal adenoma‐ and carcinoma‐derived epithelial cells. Disruption of the Rb/Bag‐1 complex through expression of E7 changes the subcellular distribution of Bag‐1, decreasing nuclear localised Bag‐1. Our work establishes that the Rb protein interacts with the Bag‐1 apoptotic regulator protein, and introduces a novel function for Rb, involving modulation of the subcellular localisation of Bag‐1 in human colonic epithelial cells.

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.

Glycogen synthase kinase-3 controls IL-10 expression in CD4 + effector T-cell subsets through epigenetic modification of the IL-10 promoter

The serine/threonine kinase glycogen synthase kinase‐3 (GSK3) plays an important role in balancing pro‐ and anti‐inflammatory cytokines. We have examined the role of GSK3 in production of IL‐10 by subsets of CD4+ T helper cells. Treatment of naive murine CD4+ T cells with GSK3 inhibitors did not affect their production of IL‐10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL‐10. GSK3 inhibition also led to upregulation of IL‐10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL‐10‐dependent mechanism. Analysis of the murine IL‐10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c‐Maf, Nfil3, and GATA3, correlating with the increase in IL‐10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease‐causing cells through GSK3 inhibition.

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over‐expression. Here we present data showing that short‐interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell‐lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN‐negative SH‐SY5Y NB cell‐line, or in two immortalized human fibroblast cell‐lines. Immunoblotting of NB cell‐lines shows that high PRMT5 expression is strongly associated with MYCN‐amplification (P < 0.004, Mann–Whitney U‐test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN‐amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN‐overexpressing cells, including the SHEP‐21N cell‐line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN‐associated cell‐death in SHEP‐21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post‐transcriptional level. Reciprocal co‐immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK‐N‐BE(2)C and NGP cell lines. By using liquid chromatography – tandem mass spectrometry (LC‐MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post‐translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small‐molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.