miRNAs are small regulatory RNAs that, due to their considerable potential to target a wide range of mRNAs, are implicated in essentially all biological process, including cancer. miR-10a is particularly interesting considering its conserved location in the Hox cluster of developmental regulators. A role for this microRNA has been described in developmental regulation as well as for various cancers. However, previous miR-10a studies are exclusively based on transient knockdowns of this miRNA and to extensively study miR-10a loss we have generated a miR-10a knock out mouse. Here we show that, in the Apcmin mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10+/+ and male controls. We further found that Lpo is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using in vitro assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of Lpo, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of miR-10a knockout mice. Lpo has previously been shown to have the capacity to oxidize estrogens into potent depurinating mutagens, creating an instable genomic environment that can cause initiation of cancer. Therefore, we postulate that Lpo upregulation in the intestinal epithelium of miR-10a deficient mice together with the predominant abundance of estrogens in female animals mainly accounts for the sex-related cancer phenotype we observed. This suggests that miR-10a could be used as a potent diagnostic marker for discovering groups of women that are at high risk of developing colorectal carcinoma, which today is one of the leading causes of cancer-related deaths.
Posttranscriptional regulation by microRNA molecules constitutes an important mechanism for gene regulation and numerous studies have demonstrated a correlation between deregulated microRNA levels and diseases, such as cancer. However, genetics studies linking individual microRNAs to the etiology of cancer remain scarce. Here, we provide causal evidence for the involvement of the conserved microRNA miR-10a in the development of intestinal adenomas in the face of activated Wnt signaling. Interestingly, we find that loss of miR-10a mediates an increase in intestinal adenomas in female mice only and delineate the pathway to involve aberrant upregulation of the miR-10a target Klf4 and subsequent transcriptional activation of the Lpo gene encoding the antibacterial protein Lactoperoxidase. Lpo, in turn, has previously been demonstrated to oxidize estrogens into DNA-damaging mutagens.
Hereditary factors are presumed to play a role in one third of colorectal cancer (CRC) cases. However, in the majority of familial CRC cases the genetic basis of predisposition remains unexplained. This is particularly true for families with few affected individuals. To identify susceptibility genes for this common phenotype, we examined familial cases derived from a consecutive series of 1514 Finnish CRC patients. Ninety-six familial CRC patients with no previous diagnosis of a hereditary CRC syndrome were included in the analysis. Eighty-six patients had one affected first-degree relative, and ten patients had two or more. Exome sequencing was utilized to search for genes harboring putative loss-of-function variants, because such alterations are likely candidates for disease-causing mutations. Eleven genes with rare truncating variants in two or three familial CRC cases were identified: UACA, SFXN4, TWSG1, PSPH, NUDT7, ZNF490, PRSS37, CCDC18, PRADC1, MRPL3, and AKR1C4. Loss of heterozygosity was examined in all respective cancer samples, and was detected in seven occasions involving four of the candidate genes. In all seven occasions the wild-type allele was lost (P = 0.0078) providing additional evidence that these eleven genes are likely to include true culprits. The study provides a set of candidate predisposition genes which may explain a subset of common familial CRC. Additional genetic validation in other populations is required to provide firm evidence for causality, as well as to characterize the natural history of the respective phenotypes.
Many individuals with a family history of colorectal cancer have no detectable germline mutation in the known cancer predisposing genes. We aimed to identify novel susceptibility genes for this common phenotype by performing exome sequencing on 96 independent cases with familial colorectal cancer. Eighty-six patients had one affected first-degree relative, and ten patients had two or more. None of the patients had a previous diagnosis of a hereditary syndrome. We focused our search on genes with rare variants, predicted to truncate the protein product, since these are likely candidates for disease predisposition. Using this approach we identified truncating germline variants in eleven genes, present in two or three independent familial colorectal cancer cases. We analyzed the respective tumor DNAs and found loss of the wild-type allele in seven out of seven occasions, involving four genes. No tumor showed loss of the mutant allele which provides us with additional evidence for disease causality. Further studies are required to provide firm evidence for pathogenicity. Genetic knowledge on confirmed predisposing genes can ultimately be translated into tools for cancer prevention and early diagnosis in individuals carrying predisposition alleles.
Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G•C bp in the context of all 64 5′-NGNN-3′ motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.
A large number of DNA mutations identified in cells from patients with cancer or human inherited disease were analyzed to address a fundamental issue in human pathology, viz, the mutational mechanisms that cause irreversible changes to DNA. By using bioinformatics and computational methods, we found that mutations do not occur randomly, but instead affect specific bases, most often guanines flanked by other guanines or adenines. We attribute this effect to electron transfer, a chemical reaction known to underlie basic biological processes such as cellular respiration and photosynthesis. Certain types of carcinogens, oxidants or radiation can interact with DNA and abstract an electron. Our results imply that the ensuing sites of electron loss can migrate from their original position in the DNA to neighboring guanines where they become trapped, leading to further chemical modifications that may eventually result in mutations. Many of the mutations known to be important for tumor growth (driver mutations), as well as passenger mutations and mutations associated with inherited disease, appear to be caused by electron transfer. Beyond pathological mutations, electron transfer may represent a universal mechanism by which genetic changes occur in all life forms to drive population fitness over evolutionary time.
