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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 β-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.

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.

Although disruption of DNA repair capacity is unquestionably associated with cancer susceptibility in humans and model organisms, it remains unclear if the inherent tumor phenotypes of DNA repair deficiency syndromes can be regulated by manipulating DNA repair pathways. Loss-of-function mutations in BLM, a member of the RecQ helicase family, cause Bloom's syndrome (BS), a rare, recessive genetic disorder that predisposes to many types of cancer. BLM functions in many aspects of DNA homeostasis, including the suppression of homologous recombination (HR) in somatic cells. We investigated whether BLM overexpression, in contrast with loss-of-function mutations, attenuated the intestinal tumor phenotypes of ApcMin/+ and ApcMin/+;Msh2−/− mice, animal models of familial adenomatous polyposis coli (FAP). We constructed a transgenic mouse line expressing human BLM (BLM-Tg) and crossed it onto both backgrounds. BLM-Tg decreased adenoma incidence in a dose-dependent manner in our ApcMin/+ model of FAP, although levels of GIN were unaffected and concomitantly increased animal survival over 50%. It did not reduce intestinal tumorigenesis in ApcMin/+;Msh2−/− mice. We used the pink-eyed unstable (pun) mouse model to demonstrate that increasing BLM dosage in vivo lowered endogenous levels of HR by 2-fold. Our data suggest that attenuation of the Min phenotype is achieved through a direct effect of BLM-Tg on the HR repair pathway. These findings demonstrate that HR can be manipulated in vivo to modulate tumor formation at the organismal level. Our data suggest that lowering HR frequencies may have positive therapeutic outcomes in the context of specific hereditary cancer predisposition syndromes, exemplified by FAP. Cancer Prev Res; 8(7); 650–6. ©2015 AACR.

Summary MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment., Highlights • miR-135b is overexpressed in mouse and human colorectal cancer • miR-135b overexpression is associated with poor clinical outcome • miR-135b activation is triggered by oncogenic pathways in colorectal cancer • miR-135b represents a therapeutic target for colorectal cancer, Valeri et al. identify miR-135b as a key oncogenic pathway effector involved in transformation and colorectal cancer (CRC) progression. Upregulation of miR-135b in human CRCs correlates with poor clinical outcome. miR-135b targets several tumor suppressor genes and is a potential target for CRC therapy.

Defects in coordinated ribosomal RNA (rRNA) transcription in the nucleolus cause cellular and organismal growth deficiencies. Bloom’s syndrome, an autosomal recessive human disorder caused by mutated recQ-like helicase BLM, presents with growth defects suggestive of underlying defects in rRNA transcription. Our previous studies showed that BLM facilitates rRNA transcription and interacts with RNA polymerase I and topoisomerase I (TOP1) in the nucleolus. The mechanisms regulating localization of BLM to the nucleolus are unknown. In this study, we identify the TOP1-interaction region of BLM by co-immunoprecipitation of in vitro transcribed and translated BLM segments and show that this region includes the highly conserved nuclear localization sequence (NLS) of BLM. Biochemical and nucleolar co-localization studies using site-specific mutants show that two serines within the NLS (S1342 and S1345) are critical for nucleolar localization of BLM but do not affect the functional interaction of BLM with TOP1. Mutagenesis of both serines to aspartic acid (phospho-mimetic), but not alanine (phospho-dead), results in approximately 80% reduction in nucleolar localization of BLM while retaining the biochemical functions and nuclear localization of BLM. Our studies suggest a role for this region in regulating nucleolar localization of BLM via modification of the two serines within the NLS.

Cells deficient in the recQ-like helicase BLM are characterized by chromosome changes that suggest the disruption of normal mechanisms needed to resolve recombination intermediates and to maintain chromosome stability. Human BLM and topoisomerase IIα interact directly via amino acids 489–587 of BLM and colocalize predominantly in late G2 and M phases of the cell cycle. Deletion of this region does not affect the inherent in vitro helicase activity of BLM but inhibits the topoisomerase IIα–dependent enhancement of its activity, based on the analysis of specific DNA substrates that represent some recombination intermediates. Deletion of the interaction domain from BLM fails to correct the elevated chromosome breakage of transfected BLM-deficient cells. Our results demonstrate that the BLM–topoisomerase IIα interaction is important for preventing chromosome breakage and elucidate a DNA repair mechanism that is critical to maintain chromosome stability in cells and to prevent tumor formation. Cancer Res; 71(2); 561–71. ©2011 AACR.

