5-Fluorouracil affects assembly of stress granules based on RNA incorporation

Abstract

The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.

Principles and Properties of Stress Granules

“…For example, many chemotherapeutic agents promote stress granule formation [84,85]. Moreover, mutations in DDX3 promoting the WNT subclass of pediatric medulloblastoma inhibit the ATPase activity of DDX3, which would be expected to trap mRNPs in stress granules based on analogous mutations in the yeast ortholog Ded1 [58].…”

Stress granules, P-bodies and cancer

“…Interestingly, another chemotherapeutic agent, the antimetabolite 5-Fluorouracil (5-FU), also induces the assembly of SGs [102]. 5-FU is used for treatment of various types of cancers including breast, colorectal, head and neck cancers [103].…”

“…5-FU cytotoxicity is caused by interference with enzymatic activities of thymidylate synthase and by its incorporation into DNA and RNA intermediates. 5-FU incorporation into RNA (5-FU-containing RNA metabolites) causes activation of PKR kinase [104], phosphorylation of eIF2α [104] and assembly of SGs that sequester RACK1, an important regulator of apoptosis [102]. RACK1-positive SGs are prosurvival and anti-apoptotic (see below).…”

“…RACK1-positive SGs are prosurvival and anti-apoptotic (see below). Other chemotherapeutic agents known to incorporate into RNA (such as 6-thioguanine and 5-azacytidine) also trigger assembly of SGs [102]. In contrast, DNA-incorporating chemotherapeutic metabolites (such as trifluorothymidine and gemcitabine) do not trigger SG formation but, surprisingly, potently increase the number of PBs in cancer cells [102].…”

“…Other chemotherapeutic agents known to incorporate into RNA (such as 6-thioguanine and 5-azacytidine) also trigger assembly of SGs [102]. In contrast, DNA-incorporating chemotherapeutic metabolites (such as trifluorothymidine and gemcitabine) do not trigger SG formation but, surprisingly, potently increase the number of PBs in cancer cells [102]. The biological significance of these observations is not clear, although recently PBs have been implicated in the cellular response to hypoxia.…”