111 research outputs found
Revealing Novel Regulators of Wildtype and Mutant p53 Stability
No full textTumour suppressor p53 (TP53) is the most frequently mutated gene in cancer. Several hotspot p53 mutants not only lose tumour suppressive capabilities, but also function in a dominant-negative manner, suppressing canonical wild-type p53 functions. Furthermore, some hotspot p53 mutants promote oncogenesis by gain-of-function mechanisms. Levels of p53 are regulated predominantly through regulation of protein stability and while wild-type p53 is normally kept at very low levels at steady-state, p53 mutants are often stabilized in tumours, which is vital for their oncogenic properties. In this thesis, I systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. I found that most proteins that regulate wild-type p53 also regulate a subset of p53 mutants with the exception of p53 R337H regulators, which are largely private to this mutant. Mechanistically, I identified FBXO42 as a novel positive regulator of a subset of p53 mutants comprising R273H, R248Q and R248W. I showed that FBXO42 acts together with CCDC6 to regulate USP28-mediated p53 stabilization. Work in this thesis also identified C16orf72 as a negative regulator of the stability of wild-type and all p53 mutants tested, and this regulation is dependent on the activity of the E3 ligase HUWE1. C16orf72 is amplified in breast cancer, and I showed that C16orf72 regulates p53 levels in mammary epithelium of mice, and its overexpression resulted in accelerated breast cancer formation with reduced p53 levels. Taken together, this thesis provides a network view of the processes that regulate p53 stability, offering insights for reinforcing wild-type p53 function or targeting mutant p53 activity in cancer.
Le suppresseur de tumeurs p53 (TP53) est le gène le plus fréquemment muté dans le cancer. Plusieurs mutations p53 récurrentes entraînent non seulement une perte des capacités de suppression des tumeurs, mais elles fonctionnent également de manière dominant-négative, réprimant les fonctions canoniques du p53 de type sauvage. De plus, certaines mutations p53 récurrentes favorisent l'oncogenèse par des mécanismes de gain de fonction. Les niveaux de p53 sont principalement régulés par la stabilité des protéines, et alors que le p53 de type sauvage est normalement maintenu à des niveaux très bas à l'état stationnaire, les mutations p53 sont souvent stabilisées dans les tumeurs, ce qui est essentiel pour leurs propriétés oncogènes. Dans cette thèse, j'ai systématiquement profilé les facteurs qui régulent la stabilité des protéines du p53 de type sauvage et des mutants en utilisant des cribles CRISPR à l'échelle du génome basés sur des marqueurs. J'ai constaté que la plupart des protéines qui régulent le p53 de type sauvage régulent également un sous-ensemble des mutants du p53, à l'exception des régulateurs du p53 R337H, qui sont largement spécifiques à cette mutation. Sur le plan mécanistique, j'ai identifié FBXO42 en tant que nouveau régulateur positif d'un sous-ensemble de mutants p53, comprenant R273H, R248Q et R248W. J'ai montré que FBXO42 agit en collaboration avec CCDC6 pour réguler la stabilisation du p53 médiée par USP28. Le travail de cette thèse a également identifié C16orf72 comme un régulateur négatif de la stabilité de la p53 sauvage et de tous les mutants de p53 testés, et cette régulation dépend de l'activité de l'E3 ligase HUWE1. C16orf72 est amplifié dans le cancer du sein, et j'ai montré que C16orf72 régule les niveaux de p53 dans l'épithélium mammaire des souris, et sa surexpression a entraîné une formation accélérée de cancer du sein avec des niveaux réduits de p53. Dans l'ensemble, cette thèse offre une vision en réseau des processus qui régulent la stabilité du p53, offrant des informations pour renforcer la fonction du p53 de type sauvage ou cibler l'activité des mutants du p53 dans le cancer.Ph.D
Establishing an In-vivo Model of Low-grade Glioma