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, 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-3β 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.

Bloom's syndrome (BS) is a recessive human chromosome breakage disorder caused by mutations in the recQ-like helicase BLM, sometimes known as RECQL2. Its clinical phenotype invariably includes male infertility, dwarfism, cancer susceptibility, and growth defects related to now characterized biochemical functions in DNA repair and rDNA transcription. The BLM protein includes two serines within a highly conserved carboxy-terminal nuclear localization sequence (NLS) that are critical for dynamic nucleolar trafficking of BLM where it facilitates rRNA transcription through direct interactions with RNA polymerase I and topoisomerase 1. CRISPR/Cas9-directed gene editing techniques generated homozygous murine models with these two conserved amino acids substituted by aspartic acids or alanines, and Blm proteins that were invariably excluded (DD) or constitutively localized (AA) within the nucleolus. Both protein isoforms retain biochemical functions and nuclear localization; BLM-/- cells transfected with the DD transgene display increases in nucleolar DNA damage compared to those transfected with the wild-type gene. BlmDD/DD mice with excluded Blm, but not BlmAA/AA mice, are smaller than their wild-type littermates at 8, 12, and 16 weeks of age; BlmDD/DD mice develop significantly more adenomas than littermates when crossed to the ApcMin/+ model of intestinal tumor formation. Finally, BlmDD/DD mice, but not their wild-type littermates, display signs of premature aging by one year of age, with weight loss, changes in coat color and quality, and spontaneous tumor formation. Our studies demonstrate functions for dynamic Blm nucleolar localization in maintaining stability of both the nucleolar and non-nucleolar genomes. A role for Blm/BLM in the prevention of aging is also exposed by these mouse models as only RecqL4 (RECQL4 mutations lead to Rothmund-Thomson syndrome in the human), but not Wrn/RecqL3 (WRN mutations lead to Werner's syndrome in the human), localizes to the murine nucleolus. Both Werner's syndrome and Rothmund-Thomson syndrome are associated with DNA repair deficiency, tumor susceptibility, and premature aging in the human. Our murine models also permit the definition of amino acid sequences and mechanisms that target or exclude proteins from the nucleolus. This abstract is also being presented as Poster A30. Citation Format: Michael McIlhatton, Max Fernandez, Samir Acharya, Vincenzo Coppola, Joanna L. Groden. Altered nucleolar trafficking of the Blm helicase in the mouse reduces size, increases DNA damage and tumor susceptibility, and facilitates premature aging [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR08.

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.

Background & Aims: The APC tumor suppressor is well known for its ability to regulate Wnt signaling through mediation of β-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 β-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.

The premature aging and cancer-prone diseases Werner and Bloom syndromes are caused by loss of function of WRN and BLM proteins, respectively. At the cellular level, WRN or BLM deficiency causes replication abnormalities, DNA damage hypersensitivity, and genome instability, suggesting that these proteins might participate in resolution of replication blockage. Although WRN and BLM are helicases belonging to the RecQ family, both have been recently shown to also facilitate pairing of complementary DNA strands. In this study, we demonstrate that both WRN and BLM (but not other selected helicases) can coordinate their unwinding and pairing activities to regress a model replication fork substrate. Notably, fork regression is widely believed to be the initial step in responding to replication blockage. Our findings suggest that WRN and/or BLM might regress replication forks in vivo as part of a genome maintenance pathway, consistent with the phenotypes of WRN- and BLM-deficient cells.

