Reduced Expression of Genes Regulating Cohesion Induces Chromosome Instability that May Promote Cancer and Impact Patient Outcomes

Abstract

chromosome instability (cin), or continual changes in chromosome complements, is an enabling feature of cancer; however, the molecular determinants of cin remain largely unknown. emerging data now suggest that aberrant sister chromatid cohesion may induce cin and contribute to cancer. to explore this possibility, we employed clinical and fundamental approaches to systematically assess the impact reduced cohesion gene expression has on cin and cancer. ten genes encoding critical functions in cohesion were evaluated and remarkably, each exhibits copy number losses in 12 common cancer types, and reduced expression is associated with worse patient survival. to gain mechanistic insight, we combined siRnA-based silencing with single cell quantitative imaging microscopy to comprehensively assess the impact reduced expression has on cin in two karyotypically stable cell lines. We show that reduced expression induces cin phenotypes, namely increases in micronucleus formation and nuclear areas. Subsequent direct tests involving a subset of prioritized genes also revealed significant changes in chromosome numbers with corresponding increases in moderate and severe cohesion defects within mitotic chromosome spreads. Collectively, our clinical and fundamental findings implicate reduced sister chromatid cohesion, resulting from gene copy number losses, as a key pathogenic event in the development and progression of many cancer types.

Reduced SKP1 Expression Induces Chromosome Instability through Aberrant Cyclin E1 Protein Turnover

“…An XY boundary exclusion filter (<7 µm) was employed to remove partial nuclei along the image periphery, while inclusion filters were employed for area (250-2800 µm 2 ) and mean Hoechst intensity (4500-5 × 10 4 au) to eliminate small nuclear debris (i.e., apoptotic bodies) and mitotic cells, respectively. MN were assessed as detailed previously [55][56][57]. Briefly, MN were operationally defined as small (<1/3 the size of the nucleus), extra-nuclear Hoechst-stained bodies exhibiting no visible attachments with the primary nucleus.…”

Chromosome Instability; Implications in Cancer Development, Progression, and Clinical Outcomes

“…Single cell approaches have also been developed to quantify and compare surrogate markers of CIN (e.g., CIN-associated phenotypes), including micronucleus formation [28,29] and changes in nuclear areas [29] or human artificial chromosomes [30]. Conceptually, micronuclei are extra nuclear bodies that are found outside the primary nucleus and are hallmarks of CIN that typically arise due to chromosome missegregation events [31][32][33], while changes in nuclear areas and human artificial chromosomes are associated with small and large (i.e., ploidy) scale changes in DNA content, respectively [30,[34][35][36][37][38]. These approaches typically involve quantitative imaging microscopy or flow cytometry that are each capable of rapidly assessing CIN-associated phenotypes in hundreds-to-thousands of cells [28,29,39].…”

“…In any case, endpoint approaches offer unparalleled insight into the level of cell-to-cell heterogeneity and population diversity associated with CIN. For example, these endpoint approaches and subsequent cytogenetic validation have been instrumental in expanding our understanding of the molecular determinants of CIN, which includes genes regulating chromosome cohesion and condensation [35,36,[40][41][42], histone modifications [43][44][45][46], microtubule motor proteins [34,47], and ubiquitin regulating complexes [37,48,49]. Only once these single cell approaches are more readily applied in both experimental and clinical contexts will we begin to expand our current understanding of the intimate and causal relationships existing between CIN and cancer so that we can ultimately realize its clinical potential in enhancing case management and predicting clinical outcomes.…”

“…In fact, of the~2300 CIN genes (i.e., genes whose aberrant expression induces CIN) predicted to exist [34,68], fewer than 150 have been identified and validated to date. Furthermore, of those that have been identified, most encode functions within intuitive pathways that orchestrate chromosome dynamics and/or DNA repair, including chromosome segregation [12,69], sister chromatid cohesion [36,40,41], chromosome condensation [35,42], mitotic spindle dynamics [69][70][71], spindle assembly/mitotic checkpoint [72][73][74][75], kinetochore-microtubule attachments [34,[76][77][78][79], centrosome dynamics [80][81][82], telomere biology [83][84][85], and DNA replication and repair [12,86,87]. Thus, significant efforts are required to greatly advance our limited understanding of the molecular determinants of CIN and their implications for cancer development, particularly as CIN pertains to intertumoral and intratumoral heterogeneity.…”

An Automated, Single Cell Quantitative Imaging Microscopy Approach to Assess Micronucleus Formation, Genotoxicity and Chromosome Instability

“…Micronuclei are typically defined as having a diameter ≤ 1/3 the size/area of the primary nucleus [1] and their appearance (i.e., micronucleus formation) is frequently employed as an indicator of an underlying genotoxic stress [2,3]. In the past, many genotoxic studies have sought to determine the impact compounds have on genome stability by manually assessing the presence of micronuclei following a treatment [4,5], while more recent studies have employed micronucleus formation assays to uncover genes with pathogenic implications in cancer [6][7][8]. In this regard, numerous cytological studies have identified micronuclei within various cancer contexts including head and neck, ovarian, and breast cancers [9][10][11].…”