No full textGliomas account for 80% of malignant central nervous system cancers and current therapies have shown limited success in treating this disease1. Characterization of the mutational landscape of these tumours has led to the identification genetic low grade glioma (LGG) subtypes within adults2 ,3,4. These are largely defined by neomorphic mutation in the genes coding for isocitrate dehydrogenase (IDH1 or IDH2), presenting in both IDH mutant astrocytoma and IDH mutant oligodendroglioma. However, these subtypes differ with respect to their mutational landscapes and clinical manifestations, with IDH mutant astrocytoma having a worse prognosis and limited therapeutic options2,3. IDH mutant oligodendroglioma harbor truncal mutations in IDH and the TERT promoter as well as co-deletion of chromosomal arms 1p and 19q. IDH mutant astrocytoma is associated with truncal mutations in IDH, TP53, and ATRX2. In addition to these canonical LGG mutations, the rs55705857 single nucleotide polymorphism (SNP) has been shown to increase the risk of IDH-mutant LGG by an odds ratio of 6 and 9 within IDH mutant astrocytoma and IDH mutant oligodendroglioma respectively1-4, however the mechanism through which this SNP confers increased LGG risk has been elusive.I have generated mouse models for IDH mutant astrocytoma using a strategy combining CRISPR gene-editing with Cre-recombinase technologies to precisely model the mutations believed to initiate LGG pathogenesis, as well as bred two rs55705857 mutant alleles into these models. Our IDH mutant astrocytoma model develops LGG-like tumours in brain tissue with a penetrance of 30% at a latency of 463 days, while the addition of mutant rs55705857 elevates tumour penetrance to ~75% while decreasing latency to 172 days. In collaboration with the Jenkins Lab at the Mayo Clinic, I have uncovered one of the mechanisms through which the rs55705857 risk allele enhances gliomagenesis. We demonstrated that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding and thus prevents OCT2/4 mediated repression of the MYC promoter, inducing increased MYC expression. Lastly, I’ve developed and executed an in-vivo CRISPR and ORF screens to identify the genes such as Notch1 and Pten that drive malignant progression of LGG into deadly glioblastoma (GBM).Ph.D
In vivo Screening Reveals Epigenetic Regulation of Mammary Epithelial Lineage Integrity and Tumorigenesis
No full textSystematic investigation of the scores of genes mutated in cancer to discern disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, I developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 genes that are recurrently mutated in ~2-10% of human breast cancers but lacked thorough study. Screen results revealed epigenetic regulation as a major tumor suppressive mechanism. I identified components of the COMPASS-like and PR-DUB complexes, including KDM6A, ASXL1/2, BAP1 and KMT2C, function as epigenetic tumor suppressor genes (EpiDrivers) that cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, I found that activation of PIK3CAH1047R and concomitant loss of EpiDrivers triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors. Mutations in EpiDrivers are found in ~39% of human breast cancers and casein expression can be seen in ~50% of ductal-carcinoma-in-situ, suggesting that lineage infidelity and alveologenic mimicry may significantly contribute to early steps of breast cancer etiology.Ph.D
Comprehensive Characterization of Tumor-Initiating Cells (TICs) in Head and Neck Squamous Cell Carcinomas (HNSCC)
No full textHead and Neck Squamous Cell Carcinomas (HNSCC) being the sixth most common cancer worldwide and a five-year survival rate of less than 50%, are a major health problem. The low survival rate is attributed to the relapse at the primary site or distal metastasis. Tumor-initiating cells (TIC), a subset of tumor cells with stem cell-like properties, are implicated in metastatic dissemination and relapse. Prognosis of patients with recurrent and metastatic HNSCC is typically unfavorable, attributed to failure of conventional therapies and limited treatment alternatives available at advanced disease stage. Several studies have identified TICs in HNSCC tumors. However, a comprehensive epigenetic and transcriptomic profiling of human HNSCC TICs has not been achieved. We hypothesized that such characterization will increase our understanding of the TIC biology and aid in identification of therapeutic targets against these cells. In this study, we demonstrated