Distinct states of nucleolar stress induced by anti-cancer drugs

Ribosome biogenesis is a vital and energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an especially high demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed "nucleolar normality score" was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I - recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development. Types of nucleolar stresses identified in this study O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/517150v3_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@18724b6org.highwire.dtl.DTLVardef@17b60e7org.highwire.dtl.DTLVardef@117004dorg.highwire.dtl.DTLVardef@114ecec_HPS_FORMAT_FIGEXP M_FIG (1) DNA intercalators and RNA Pol inhibitors induced canonical nucleolar stress manifested by partial dispersion of granular component (GC) and segregation of rDNA and fibrillar center (FC) components UBF, Treacle, and POLR1A within nucleolar stress caps. (2) Inhibition of mTOR and PI3K growth pathways induced a metabolic suppression of function accompanied by the decrease in nucleolar normality score, size, and rRNA production, without dramatic re-organization of nucleolar anatomy. (3) Inhibitors targeting HSP90 and proteasome induced proteotoxicity, resulting in the disruption of protein homeostasis and the accumulation of misfolded and/or undegraded proteins. These effects were accompanied by a decrease in nucleolar normality score, rRNA output, and in some cases formation of protein aggregates (aggresomes) inside the nucleolus. (4) Inhibition of transcriptional CDK activity led to the disruption of interactions between rDNA, RNA Pol I, and GC proteins. This resulted in almost complete nucleolar dissolution, leaving behind an extended bare rDNA scaffold with only a few associated FC proteins remaining. UBF and PolI-recruiting protein Treacle remained associated with the rDNA, while POLR1A and GC dispersed in the nucleoplasm. rRNA production ceased and the nucleolar normality score was greatly reduced. C_FIG