Mutation of the APC tumor suppressor gene is one of the earliest events in the development of most colorectal tumors. The APC gene encodes multiple protein isoforms through a complicated pattern of expression and alternative splicing. The role that each isoform plays in cellular physiology is unknown, although the presence of some of these isoforms in postmitotic cells suggests a role in controlling cell growth or promoting differentiation. Three APC isoforms that differ in their amino-terminal domains were evaluated by gene transfer experiments using a colon cancer cell line that lacks functional APC. All three isoforms alter cellular morphology and affect cell growth by elongating the G1 phase of the cell cycle. The conventional APC and brain-specific APC isoforms suppress the tumorigenic phenotype of cultured cells, while the 0.3 APC isoform does not. In support of these experiments, BrdU incorporation as a marker for S-phase entry occurs at a higher level in transiently transfected cells with 0.3 APC when compared to cells transfected with the other isoforms. All three APC isoforms colocalize with microtubules and dramatically reduce beta-catenin activity to the same extent in transiently transfected cancer cells, suggesting that the different effects of each isoform on tumorigenesis may be nontranscriptional in origin.

Gastrointestinal cancer affects 250,000 Americans a year with nearly half of those cases being colorectal cancer. The Wnt pathway is activated in most spontaneous and familial colorectal cancers and has been implicated in tumor formation at other sites in the gastrointestinal tract. In human tumors, the Wnt pathway is most often altered by mutations affecting certain components of this signal transduction cascade-the adenomatous polyposis coli (APC) tumor suppressor gene or the ss-catenin gene. Perturbations in the function of either protein lead to altered gene regulation through the interaction of ss-catenin with T-cell factor (Tcf)/lymphoid enhancer binding protein (Lef) transcription factors. This review will discuss the Wnt pathway, examine the mutations of its components that are found in human cancer, and discuss the known downstream gene targets.

Background & Aims: APC gene mutation is an early alteration in most colorectal tumors. In an attempt to determine its role in tumor development, we asked whether reintroducing wild-type APC into colorectal cancer cells with mutant APC affected cell cycle progression. Methods: Using transient transfection, a plasmid containing the APC complementary DNA and DNA encoding the green fluorescent protein was expressed in SW480 cells. In addition, several other constructs were co-expressed with APC to determine their combined effects. Results: We report that colorectal cancer cell lines transfected with wild-type APC arrest in the G 1 - phase of the cell cycle and that this arrest is abrogated by cotransfecting constitutively active β-catenin or cyclin D1 and cMYC together. This APC-induced cell cycle arrest involves the disruption of β-catenin–mediated transcription and depends on components of the G 1 /S regulatory machinery, as overexpression of E1a or E2F-1 , - 2 , or - 3 overrides the G 1 arrest. Consistent with this, APC transfection inhibits RB phosphorylation and reduces levels of cyclin D1. Conclusions: Our results suggest that APC functions upstream of RB in the G 1 /S regulatory pathway, cyclin D1 and cMYC affect APC-mediated arrest equivalently to oncogenic β-catenin , and most colon tumors disrupt control of G 1 /S progression by APC mutation. GASTROENTEROLOGY 2002;123:751-763

Telomere maintenance can occur in the presence of telomerase or in its absence, termed alternative lengthening of telomeres (ALT). ALT adds telomere repeats using recombination-based processes and DNA repair proteins that function in homologous recombination. Our previous work reported that the RecQ-like BLM helicase is required for ALT and that it unwinds telomeric substrates in vitro. WRN is also a RecQ-like helicase that shares many biochemical functions with BLM. WRN interacts with BLM, unwinds telomeric substrates, and co-localizes to ALT-associated PML bodies (APBs), suggesting that it may also be required for ALT processes. Using long-term siRNA knockdown of WRN in three ALT cell lines, we show that some, but not all, cell lines require WRN for telomere maintenance. VA-13 cells require WRN to prevent telomere loss and for the formation of APBs; Saos-2 cells do not. A third ALT cell line, U-2 OS, requires WRN for APB formation, however WRN loss results in p53-mediated apoptosis. In the absence of WRN and p53, U-2 OS cells undergo telomere loss for an intermediate number of population doublings (50–70), at which point they maintain telomere length even with the continued loss of WRN. WRN and the tumor suppressor BRCA1 co-localize to APBs in VA-13 and U-2 OS, but not in Saos-2 cells. WRN loss in U-2 OS is associated with a loss of BRCA1 from APBs. While the loss of WRN significantly increases telomere sister chromatid exchanges (T-SCE) in these three ALT cell lines, loss of both BRCA1 and WRN does not significantly alter T-SCE. This work demonstrates that ALT cell lines use different telomerase-independent maintenance mechanisms that variably require the WRN helicase and that some cells can switch from one mechanism to another that permits telomere elongation in the absence of WRN. Our data suggest that BRCA1 localization may define these mechanisms.

