Genetic disruption of USP9X sensitizes colorectal cancer cells to 5-fluorouracil

Abstract

The X-linked deubiquitinase USP9X affects the stability and activity of numerous regulatory proteins that influence cell survival. Recent studies suggest that decreased USP9X expression can confer a selective advantage onto developing cancer cells and thereby promotes disease progression. To examine the effect of USP9X on the cellular responses to anticancer therapies, we derived cancer cell lines in which the USP9X locus was disrupted by homologous recombination. The resulting USP9X-deficient cancer cells exhibited increased activation of apoptotic pathways and markedly decreased clonogenic survival in response to 5-fluorouracil, a chemotherapeutic drug that is widely used for treatment of gastrointestinal malignancies. These unexpected results suggest that cancers with low USP9X expression might be specifically sensitized to some conventional therapeutic agents.

Figure 1. Targeted inactivation of human USP9X. (A) A targeting construct with two regions of homology flanking a selectable marker cassette (Neo^r) was designed to delete a 0.8 kb region containing exons 7 and 8 upon homologous integration. Primers that anneal outside the region of homology, within the region of deletion and within the selectable marker were used to identify recombinant clones. Following infection of target cells with the targeting rAAV and screening, multiple homologous recombinant clones were identified. (B) Shown are representative PCR products obtained with the indicated primer pairs. In this experiment, template DNAs were derived from one nontargeted clone (NT) and two homologous recombinants (1 and 2), all derived from HCT116 cells.

Figure 2. Levels and stability of MCL1 protein. (A) Cells of the indicated genotypes were treated for 2h with 10 μM MG132 (+MG132) or with DMSO (-MG132), and then harvested for protein analysis. Immunoblots were probed with the indicated antibodies. *Indicates a cross-reacting protein. (B) Wild type HCT116 cells and USP9X-deficient derivatives were incubated with the protein synthesis inhibitor cycloheximde (CHX; 20 μg/ml) for the times shown, and then harvested for protein analysis. The levels of α-tubulin were assessed as a loading control.

Figure 3. Apoptosis induced by 5-FU. (A) HCT116 cells of the indicated genotypes were incubated with 5-FU (10 mM or 50 mM) for 72 h. Cell nuclei were scored for morphological evidence of apoptosis. (B) Cells were treated with 10 μM 5-FU for 72 h and then harvested for protein analysis. Immunoblots were probed with the indicated antibodies. (C) Cells were treated with 50 μM 5-FU for the times indicated. Subcellular fractions enriched for cytosolic (Cyto) or mitochondrial (Mito) proteins were assessed by immunoblot. The mitochondrial protein TOM20 and the cytosolic protein α-tubulin were assessed as loading controls. (*p ≤ 0.002).

Figure 4. Effects of USP9X on clonogenic survival. (A) The survival of HCT116 cells and the USP9X-deficient derivative (USP9X ^-/o) was assessed following treatment with 5-FU at the indicated concentrations for 24 h. (B) Representative plates from experiment described in (A). Colonies are stained with crystal violet. (C) The survival of DLD-1 cells and the USP9X-deficient derivative (USP9X ^-/o) was assessed following treatment with 5-FU at the indicated concentrations for 24 h. (D) The survival of HCT116 cells and the USP9X-deficient derivative (USP9X ^-/o) was assessed following treatment with a single dose of ionizing radiation (IR) as indicated.

Figure 5. Apoptosis and survival mediated by USP9X in p53-nullizygous HCT116 cells. (A) Cells of the indicated genotypes were treated with 10 μM or 200 μM 5-FU for 72 h, and then harvested for immunoblot analysis. (B) Clonogenic survival was assessed in USP9X-proficient and -deficient cells, in the p53^−/− background, after treatment with 10 μM 5-FU for 72 h. (*p < 0.001).