Understanding cell proliferation mechanisms has been a long-lasting goal of the scientific community and specifically of cancer researchers. Previous genome-scale studies of cancer proliferation determinants have mainly relied on knockdown screens aimed to gauge their effects on cancer growth. This powerful approach has several limitations such as off-target effects, partial knockdown, and masking effects due to functional backups. Here we employ a complementary approach and assign each gene a cancer Proliferation Index (cPI) that quantifies the association between its expression levels and growth rate measurements across 60 cancer cell lines. Reassuringly, genes found essential in cancer gene knockdown screens exhibit significant positive cPI values, while tumor suppressors exhibit significant negative cPI values. Cell cycle, DNA replication, splicing and protein production related processes are positively associated with cancer proliferation, while cellular migration is negatively associated with it – in accordance with the well known “go or grow” dichotomy. A parallel analysis of genes' non-cancerous proliferation indices (nPI) across 224 lymphoblastoid cell lines reveals surprisingly marked differences between cancerous and non-cancerous proliferation. These differences highlight genes in the translation and spliceosome machineries as selective cancer proliferation-associated proteins. A cross species comparison reveals that cancer proliferation resembles that of microorganisms while non-cancerous proliferation does not. Furthermore, combining cancerous and non-cancerous proliferation signatures leads to enhanced prediction of patient outcome and gene essentiality in cancer. Overall, these results point to an inherent difference between cancerous and non-cancerous proliferation determinants, whose understanding may contribute to the future development of novel cancer-specific anti-proliferative drugs.
One of the hallmarks of cancer is uncontrolled cellular proliferation, and therefore many anti-cancer drugs aim to disrupt cancer proliferation. However, some of these drugs (e.g., chemotherapeutic agents) affect normal proliferating cells as well, resulting in undesirable side effects. Understanding the differences between cancerous and non-cancerous proliferation can help us design new selective drugs that kill cancer cells without harming normal cells. In this work, we use genome scale gene expression and growth rate measurements across 60 cancer cell lines (NCI-60) to uncover genetic determinants of cancerous proliferation. In parallel, gene expression and growth rate measurements of non-cancerous cell lines allow us to uncover determinants of non-cancerous proliferation. Notably, we find marked differences between the cancerous and non-cancerous proliferation. The two proliferation signatures can be used jointly to enhance the prediction of patient outcome in cancer. Notably, we find that certain genes in the translation and spliceosome machineries are involved in cancerous proliferation but not in non-cancerous proliferation, highlighting them as putative selective anti-cancer drug targets.
Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted.
There is increasing interest in developing methods to treat cancer by targeting non-cancer cells that play supportive roles in the tumor microenvironment. One type of non-cancer cell that has received considerable attention along these lines is cancer-associated fibroblasts, which can promote tumor formation and tumor growth. There have been several studies showing that inhibition of individual fibroblast genes or proteins dramatically reduces the tumor supportive function of fibroblasts. From the perspective of developing a therapeutic strategy, what remains unclear is whether the several different important factors discovered to date reflect the requirement of a multitude of fibroblast factors to promote tumorigenicity, or whether it reflects the diversity of the epithelial cancer cells and fibroblasts used in these different studies. Here, we addressed this question directly using a single system of fibroblasts and breast cancer epithelial cells. Importantly, we found that a multitude of fibroblast factors are indeed required to promote tumorigenicity, and that they have different effects on the tumor microenvironment. Furthermore, we found that inhibiting multiple fibroblast-secreted factors is more efficacious than blocking individual factors. These results suggest that fibroblasts and cancer cells act through multiple parallel pathways and that effective anti-stromal therapeutic strategies will need to be multi-targeted.
The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers.
Cancer is essentially a genetic disease, caused by serial genetic changes including point mutations and chromosome number abnormalities. The latter leads to copy number alterations of many genes. While there are usually thousands of these genetic changes in a given tumor, only a small fraction likely contribute to cancer development. One of the major challenges is to distinguish these cancer “driver” genes from “passenger” mutations that do not contribute to the cancer phenotype. In particular, identifying the driver genes on entire chromosomes that are frequently gained or lost in tumors remains a recalcitrant problem as these alterations contain so many genes. We demonstrate that, because the chromosomal location of genes is highly scrambled between zebrafish and human, the number of passenger genes can be dramatically reduced by comparing the genes in copy number alterations found in zebrafish and human tumors. Thus, our approach dramatically narrows down the list of candidate cancer drivers, and can accelerate discovery of novel cancer drivers and pathways that could inform future targeted therapy and personalized medicine.
