Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics1. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.

We demonstrate that the binding sites for highly conserved transcription factors vary extensively between human and mouse. We mapped the binding of four tissue-specific transcription factors (FOXA2, HNF1A, HNF4A, HNF6) to 4,000 orthologous gene pairs in hepatocytes purified from human and mouse livers. Despite the conserved function of these factors, from 41% to 89% of their binding events appear to be species-specific. When the same protein binds the promoters of orthologous genes, approximately two-thirds of the binding sites do not align.Elements of transcriptional regulation have central roles in evolution [1][2][3] . In many cases, conserved biological processes are controlled by evolutionarily conserved regulatory programs while evolving phenotypes are associated with cross-species variation in transcription regulation 4 . However, in the absence of suitable genome-wide data, it is unclear what fraction of all protein-DNA interactions are under either positive or negative selective pressure 1 . A preliminary effort to compare genome-wide binding sites for two stem cell-specific transcription factors in human and mouse has suggested that large differences exist between mouse and human 5, 6 yet because the data were obtained using different To compare systematically the binding of transcriptional regulators to promoter regions across species, we designed carefully matched ChIP-chip experiments 7 in human and mouse. We created custom DNA microarrays that array ten kilobases of sequence surrounding the known transcription start sites of over 4,000 orthologous pairs of mouse and human genes. These genes were selected because their orthology could be unambiguously assigned and oligonucleotides could be designed to represent the putative regulatory regions at high density ( Figure 1A, Supplementary Methods). Forty-seven hand-curated, tissuespecific genes were included in the array design as controls.Chromatin immunoprecipitations were performed independently in primary hepatocytes directly isolated from mouse and human liver using antibodies against four tissue-specific transcription factors (FOXA2, HNF1A, HNF4A, HNF6) involved in liver development and regulation ( Figure 1B, Table S1) 7 . Hepatocytes were chosen as a representative tissue for these experiments because (1) they are functionally and structurally conserved among mammals 8 ; (2) their gene expression programs are similar across species (Table S1); (3) their gene expression patterns are largely unperturbed by isolation procedures 9 ; and (4) the transcription factors responsible for hepatocyte development and function are highly conserved 8 . We amplified and fluorescently labeled the DNA from these binding experiments, hybridized it to the microarrays, and then scored binding events 10 .Several possible outcomes can be distinguished when comparing a binding event in one species with the data from the second species (Figure 1). First, one can determine if a particular transcription factor binds anywhere within the arrayed region of the human ...

Homologous sets of transcription factors direct conserved tissue-specific gene expression, yet transcription factor binding events diverge rapidly between closely related species. We used hepatocytes from an aneuploid mouse strain carrying human chromosome 21 to determine on a chromosomal scale whether interspecies differences in transcriptional regulation are primarily directed by human genetic sequence or mouse nuclear environment. Virtually all transcription factor binding locations, landmarks of transcription initiation, and the resulting gene expression observed in human hepatocytes were recapitulated across the entire human chromosome 21 in the mouse hepatocyte nucleus. Thus, in homologous tissues, genetic sequence is largely responsible for directing transcriptional programs; interspecies differences in epigenetic machinery, cellular environment, and transcription factors themselves play secondary roles.

Figure 1. TAMs are the predominant source of PD-L1 in CCA. (A) Representative images (left and middle panels) of PD-L1 (brown staining, black arrowhead) plus CD68 (red staining, red arrowhead) coimmunostaining (n = 33) and PD-L1 (brown staining) plus CK-19 (red staining) coimmunostaining (n = 18) in human resected CCA specimens. Percentage of patients with positive PD-L1/CD68 costaining and PD-L1/CK19 costaining, respectively (right panel). Scale bars: 40 μm. (B) Histograms show expression of PD-L1 + macrophages in human CCA tumors. (C-F) Flow cytometry analysis of normal WT mouse livers (from WT mice without tumors) as well as adjacent livers and tumors of mice 28 days after orthotopic implantation of 1 × 10 6 SB (murine CCA) cells. (C) Percentage of PD-L1 + macrophages (Mφ) of total macrophages (CD45 + CD11b + F4/80 + ) in WT mouse normal liver, tumor-adjacent liver, or tumor. Fluorescence Minus One (FMO) controls were used for each independent experiment to establish gates (See Supplemental Figure 1A for gating strategy) (n ≥ 8). Representative histograms show expression of PD-L1 + macrophages. (D) Percentage of CD206 + TAMs (left panel) and PD-L1 + CD206 + TAMs (middle panel) of F4/80 int macrophages (CD45 + CD11b + F4/80 int ) in WT mouse liver, tumor-adjacent liver, or tumor. Representative contour plots (right panel) show CD206 and PD-L1 expression of F4/80 int macrophages (n ≥ 7). (E) Percentage of PD-L1 + CD206macrophages or PD-L1 + CD206 + macrophages (CD11b + F4/80 + ) of CD45 + cells from SB tumors (n = 28). (F) Percentage of PD-L1 expression in myeloid cells from SB tumors.