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.

ABSTRACT: The adenomatous polyposis coli (APC) gene was first identified as the gene mutated in an inherited syndrome of colon cancer predisposition known as familial adenomatous polyposis coli (FAP). Mutation of APC is also found in 80% of all colorectal adenomas and carcinomas and is one of the earliest mutations in colon cancer progression. Similar to other tumor suppressor genes, both APC alleles are inactivated by mutation in colon tumors, resulting in the loss of full-length protein in tumor cells. The functional significance of altering APC is the dysregulation of several physiologic processes that govern colonic epithelial cell homeostasis, which include cell cycle progression, migration, differentiation, and apoptosis. Roles for APC in some of these processes are in large part attributable to its ability to regulate cytosolic levels of the signaling molecule beta-catenin and to affect the transcriptional profile in cells. This article summarizes numerous genetic, biochemical, and cell biologic studies on the mechanisms of APC-mediated tumor suppression. Mouse models of FAP, in which the APC gene has been genetically inactivated, have been particularly useful in testing therapeutic and chemopreventive strategies. These data have significant implications for colorectal cancer treatment approaches as well as for understanding other disease genes and cancers of other tissue types.

Aims and background The phenotypic expression of different APC mutations in familial adenomatous polyposis (FAP) is variable: two to three variants of the disease have been defined based on the severity of colonic manifestations. Age of onset and number of polypectomies per person-year of postsurgical follow-up were compared in two FAP families with very close mutation sites in the APC gene, in order to ascertain mutation-specific variation of expressivity. Families and APC mutations Family A (5 patients) carried a newly characterized mutation, a four bp deletion at codon 843. Family B (5 patients) carried a previously identified mutation at codon 835. Results Mean age of onset was 49.7 years in family A and 30.5 years in family B; number of polypectomies per person-year of follow-up was 1.05 for family A and 10.1 for family B (P Conclusions There is significant variation of expressivity (allelic heterogeneity) in FAP between two mutations separated by only eight codons, located at the 5’ extremity of APC gene exon 15.

A method for concatenating exons from genomic DNA, thereby skipping large stretches of intron sequence, has been developed using the polymerase chain reaction (PCR) with primers based on known intron–exon junction sequences. The use of genomic DNA circumvents the need for cDNA preparation for many purposes, including cDNA construction and mutational analysis. This PCR method also facilitates the concatenation of nonconsecutive exons, allowing different (known or hypothetical) splice-forms to be amplified. We have used this technique to obtain concatamers of exons 3–9A of APC, a tumor suppressor gene that is mutated in sporadic colorectal cancers and in the germline of individuals with adenomatous polyposis coli. This method also facilitates the generation of any polymorphic derivative of a known sequence, even where the derivative differs from the available sequence at several positions. Hum Mutat 12:122–127, 1998. © 1998 Wiley-Liss, Inc.

Fifteen percent of tumors utilize recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. The mechanisms underlying ALT are unclear but involve several proteins involved in homologous recombination including the BLM helicase, mutated in Bloom's syndrome, and the BRCA1 tumor suppressor. Cells deficient in either BLM or BRCA1 have phenotypes consistent with telomere dysfunction. Although BLM associates with numerous DNA damage repair proteins including BRCA1 during DNA repair, the functional consequences of BLM-BRCA1 association in telomere maintenance are not completely understood. Our earlier work showed the involvement of BRCA1 in different mechanisms of ALT, and telomere shortening upon loss of BLM in ALT cells. In order to delineate their roles in telomere maintenance, we studied their association in telomere metabolism in cells using ALT. This work shows that BLM and BRCA1 co-localize with RAD50 at telomeres during S- and G2-phases of the cell cycle in immortalized human cells using ALT but not in cells using telomerase to maintain telomeres. Co-immunoprecipitation of BRCA1 and BLM is enhanced in ALT cells at G2. Furthermore, BRCA1 and BLM interact with RAD50 predominantly in S- and G2-phases, respectively. Biochemical assays demonstrate that full-length BRCA1 increases the unwinding rate of BLM three-fold in assays using a DNA substrate that models a forked structure composed of telomeric repeats. Our results suggest that BRCA1 participates in ALT through its interactions with RAD50 and BLM.

