Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing - PubMed (original) (raw)

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing

Kirk J McManus et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Mutations that cause chromosome instability (CIN) in cancer cells produce "sublethal" deficiencies in an essential process (chromosome segregation) and, therefore, may represent a major untapped resource that could be exploited for therapeutic benefit in the treatment of cancer. If second-site unlinked genes can be identified, that when knocked down, cause a synthetic lethal (SL) phenotype in combination with a somatic mutation in a CIN gene, novel candidate therapeutic targets will be identified. To test this idea, we took a cross species SL candidate gene approach by recapitulating a SL interaction observed between rad54 and rad27 mutations in yeast, via knockdown of the highly sequence- and functionally-related proteins RAD54B and FEN1 in a cancer cell line. We show that knockdown of RAD54B, a gene known to be somatically mutated in cancer, causes CIN in mammalian cells. Using high-content microscopy techniques, we demonstrate that RAD54B-deficient human colorectal cancer cells are sensitive to SL killing by reduced FEN1 expression, while isogenic RAD54B proficient cells are not. This conserved SL interaction suggests that extrapolating SL interactions observed in model organisms for homologous genes mutated in human cancers will aid in the identification of novel therapeutic targets for specific killing of cancerous cells exhibiting CIN.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Synthetic lethality in model organisms and human cancer. (A) A SL interaction occurs when 2 independently viable gene mutations/deletions [i.e., yfg1 (e.g., rad54) and yfg2 (e.g., rad27)] are combined to produce a lethal phenotype. If slow growth is observed, a synthetic growth defect (SGD) is defined. (B) A representative example of a genetic interaction network generated from yeast data available in Biogrid (49), where circles identify genes and lines represent SL/SGD interactions. Note that rad27 intersects with both rad54 and rdh54. (C) Schematic representation of SL/SGD in a human cancer context. A mutation or deletion of yfg1 (e.g., RAD54B CIN mutation) genetically sensitizes a cancer cell to SL attack through down-regulation of a second unlinked gene product [yfg2 (e.g., FEN1)], while leaving the normal adjacent cell(s) unaffected. (D) The yeast network presented in (B) has been humanized by identifying the top hit human homolog for the respective yeast genes and is presented. Note that the lines only identify candidate interactions assuming evolutionary conservation. (E) Haploid rad54::URA3 and rad27::KanMX were mated and induced to undergo meiosis. The resulting tetrads were dissected on YPD and later replica plated to additional selection media to identify the genotypes indicated on the right. The combination of rad54::URA3 rad27::KanMX within the same spore resulted in SL (indicated by boxes).

Fig. 2.

Fig. 2.

RAD54B depletion underlies CIN. (A) Western blots depicting RAD54B expression levels in knockout (RAD54B+/+/− and RAD54B−/−/−), knockdown (RAD54B-1, RAD54B-2, and RAD54B-3), and control (Untransfected, Non-silencing, and eGFP) cells. An α-tubulin loading control has been included. (B) DNA content analysis of RAD54B knockout and knockdown cells. Asynchronous cells were PI-labeled and subjected to flow cytometry. The diploid 2N (G0/G1), 4N (G2/M) and >4N (aneuploid/polyploid) populations have been identified. The various cell lines/conditions are indicated in the legend. (C) Representative images of DAPI counterstained chromosomes found in mitotic spreads generated from untransfected HCT116 (top left), RAD54B-1 transfected (top right and bottom left) and RAD54B−/−/− (bottom right) cells. The total chromosome numbers are indicated. (D) Scatter plots depicting the total chromosome distribution for cells RAD54B knockout and knockdown cells and controls. (E) Graphical representation of the mean chromosomes numbers determined for each of the conditions indicated on the x axis as quantified from the mitotic spreads (± SEM). Student's t tests were performed between the mean chromosome number of the untransfected HCT and each of the conditions. Conditions with statistically significant differences in means are identified by *, P <0.05 and ***, P <0.001.

Fig. 3.

Fig. 3.

Ectopic RAD54B expression rescues CIN. (A) Western blot analysis of RAD54B expression in the RAD54B−/−/− cells ectopically expressing V5-RAD54B was determined to be near wild-type levels (HCT116) at the populations level. An α-tubulin loading control is included. (B) GFP-RAD54B expression levels at single cell resolution as determined by QIM. Note that the entire range of the normalized RAD54B signal intensities are shown for both the wild-type HCT116 cells and the isogenic RAD54B−/−/− cells ectopically expressing EmGFP-RAD54B. Although the distribution range is larger in the transfected cells, the regions indicated in the boxes [25th percentile (bottom line), mean (middle line), and 75th percentile (top line)] overlap to a large degree, indicating that protein expression levels are similar, albeit slightly elevated. (C) Asynchronous and sub-confluent cells were PI-labeled and subjected to flow cytometry. The DNA content profiles were determined for wild-type HCT116 cells (red) and RAD54B−/−/− cells (green) ectopically expressing V5-RAD54B (blue), EmGFP-RAD54B (brown), or empty EmGFP vector alone (purple). The arrows highlight the near polyploid populations that exist within the RAD54B-deficient cells.

Fig. 4.

Fig. 4.

FEN1 down-regulation underlies synthetic lethality in RAD54B-deficient human cells. (A) Graphical representation of the percentages of cells relative to GAPD knockdown (± SEM) are shown for the isogenic RAD54B+/+/+ and RAD54B−/−/− cells treated with the various siRNAs indicated (_x_-axis). A single representative data series collected in sextuplet and compiled from 1 of 3 experiments is shown (see

Table S3

). Highly statistically significant differences (P <0.0001) in the mean percentage of cells relative to GAPD knockdown as determined by Student's _t_- test (see

Table S4

) are identified (***). (B) Live cell imaging coupled with PI incorporation into dead/dying cells reveals an increase in death in RAD54B deficient (black) cells treated with FEN1 siRNAs versus RAD54B-proficient (gray) cells treated similarly. Five non-overlapping images from each well were collected every 2 h for 48 h and the total number of PI-positive nuclei were scored. All data were normalized to the first time-point (t = 0) to permit easy comparisons between the respective siRNA treatments indicated at the top. Each graph depicts a single representative experiment performed in triplicate and repeated at least once. Note that the relative death index (_y_-axis) scale is different for the PLK1 positive control.

Similar articles

Cited by

References

    1. Komarova NL, Lengauer C, Vogelstein B, Nowak MA. Dynamics of genetic instability in sporadic and familial colorectal cancer. Cancer Biol Ther. 2002;1:685–692. - PubMed
    1. Cahill DP, Kinzler KW, Vogelstein B, Lengauer C. Genetic instability and darwinian selection in tumours. Trends Cell Biol. 1999;9:M57–60. - PubMed
    1. Delhanty JD, Davis MB, Wood J. Chromosome instability in lymphocytes, fibroblasts, and colon epithelial-like cells from patients with familial polyposis coli. Cancer Genet Cytogenet. 1983;8:27–50. - PubMed
    1. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature. 1997;386:623–627. - PubMed
    1. Rajagopalan H, et al. Inactivation of hCDC4 can cause chromosomal instability. Nature. 2004;428:77–81. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources