Cell Invasion through Stiff Constrictions causes Mutations While Damaging the Nucleus

Abstract

Solid tumors in stiff tissues such as human lung and liver have been reported to exhibit average mutation rates that are ∼100-fold higher than the 0.1 per Mb rates in cancers in soft tissues such as bone marrow and brain. Here, migration of human cancer derived cells through stiff, constricting micro-pores greatly increases both the number and variation of single nucleotide variations (SNV) among clones that otherwise exhibit normal proliferation and migration. Clonal growth in standard 2D cultures gives consistently low rates of detectable SNV's, and dysmorphic nuclei are also rare, which suggests such cultures are effectively ‘soft’ in minimizing nuclear stress. In contrast, constricted migration through small micro-pores but not through large pores (in the absence of any gradients) causes nuclear blebs with segregated lamins in almost all cells plus many cells with extra-nuclear tethers of DNA and an increase in DNA double-strand breaks (DSBs). While lamin-A knockdown increases DSBs, nuclear blebs are enriched in lamin-A but deficient in DNA, lamin-B, and a progeria mutant of lamin-A that was studied because aging increases stiffening-associated fibrosis. Migration-enhanced DSBs and nuclear blebs return eventually in 2D cultures to basal levels, but constriction of nuclei cause local deficits in DNA repair factors plus other mobile proteins relative to DNA, which is consistent with squeeze-out by steric exclusion and with occasional nuclear envelope rupture. A specific DSB site on chromosome-1 is also seen to re-orient and extend into constrictions, modulating the bound density of DSB repair factors. Mutations might thus be caused by stress-inhibited repair of DNA damage in nuclear migration through stiff microenvironments in cancer and aging.