p53 protects us from cancer by transcriptionally regulating tumor suppressive programs designed to either prevent the development or clonal expansion of malignant cells. How p53 selects target genes in the genome in a context- and tissue-specific manner remains largely obscure. There is growing evidence that the ability of p53 to bind DNA in a cooperative manner prominently influences target gene selection with activation of the apoptosis program being completely dependent on DNA binding cooperativity. Here, we used ChIP-seq to comprehensively profile the cistrome of p53 mutants with reduced or increased cooperativity. The analysis highlighted a particular relevance of cooperativity for extending the p53 cistrome to non-canonical binding sequences characterized by deletions, spacer insertions and base mismatches. Furthermore, it revealed a striking functional separation of the cistrome on the basis of cooperativity; with low cooperativity genes being significantly enriched for cell cycle and high cooperativity genes for apoptotic functions. Importantly, expression of high but not low cooperativity genes was correlated with superior survival in breast cancer patients. Interestingly, in contrast to most p53-activated genes, p53-repressed genes did not commonly contain p53 binding elements. Nevertheless, both the degree of gene activation and repression were cooperativity-dependent, suggesting that p53-mediated gene repression is largely indirect and mediated by cooperativity-dependently transactivated gene products such as CDKN1A, E2F7 and non-coding RNAs. Since both activation of apoptosis genes with non-canonical response elements and repression of pro-survival genes are crucial for p53's apoptotic activity, the cistrome analysis comprehensively explains why p53-induced apoptosis, but not cell cycle arrest, strongly depends on the intermolecular cooperation of p53 molecules as a possible safeguard mechanism protecting from accidental cell killing.
The tumor suppressor gene p53 counteracts tumor growth by activating genes that prevent cell proliferation or induce cell death. How p53 selects genes in the genome to direct cell fate specifically into one or the other direction remains unclear. We show that the ability of p53 molecules to interact and thereby cooperate, influences which genes in the genome p53 is regulating. In the absence of cooperation, p53 only binds and regulates a limited ‘default’ set of genes that is proficient to stop cell proliferation but insufficient to induce cell death. Cooperation increases p53's DNA binding and enables context-dependent activation of apoptosis genes and repression of pro-survival genes which together triggers cell death. As the concerted effort of p53 molecules is needed, the threshold for cell killing is raised possibly to protect us from accidental cell loss. Thus, by shaping the genomic binding pattern, p53 cooperation fine-tunes the gene activity pattern to steer cell fate into the most appropriate, context-dependent direction. The genome-wide binding patterns of cooperating and non-cooperating p53 proteins generated in this study provide a comprehensive list of p53 binding sites as a resource for the scientific community to further explore mechanisms of tumor suppression by p53.
ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR
in vitro. All three can also independently activate Mec1ATR
in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.
DNA damage checkpoint signalling is an essential component of the DNA damage response. Many of the key proteins initiating the checkpoint signal have been identified and characterised in yeast. Here we explore the role of the ATR activating domain (AAD) of TopBP1 in embryonic development, cell growth and checkpoint activation using a mouse model. In contrast to yeasts, where the TopBP1 AAD plays a redundant, and thus phenotypically minor, role in ATR activation, our data demonstrate that the mouse TopBP1 AAD is essential for cellular proliferation. Interestingly, this suggests evolution has provided a simpler ATR activation mechanism in metazoans than it has in yeasts.
Arsenic is a well-established human carcinogen of poorly understood mechanism of genotoxicity. It is generally accepted that arsenic acts indirectly by generating oxidative DNA damage that can be converted to replication-dependent DNA double-strand breaks (DSBs), as well as by interfering with DNA repair pathways and DNA methylation. Here we show that in budding yeast arsenic also causes replication and transcription-independent DSBs in all phases of the cell cycle, suggesting a direct genotoxic mode of arsenic action. This is accompanied by DNA damage checkpoint activation resulting in cell cycle delays in S and G2/M phases in wild type cells. In G1 phase, arsenic activates DNA damage response only in the absence of the Yku70–Yku80 complex which normally binds to DNA ends and inhibits resection of DSBs. This strongly indicates that DSBs are produced by arsenic in G1 but DNA ends are protected by Yku70–Yku80 and thus invisible for the checkpoint response. Arsenic-induced DSBs are processed by homologous recombination (HR), as shown by Rfa1 and Rad52 nuclear foci formation and requirement of HR proteins for cell survival during arsenic exposure. We show further that arsenic greatly sensitizes yeast to phleomycin as simultaneous treatment results in profound accumulation of DSBs. Importantly, we observed a similar response in fission yeast Schizosaccharomyces pombe, suggesting that the mechanisms of As(III) genotoxicity may be conserved in other organisms.
