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 β-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 β-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.
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.
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.
DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in ...living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP) can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed.
Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA) were compared to MethylCap-seq data.
Analysis of methylation in promoter CpG islands (CGIs) identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a "hypermethylator state." High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA.
We identified a methylation signature for a "hypermethylator phenotype" in endometrial cancer and developed methods that may prove useful for identifying extreme methylation phenotypes in other cancers.
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 p16(INK4A), pRB, HDM2 and p21(WAF1).
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 p21
-mediated signaling to limit proliferation in TEP cells.
•Threaded structure of human HsRAD51B and HsRAD51C proteins suggests they are capable of integrating into the HsRAD51 NPF.•Purified HsRAD51B–HsRAD51C shows extremely stable binding to ssDNA and ...dsDNA.•HsRAD51B–HsRAD51C enhances the stability of the HsRAD51 NPF.•HsRAD51B–HsRAD51C enhances HsRAD51 catalyzed D-loop formation at substoichiometric concentrations.•HsRAD51B–HsRAD51C protects the HsRAD51 NPF from dissociation by the RecQ family helicase BLM.
There are six human RAD51 related proteins (HsRAD51 paralogs), HsRAD51B, HsRAD51C, HsRAD51D, HsXRCC2, HsXRCC3 and HsDMC1, that appear to enhance HsRAD51 mediated homologous recombinational (HR) repair of DNA double strand breaks (DSBs). Here we model the structures of HsRAD51, HsRAD51B and HsRAD51C and show similar domain orientations within a hypothetical nucleoprotein filament (NPF). We then demonstrate that HsRAD51B–HsRAD51C heterodimer forms stable complex on ssDNA and partially stabilizes the HsRAD51 NPF against the anti-recombinogenic activity of BLM. Moreover, HsRAD51B–HsRAD51C stimulates HsRAD51 mediated D-loop formation in the presence of RPA. However, HsRAD51B–HsRAD51C does not facilitate HsRAD51 nucleation on a RPA coated ssDNA. These results suggest that the HsRAD51B–HsRAD51C complex plays a role in stabilizing the HsRAD51 NPF during the presynaptic phase of HR, which appears downstream of BRCA2-mediated HsRAD51 NPF formation.
The expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may ...represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5.
We report here that 39 colon tumors from four independent mouse models and 100 human CRCs encompassing all clinical stages shared a striking recapitulation of embryonic colon gene expression. Compared to normal adult colon, all mouse and human tumors over-expressed a large cluster of genes highly enriched for functional association to the control of cell cycle progression, proliferation, and migration, including those encoding MYC, AKT2, PLK1 and SPARC. Mouse tumors positive for nuclear beta-catenin shifted the shared embryonic pattern to that of early development. Human and mouse tumors differed from normal embryonic colon by their loss of expression modules enriched for tumor suppressors (EDNRB, HSPE, KIT and LSP1). Human CRC adenocarcinomas lost an additional suppressor module (IGFBP4, MAP4K1, PDGFRA, STAB1 and WNT4). Many human tumor samples also gained expression of a coordinately regulated module associated with advanced malignancy (ABCC1, FOXO3A, LIF, PIK3R1, PRNP, TNC, TIMP3 and VEGF).
Cross-species, developmental, and multi-model gene expression patterning comparisons provide an integrated and versatile framework for definition of transcriptional programs associated with oncogenesis. This approach also provides a general method for identifying pattern-specific biomarkers and therapeutic targets. This delineation and categorization of developmental and non-developmental activator and suppressor gene modules can thus facilitate the formulation of sophisticated hypotheses to evaluate potential synergistic effects of targeting within- and between-modules for next-generation combinatorial therapeutics and improved mouse models.
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.
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.
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