Anil K. Rustgi

The landscape of somatic copy-number alteration across human cancers

A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κΒ pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types.

SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas

Lineage-survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development. Here we show that a peak of genomic amplification on chromosome 3q26.33 found in squamous cell carcinomas (SCCs) of the lung and esophagus contains the transcription factor gene SOX2, which is mutated in hereditary human esophageal malformations, is necessary for normal esophageal squamous development, promotes differentiation and proliferation of basal tracheal cells and cooperates in induction of pluripotent stem cells. SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 here cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These characteristics identify SOX2 as a lineage-survival oncogene in lung and esophageal SCC.

A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4).

BACKGROUND Juvenile polyposis and hereditary haemorrhagic telangiectasia are autosomal dominant disorders with distinct and non-overlapping clinical features. The former, an inherited gastrointestinal malignancy predisposition, is caused by mutations in MADH4 (encoding SMAD4) or BMPR1A, and the latter is a vascular malformation disorder caused by mutations in ENG (endoglin) or ACVRL1 (ALK1). All four genes encode proteins involved in the transforming-growth-factor-beta signalling pathway. Although there are reports of patients and families with phenotypes of both disorders combined, the genetic aetiology of this association is unknown. METHODS Blood samples were collected from seven unrelated families segregating both phenotypes. DNA from the proband of each family was sequenced for the ACVRL1, ENG, and MADH4 genes. Mutations were examined for familial cosegregation with phenotype and presence or absence in population controls. Findings No patient had mutations in the ENG or ACVRL1 genes; all had MADH4 mutations. Three cases of de-novo MADH4 mutations were found. In one, the mutation was passed on to a similarly affected child. Each mutation cosegregated with the syndromic phenotype in other affected family members. INTERPRETATION Mutations in MADH4 can cause a syndrome consisting of both juvenile polyposis and hereditary haemorrhagic telangiectasia phenotypes. Since patients with these disorders are generally ascertained through distinct medical specialties, genetic testing is recommended for patients presenting with either phenotype to identify those at risk of this syndrome. Patients with juvenile polyposis who have an MADH4 mutation should be screened for the vascular lesions associated with hereditary haemorrhagic telangiectasia, especially occult arteriovenous malformations in visceral organs that may otherwise present suddenly with serious medical consequences.

Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer

MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators. miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. We studied miRNA deregulation in human epithelial ovarian cancer by integrative genomic approach, including miRNA microarray (n = 106), array-based comparative genomic hybridization (n = 109), cDNA microarray (n = 76), and tissue array (n = 504). miRNA expression is markedly down-regulated in malignant transformation and tumor progression. Genomic copy number loss and epigenetic silencing, respectively, may account for the down-regulation of ≈15% and at least ≈36% of miRNAs in advanced ovarian tumors and miRNA down-regulation contributes to a genome-wide transcriptional deregulation. Last, eight miRNAs located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumor suppressor genes. Therefore, our results suggest that miRNAs may offer new biomarkers and therapeutic targets in epithelial ovarian cancer.

The Hereditary Nonpolyposis Colorectal Cancer Syndrome: Genetics and Clinical Implications