Arsenic is a highly toxic compound which causes several types of cancer in humans. However, precise mechanisms of arsenic carcinogenesis remain elusive and are still a matter of debate. For example, the oxidative stress theory of arsenic proposes that arsenic generates reactive oxygen species producing oxidative DNA damage that can be converted to DNA double-strand breaks (DSBs) during replication. Using budding yeast as a model organism, we show that arsenic is able to induce DSBs in the absence of transcription, replication and pronounced oxidative stress. Importantly, we also demonstrate that arsenic greatly enhances cytotoxic activity of antitumor drug phleomycin, as evidenced by increased sensitivity and DNA fragmentation visible upon co-treatment. Our work suggests that arsenic acts as a direct inducer of DNA breaks and could be potentially used with other anticancer drugs, like phleomycin-related bleomycin, as a new combinatory therapy to treat cancers that poorly respond to these drugs. Additionally, since in many countries millions of people are exposed to high doses of arsenic in drinking water, we believe that our findings about genotoxicity of arsenic are important not only to geneticists but also to the general public.
Hypomethylating agents reactivate tumor suppressor genes that are epigenetically silenced in cancer. Inevitably these genes are resilenced, leading to drug resistance. Using the MLH1 tumor suppressor gene as a model, we showed that decitabine-induced re-expression was dependent upon demethylation and eviction of promoter nucleosomes. Following decitabine withdrawal, MLH1 was rapidly resilenced despite persistent promoter demethylation. Single molecule analysis at multiple time points showed that gene resilencing was initiated by nucleosome reassembly on demethylated DNA and only then was followed by remethylation and stable silencing. Taken together, these data establish the importance of nucleosome positioning in mediating resilencing of drug-induced gene reactivation and suggest a role for therapeutic targeting of nucleosome assembly as a mechanism to overcome drug resistance.
Hypomethylating agents are emerging as effective cancer therapies. However, their therapeutic effects are transient and drug resistance inevitably develops. While resistance is associated with resilencing of genes initially demethylated by the drug, the mechanism underlying this resilencing is unknown. We provide evidence that the rapid reassembly of nucleosomes at transcription start sites initiates resilencing and is a prerequisite for promoter remethylation. This finding shows reassembly of nucleosomes at the promoter of critical genes is a potential early marker of resistance to hypomethylating agents. Our findings have implications for the treatment of cancer using epigenetic therapies that target DNA methylation alone, and suggest that overcoming drug resistance will require therapeutic strategies which prevent nucleosome deposition.
The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state.
Cancer is a multigenic process, involving cooperative interactions between oncogenes or tumour suppressors. In this study, in a genetic screen in the vinegar fly, Drosophila melanogaster, for genes that cooperate with a mutation in the cell polarity (shape) regulator, scribbled (scrib), we identify a novel cooperative oncogene, abrupt. Expression of abrupt in scrib mutant tissue in the developing eye/antennal epithelium results in overgrown invasive tumours. abrupt encodes a BTB-zinc finger transcription factor, which has homology to several cancer-causing proteins in humans, such as BCL6. Analysis of the Abrupt targets and misexpressed genes in abrupt expressing-tissue and abrupt-expressing scrib mutant tumours, revealed cell fate regulators as a major class of targets. Thus, our results reveal that deregulation of multiple cell fate factors by Abrupt expression in the context of polarity disruption is associated with a progenitor-like cell state and the formation of overgrown invasive tumours. Our findings suggest that defective polarity may also be a critical factor in BTB-zinc finger-driven human cancers, and warrants further investigation into this issue.
Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b+, ascl1+, elavl3− cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.
Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most common cancer diagnosed in infancy. Although most cases arise sporadically, familial predisposition also occurs in association with mutations in a single copy of the PHOX2B gene, a “master regulator” of sympathetic neuronal development. The exact mechanisms by which these mutations increase susceptibility to neuroblastoma are unclear, primarily because of the paucity of optimal models in which to study very early development of the sympathetic nervous system. We took advantage of the ex vivo development and transparent nature of zebrafish embryos to study the roles of both normal and mutated PHOX2B in development of the sympathetic nervous system. We present data indicating that aberrant PHOX2B expression causes an arrest in the normal maturation of sympathetic neurons, leading to immature cells that are resistant to drug-induced differentiation. Indeed, we demonstrate that phox2b gene “dosage” is important for normal differentiation of sympathetic neurons in the zebrafish and suggest that the population of immature cells resulting from a decreased dosage of this pivotal factor may be susceptible to secondary mutations that could ultimately lead to neuroblastoma.
The Reed-Sternberg (RS) cell is the driving force behind Hodgkin lymphoma (HL), a unique malignancy in which the rare RS cell creates an inflammatory microenvironment that recruits a reactive tumor infiltrate. Well-known oncogenic factors such as nuclear factor kappa B (NFκB) signaling and Epstein-Barr virus infection are linked to HL pathogenesis but do not adequately explain the RS cell’s key pathologic features of multi-nucleation, abnormalities of centrosome function and number and aneuploidy. Chromosomal instability is also considered a key pathway in the origin of the RS cell, though the molecular mechanisms have largely been a “black box.” We demonstrated that the midbody kelch domain protein KLHDC8B protects against mitotic errors, centrosomal amplification and chromosomal instability. Here we discuss how the new findings linking KLHDC8B to mitotic integrity and faithful chromosomal segregation are providing mechanistic explanations for the origin of the RS cell and the molecular pathogenesis of chromosomal instability in HL.
