Heterozygous germline BLM mutations increase susceptibility to asbestos and mesothelioma.

Rare biallelic BLM gene mutations cause Bloom syndrome. Whether BLM heterozygous germline mutations (BLM+/-) cause human cancer remains unclear. We sequenced the germline DNA of 155 mesothelioma patients (33 familial and 122 sporadic). We found 2 deleterious germline BLM+/- mutations within 2 of 33 families with multiple cases of mesothelioma, one from Turkey (c.569_570del; p.R191Kfs*4) and one from the United States (c.968A>G; p.K323R). Some of the relatives who inherited these mutations developed mesothelioma, while none with nonmutated BLM were affected. Furthermore, among 122 patients with sporadic mesothelioma treated at the US National Cancer Institute, 5 carried pathogenic germline BLM+/- mutations. Therefore, 7 of 155 apparently unrelated mesothelioma patients carried BLM+/- mutations, significantly higher (P = 6.7E-10) than the expected frequency in a general, unrelated population from the gnomAD database, and 2 of 7 carried the same missense pathogenic mutation c.968A>G (P = 0.0017 given a 0.00039 allele frequency). Experiments in primary mesothelial cells from Blm+/- mice and in primary human mesothelial cells in which we silenced BLM revealed that reduced BLM levels promote genomic instability while protecting from cell death and promoted TNF-α release. Blm+/- mice injected intraperitoneally with asbestos had higher levels of proinflammatory M1 macrophages and of TNF-α, IL-1ß, IL-3, IL-10, and IL-12 in the peritoneal lavage, findings linked to asbestos carcinogenesis. Blm+/- mice exposed to asbestos had a significantly shorter survival and higher incidence of mesothelioma compared to controls. We propose that germline BLM+/- mutations increase the susceptibility to asbestos carcinogenesis, enhancing the risk of developing mesothelioma.

A tri-serine cluster within the topoisomerase IIα-interaction domain of the BLM helicase is required for regulating chromosome breakage in human cells.

The recQ-like helicase BLM interacts directly with topoisomerase IIα to regulate chromosome breakage in human cells. We demonstrate that a phosphosite tri-serine cluster (S577/S579/S580) within the BLM topoisomerase IIα-interaction region is required for this function. Enzymatic activities of BLM and topoisomerase IIα are reciprocally stimulated in vitro by ten-fold for topoisomerase IIα decatenation/relaxation activity and three-fold for BLM unwinding of forked DNA duplex substrates. A BLM transgene encoding alanine substitutions of the tri-serine cluster in BLM-/- transfected cells increases micronuclei, DNA double strand breaks and anaphase ultra-fine bridges (UFBs), and decreases cellular co-localization of BLM with topoisomerase IIα. In vitro, these substitutions significantly reduce the topoisomerase IIα-mediated stimulation of BLM unwinding of forked DNA duplexes. Substitution of the tri-serine cluster with aspartic acids to mimic serine phosphorylation reverses these effects in vitro and in vivo. Our findings implicate the modification of this BLM tri-serine cluster in regulating chromosomal stability.

Functions of the APC tumor suppressor protein dependent and independent of canonical WNT signaling: implications for therapeutic targeting.

The acquisition of biallelic mutations in the APC gene is a rate-limiting step in the development of most colorectal cancers and occurs in the earliest lesions. APC encodes a 312-kDa protein that localizes to multiple subcellular compartments and performs diverse functions. APC participates in a cytoplasmic complex that promotes the destruction of the transcriptional licensing factor ß-catenin; APC mutations that abolish this function trigger constitutive activation of the canonical WNT signaling pathway, a characteristic found in almost all colorectal cancers. By negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis, and suppresses invasion and tumor progression. APC further antagonizes canonical WNT signaling by interacting with and counteracting ß-catenin in the nucleus. APC also suppresses tumor initiation and progression in the colorectal epithelium through functions that are independent of canonical WNT signaling. APC regulates the mitotic spindle to facilitate proper chromosome segregation, localizes to the cell periphery and cell protrusions to establish cell polarity and appropriate directional migration, and inhibits DNA replication by interacting directly with DNA. Mutations in APC are often frameshifts, insertions, or deletions that introduce premature stop codons and lead to the production of truncated APC proteins that lack its normal functions and possess tumorigenic properties. Therapeutic approaches in development for the treatment of APC-deficient tumors are focused on the inhibition of canonical WNT signaling, especially through targets downstream of APC in the pathway, or on the restoration of wild-type APC expression.

