A general calculus of fitness landscapes finds genes under selection in cancers.

Genetic variants drive the evolution of traits and diseases. We previously modeled these variants as small displacements in fitness landscapes and estimated their functional impact by differentiating the evolutionary relationship between genotype and phenotype. Conversely, here we integrate these derivatives to identify genes steering specific traits. Over cancer cohorts, integration identified 460 likely tumor-driving genes. Many have literature and experimental support but had eluded prior genomic searches for positive selection in tumors. Beyond providing cancer insights, these results introduce a general calculus of evolution to quantify the genotype-phenotype relationship and discover genes associated with complex traits and diseases.

EPIMUTESTR: a nearest neighbor machine learning approach to predict cancer driver genes from the evolutionary action of coding variants.

Discovering rare cancer driver genes is difficult because their mutational frequency is too low for statistical detection by computational methods. EPIMUTESTR is an integrative nearest-neighbor machine learning algorithm that identifies such marginal genes by modeling the fitness of their mutations with the phylogenetic Evolutionary Action (EA) score. Over cohorts of sequenced patients from The Cancer Genome Atlas representing 33 tumor types, EPIMUTESTR detected 214 previously inferred cancer driver genes and 137 new candidates never identified computationally before of which seven genes are supported in the COSMIC Cancer Gene Census. EPIMUTESTR achieved better robustness and specificity than existing methods in a number of benchmark methods and datasets.

Literature-based automated discovery of tumor suppressor p53 phosphorylation and inhibition by NEK2.

Scientific progress depends on formulating testable hypotheses informed by the literature. In many domains, however, this model is strained because the number of research papers exceeds human readability. Here, we developed computational assistance to analyze the biomedical literature by reading PubMed abstracts to suggest new hypotheses. The approach was tested experimentally on the tumor suppressor p53 by ranking its most likely kinases, based on all available abstracts. Many of the best-ranked kinases were found to bind and phosphorylate p53 (P value = 0.005), suggesting six likely p53 kinases so far. One of these, NEK2, was studied in detail. A known mitosis promoter, NEK2 was shown to phosphorylate p53 at Ser315 in vitro and in vivo and to functionally inhibit p53. These bona fide validations of text-based predictions of p53 phosphorylation, and the discovery of an inhibitory p53 kinase of pharmaceutical interest, suggest that automated reasoning using a large body of literature can generate valuable molecular hypotheses and has the potential to accelerate scientific discovery.

miR-130b directly targets ARHGAP1 to drive activation of a metastatic CDC42-PAK1-AP1 positive feedback loop in Ewing sarcoma.

Ewing Sarcoma (ES) is a highly aggressive bone tumor with peak incidence in the adolescent population. It has a high propensity to metastasize, which is associated with dismal survival rates of approximately 25%. To further understand mechanisms of metastasis we investigated microRNA regulatory networks in ES. Our studies focused on miR-130b due to our analysis that enhanced expression of this microRNA has clinical relevance in multiple sarcomas, including ES. Our studies provide insights into a novel positive feedback network involving the direct regulation of miR-130b and activation of downstream signaling events contributing toward sarcoma metastasis. Specifically, we demonstrated miR-130b induces proliferation, invasion, and migration in vitro and increased metastatic potential in vivo. Using microarray analysis of ES cells with differential miR-130b expression we identified alterations in downstream signaling cascades including activation of the CDC42 pathway. We identified ARHGAP1, which is a negative regulator of CDC42, as a novel, direct target of miR-130b. In turn, downstream activation of PAK1 activated the JNK and AP-1 cascades and downstream transcriptional targets including IL-8, MMP1 and CCND1. Furthermore, chromatin immunoprecipitation of endogenous AP-1 in ES cells demonstrated direct binding to an upstream consensus binding site within the miR-130b promoter. Finally, small molecule inhibition of PAK1 blocked miR-130b activation of JNK and downstream AP-1 target genes, including primary miR-130b transcripts, and miR-130b oncogenic properties, thus identifying PAK1 as a novel therapeutic target for ES. Taken together, our findings identify and characterize a novel, targetable miR-130b regulatory network that promotes ES metastasis.

Pla2g16 phospholipase mediates gain-of-function activities of mutant p53.

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis.

Secreted Frizzled-Related Protein 2 (sFRP2) promotes osteosarcoma invasion and metastatic potential.