Hodgkin lymphoma; Reed-Sternberg cell; aneuploidy; micronuclei; chromothripsis; kelch proteins; mitosis
Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA) and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing) and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1–4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson “two hit” hypothesis.
Polyps in the bowel are the precursors of development of bowel cancer. Much is known about the biology and pathology of these polyps. These polyps have classically been divided into two main types: adenomas and hyperplastic polyps. The former has pre-malignant potential, whereas it is thought the second does not. Within the last 10 years, a third type of polyp has been discovered, called the “serrated adenoma,” that has features of both adenomas and hyperplastic polyps. However, they seem to have very different characteristics, and we are not certain of the distinct molecular event that leads to their development. In this study we investigate the genetic differences that set apart serrated adenomas from other types of polyps. We report on a gene change within these polyps that seems to be associated with their development. The gene change is highly unusual as it consists of the loss of one copy of the gene as well as methylation of the other gene. This methylation switches off the remaining copy of the gene and stops its associated protein from being expressed in the polyp. To our knowledge, this is the first time this phenomenon has been seen.
LXR (Liver X Receptors) act as “sensor” proteins that regulate cholesterol uptake, storage, and efflux. LXR signaling is known to influence proliferation of different cell types including human prostatic carcinoma (PCa) cell lines. This study shows that deletion of LXR in mouse fed a high-cholesterol diet recapitulates initial steps of PCa development. Elevation of circulating cholesterol in Lxrαβ-/- double knockout mice results in aberrant cholesterol ester accumulation and prostatic intra-epithelial neoplasia. This phenotype is linked to increased expression of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2), which results in the down-regulation of the tumor suppressors Msmb and Nkx3.1 through increased methylation of lysine 27 of histone H3 (H3K27) on their promoter regions. Altogether, our data provide a novel link between LXR, cholesterol homeostasis, and epigenetic control of tumor suppressor gene expression.
Cholesterol is one of the major metabolic molecules required for a broad range of cellular processes. Recent advances in prostate cancer research have demonstrated that tumor cells need to increase their supply of cholesterol to sustain membrane building, proliferation, and survival capacities. Liver X receptors, which belong to the nuclear receptor superfamily, are central mediators of cholesterol homeostasis. Indeed, they regulate the expression of many genes involved in cholesterol uptake storage and efflux. Here, we show that genetic ablation of LXRs in mice results in the formation of precancerous lesions in the prostate, called prostatic intra-epithelial neoplasia. These are only observed when mice are fed a high-cholesterol diet. Hence, LXRs regulate cholesterol homeostasis in the prostate and protect cells from abnormal proliferation when exposed to high dietary cholesterol.
Gene fusions, like BCR/ABL1 in chronic myelogenous leukemia, have long been recognized in hematologic and mesenchymal malignancies. The recent finding of gene fusions in prostate and lung cancers has motivated the search for pathogenic gene fusions in other malignancies. Here, we developed a “breakpoint analysis” pipeline to discover candidate gene fusions by tell-tale transcript level or genomic DNA copy number transitions occurring within genes. Mining data from 974 diverse cancer samples, we identified 198 candidate fusions involving annotated cancer genes. From these, we validated and further characterized novel gene fusions involving ROS1 tyrosine kinase in angiosarcoma (CEP85L/ROS1), SLC1A2 glutamate transporter in colon cancer (APIP/SLC1A2), RAF1 kinase in pancreatic cancer (ATG7/RAF1) and anaplastic astrocytoma (BCL6/RAF1), EWSR1 in melanoma (EWSR1/CREM), CDK6 kinase in T-cell acute lymphoblastic leukemia (FAM133B/CDK6), and CLTC in breast cancer (CLTC/VMP1). Notably, while these fusions involved known cancer genes, all occurred with novel fusion partners and in previously unreported cancer types. Moreover, several constituted druggable targets (including kinases), with therapeutic implications for their respective malignancies. Lastly, breakpoint analysis identified new cell line models for known rearrangements, including EGFRvIII and FIP1L1/PDGFRA. Taken together, we provide a robust approach for gene fusion discovery, and our results highlight a more widespread role of fusion genes in cancer pathogenesis.