through analysis of fourteen primary HNSCC patient tumors, that the epigenetic and transcriptomic profiles of TICs were distinct from non-TIC tumor cells and normal keratinocytes (KT). A quantitative analysis of epigenetic data showed the presence of variable regulatory elements (VREs) in TICs compared to KT. A subset of these VREs were recurrently upregulated or downregulated in multiple (10 or more) independent TIC samples and were termed recurrent VREs (rVREs). Motif analysis of upregulated rVREs revealed that they were significantly enriched for binding sites for AP-1 and TP63 transcription factors when compared to non-recurrent VREs and background genomic regions. Next, we employed chromatin captured conformation (3C) based experiments to identify the gene promoters that might be interacting with rVREs. 5,916 genes were assigned to upregulated rVREs whereas 9,684 genes were assigned to downregulated rVREs. Functional genetic CRISPR and CRISPRi screens were performed to investigate the role of upregulated rVREs and upregulated rVRE-associated genes in TICs biology. We also performed single-cell RNA sequencing experiments to define the heterogeneity within the TIC population. We showed that TICs are a heterogeneous mix of proliferating and quiescent cells. Quiescent TICs in HNSCC have not been reported before. By integrating the data generated from multiple omics approaches we hope to advance the understanding of TIC biology and contribute to improvement of HNSCC outcomes.Ph.D
Abstract 4766: Translation from unconventional 5' start sites drives tumor initiation
No full textAbstract
How translational control impacts tumor-initiation and malignancy is just beginning to unfold. Here, we devise an epidermis-specific, in vivo ribosome profiling strategy to interrogate the translational landscape during the transition from normal homeostasis to malignancy. Inducing SOX2, broadly expressed in oncogenic RAS-associated cancers, we find that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. Seeking mechanism, we find that during tumor-initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing translational efficiency of oncogenic mRNAs. An in vivo RNAi screen of translational regulators revealed that dampening conventional EIF2 complexes has dire consequences for normal but not oncogenic growth. Conversely, we identify alternative initiation factors essential for cancer progression, where they mediate initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a hitherto unappreciated role of 5’UTR translation in cancer, and expose new targets for therapeutic intervention.
Citation Format: Ataman Sendoel, Joshua G. Dunn, Edwin H. Rodriguez, Shruti Naik, Nicholas C. Gomez, Brian Hurwitz, John Levorse, Brian D. Dill, Daniel Schramek, Henrik Molina, Jonathan S. Weissman, Elaine Fuchs. Translation from unconventional 5' start sites drives tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4766. doi:10.1158/1538-7445.AM2017-4766</jats:p
Disruption of STAT3 signaling promotes KRAS induced lung tumorigenesis
No full textSTAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased KrasG12D-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3–NF-κB–IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance.Beatrice Grabner, Daniel Schramek, Kristina M. Mueller, Herwig P. Moll, Jasmin Svinka, Thomas Hoffmann, Eva Bauer, Leander Blaas, Natascha Hruschka, Katalin Zboray, Patricia Stiedl, Harini Nivarthi, Edith Bogner, Wolfgang Gruber, Thomas Mohr, Ralf Harun Zwick, Lukas Kenner, Valeria Poli, Fritz Aberger, Dagmar Stoiber, Gerda Egger, Harald Esterbauer, Johannes Zuber, Richard Moriggl, Robert Eferl, Balázs Györffy, Josef M. Penninger, Helmut Popper & Emilio Casanov
In vivo CRISPR screens reveal potent driver mutations in head and neck cancers
No full textWe have recently tested the transforming potential of 484 ‘long-tail’ genes, which are recurrently albeit infrequently mutated in head and neck cancers (HNSCC). We identified 15 novel tumor suppressors and our top hits converge on regulating the NOTCH signaling pathway. Therapeutic approaches activating NOTCH signaling could be a promising strategy to treat two-thirds of human HNSCC patients
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