BACKGROUND: Osteosarcoma (OS), which has a high potential for developing metastatic disease, is the most frequent malignant bone tumor in children and adolescents. Molecular analysis of a metastatic genetically engineered mouse model of osteosarcoma identified enhanced expression of Secreted Frizzled-Related Protein 2 (sFRP2), a putative regulator of Wnt signaling within metastatic tumors. Subsequent analysis correlated increased expression in the human disease, and within highly metastatic OS cells. However, the role of sFRP2 in osteosarcoma development and progression has not been well elucidated. METHODS: Studies using stable gain or loss-of-function alterations of sFRP2 within human and mouse OS cells were performed to assess changes in cell proliferation, migration, and invasive ability in vitro, via both transwell and 3D matrigel assays. In additional, xenograft studies using overexpression of sFRP2 were used to assess effects on in vivo metastatic potential. RESULTS: Functional studies revealed stable overexpression of sFRP2 within localized human and mouse OS cells significantly increased cell migration and invasive ability in vitro and enhanced metastatic potential in vivo. Additional studies exploiting knockdown of sFRP2 within metastatic human and mouse OS cells demonstrated decreased cell migration and invasion ability in vitro, thus corroborating a critical biological phenotype carried out by sFRP2. Interestingly, alterations in sFRP2 expression did not alter OS proliferation rates or primary tumor development. CONCLUSIONS: While future studies further investigating the molecular mechanisms contributing towards this sFRP2-dependent phenotype are needed, our studies clearly provide evidence that aberrant expression of sFRP2 can contribute to the invasive and metastatic potential for osteosarcoma.

REGγ deficiency promotes premature aging via the casein kinase 1 pathway.

Our recent studies suggest a role for the proteasome activator REG (11S regulatory particles, 28-kDa proteasome activator)γ in the regulation of tumor protein 53 (p53). However, the molecular details and in vivo biological significance of REGγ-p53 interplay remain elusive. Here, we demonstrate that REGγ-deficient mice develop premature aging phenotypes that are associated with abnormal accumulation of casein kinase (CK) 1δ and p53. Antibody array analysis led us to identify CK1δ as a direct target of REGγ. Silencing CK1δ or inhibition of CK1δ activity prevented decay of murine double minute (Mdm)2. Interestingly, a massive increase of p53 in REGγ(-/-) tissues is associated with reduced Mdm2 protein levels despite that Mdm2 transcription is enhanced. Allelic p53 haplodeficiency in REGγ-deficient mice attenuated premature aging features. Furthermore, introducing exogenous Mdm2 to REGγ(-/-) MEFs significantly rescues the phenotype of cellular senescence, thereby establishing a REGγ-CK1-Mdm2-p53 regulatory pathway. Given the conflicting evidence regarding the "antiaging" and "proaging" effects of p53, our results indicate a key role for CK1δ-Mdm2-p53 regulation in the cellular aging process. These findings reveal a unique model that mimics acquired aging in mammals and indicates that modulating the activity of the REGγ-proteasome may be an approach for intervention in aging-associated disorders.

Coamplification of Myc/Pvt1 and homozygous deletion of Nlrp1 locus are frequent genetics changes in mouse osteosarcoma.

Osteosarcomas (OSs) are characterized by high levels of genomic instability (GI). To gain insights into the GI and its contribution toward understanding the genetic basis of OS, we characterized 19 primary and 13 metastatic mouse tumors in a genetically engineered novel mouse model of OS by a combination of genomic techniques. Through the bone-specific deletion of the wild-type Trp53 locus or activation of a metastatic-promoting missense R172Hp53 allele, C57BL/6 mice developed either localized or metastatic OS. Subsequent tumors were isolated and primary cultures created from primary bone and/or distal metastatic lesions, for example, lung and liver. These tumors exhibited high levels of GI with complex chromosomal rearrangements, amplifications, and deletions comparable to human OS. The combined genomic approaches identified frequent amplification of chromosome 15D1 and loss of 11B4 by CGH and/or SKY. Both 15D1 and 11B4 have homology with frequently altered chromosomal bands 8q24 and 17p13 in human OS, respectively. Subsequent array CGH, FISH, and qRT-PCR analysis identified coamplification and overexpression of Myc/Pvt1 transcripts from the 15D1 amplicon and loss and decreased expression of the Nlrp1b from 11B4. The Nlrp1 gene is the key mediator of apoptosis and interacts strongly with caspase 2.