Differential requirements for DNA repair proteins in immortalized cell lines using alternative lengthening of telomere mechanisms.

Cancer cells require telomere maintenance to enable uncontrolled growth. Most often telomerase is activated, although a subset of human cancers are telomerase-negative and depend on recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). ALT depends on proteins that are essential for homologous recombination, including BLM and the MRN complex, to extend telomeres. This study surveyed the requirement for requisite homologous recombination proteins, yet to be studied in human ALT cell lines, by protein depletion using RNA interference. Effects on ALT were evaluated by measuring C-circle abundance, a marker of ALT. Surprisingly, several proteins essential for homologous recombination, BARD1, BRCA2, and WRN, were dispensable for C-circle production, while PALB2 had varying effects on C-circles among ALT cell lines. Depletion of homologous recombination proteins BRCA1 and BLM, which have been previously studied in ALT, decreased C-circles in all ALT cell lines. Depletion of the non-homologous end joining proteins 53BP1 and LIG4 had no effect on C-circles in any ALT cell line. Proteins such as chromatin modifiers that recruit double-strand break proteins, RNF8 and RNF168, and other proteins loosely grouped into excision DNA repair processes, XPA, MSH2, and MPG, reduced C-circles in some ALT cell lines. MSH2 depletion also reduced recombination at telomeres as measured by intertelomeric exchanges. Collectively, the requirement for DNA repair proteins varied between the ALT cell lines compared. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways.

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription.

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

Human sarcomas are mosaic for telomerase-dependent and telomerase-independent telomere maintenance mechanisms: implications for telomere-based therapies.

Telomere shortening necessitates that tumor cells activate a telomere maintenance mechanism (TMM) to support immortalization. Although most tumor cells activate expression of the enzyme telomerase, some cells elongate telomeres in a telomerase-independent manner, termed alternative lengthening of telomeres (ALT). Previous studies have evaluated the presence of telomerase or ALT mechanisms or both in a variety of tumor types. Our studies also show that TMMs are not mutually exclusive in some tumors. In contrast, our IHC analyses of human sarcomas identified a subset of tumors with some cells containing ALT-associated PML bodies, a hallmark of ALT, and separate cells expressing telomerase in the same tumor. By using a second set of human osteosarcomas, we merged IHC and biochemical analyses to characterize more fully the tumor TMM. The IHC data reveal the presence of both telomerase- and ALT-positive tumor cells in samples that demonstrate characteristics of both telomerase and ALT in biochemical assays. These assays, which measure telomere length and telomerase activity of tumor extracts, are conventionally used to classify tumor TMM. Our results suggest that TMM is not a single or perhaps static characteristic of some tumors and that TMM heterogeneity should be considered in tumor stratification. Furthermore, clinical interest in telomere-based therapies may necessitate accurate characterization of tumor TMM before treatment to maximize therapeutic efficacy.

Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription.

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

Alternative mechanisms of telomere lengthening: permissive mutations, DNA repair proteins and tumorigenic progression.

Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor cells are unknown, although cellular origin may favor one or the other mechanisms. ALT pathways are incompletely understood to date; however, recent publications have increasingly broadened our understanding of how ALT is activated, how it proceeds, and how it influences tumor growth. Specific mutational events influence ALT activation, as mutations in genes that suppress recombination and/or alterations in the regulation of telomerase expression are associated with ALT. Once engaged, ALT uses DNA repair proteins to maintain telomeres in the absence of telomerase; experiments that manipulate the expression of specific proteins in cells using ALT are illuminating some of its mechanisms. Furthermore, ALT may influence tumor growth, as experimental and clinical data suggest that telomerase expression may favor tumor progression. This review summarizes recent findings in mammalian cells and models, as well as clinical data, that identify the genetic mutations permissive to ALT, the DNA repair proteins involved in ALT mechanisms and the importance of telomere maintenance mechanisms for tumor progression. A comprehensive understanding of the mechanisms that permit tumor cell immortalization will be important for identifying novel therapeutic targets in cancer.

Crosslinks and crosstalk: human cancer syndromes and DNA repair defects.