In the United States, an individuals lifetime risk for developing some form of cancer is estimated to be as high as 40% (1). Cancer is fundamentally a genetic disorder, meaning that mutations in specific genes confer a selective growth advantage for tumor cells. Such mutations may develop sporadically or may be inherited through the germline, which results in a hereditary predisposition to multiple cases of early-onset cancer. Identification of these cancer-susceptibility genes has revealed the cellular processes that ordinarily guard against tumor formation. One particularly instructive example is the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. Defining the genetic basis for HNPCC has demonstrated a fascinating connection between the cellular machinery that regulates the replication and repair of DNA and cancer formation. Replication is the process by which DNA is copied in preparation for cell division. Errors of replication are inevitable, but specialized repair systems have evolved to prevent the accumulation of harmful mutations in the genome. The DNA mismatch repair system functions as a critical spell checker that identifies and then corrects mismatched base pairs of DNA. A germline mutation in a DNA mismatch repair gene results in the HNPCC syndrome, which is inherited as an autosomal dominant disorder. The 1990 Amsterdam criteria strictly define HNPCC as occurring when colon cancer is diagnosed in at least three family members, one a first-degree relative of the other two. The cases must span two generations, and one case must be diagnosed before 50 years of age (2). Less stringent criteria, including the Amsterdam II, modified Amsterdam, and Bethesda criteria, have also been developed. This review first highlights the key features of normal DNA replication and the mechanisms that ensure the fidelity of this process. The next section focuses on the genes involved in DNA mismatch repair and illustrates the biological consequences that result when mutations arise in these genes. Finally, we discuss the clinical implications of these genetic insights in the management of HNPCC families as well as persons with sporadic colon cancer. DNA Replication Replication of DNA takes place in preparation for mitosis. Consequently, each daughter cell carries an exact replica of the entire parental genome. Many nuclear proteins work in concert to execute the three phases of initiation, elongation, and termination. These mechanisms of DNA replication have been defined primarily from studies in bacteria and yeast, but the fundamental processes are relevant to higher eukaryotic cells. Replication is always initiated at specific points of origin in a chromosome when a cell enters the S (or DNA Synthesis) phase of the cell cycle (3). The helicase enzyme separates the double strands of DNA at these replication origin points. This unwinding of DNA results in an open loop structure. Each of the two parental strands can then serve as a template for the synthesis of a new complementary strand. Because each daughter chromosome contains one parental strand and one newly synthesized strand, the replication process is termed semi-conservative. The DNA polymerase enzyme carries out new DNA synthesis, but it cannot begin until a primer is available. Most commonly, a specific RNA polymerase called primase synthesizes a short RNA primer at the replication origin. Two replication forks are created as DNA is synthesized in a bi-directional manner away from the point of origin (4). A multicomponent complex containing DNA polymerase carries out the elongation process that adds each complementary nucleotide to the primer. Replication always proceeds in a 5 to 3 direction. This results in continuous DNA synthesis along one strand (the leading strand), but it is necessarily discontinuous along the other template strand (the lagging strand) (5). The lagging strand is thus composed of many Okazaki fragments that are initiated from discontinuous primers (Figure 1), and DNA ligase joins each of these fragments to form one continuous strand. Figure 1. Events at the replication fork. Replication is terminated when the entire length of a chromosome has been synthesized, resulting in two complete copies of each chromosome. In circular bacterial chromosomes, termination occurs simply when the origin of replication has been reached. The mechanisms that control termination of linear eukaryotic chromosomes are far more complex. DNA Damage: Errors of DNA Replication Maintaining the integrity of DNA is essential for normal cellular function. Chemical carcinogens or ultraviolet radiation can directly induce structural alterations, such as thymidine dimers, single- or double-stranded breaks, and covalent cross-linking. The DNA excision repair system recognizes and excises such structurally altered nucleotides; specific details are available in a recent excellent review (6). The current review focuses instead on a different type of error that occurs during replication. Errors of DNA replication are observed primarily during the elongation phase. The most common error is a simple mispairing of nucleotides. Patterns of hydrogen bonding govern the pairings of nucleotides. An adenine (A) is always paired with a thymidine (T), and a cytosine (C) is always paired with a guanine (G). The DNA polymerase may incorrectly pair an adenine with a guanine, for example, and such mispairings are predicted to occur once every 103 to 104 base pairs. Repair mechanisms keep the actual error rate much lower. In bacteria, de novo mutations occur only once every 1010 base pairs (7). A second type of error can occur during DNA replication. There can be slippage of the DNA polymerase complex, a process analogous to a loose bicycle chain transiently slipping but then reengaging. This slippage occurs during the replication of microsatellite DNA sequences, which are defined as short dinucleotide (5-TCAATGCCACACACACACACACCTGAGGC-) or mononucleotide (5-TCTAGGCTAAAAAAAAATGCCGAGT-) repeats. The daughter strands may then contain either too many or too few copies of these repeated sequences. With intact mismatch repair mechanisms, these errors of slippage are quickly corrected, and microsatellite DNA sequences are considered stable. However, in the presence of deficient mismatch repair function, these errors are not corrected. This phenomenon is called microsatellite instability (MSI) (Figure 2). Figure 2. Slippage during DNA replication. MSS MSI The DNA polymerase complex itself provides the first line of defense against errors of replication. Through poorly defined mechanisms, DNA polymerase can immediately recognize a mismatched pair of nucleotides. An intrinsic enzyme subunit with 3 to 5 exonuclease activity excises the mispaired nucleotide from the newly synthesized strand, and the excised sequences are replaced with the correct nucleotides. The DNA Mismatch Repair System For errors that are not immediately corrected by DNA polymerase, the DNA mismatch repair system provides a secondary system of proofreading. This system corrects not only single base-pair mismatches but also small mispaired loops of DNA that result from replication errors of microsatellite tracts. Studies in yeast have demonstrated that defective mismatch repair specifically leads to a 100- to 700-fold increase in the instability of these microsatellite tracts (8). The DNA mismatch repair system requires the cooperation of many genes from the mutS (hMSH2, hMSH3, hMSH6) and mutL (hMLH1, hMLH3, hPMS1, and hPMS2) families. Briefly, hMSH2 serves as the scout that recognizes and binds directly to the mismatched DNA sequence (9, 10). It forms a heterodimeric complex with hMSH6 if a single base-pair mismatch is recognized or with hMSH3 if there is a larger two- to eight-nucleotide insertion or deletion (Figure 3). A second heterodimeric complex of hMLH1 and hPMS2 is then recruited to excise the mismatched nucleotides. Although heterodimers of hMLH1/hPMS1 and hMLH1/hMLH3 also form, their specific roles remain to be defined. Figure 3. Components of the DNA mismatch repair system. Mutations in DNA Mismatch Repair Genes A mutation in one of several mismatch repair genes results in deficient DNA mismatch repair activity. The extent of the mismatch repair deficiency depends on the specific gene that is altered. If hMSH2 or hMLH1 is inactivated, then a high level of MSI is typically observed, the so-called MSI-H phenotype (11). Mutations in genes such as hMSH6 result in only a partial deficiency of mismatch repair and low levels of MSI (the MSI-L phenotype) (12, 13). The U.S. National Cancer Institute defines the MSI-H phenotype as present when two of five microsatellite markers from a standard panel display instability and the MSI-L phenotype as present when one of the five markers is unstable. Mutations in mismatch repair genes may occur in germline or somatic DNA. Of interest, an inherited germline alteration does not result in widespread developmental anomalies. One possible explanation for this is that the second wild-type allele may provide sufficient DNA mismatch repair function. The observed phenotype is the predisposition to early-onset tumors, primarily of the colon and endometrium. The biological basis for this organ specificity is unknown. In each tumor that forms, the second copy of the affected DNA mismatch repair gene has been somatically mutated, thereby leading to bi-allelic gene inactivation (14, 15). The direct consequence of defective DNA mismatch repair is the so-called mutator or replication-error phenotype. Rather than directly causing malignant transformation, DNA mismatch repair deficiency creates the milieu that permits mutations to accumulate in other growth-regulatory genes. Colon tumors that develop in persons with germline mutations in hMSH2 or hMLH1 characteristically display the MSI-H phenotype (16, 17). Most microsatellite sequences in the genome are located within noncoding, or intr

Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates

Epithelial metaplasia occurs when one predominant cell type in a tissue is replaced by another, and is frequently associated with an increased risk of subsequent neoplasia. In both mouse and human pancreas, acinar-to-ductal metaplasia has been implicated in the generation of cancer precursors. We show that pancreatic epithelial explants undergo spontaneous acinar-to-ductal metaplasia in response to EGFR signaling, and that this change in epithelial character is associated with the appearance of nestin-positive transitional cells. Lineage tracing involving Cre/lox-mediated genetic cell labeling reveals that acinar-to-ductal metaplasia represents a true transdifferentiation event, mediated by initial dedifferentiation of mature exocrine cells to generate a population of nestin-positive precursors, similar to those observed during early pancreatic development. These results demonstrate that a latent precursor potential resides within mature exocrine cells, and that this potential is regulated by EGF receptor signaling. In addition, these observations provide a novel example of rigorously documented transdifferentiation within mature mammalian epithelium, and suggest that plasticity of mature cell types may play a role in the generation of neoplastic precursors.

Bile Acid and Inflammation Activate Gastric Cardia Stem Cells in a Mouse Model of Barrett-Like Metaplasia

Esophageal adenocarcinoma (EAC) arises from Barrett esophagus (BE), intestinal-like columnar metaplasia linked to reflux esophagitis. In a transgenic mouse model of BE, esophageal overexpression of interleukin-1β phenocopies human pathology with evolution of esophagitis, Barrett-like metaplasia and EAC. Histopathology and gene signatures closely resembled human BE, with upregulation of TFF2, Bmp4, Cdx2, Notch1, and IL-6. The development of BE and EAC was accelerated by exposure to bile acids and/or nitrosamines, and inhibited by IL-6 deficiency. Lgr5(+) gastric cardia stem cells present in BE were able to lineage trace the early BE lesion. Our data suggest that BE and EAC arise from gastric progenitors due to a tumor-promoting IL-1β-IL-6 signaling cascade and Dll1-dependent Notch signaling.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc.

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.

Epidermal Growth Factor Receptor Mediates Increased Cell Proliferation, Migration, and Aggregation in Esophageal Keratinocytes in Vitro and in Vivo

Epidermal growth factor receptor (EGFR) overexpression is observed in a number of malignancies, especially those of esophageal squamous cell origin. However, little is known about the biological functions of EGFR in primary esophageal squamous epithelial cells. Using newly established primary human esophageal squamous epithelial cells as a platform, we overexpressed EGFR through retroviral transduction and established novel three-dimensional organotypic cultures. Additionally, EGFR was targeted in a cell type- and tissue-specific fashion to the esophageal epithelium in transgenic mice. EGFR overexpression in primary esophageal keratinocytes resulted in the biochemical activation of Akt and STAT pathways and induced enhanced cell migration and cell aggregation. When established in organotypic culture, EGFR-overexpressing cells had evidence of epithelial cell hyperproliferation and hyperplasia. These effects were also observed in EGFR-overexpressing transgenic mice and the esophageal cell lines established thereof. In particular, EGFR-induced effects upon aggregation appear to be mediated through the relocalization of p120 from the cytoplasm to the membrane and increased interaction with E-cadherin. EGFR modulates cell migration through the up-regulation of matrix metalloproteinase 1. Taken together, the functional effects of EGFR overexpression help to explain its role in the initiating steps of esophageal squamous carcinogenesis.