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis.

BACKGROUND: Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear. METHODS: si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role. RESULTS: Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways. CONCLUSIONS: Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS.

Ghrelin Prevents Cisplatin-Induced Testicular Damage by Facilitating Repair of DNA Double Strand Breaks Through Activation of p53 in Mice.

Cisplatin administration induces DNA damage resulting in germ cell apoptosis and subsequent testicular atrophy. Although 50 percent of male cancer patients receiving cisplatin-based chemotherapy develop long-term secondary infertility, medical treatment to prevent spermatogenic failure after chemotherapy is not available. Under normal conditions, testicular p53 promotes cell cycle arrest, which allows time for DNA repair and reshuffling during meiosis. However, its role in the setting of cisplatin-induced infertility has not been studied. Ghrelin administration ameliorates the spermatogenic failure that follows cisplatin administration in mice, but the mechanisms mediating these effects have not been well established. The aim of the current study was to characterize the mechanisms of ghrelin and p53 action in the testis after cisplatin-induced testicular damage. Here we show that cisplatin induces germ cell damage through inhibition of p53-dependent DNA repair mechanisms involving gamma-H2AX and ataxia telangiectasia mutated protein kinase. As a result, testicular weight and sperm count and motility were decreased with an associated increase in sperm DNA damage. Ghrelin administration prevented these sequelae by restoring the normal expression of gamma-H2AX, ataxia telangiectasia mutated, and p53, which in turn allows repair of DNA double stranded breaks. In conclusion, these findings indicate that ghrelin has the potential to prevent or diminish infertility caused by cisplatin and other chemotherapeutic agents by restoring p53-dependent DNA repair mechanisms.

Combinatorial therapy discovery using mixed integer linear programming.

MOTIVATION: Combinatorial therapies play increasingly important roles in combating complex diseases. Owing to the huge cost associated with experimental methods in identifying optimal drug combinations, computational approaches can provide a guide to limit the search space and reduce cost. However, few computational approaches have been developed for this purpose, and thus there is a great need of new algorithms for drug combination prediction. RESULTS: Here we proposed to formulate the optimal combinatorial therapy problem into two complementary mathematical algorithms, Balanced Target Set Cover (BTSC) and Minimum Off-Target Set Cover (MOTSC). Given a disease gene set, BTSC seeks a balanced solution that maximizes the coverage on the disease genes and minimizes the off-target hits at the same time. MOTSC seeks a full coverage on the disease gene set while minimizing the off-target set. Through simulation, both BTSC and MOTSC demonstrated a much faster running time over exhaustive search with the same accuracy. When applied to real disease gene sets, our algorithms not only identified known drug combinations, but also predicted novel drug combinations that are worth further testing. In addition, we developed a web-based tool to allow users to iteratively search for optimal drug combinations given a user-defined gene set. AVAILABILITY: Our tool is freely available for noncommercial use at http://www.drug.liuzlab.org/. CONTACT: zhandong.liu@bcm.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effects of TP53 mutational status on gene expression patterns across 10 human cancer types.

Mutations in the TP53 tumour suppressor gene occur in half of all human cancers, indicating its critical importance in inhibiting cancer development. Despite extensive studies, the mechanisms by which mutant p53 enhances tumour progression remain only partially understood. Here, using data from the Cancer Genome Atlas (TCGA), genomic and transcriptomic analyses were performed on 2256 tumours from 10 human cancer types. We show that tumours with TP53 mutations have altered gene expression profiles compared to tumours retaining two wild-type TP53 alleles. Among 113 known p53-up-regulated target genes identified from cell culture assays, 10 were consistently up-regulated in at least eight of 10 cancer types that retain both copies of wild-type TP53. RPS27L, CDKN1A (p21(CIP1)) and ZMAT3 were significantly up-regulated in all 10 cancer types retaining wild-type TP53. Using this p53-based expression analysis as a discovery tool, we used cell-based assays to identify five novel p53 target genes from genes consistently up-regulated in wild-type p53 cancers. Global gene expression analyses revealed that cell cycle regulatory genes and transcription factors E2F1, MYBL2 and FOXM1 were disproportionately up-regulated in many TP53 mutant cancer types. Finally, > 93% of tumours with a TP53 mutation exhibited greatly reduced wild-type p53 messenger expression, due to loss of heterozygosity or copy neutral loss of heterozygosity, supporting the concept of p53 as a recessive tumour suppressor. The data indicate that tumours with wild-type TP53 retain some aspects of p53-mediated growth inhibitory signalling through activation of p53 target genes and suppression of cell cycle regulatory genes.