A subset of human cancer syndromes result from inherited defects in genes responsible for DNA repair. During the past few years, discoveries concerning the intersection of certain DNA repair processes have increased our understanding of how the disruption of specific DNA repair mechanisms leads to genomic instability and tumorigenesis. This review focuses on the human genes MUTYH, BRCA2/FANCD1, and BLM.

Phosphatidylinositol 3-kinase (PI3K) signaling via glycogen synthase kinase-3 (Gsk-3) regulates DNA methylation of imprinted loci.

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3ß, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3ß in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.

The mitochondrial protein hTID-1 partners with the caspase-cleaved adenomatous polyposis cell tumor suppressor to facilitate apoptosis.

BACKGROUND & AIMS: The adenomatous polyposis cell (APC) tumor suppressor is a multifunctional protein involved in cell migration, proliferation, differentiation, and apoptosis. Cleavage of APC and the subsequent release of an amino-terminal segment are necessary for a transcription-independent mechanism of APC-mediated apoptosis. The aim of the current study is to elucidate the mechanism by which the amino-terminus of APC contributes to the enhancement of apoptosis. METHODS: Previous yeast 2-hybrid screens, using the armadillo repeat domain of APC as bait, identified hTID-1 as a potential binding partner. Coimmunoprecipitations, coimmunofluorescence, and binding assays confirm a direct interaction between caspase-cleaved APC and hTID-1 in vivo at the mitochondria. Overexpression and small interfering RNA (siRNA) knockdown studies were designed to determine the significance of this interaction. RESULTS: These experiments have identified hTID-1 as a directly interacting protein partner of caspase-cleaved APC. hTID-1 is an apoptosis modulator: 2 of its known mitochondrial protein isoforms, 43-kilodaltons and 40-kilodaltons, have opposing effects in apoptosis. We demonstrate that the amino-terminal segment of APC interacts with both hTID-1 isoforms directly, although there is a stronger association with the apoptotic suppressor 40-kilodalton isoform in vitro. This interaction localizes to amino acids 202-512 of APC, a region including 2 of the 7 armadillo repeats. Overexpression of the 40-kilodalton hTID-1 isoform partially rescues cells from apoptosis mediated by APC 1-777, whereas siRNA knockdown of this hTID-1 isoform enhances apoptosis. CONCLUSIONS: These data suggest that the amino-terminal segment of APC promotes cell sensitivity to apoptosis modulated through its binding to 40- and 43-kilodalton hTID-1 isoforms.

Functional characterization of miR-708 microRNA in telomerase positive and negative human cancer cells.

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.

Unwinding protein complexes in ALTernative telomere maintenance.

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA-binding proteins and a number of three dimensional nucleic acid structures including G-quartets and D-loops. A number of studies suggest that the BLM and WRN recQ-like helicases play important roles in recombination-mediated mechanisms of telomere elongation or Alternative Lengthening of Telomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT-associated nuclear bodies in telomerase-negative immortalized cell lines and interact with the telomere-specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double-strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, resolution of stalled replication forks and synthesis-dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ-like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec-Q-like helicases and their role in telomere elongation during ALT processes.

Tumour predisposition and cancer syndromes as models to study gene-environment interactions.

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.

The APC tumor suppressor inhibits DNA replication by directly binding to DNA via its carboxyl terminus.

BACKGROUND & AIMS: The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of beta-catenin levels in the cell. Transient over expression of the tumor suppressor gene APC in colon cancer cells prevents entry into S phase of the cell cycle, a phenotype only partially restored by cotransfection of a transcriptionally active form of beta-catenin. In an attempt to define its transcription-independent tumor suppressor functions, we tested whether APC directly affects DNA replication. METHODS: A transcriptionally quiescent in vitro DNA replication system, the polymerase chain reaction, DNA binding assays, and transient transfections in colon cancer cell lines were used to determine the effects of APC on DNA replication and the mechanism by which it works. RESULTS: We report that exogenous full-length APC inhibits replication of template DNA through a function that maps to amino acids 2140-2421, a region of the protein commonly lost by somatic or germline mutation. This segment of APC directly interacts with DNA, while mutation of the DNA-binding S(T)PXX motifs within it abolishes DNA binding and reduces inhibition of DNA replication. Phosphorylation of this segment by cyclin-dependent kinases also reduces inhibition of DNA replication. Furthermore, transient transfection of an APC segment encoding amino acids 2140-2421 into a colon cancer cell line with mutant APC prevents cell cycle progression into or through S phase. CONCLUSIONS: Our results suggest that APC can negatively regulate cell cycle progression through inhibition of DNA replication by direct interaction with DNA.

beta-Catenin/Wnt signaling regulates expression of the membrane type 3 matrix metalloproteinase in gastric cancer.