Significance of microRNAs (miRs), small non-coding molecules, has been implicated in a variety of biological processes. Here, we recruited retroviral insertional mutagenesis to obtain induction of an arbitrary noncoding RNAs and coupled it with a cell based loss-of-function (5-Aza-2′-deoxycytidine (5Aza-dC)-induced senescence bypass) screening system. Cells that escaped 5-Aza-dC-induced senescence were subjected to miR-microarray analysis with respect to the untreated control. We identified miR-335 as one of the upregulated miRs. In order to characterize the functional significance, we overexpressed miR-335 in human cancer cells and found that it caused growth suppression. We demonstrate that the latter accounted for inhibition of 5-Aza-dC incorporation into the cell genome, enabling them to escape from induction of senescence. We also report that CARF (Collaborator of ARF) is a new target of miR-335 that regulates its growth suppressor function by complex crosstalk with other proteins including p16INK4A, pRB, HDM2 and p21WAF1.

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.

Adenomatous polyposis coli (APC) is an autosomal dominant disease characterized by the development of hundreds of colorectal adenomatous polyps during the first decades of life. The expression of the disease varies, as the age of onset of colonic cancer and the severity of extracolonic manifestations often differ between affected families. An attenuated form of APC has also been described in which a small number of polyps and a later age of onset of colonic cancer is observed. Cloning of the APC gene has allowed disease-causing mutations in APC families to be identified. Here, we report a novel splice site mutation (a G to T transversion at position +5 of the splice donor site in intron 9) in the APC gene of affected individuals in an Italian family. Characterization of the transcription products from this mutant APC allele revealed that normal splicing was disrupted: a shorter mRNA was expressed in which exon 8 was connected directly to exon 10. This created a shift in the reading frame and the introduction of a stop codon at position 1358. In addition, some normal APC transcript was produced from the mutant allele in lymphoblastoid cells. A comparison of the clinical features of affected members of this family with four unrelated Italian APC kindreds, in which the same AAAAG deletion at position 3926 has been found, showed a significant difference in the onset of disease symptoms and in the age of death attributable to colorectal cancer. Inefficient exon skipping may be, at least in part, responsible for the delay in the development of the disease in the reported family.

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.

An attenuated form of familial adenomatous polyposis coli, AAPC, causes relatively few colonic polyps, but still carries a significant risk of colon cancer. The mutant alleles responsible for this attenuated phenotype have been mapped in several families to the adenomatous polyposis coli ( APC ) locus on human chromosome 5q. Four distinct mutations in the APC gene have now been identified in seven AAPC families. These mutations that predict truncation products, either by single base pair changes or frameshifts, are similar to mutations identified in families with classical APC. However, they differ in that the four mutated sites are located very close to one another and nearer the 5′ end of the APC gene than any base substitutions or small deletions yet discovered in patients with classical APC.

Germ-line mutations in the adenomatous polyposis coli (APC) gene result in familial adenomatous polyposis coli (APC), an inherited syndrome that predisposes affected individuals to early onset of colorectal cancer. Somatic APC mutations also have been detected in sporadic colon tumors. We have used single strand conformational polymorphism (SSCP) analysis to scan a region of the APC gene that frequently is mutated in both APC and sporadic colorectal cancer. Four truncating mutations were found between codons 1060 and 1327 in 17 of 68 unrelated APC individuals. Fourteen of these persons carried either of two previously described five-nucleotide deletions which represent about 20% of APC mutations in these pedigrees. Patients with mutations in this region of exon 15 develop a classic APC colonic phenotype with multiple, diffuse adenomas developing by the second or third decade. However, the density of adenomas and the extracolonic disease manifestations associated with this syndrome are variable among individuals with identical APC mutations.