The Wip1 Phosphatase acts as a gatekeeper in the p53-Mdm2 autoregulatory loop.

The tumor suppressor p53 is a transcription factor that responds to cellular stresses by initiating cell cycle arrest or apoptosis. One transcriptional target of p53 is Mdm2, an E3 ubiquitin ligase that interacts with p53 to promote its proteasomal degradation in a negative feedback regulatory loop. Here we show that the wild-type p53-induced phosphatase 1 (Wip1), or PPM1D, downregulates p53 protein levels by stabilizing Mdm2 and facilitating its access to p53. Wip1 interacts with and dephosphorylates Mdm2 at serine 395, a site phosphorylated by the ATM kinase. Dephosphorylated Mdm2 has increased stability and affinity for p53, facilitating p53 ubiquitination and degradation. Thus, Wip1 acts as a gatekeeper in the Mdm2-p53 regulatory loop by stabilizing Mdm2 and promoting Mdm2-mediated proteolysis of p53.

Insights into wild-type and mutant p53 functions provided by genetically engineered mice.

Recent whole-exome sequencing studies of numerous human cancers have now conclusively shown that the TP53 tumor-suppressor gene is the most frequently mutated gene in human cancers. Despite extensive studies of the TP53 gene and its encoded protein (p53), our understanding of how TP53 mutations contribute to cancer initiation and progression remain incomplete. Genetically engineered mice with germline or inducible Trp53 somatic mutations have provided important insights into the mechanisms by which different types of p53 mutation influence cancer development. Trp53 germline mutations that alter specific p53 structural domains or posttranslation modification sites have benefitted our understanding of wild-type p53 functions in a whole organism context. Moreover, genetic approaches to reestablish functional wild-type p53 to p53-deficient tissues and tumors have increased our understanding of the therapeutic potential of restoring functional p53 signaling to cancers. This review outlines many of the key insights provided by the various categories of Trp53 mutant mice that have been generated by multiple genetic engineering approaches.

MLH1-silenced and non-silenced subgroups of hypermutated colorectal carcinomas have distinct mutational landscapes.

Approximately 15% of colorectal carcinomas (CRCs) exhibit a hypermutated genotype accompanied by high levels of microsatellite instability (MSI-H) and defects in DNA mismatch repair. These tumours, unlike the majority of colorectal carcinomas, are often diploid, exhibit frequent epigenetic silencing of the MLH1 DNA mismatch repair gene, and have a better clinical prognosis. As an adjunct study to The Cancer Genome Atlas consortium that recently analysed 224 colorectal cancers by whole exome sequencing, we compared the 35 CRCs (15.6%) with a hypermutated genotype to those with a non-hypermutated genotype. We found that 22 (63%) of the hypermutated CRCs exhibited transcriptional silencing of the MLH1 gene, a high frequency of BRAF V600E gene mutations, and infrequent APC and KRAS mutations, a mutational pattern significantly different from their non-hypermutated counterparts. However, the remaining 13 (37%) hypermutated CRCs lacked MLH1 silencing, contained tumours with the highest mutation rates ('ultramutated' CRCs), and exhibited higher incidences of APC and KRAS mutations, but infrequent BRAF mutations. These patterns were confirmed in an independent validation set of 250 exome-sequenced CRCs. Analysis of mRNA and microRNA expression signatures revealed that hypermutated CRCs with MLH1 silencing had greatly reduced levels of WNT signalling and increased BRAF signalling relative to non-hypermutated CRCs. Our findings suggest that hypermutated CRCs include one subgroup with fundamentally different pathways to malignancy than the majority of CRCs. Examination of MLH1 expression status and frequencies of APC, KRAS, and BRAF mutation in CRC may provide a useful diagnostic tool that could supplement the standard microsatellite instability assays and influence therapeutic decisions.

Widespread and tissue specific age-related DNA methylation changes in mice.