Reactive oxygen species (ROS) such as superoxide radicals are responsible for the pathogenesis of various human diseases. ROS are generated during normal metabolic process in all of the oxygen-utilizing organisms. The copper-zinc-containing SOD (SOD1) acts as a major defense against ROS by detoxifying the superoxide anion. In model organisms, SOD1 has been shown to play a role in the aging process. However, the exact role of the SOD1 protein in the human aging process remains to be resolved. We show that SOD1 RNA interference (RNAi) induces senescence in normal human fibroblasts. This premature senescence depends on p53 induction. In contrast, in human fibroblastic cells with inactivated p53, the SOD1 RNAi is without effect. Surprisingly, in cancer cells (HeLa), the SOD1 RNAi induces cell death rather then senescence. Together, these findings support the notion that in normal human cells the SOD1 protein may play a role in the regulation of cellular lifespan by p53 and may also regulate the death signals in cancer cells. ROS1 are generated by normal metabolic processes in all of the oxygen-utilizing organisms. It is estimated that around 1% of the total oxygen consumed in the mitochondria becomes the superoxide anion (1). Damage induced by ROS includes DNA mutation, protein oxidation, and lipid peroxidation. ROS may contribute to the development of various diseases, such as cancer, diabetes, atherosclerosis, inflammation, and premature aging (2, 3).The superoxide dismutases (SODs) constitute a family of antioxidant enzymes that catalyzes the conversion of superoxide anions to oxygen and hydrogen peroxide (for recent review on SODs, see Ref. 4). They include the manganese-containing SOD (SOD2) in the mitochondria (5) and the copper-zinc-containing SOD (SOD1) in the cytoplasm (5) with a small fraction in the mitochondria intermembrane space (6). Ͼ90 different mutations in the SOD1 gene have been found to associate with amyotrophic lateral sclerosis, a disease that causes the degradation of motor neurons (7).The free radical theory of aging states that ROS generation during metabolism can cause damage to cellular constituents (8). During the life of a cell or an organism, SODs and catalase detoxify ROS. However this process is not perfect and ROS species can cause cumulative damage, which causes the physiological decline characteristic of aging (for review, see Ref. 9). In yeast survival, the stationary phase appears to be limited by ROS because deletion of either SOD1 or SOD2 shortens survival time (10). In Drosophila, overexpression of the human SOD1 extends the fly life span by 40% over controls (11).After a finite number of divisions, primary human cells in culture enter into a state of replicative senescence in which they are growth-arrested and resistant to mitogenic stimulation. Senescence is considered a mechanism to suppress tumorigenesis because it inhibits cell proliferation (12). Oxidants appear to be important in the development of the senescent phenotype. Cells grown in low oxygen tension exhibit a pr...

Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with down-regulation of β-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties to immortalized human Schwann cells, and down-regulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small molecule inhibition of Wnt signaling effectively reduced viability of MPNST cell lines, and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors.

Cholangiocarcinoma (CCA) represents a heterogeneous group of epithelial tumours that are classified according to anatomical location as intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA). Although surgical resection and liver transplantation following neoadjuvant therapy are potentially curative options for a subset of patients with early-stage disease, the currently available medical therapies for CCA have limited efficacy. Immunotherapeutic strategies such as immune checkpoint blockade (ICB) harness the host immune system to unleash an effective and durable antitumour response in a subset of patients with a variety of malignancies. However, response to ICB monotherapy has been relatively disappointing in CCA. CCAs are desmoplastic tumours with an abundant tumour immune microenvironment (TIME) that contains immunosuppressive innate immune cells such as tumour-associated macrophages and myeloid-derived suppressor cells. A subset of CCAs may be classified as immune 'hot' tumours with a high density of CD8 + T cells and enhanced expression of immune checkpoint molecules. Immune 'hot' tumour types are associated with higher response rates to ICB. However, the suboptimal response rates to ICB monotherapy in human clinical trials of CCA imply that the preponderance of CCAs are immune 'cold' tumours with a non-T cell infiltrated TIME. An enhanced comprehension of the immunobiology of CCA, particularly the innate immune response to CCA, is essential in the effort to develop effective combination immunotherapeutic strategies that can target a larger subset of CCAs.

Maintaining quiescent cells in G0 phase is achieved in part through the multiprotein subunit complex known as DREAM, and in human cell lines the transcription factor E2F4 directs this complex to its cell cycle targets. We found that E2F4 binds a highly overlapping set of human genes among three diverse primary tissues and an asynchronous cell line, which suggests that tissue-specific binding partners and chromatin structure have minimal influence on E2F4 targeting. To investigate the conservation of these transcription factor binding events, we identified the mouse genes bound by E2f4 in seven primary mouse tissues and a cell line. E2f4 bound a set of mouse genes that was common among mouse tissues, but largely distinct from the genes bound in human. The evolutionarily conserved set of E2F4 bound genes is highly enriched for functionally relevant regulatory interactions important for maintaining cellular quiescence. In contrast, we found minimal mRNA expression perturbations in this core set of E2f4 bound genes in the liver, kidney, and testes of E2f4 null mice. Thus, the regulatory mechanisms maintaining quiescence are robust even to complete loss of conserved transcription factor binding events.