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.

The BLM helicase associates with the telomere structural proteins TRF1 and TRF2 in immortalized cells using the alternative lengthening of telomere (ALT) pathways. This work focuses on identifying protein partners of BLM in cells using ALT. Mass spectrometry and immunoprecipitation techniques have identified three proteins that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIalpha (TOPOIIalpha). BLM predominantly co-localizes with these proteins in foci actively synthesizing DNA during late S and G(2)/M phases of the cell cycle when ALT is thought to occur. Immunoprecipitation studies also indicate that only HSP90 and TOPOIIalpha are components of a specific complex containing BLM, TRF1, and TRF2 but that this complex does not include TEP1. TEP1, TOPOIIalpha, and HSP90 interact directly with BLM in vitro and modulate its helicase activity on telomere-like DNA substrates but not on non-telomeric substrates. Initial studies suggest that knockdown of BLM in ALT cells reduces average telomere length but does not do so in cells using telomerase.

Bloom syndrome (BS) was first described by Dr. David Bloom in 1954 after he observed a small number of patients of Ashkenazi Jewish origin with erythematous lesions of the face and small stature (Bloom 1954). Approximately 10 years later, the chromosomal instability and cancer predisposition of BS were reported (German et al. 1965). BS is a rare autosomal recessive disorder in which affected individuals show preand postnatal growth retardation, sun-sensitive facial erythema, immunodeficiency, and male infertility. Those affected are predisposed to a plethora of cancers, most commonly occurring before the age of 25 (German 1993). Carcinomas are observed with highest frequency, followed by leukemias and lymphomas (German and Ellis 1998). Cytologically, the hallmark of BS is elevated sister chromatid exchange (SCE), approximately 5to 10-fold higher than in cells from unaffected individuals. SCE is often used as a diagnostic marker of BS, although molecular genetic mutational analysis is available. Other cytological features of BS include quadriradial structures, telomere associations, and chromosome breaks (German 1964; German et al. 1965; Chaganti et al. 1974). The disease gene for BS, known as BLM, maps to chromosome 15q26.1 and encodes a 159-kDa protein that is a member of the recQ family of helicases (Ellis et al. 1995). This family of helicases is highly conserved throughout evolution; multicellular organisms have multiple recQ-like helicase genes in contrast to unicellular organisms that have only one. The BLM gene encodes a protein that contains a central helicase domain. Carboxy terminal to the helicase domain is the conserved RQC domain (recQC-terminal) that defines this family of helicases, and the HRDC domain (helicase, RNaseD and C-terminal), a domain common to RNA helicases. A nuclear localization signal is also present in the C terminus of most eukaryotic recQ-like helicases. The human recQ-like helicases have very little Chapter 8.6 BLM Mutation and Colorectal Cancer Susceptibility

DNA from 61 unrelated patients with adenomatous polyposis coli (APC) was examined for mutations in three genes (DP1, SRP19, and DP2.5) located within a 100 kb region deleted in two of the patients. The intron-exon boundary sequences were defined for each of these genes, and single-strand conformation polymorphism analysis of exons from DP2.5 identified four mutations specific to APC patients. Each of two aberrant alleles contained a base substitution changing an amino acid to a stop codon in the predicted peptide; the other mutations were small deletions leading to frameshifts. Analysis of DNA from parents of one of these patients showed that his 2 bp deletion is a new mutation; furthermore, the mutation was transmitted to two of his children. These data have established that DP2.5 is the APC gene.

Recent studies have suggested the existence of a tumor suppressor gene located at chromosome region 5q21. DNA probes from this region were used to study a panel of sporadic colorectal carcinomas. One of these probes, cosmid 5.71, detected a somatically rearranged restriction fragment in the DNA from a single tumor. Further analysis of the 5.71 cosmid revealed two regions that were highly conserved in rodent DNA. These sequences were used to identify a gene, MCC (mutated in colorectal cancer), which encodes an 829-amino acid protein with a short region of similarity to the G protein-coupled m3 muscarinic acetylcholine receptor. The rearrangement in the tumor disrupted the coding region of the MCC gene. Moreover, two colorectal tumors were found with somatically acquired point mutations in MCC that resulted in amino acid substitutions. MCC is thus a candidate for the putative colorectal tumor suppressor gene located at 5q21. Further studies will be required to determine whether the gene is mutated in other sporadic tumors or in the germ line of patients with an inherited predisposition to colonic tumorigenesis.