Aberrant methylation of promoter CpG islands in cancer is associated with silencing of tumor-suppressor genes, and age-dependent hypermethylation in normal appearing mucosa may be a risk factor for human colon cancer. It is not known whether this age-related DNA methylation phenomenon is specific to human tissues. We performed comprehensive DNA methylation profiling of promoter regions in aging mouse intestine using methylated CpG island amplification in combination with microarray analysis. By comparing C57BL/6 mice at 3-mo-old versus 35-mo-old for 3627 detectable autosomal genes, we found 774 (21%) that showed increased methylation and 466 (13%) that showed decreased methylation. We used pyrosequencing to quantitatively validate the microarray data and confirmed linear age-related methylation changes for all 12 genomic regions examined. We then examined 11 changed genomic loci for age-related methylation in other tissues. Of these, three of 11 showed similar changes in lung, seven of 11 changed in liver, and six of 11 changed in spleen, though to a lower degree than the changes seen in colon. There was partial conservation between age-related hypermethylation in human and mouse intestines, and Polycomb targets in embryonic stem cells were enriched among the hypermethylated genes. Our findings demonstrate a surprisingly high rate of hyper- and hypomethylation as a function of age in normal mouse small intestine tissues and a strong tissue-specificity to the process. We conclude that epigenetic deregulation is a common feature of aging in mammals.

Mdm2-p53 signaling regulates epidermal stem cell senescence and premature aging phenotypes in mouse skin.

The p53 transcription factor is activated by various types of cell stress or DNA damage and induces the expression of genes that control cell growth and inhibit tumor formation. Analysis of mice that express mutant forms of p53 suggest that inappropriate p53 activation can alter tissue homeostasis and life span, connecting p53 tumor suppressor functions with accelerated aging. However, other mouse models that display increased levels of wildtype p53 in various tissues fail to corroborate a link between p53 and aging phenotypes, possibly due to the retention of signaling pathways that negatively regulate p53 activity in these models. In this present study, we have generated mice lacking Mdm2 in the epidermis. Deletion of Mdm2, the chief negative regulator of p53, induced an aging phenotype in the skin of mice, including thinning of the epidermis, reduced wound healing, and a progressive loss of fur. These phenotypes arise due to an induction of p53-mediated senescence in epidermal stem cells and a gradual loss of epidermal stem cell function. These results reveal that activation of endogenous p53 by ablation of Mdm2 can induce accelerated aging phenotypes in mice.

Rpl27a mutation in the sooty foot ataxia mouse phenocopies high p53 mouse models.

Ribosomal stress is an important, yet poorly understood, mechanism that results in activation of the p53 tumour suppressor. We present a mutation in the ribosomal protein Rpl27a gene (sooty foot ataxia mice), isolated through a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for p53 pathway defects, that shares striking phenotypic similarities with high p53 mouse models, including cerebellar ataxia, pancytopenia and epidermal hyperpigmentation. This phenocopy is rescued in a haploinsufficient p53 background. A detailed examination of the bone marrow in these mice identified reduced numbers of haematopoietic stem cells and a p53-dependent c-Kit down-regulation. These studies suggest that reduced Rpl27a increases p53 activity in vivo, further evident with a delay in tumorigenesis in mutant mice. Taken together, these data demonstrate that Rpl27a plays a crucial role in multiple tissues and that disruption of this ribosomal protein affects both development and transformation.

K-Ras and p53 mouse model with molecular characteristics of human rhabdomyosarcoma and translational applications.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of ∼65-70%. Thus, additional molecular insights and representative models are critical for identifying and evaluating new treatment modalities. Using MyoD-Cre-mediated introduction of mutant K-RasG12D and perturbations in p53, we developed a novel genetically engineered mouse model (GEMM) for RMS. The anatomic sites of primary RMS development recapitulated human disease, including tumors in the head, neck, extremities and abdomen. We confirmed RMS histology and diagnosis through Hematoxylin and Eosin staining, and positive immunohistochemical staining for desmin, myogenin, and phosphotungstic acid-Hematoxylin. Cell lines from GEMM tumors were established with the ability to engraft in immunocompetent mice with comparable histological and staining features as the primary tumors. Tail vein injection of cell lines had high metastatic potential to the lungs. Transcriptomic analyses of p53R172H/K-RasG12D GEMM-derived tumors showed evidence of high molecular homology with human RMS. Finally, pre-clinical use of these murine RMS lines showed similar therapeutic responsiveness to chemotherapy and targeted therapies as human RMS cell lines.