Gene fusions represent an important class of cancer genes, created by rearrangements of the genome that bring together two different genes. Because they are unique to cancer cells, gene fusions are ideal diagnostic markers and therapeutic targets. While gene fusions were once thought restricted mainly to blood cancers, recent discoveries suggest they are more widespread. Here, we have developed an approach for mining DNA microarray data to detect the tell-tale signatures of gene fusions, as “breakpoints” occurring within the encoding DNA or expressed transcripts. We apply this approach to a large collection of nearly 1,000 human cancer specimens. From this analysis, we discover and verify twelve new gene fusions occurring in diverse cancer types. We verify that some of these rearrangements recur in other samples of the same cancer type (supporting a causal role) and that the cancers show dependency on the fusion for cancer cell growth. Notably, some of these fusions (e.g. CEP85L/ROS1 in angiosarcoma) represent the first for that cancer type and thus provide important new biological insight. Some are also good drug targets (including rearrangements of ROS1, RAF1, and CDK6 kinases), with clear implications for therapy.
Rare variation in protein coding sequence is poorly captured by GWAS arrays and has been hypothesized to contribute to disease heritability. Using the Illumina HumanExome SNP array, we successfully genotyped 191,032 common and rare non-synonymous, splice site, or nonsense variants in a multiethnic sample of 2,984 breast cancer cases, 4,376 prostate cancer cases, and 7,545 controls. In breast cancer, the strongest associations included either SNPs in or gene burden scores for genes LDLRAD1, SLC19A1, FGFBP3, CASP5, MMAB, SLC16A6, and INS-IGF2. In prostate cancer, one of the most associated SNPs was in the gene GPRC6A (rs2274911, Pro91Ser, OR = 0.88, P = 1.3×10−5) near to a known risk locus for prostate cancer; other suggestive associations were noted in genes such as F13A1, ANXA4, MANSC1, and GP6. For both breast and prostate cancer, several of the most significant associations involving SNPs or gene burden scores (sum of minor alleles) were noted in genes previously reported to be associated with a cancer-related phenotype. However, only one of the associations (rs145889899 in LDLRAD1, p = 2.5×10−7 only seen in African Americans) for overall breast or prostate cancer risk was statistically significant after correcting for multiple comparisons. In addition to breast and prostate cancer, other cancer-related traits were examined (body mass index, PSA level, and alcohol drinking) with a number of known and potentially novel associations described. In general, these findings do not support there being many protein coding variants of moderate to high risk for breast and prostate cancer with odds ratios over a range that is probably required for protein coding variation to play a truly outstanding role in risk heritability. Very large sample sizes will be required to better define the role of rare and less penetrant coding variation in prostate and breast cancer disease genetics.
For breast and prostate cancer, GWAS have revealed many risk variants (>70 for each cancer as of this report). All together the common variants in these regions explain only a minority of familial risk of these cancers. Using the Illumina HumanExome SNP array, we explored the hypothesis of rare coding variation contributing to breast and prostate cancer risk in a sample of African American, Latino, Japanese, Native Hawaiian, and European American breast and prostate cancer cases and controls from the Multiethnic Cohort study. While only one association exceeded significance thresholds after correcting for multiple comparisons, a number of suggestive associations involving genes previously reported to be associated with a cancer-related phenotype were noted. Our results do not generally support a major role of protein-coding variants with odds ratios over a range that is probably required for protein coding variation to play a truly outstanding role in risk heritability. If very rare and/or less penetrant coding variants underlie disease heritability of these cancers, then very large sample sizes (i.e. consortia) will be required for their discovery.
Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.
The ends of chromosomes, called telomeres, prevent chromosome ends from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion by forming a specialized nucleo-protein complex. The critical function of telomeres in end protection has a downside, in that it interferes with the repair of DSBs that occur near telomeres. DSBs are critical DNA lesions, because if they are not repaired correctly they can result in gross chromosome rearrangements (GCRs). As a result, the deficiency in DSB repair near telomeres has now been implicated in ageing, by promoting cell senescence, and cancer, by promoting telomere dysfunction due to oncogene-induced replication stress. The studies presented here demonstrate that DSBs near telomeres commonly result in GCRs in a human tumor cell line. Moreover, our results demonstrate that the mechanism of repair of telomeric DSBs is very different from the mechanism of repair of DSBs at other locations, supporting our hypothesis that the deficiency in repair of DSBs near telomeres is a result of the abnormal processing of DSBs due to the presence of telomeric proteins. Understanding the mechanism responsible for the deficiency in DSB repair near telomeres will provide important insights into critical human disease pathways.