Activation of Wnt signaling through β-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 β-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 β-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. (Cancer Res 2006; 66(9): 4734-41)

Recombination-mediated pathways for telomere lengthening may be utilized in the absence of telomerase activity. The RecQ-like helicases, BLM and Sgs1, are implicated in recombination-mediated telomere lengthening in human cells and budding yeast, respectively. Here, we show that BLM expression rescues disrupted telomere lengthening in telomerase-negative sgs1 yeast. BLM helicase activity is required for this complementation, indicating BLM and Sgs1 resolve the same telomeric structures. These data support a conserved function for BLM and Sgs1 in recombination-mediated telomere lengthening.

RecQ helicases are critical for maintaining genomic integrity. In this study, we show that three RecQ members (WRN, deficient in the Werner syndrome; BLM, deficient in the Bloom syndrome; and Drosophila melanogaster RecQ5b (dmRecQ5b)) possess a novel strand pairing activity. Furthermore, each of these enzymes combines this strand pairing activity with its inherent DNA unwinding capability to perform coordinated strand exchange. In this regard, WRN and BLM are considerably more efficient than dmRecQ5b, apparently because dmRecQ5b lacks conserved sequences C-terminal to the helicase domain that contribute to DNA binding, strand pairing, and strand exchange. Based on our findings, we postulate that certain RecQ helicases are structurally designed to accomplish strand exchange on complex replication and recombination intermediates. This is highly consistent with proposed roles for RecQ members in DNA metabolism and the illegitimate recombination and cancer-prone phenotypes associated with RecQ defects.

In addition to increased DNA-strand exchange, a cytogenetic feature of cells lacking the RecQ-like BLM helicase is a tendency for telomeres to associate. We also report additional cellular and biochemical evidence for the role of BLM in telomere maintenance. BLM co-localizes and complexes with the telomere repeat protein TRF2 in cells that employ the recombination-mediated mechanism of telomere lengthening known as ALT (alternative lengthening of telomeres). BLM co-localizes with TRF2 in foci actively synthesizing DNA during late S and G2/M; co-localization increases in late S and G2/M when ALT is thought to occur. Additionally, TRF1 and TRF2 interact directly with BLM and regulate BLM unwinding activity in vitro. Whereas TRF2 stimulates BLM unwinding of telomeric and non-telomeric substrates, TRF1 inhibits BLM unwinding of telomeric substrates only. Finally, TRF2 stimulates BLM unwinding with equimolar concentrations of TRF1, but not when TRF1 is added in molar excess. These data suggest a function for BLM in recombination-mediated telomere lengthening and support a model for the coordinated regulation of BLM activity at telomeres by TRF1 and TRF2.

Guanylin is a pro-secretory hormone that is expressed in intestinal epithelia. Previously, we mapped the guanylin gene to mouse and human chromosomal regions containing multiple intestinal tumor-modifying loci. Here, we investigate whether guanylin expression is downregulated in precancerous human and mouse intestinal adenomas and whether diminished guanylin expression increases adenoma susceptibility in an animal model of intestinal cancer, the multiple intestinal neoplasia (Min) mouse. In situ hybridization analysis indicated diminished guanylin expression in both mouse and human adenomas. Northern analysis of mouse intestinal tissues showed strain-specific levels of guanylin expression but no correlation with the resistance or susceptibility of each strain to adenoma formation. Similarly, cDNA sequence analysis indicated no inactivating mutations or polymorphisms common to either the high or low adenoma-risk groups. Nonetheless, we have shown that significant loss of guanylin RNA in adenomas of mouse and human is a marker of intestinal epithelial cell transformation.

