Diminished Condensin Gene Expression Drives Chromosome Instability That May Contribute to Colorectal Cancer Pathogenesis

Abstract

Chromosome instability (CIN), or constantly evolving chromosome complements, is a form of genome instability implicated in the development and progression of many cancer types, however, the molecular determinants of CIN remain poorly understood. Condensin is a protein complex involved in chromosome compaction, and recent studies in model organisms show that aberrant compaction adversely impacts mitotic fidelity. To systematically assess the clinical and fundamental impacts that reduced condensin gene expression have in cancer, we first assessed gene copy number alterations of all eight condensin genes. Using patient derived datasets, we show that shallow/deep deletions occur frequently in 12 common cancer types. Furthermore, we show that reduced expression of each gene is associated with worse overall survival in colorectal cancer patients. To determine the overall impact that reduced condensin gene expression has on CIN, a comprehensive siRNA-based screen was performed in two karyotypically stable cell lines. Following gene silencing, quantitative imaging microscopy identified increases in CIN-associated phenotypes, including changes in nuclear areas, micronucleus formation, and chromosome numbers. Although silencing corresponded with increases in CIN phenotypes, the most pronounced phenotypes were observed following SMC2 and SMC4 silencing. Collectively, our clinical and fundamental findings suggest reduced condensin expression and function may be a significant, yet, underappreciated driver of colorectal cancer.

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.…”

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

“…To determine the impact that reduced expression of all 10 cohesion genes has on CIN, a scQuantIM screen was performed, in which each gene was independently silenced and assessed for changes in two CIN-associated phenotypes (micronucleus formation and changes in nuclear area; Fig. 2A) in a karyotypically stable cell line, HCT116, which has been used extensively in CIN studies 8,18,[28][29][30][31] . Conceptually, micronuclei are extranuclear bodies found outside of the primary nucleus and are hallmarks of CIN [32][33][34] , while changes in nuclear areas are associated with changes in DNA/chromosome complements 18,31 .…”