The domestic dog is a robust model for studying the genetics of complex disease susceptibility. The strategies used to develop and propagate modern breeds have resulted in an elevated risk for specific diseases in particular breeds. One example is that of Standard Poodles (STPOs), who have increased risk for squamous cell carcinoma of the digit (SCCD), a locally aggressive cancer that causes lytic bone lesions, sometimes with multiple toe recurrence. However, only STPOs of dark coat color are at high risk; light colored STPOs are almost entirely unaffected, suggesting that interactions between multiple pathways are necessary for oncogenesis. We performed a genome-wide association study (GWAS) on STPOs, comparing 31 SCCD cases to 34 unrelated black STPO controls. The peak SNP on canine chromosome 15 was statistically significant at the genome-wide level (Praw = 1.60×10−7; Pgenome = 0.0066). Additional mapping resolved the region to the KIT Ligand (KITLG) locus. Comparison of STPO cases to other at-risk breeds narrowed the locus to a 144.9-Kb region. Haplotype mapping among 84 STPO cases identified a minimal region of 28.3 Kb. A copy number variant (CNV) containing predicted enhancer elements was found to be strongly associated with SCCD in STPOs (P = 1.72×10−8). Light colored STPOs carry the CNV risk alleles at the same frequency as black STPOs, but are not susceptible to SCCD. A GWAS comparing 24 black and 24 light colored STPOs highlighted only the MC1R locus as significantly different between the two datasets, suggesting that a compensatory mutation within the MC1R locus likely protects light colored STPOs from disease. Our findings highlight a role for KITLG in SCCD susceptibility, as well as demonstrate that interactions between the KITLG and MC1R loci are potentially required for SCCD oncogenesis. These findings highlight how studies of breed-limited diseases are useful for disentangling multigene disorders.
Domesticated dogs offer a unique mechanism for disentangling complex genetic traits, such as cancer. Over 300 breeds exist worldwide, each selected for particular morphologic and behavioral traits. Unfortunately the breeding programs used to generate such diversity are associated with breed-specific increase in disease. Squamous cell carcinoma of the digit (SCCD) is a locally aggressive cancer that causes lytic bone lesions and, occasionally, death. Among the breeds with the highest risk is the Standard Poodle (STPO), where the disease is found only in dark-coated dogs. We show that the KITLG locus is highly associated with SCCD and that a 5.7-Kb copy number variant is likely causative for the disease when in an expanded form. Interestingly, light-colored STPO carry the putative causal variant at the same frequency as black STPOs, but are protected from SCCD. We show this is likely due to a compensatory mutation in the well-known coat color locus, MC1R. This work demonstrates the utility of dog breeds for understanding the genetic causes of complex diseases of interest to both human and animal health.
The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.
Gene expression can be modulated at different steps on the way from DNA to protein including control of transcription, translation, and post-translational modifications. An abnormality in the regulation of mRNA and protein expression is a hallmark of many human diseases, including cancer. In some eukaryotic genes translation can be influenced by small DNA sequences termed upstream open reading frames (uORFs). These elements located upstream to the gene start codon may either negatively influence the ability of the translational machinery to reinitiate translation of the main protein or, much less frequently, stimulate protein translation by enabling the ribosomes to bypass cis-acting inhibitory elements. CDKN1B, which encodes the cell cycle inhibitor p27KIP1, includes an uORF in its 5′UTR sequence. p27KIP1 expression is often reduced in cancer, and germline mutations have been identified in CDKN1B in patients affected with a syndrome (MEN4) characterized by varying combinations of tumors in endocrine glands. Here we show that a small deletion in the uORF upstream to CDKN1B reduces translation reinitiation efficiency, leading to underexpression of p27KIP1 and coinciding with tumorigenesis. This study describes a novel mechanism by which p27KIP1 could be underexpressed in human tumors. In addition, our data provide a new insight to the unique pathogenic potential of uORFs in human diseases.
Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease FEN1, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through in vitro biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in CDC4, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between FEN1 and MRE11A, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model.
Anticancer therapeutic discovery is a major challenge in cancer research. Because cancer is a disease caused by somatic genetic mutations, the search for anticancer therapeutics is often driven by the ability to exploit genetic differences specific to tumor cells. Recently, cancer therapeutic development has sought to exploit synthetic lethality, a situation in which the combination of two independently viable mutations results in lethality. If a compound can be found to selectively kill a specific genotype via inhibition of a specific gene product, this is known as a chemical-genetic interaction, and it mimics a synthetic lethal genetic interaction. The ideal therapeutic would be broad spectrum, that is, active against multiple cancer genotypes within a tumor type and/or across a variety of cancers. We have developed an approach, taking advantage of the evolutionary conservation of synthetic lethal interactions, to identify “second-site” targets in cancer: genes whose chemical inhibition leads to selective killing of tumor cells across a broad spectrum of cancer genotypes. We identified small-molecule inhibitors of one such target, FEN1, and showed that these compounds were able to selectively kill human cells carrying cancer-relevant mutations. This approach will facilitate the development of anticancer therapeutics active against a variety of cancer genotypes.
The C. elegans cell fate map, in which the lineage of its approximately 1000 cells is visibly charted beginning from the zygote, represents a developmental biology milestone. Nematode development is invariant from one specimen to the next, whereas in mammals, aspects of development are probabilistic, and development exhibits variation between even genetically identical individuals. Consequently, a single defined cell fate map applicable to all individuals cannot exist.