Activation of Wnt signaling through beta-catenin dysregulation occurs in numerous human tumors, including gastric cancer. The specific consequences of Wnt signaling in gastric cancer, however, are not well characterized. This study shows that the introduction of mutant beta-catenin into gastric cancer cell lines by adenoviral infection enhances invasiveness and proliferation and up-regulates the expression of the gene encoding the matrix metalloproteinase (MMP) family member membrane type 3 MMP (MT3-MMP). Up-regulation of MT3-MMP is critical to the invasive phenotype as shown by small interfering RNA (siRNA) studies. Immunohistochemical staining also showed that MT3-MMP was highly expressed in gastric cancers with activating beta-catenin mutations. These observations suggest that Wnt activation may contribute to gastric cancer progression by increasing the invasiveness of neoplastic cells in the stomach via up-regulation of MT3-MMP expression.

Chromatin-associated APC regulates gene expression in collaboration with canonical WNT signaling and AP-1.

Mutation of the APC gene occurs in a high percentage of colorectal tumors and is a central event driving tumor initiation in the large intestine. The APC protein performs multiple tumor suppressor functions including negative regulation of the canonical WNT signaling pathway by both cytoplasmic and nuclear mechanisms. Published reports that APC interacts with ß-catenin in the chromatin fraction to repress WNT-activated targets have raised the possibility that chromatin-associated APC participates more broadly in mechanisms of transcriptional control. This screening study has used chromatin immunoprecipitation and next-generation sequencing to identify APC-associated genomic regions in colon cancer cell lines. Initial target selection was performed by comparison and statistical analysis of 3,985 genomic regions associated with the APC protein to whole transcriptome sequencing data from APC-deficient and APC-wild-type colon cancer cells, and two types of murine colon adenomas characterized by activated Wnt signaling. 289 transcripts altered in expression following APC loss in human cells were linked to APC-associated genomic regions. High-confidence targets additionally validated in mouse adenomas included 16 increased and 9 decreased in expression following APC loss, indicating that chromatin-associated APC may antagonize canonical WNT signaling at both WNT-activated and WNT-repressed targets. Motif analysis and comparison to ChIP-seq datasets for other transcription factors identified a prevalence of binding sites for the TCF7L2 and AP-1 transcription factors in APC-associated genomic regions. Our results indicate that canonical WNT signaling can collaborate with or antagonize the AP-1 transcription factor to fine-tune the expression of shared target genes in the colorectal epithelium. Future therapeutic strategies for APC-deficient colorectal cancers might be expanded to include agents targeting the AP-1 pathway.

Mutational Mechanisms That Activate Wnt Signaling and Predict Outcomes in Colorectal Cancer Patients.

APC biallelic loss-of-function mutations are the most prevalent genetic changes in colorectal tumors, but it is unknown whether these mutations phenocopy gain-of-function mutations in the CTNNB1 gene encoding ß-catenin that also activate canonical WNT signaling. Here we demonstrate that these two mutational mechanisms are not equivalent. Furthermore, we show how differences in gene expression produced by these different mechanisms can stratify outcomes in more advanced human colorectal cancers. Gene expression profiling in Apc-mutant and Ctnnb1-mutant mouse colon adenomas identified candidate genes for subsequent evaluation of human TCGA (The Cancer Genome Atlas) data for colorectal cancer outcomes. Transcriptional patterns exhibited evidence of activated canonical Wnt signaling in both types of adenomas, with Apc-mutant adenomas also exhibiting unique changes in pathways related to proliferation, cytoskeletal organization, and apoptosis. Apc-mutant adenomas were characterized by increased expression of the glial nexin Serpine2, the human ortholog, which was increased in advanced human colorectal tumors. Our results support the hypothesis that APC-mutant colorectal tumors are transcriptionally distinct from APC-wild-type colorectal tumors with canonical WNT signaling activated by other mechanisms, with possible implications for stratification and prognosis.Significance: These findings suggest that colon adenomas driven by APC mutations are distinct from those driven by WNT gain-of-function mutations, with implications for identifying at-risk patients with advanced disease based on gene expression patterns. Cancer Res; 78(3); 617-30. ©2017 AACR.