The conventional protein isoform of the APC tumor suppressor is 310 kD and is encoded by exons 1 - 15 of the APC gene. Other RNAs are expressed from the APC gene and include one form that contains an exon upstream of exon 1, designated BS, but this transcript does not include exon 1. This transcript recently has been shown to be enriched in non-dividing, terminally-differentiated cells (Santoro and Groden, 1997). To determine if the BS-containing transcript encoded an alternate APC protein isoform, we generated and affinity-purified a polyclonal antibody directed to protein sequence predicted by exon BS. The BS antibody labeled a band of approximately 300 kD on immunoblots of cerebral and cerebellar tissue from adult human, baboon, rat and mouse. These same tissue lysates also contained prominent BS-reactive proteins of 290 kD, 200 kD and 150 kD. Lysates from mitotically active cells did not contain these APC isoforms. To verify that BS-reactive proteins were APC isoforms, BS-immunoprecipitates were blotted and labeled with commercially available APC antibodies. All four high molecular weight BS-antibody-precipitated proteins were recognized by antibodies directed against epitopes encoded by APC exons 2 and 15. BS isoforms were not, however, labeled with antibodies to an epitope encoded by APC exon 1, consistent with the prediction that BS - APC isoforms lack the domain encoded by these sequences. Like conventional APC, at least one of the four BS-APC protein isoforms also interacts with beta-catenin. BS-APC isoforms that lack exon 1-encoded sequences are incapable of dimerization with the conventional form of APC, yet retain the ability to bind beta-catenin. Such isoforms are likely to be functionally distinct from the conventional APC protein.

Mutations in the tumor suppressor gene APC invariably lead to the development of colorectal cancer. The vast majority of these mutations are nonsense or frameshifts resulting in nonfunctional, truncated APC protein products. Eleven cyclin-dependent kinase (CDK) consensus phosphorylation sites have been identified in the frequently deleted carboxyl-terminal region of APC; loss of these phosphorylation sites by mutation could therefore compromise the ability of APC to inhibit cell growth. This report demonstrates that immunoprecipitates of full-length, but not truncated, APC protein include a mitosis-specific kinase activity in vivo. Biochemical and Western analysis of these immunoprecipitates confirms the presence of the CDK p34(cdc2). We also show that APC is a substrate for recombinant human p34(cdc2)-cyclin B1. Modification of APC by p34(cdc2) implicates phosphorylation as a mechanism for regulating APC function via a link to the cell cycle.

This report describes the physical mapping of the 1100-kb NotI restriction fragment harboring the gene for adenomatous polyposis coli (APC) gene to human chromosome 5q. 6 refs., 1 fig.

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by cancer predisposition and idiopathic growth retardation. The BLM helicase, mutations of which lead to BS, localizes to the nucleolus of the cell, the site of RNA polymerase l-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. Disorders with defective ribosome biogenesis, such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome, display similar cancer predisposition and growth abnormalities to BS. The objective of this study was to investigate a potential mechanism underlying the proportional dwarfism of BS. We tested the hypothesis that the BLM helicase facilitates rRNA transcription as a component of an RNA polymerase I-associated complex, which when deficient has the potential to lead to proportional dwarfism. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription using H3-uridine pulse-chase assays and nuclear run-on assays in cells with an innate or experimentally induced BLM deficiency. In vitro helicase assays demonstrate that BLM unwinds GC-rich hybrid RNA/DNA nucleic acid substrates predicted to form in the nucleolus during rRNA transcription; the persistence of such structures inhibits progression of the RNA polymerase I transcription complex. Immunofluorescence studies using actinomycin D at a concentration that inhibits RNA polymerase I demonstrate the dependence of BLM nucleolar localization upon ongoing RNA polymerase l-mediated rRNA transcription. Finally, in vivo protein co-immunoprecipitation shows that BLM physically associates with RNA polymerase I in the nucleolus. These studies suggest that nucleolar BLM interacts with RNA polymerase I and facilitates rRNA transcription by modulating rDNA structure. Given the intricate relationship between rDNA metabolism and growth, our data may help to understand the etiology of the proportional dwarfism in Bloom's syndrome. Furthermore, this work provides greater understanding of the mechanisms regulating rRNA transcription and ribosome biogenesis, processes currently being exploited in cancer therapeutics. This abstract is also presented as Poster B34. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR3.