To determine the extent to which patterns of cell lineage are conserved between different mice, we have employed the recently developed method of “phylogenetic fate mapping” to compare cell fate maps in siblings. In this approach, somatic mutations arising in individual cells are used to retrospectively deduce lineage relationships through phylogenetic and—as newly investigated here—related analytical approaches based on genetic distance. We have cataloged genomic mutations at an average of 110 mutation-prone polyguanine (polyG) tracts for about 100 cells clonally isolated from various corresponding tissues of each of two littermates of a hypermutable mouse strain.
We find that during mouse development, muscle and fat arise from a mixed progenitor cell pool in the germ layer, but, contrastingly, vascular endothelium in brain derives from a smaller source of progenitor cells. Additionally, formation of tissue primordia is marked by establishment of left and right lateral compartments, with restricted cell migration between divisions. We quantitatively demonstrate that development represents a combination of stochastic and deterministic events, offering insight into how chance influences normal development and may give rise to birth defects.
Fate map; Cell lineage; Differentiation
Nasopharyngeal carcinoma (NPC) is an epithelial malignancy facilitated by Epstein-Barr Virus infection. Here we resolve the major genetic influences for NPC incidence using a genome-wide association study (GWAS), independent cohort replication, and high-resolution molecular HLA class I gene typing including 4,055 study participants from the Guangxi Zhuang Autonomous Region and Guangdong province of southern China. We detect and replicate strong association signals involving SNPs, HLA alleles, and amino acid (aa) variants across the major histocompatibility complex-HLA-A, HLA –B, and HLA -C class I genes (PHLA-A-aa-site-62 = 7.4×10−29; P HLA-B-aa-site-116 = 6.5×10−19; P HLA-C-aa-site-156 = 6.8×10−8 respectively). Over 250 NPC-HLA associated variants within HLA were analyzed in concert to resolve separate and largely independent HLA-A, -B, and -C gene influences. Multivariate logistical regression analysis collapsed significant associations in adjacent genes spanning 500 kb (OR2H1, GABBR1, HLA-F, and HCG9) as proxies for peptide binding motifs carried by HLA- A*11:01. A similar analysis resolved an independent association signal driven by HLA-B*13:01, B*38:02, and B*55:02 alleles together. NPC resistance alleles carrying the strongly associated amino acid variants implicate specific class I peptide recognition motifs in HLA-A and -B peptide binding groove as conferring strong genetic influence on the development of NPC in China.
NPC is a deadly throat cancer in China that is dependent on EBV infection. Here, we performed a 1 M SNP genome-wide association study using a large cohort of Chinese study participants at risk for NPC. Although several putative gene regions show significant associations, the strongest statistical signals involved scores of variants within the HLA region on chromosome 6. HLA poses a formidable association-genetics challenge because of extensive linkage disequilibrium, rather low allele frequencies, and multiple physically close interacting genes of diverse function. We examined over 250 NPC-HLA associated variants detected with sequence-based nucleotide alleles and amino acid variants. The multiple associations were collapsed to implicate causal signals by multivariate logistical regression to resolve allele association interaction. One operative variant was identified as the HLA-A*11:01 allele motif, specifically in the peptide binding groove, which recognizes invading antigens; a second involved two aa sites with HLA-B tracking B*13:01 and B*55:02 alleles. We synthesize these new and previous discoveries to help resolve the important gene influences on this disease.
Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.
Normal cells have robust mechanisms to maintain the proper sequence of their DNA; in cancer cells these mechanisms are compromised, resulting in complex changes in the DNA of tumors. How this genome instability begins has not been defined, except in cases of familial cancers, which often have mutations in genes called “caretaker” genes, necessary to preserve DNA stability. We have defined a mechanism for genome instability in non-familial tumors that occur sporadically in the population. Certain fragile regions of our DNA are more difficult to duplicate during cell division and are prone to breakage. A fragile region, FRA3B, lies within the FHIT gene, and deletions within FRA3B are common in precancer cells, causing loss of Fhit protein expression. We find that loss of Fhit protein causes defective DNA replication, leading to further DNA breaks. Cells that continue DNA replication in the absence of Fhit develop numerous chromosomal aberrations. Importantly, cells established from tissues of mice that are missing Fhit undergo selection for increasing DNA alterations that can promote immortality, a cancer cell hallmark. Thus, loss of Fhit expression in precancer cells is the first step in the initiation of genomic instability and facilitates cancer development.
Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1HI cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24LOCD44HI cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDHHI and dye effluxHI cells, and increasing Oct1 increases the proportion of ALDHHI cells. Normal ALDHHI cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.
Understanding the mechanisms that control stem cell function is a fundamental prerequisite both for the full application of stem cells to regenerative medicine and for a full understanding of the relationship between stem cells and cancer. In this study we show that a transcription factor known as Oct1 is a central regulator of normal and cancer stem cell function. We show that high Oct1 levels are associated with stem cells in multiple normal and malignant settings. Altering Oct1 expression, up or down, correspondingly alters multiple stem cell parameters, as well as stem cell function. We highlight known and identify new target genes Oct1 binds to that are consistent with a role in stem cell function.