Hongwu Zheng

p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation.

Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53-/- Pten-/-) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53-/- Pten-/- TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP–SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis

The PI3K-AKT-FoxO pathway is integral to lifespan regulation in lower organisms and essential for the stability of long-lived cells in mammals. Here, we report the impact of combined FoxO1, 3, and 4 deficiencies on mammalian brain physiology with a particular emphasis on the study of the neural stem/progenitor cell (NSC) pool. We show that the FoxO family plays a prominent role in NSC proliferation and renewal. FoxO-deficient mice show initial increased brain size and proliferation of neural progenitor cells during early postnatal life, followed by precocious significant decline in the NSC pool and accompanying neurogenesis in adult brains. Mechanistically, integrated transcriptomic, promoter, and functional analyses of FoxO-deficient NSC cultures identified direct gene targets with known links to the regulation of human brain size and the control of cellular proliferation, differentiation, and oxidative defense. Thus, the FoxO family coordinately regulates diverse genes and pathways to govern key aspects of NSC homeostasis in the mammalian brain.

PLAGL2 Regulates Wnt Signaling to Impede Differentiation in Neural Stem Cells and Gliomas

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state, which contributes to its plasticity and therapeutic resistance. Here, integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells.

Genomic alterations link Rho family of GTPases to the highly invasive phenotype of pancreas cancer

Pancreas ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically presents as advanced, unresectable disease. This invasive tendency, coupled with intrinsic resistance to standard therapies and genome instability, are major contributors to poor long-term survival. The genetic elements governing the invasive propensity of PDAC have not been well elucidated. Here, in the course of validating resident genes in highly recurrent and focal amplifications in PDAC, we have identified Rio Kinase 3 (RIOK3) as an amplified gene that alters cytoskeletal architecture as well as promotes pancreatic ductal cell migration and invasion. We determined that RIOK3 promotes its invasive activities through activation of the small G protein, Rac. This genomic and functional link to Rac signaling prompted a genome wide survey of other components of the Rho family network, revealing p21 Activated Kinase 4 (PAK4) as another amplified gene in PDAC tumors and cell lines. Like RIOK3, PAK4 promotes pancreas ductal cell motility and invasion. Together, the genomic and functional profiles establish the Rho family GTP-binding proteins as integral to the hallmark invasive nature of this lethal disease.

Feedback circuit among INK4 tumor suppressors constrains human glioblastoma development.

Summary We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.

Pten Is a Major Tumor Suppressor in Pancreatic Ductal Adenocarcinoma and Regulates an NF-κB-Cytokine Network

Initiation of pancreatic ductal adenocarcinoma (PDAC) is driven by oncogenic KRAS mutation, and disease progression is associated with frequent loss of tumor suppressors. In this study, human PDAC genome analyses revealed frequent deletion of the PTEN gene as well as loss of expression in primary tumor specimens. A potential role for PTEN as a haploinsufficient tumor suppressor is further supported by mouse genetic studies. The mouse PDAC driven by oncogenic Kras mutation and Pten deficiency also sustains spontaneous extinction of Ink4a expression and shows prometastatic capacity. Unbiased transcriptomic analyses established that combined oncogenic Kras and Pten loss promotes marked NF-κB activation and its cytokine network, with accompanying robust stromal activation and immune cell infiltration with known tumor-promoting properties. Thus, PTEN/phosphoinositide 3-kinase (PI3K) pathway alteration is a common event in PDAC development and functions in part to strongly activate the NF-κB network, which may serve to shape the PDAC tumor microenvironment.

FoxOs Enforce a Progression Checkpoint to Constrain mTORC1-Activated Renal Tumorigenesis

mTORC1 is a validated therapeutic target for renal cell carcinoma (RCC). Here, analysis of Tsc1-deficient (mTORC1 hyperactivation) mice uncovered a FoxO-dependent negative feedback circuit constraining mTORC1-mediated renal tumorigenesis. We document robust FoxO activation in Tsc1-deficient benign polycystic kidneys and FoxO extinction on progression to murine renal tumors; murine renal tumor progression on genetic deletion of both Tsc1 and FoxOs; and downregulated FoxO expression in most human renal clear cell and papillary carcinomas, yet continued expression in less aggressive RCCs and benign renal tumor subtypes. Mechanistically, integrated analyses revealed that FoxO-mediated block operates via suppression of Myc through upregulation of the Myc antagonists, Mxi1-SRα and mir-145, establishing a FoxO-Mxi1-SRα/mir-145 axis as a major progression block in renal tumor development.

Mig-6 controls EGFR trafficking and suppresses gliomagenesis

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

microRNA Regulatory Network Inference Identifies miR-34a as a Novel Regulator of TGF-β Signaling in Glioblastoma

UNLABELLED Leveraging The Cancer Genome Atlas (TCGA) multidimensional data in glioblastoma, we inferred the putative regulatory network between microRNA and mRNA using the Context Likelihood of Relatedness modeling algorithm. Interrogation of the network in context of defined molecular subtypes identified 8 microRNAs with a strong discriminatory potential between proneural and mesenchymal subtypes. Integrative in silico analyses, a functional genetic screen, and experimental validation identified miR-34a as a tumor suppressor in proneural subtype glioblastoma. Mechanistically, in addition to its direct regulation of platelet-derived growth factor receptor-alpha (PDGFRA), promoter enrichment analysis of context likelihood of relatedness-inferred mRNA nodes established miR-34a as a novel regulator of a SMAD4 transcriptional network. Clinically, miR-34a expression level is shown to be prognostic, where miR-34a low-expressing glioblastomas exhibited better overall survival. This work illustrates the potential of comprehensive multidimensional cancer genomic data combined with computational and experimental models in enabling mechanistic exploration of relationships among different genetic elements across the genome space in cancer. SIGNIFICANCE We illustrate here that network modeling of complex multidimensional cancer genomic data can generate a framework in which to explore the biology of cancers, leading to discovery of new pathogenetic insights as well as potential prognostic biomarkers. Specifically in glioblastoma, within the context of the global network, promoter enrichment analysis of network edges uncovered a novel regulation of TGF-β signaling via a Smad4 transcriptomic network by miR-34a.