Fragile sites in cancer: more than meets the eye

Abstract

Ever since initial suggestions that instability at common fragile sites (CFSs) could be responsible for chromosome rearrangements in cancers, CFSs and associated genes have been the subject of numerous studies, leading to questions and controversies about their role and importance in cancer. It is now clear that CFSs are not frequently involved in translocations or other cancer-associated recurrent gross chromosome rearrangements. However, recent studies have provided new insights into the mechanisms of CFS instability, their effect on genome instability, and their role in generating focal copy number alterations that affect the genomic landscape of many cancers.

Figure 1. Possible genomic outcomes of replication stress

DNA replication stress during S-phase leads to stalled or collapsed replication forks, which may be resolved in several ways. Clockwise, starting from top left: (a) Successful restart and completion of replication leads to an intact, normal genome. (b) If regions of unreplicated DNA persist through late-S phase, replication may be completed as late as M-phase, resulting in an apparent common fragile site (CFS) gap/break due to a lack of normal chromosome condensation. (c) If the unreplicated DNA is not resolved and persists to anaphase, ultrafine anaphase bridges can form at these sites. (d) If the stalled/collapsed forks are repaired and restarted through an error-prone mechanism, genome rearrangements, including copy number variants (CNVs) can occur. Alt-EJ, alternative end-joining; FoSTeS, fork stalling and template switching; MMBIR, microhomology-mediated break-induced replication; SMC2, structural maintenance of chromosomes protein 2.

Figure 2. Model of genomic instability at active large transcription units

Replication fork failures resulting from replication stress that occur at most genomic loci, including non-transcribed large genes, can be rescued by the firing of late, or “dormant” origins within the unreplicated region leading to complete replication (left). Large genes in which transcription persists into S-phase, are at high risk for incomplete replication leading to copy number variants (CNVs), common fragile sites (CFSs) and ultrafine anaphase bridges (UFBs; right). The Transcription-dependent Double-Fork Failure (TrDoFF) model for extreme locus instability under replication stress proposes that this results from the simultaneous failure of two converging forks, e.g., through the formation of R-loops, and that this creates large, late-replicating domains where displacement of pre-replication complexes (pre-RCs) by prolonged transcription into S-phase prevents dormant origin firing. CFS breaks and deletion CNVs arise within the resulting unreplicated DNA region while duplications arise on the flanks, (red arrows), likely due to fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR) or alternative end-joining (Alt-EJ), Modified with permission from Wilson et al..

Figure 3. Gene content of pan-cancer focal deletions showing strong association with large genes

Summary of The Cancer Genome Atlas (TCGA) somatic focal deletions in 30 tumor types representing 10,221 tumor specimens. GISTIC2-derived interstitial focal deletion calls with size <= 5 Mb and Q value <= 10⁻¹⁰ were merged into overlapping deletion regions. The maximum -log₁₀(Q) (i.e. strongest focal deletion) in each region is reported. A summary of the properties of genes in each region is shown in column “All Tumor Types”, where “T” indicates the presence of a gene in the Tumor Suppressor Gene Database (TSGene) 2.0 and “L” indicates the presence of a Large gene >= 500 kb. Similar entries are shown for each contributing tumor type, where genes had to lie within a focal deletion and have been identified as a down-regulated tumor suppressor in TSGene 2.0 for that tumor type (“-” indicates a focal deletion with no down-regulated tumor suppressor or large genes). Only tumor types with two or more focal deletions and regions with two or more contributing tumor types are shown. Of these 28 strongly recurrent focal deletions, 19 were at genes > 500 kb, nine of which were listed in TSGene 2.0, and the remaining nine all included smaller tumor suppressor gene loci. A number of the large genes are known CFSs, including WWOX, FHIT, PARK2, IMMP2L and LSAMP. Data summarized in this figure were generated in part by the TCGA Research Network: http://cancergenome.nih.gov/ and obtained from http://firebrowse.org/. CDKN2A, cyclin dependent kinase inhibitor 2A; PTEN, Phosphatase And Tensin Homolog; CCSER1, Coiled-Coil Serine Rich Protein 1; GRID2, Glutamate Ionotropic Receptor Delta Type Subunit 2; PDE4D, Phosphodiesterase 4D; RB1, RB Transcriptional Corepressor 1; WWOX, WW domain containing oxidoreductase; LRP1B, LDL Receptor Related Protein 1B; CSMD1, CUB And Sushi Multiple Domains 1; PTPRD, Protein Tyrosine Phosphatase, Receptor Type D; PLK5, Polo Like Kinase 5; FHIT, fragile histidine triad; DMD, Dystrophin; ARID1A, AT-Rich Interaction Domain 1A; SMAD4, SMAD Family Member 4; TENM3, Teneurin Transmembrane Protein 3; RBFOX1, RNA Binding Protein, Fox-1 Homolog 1; FOXC1, Forkhead Box C1; GMDS, GDP-Mannose 4,6-Dehydratase; PARK2, Parkin RBR E3 Ubiquitin Protein Ligase (also known as PRKN); MACROD2, MACRO Domain Containing 2; SOX6, SRY-Box 6; TP53, Tumor Protein P53; IMMP2L, inner mitochondrial membrane peptidase subunit 2; ZFHX3, Zinc Finger Homeobox 3; NAALADL2, N-Acetylated Alpha-Linked Acidic Dipeptidase Like 2; NF1, Neurofibromin 1; PTPRN2, Protein Tyrosine Phosphatase, Receptor Type N2; LSAMP, limbic system-associated membrane protein.

Figure 4. Experimentally-induced CNVs and focal deletions in cancer arise in the centers of large genes

(A) Example comparison of experimentally induced (in 090 human fibroblasts) and in vivo cancer focal deletion copy number variants (CNVs) at the limbic system-associated membrane protein (LSAMP) gene in in 539 endometrial carcinomas (focal deletion -log10(Q) value = 7.8, which is below the threshold used in Figure 3) and 579 ovarian serous cystadenocarcinomas. Both sets cluster near the center of this large, 2.2 Mb gene. (B) Aggregate metagene analysis of all deletions <= 1Mb in or near genes >= 1Mb in 10,221 The Cancer Gene Atlas (TCGA) tumors representing 30 tumor types. Experimentally induced (blue) and a large proportion of cancer (red) deletion CNVs accumulate specifically and precisely at the centers of these large genes in a manner consistent with the model in Figure 2. (C) Examples of deletion CNV hotspot specificity by tumor type. Differences between ovarian serous cystadenocarcinoma (blue) and head and neck squamous carcinoma (red) are shown with respect to acquired deletion CNV occurrence in genes LSAMP and CUB and Sushi multiple domains 1 (CSMD1). Data summarized in this figure were generated in part by the TCGA Research Network, http://cancergenome.nih.gov/, and obtained from http://firebrowse.org/.