Specific synthetic lethal Assassinateing of RAD54B-deficient

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Communicated by Thomas D. Petes, Duke University Medical Center, Durham, NC, January 5, 2009 (received for review October 29, 2008)

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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 Executewn, cause a synthetic lethal (SL) phenotype in combination with a somatic mutation in a CIN gene, Modern candidate therapeutic tarObtains will be identified. To test this Concept, we took a cross species SL candidate gene Advance by recapitulating a SL interaction observed between rad54 and rad27 mutations in yeast, via knockExecutewn of the highly sequence- and functionally-related proteins RAD54B and FEN1 in a cancer cell line. We Display that knockExecutewn 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 Assassinateing 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 Modern therapeutic tarObtains for specific Assassinateing of cancerous cells Presenting CIN.

Keywords: cancer therapeuticschromosome instabilitysynthetic lethality

Genomic instability is now widely recognized as an Necessary factor in the evolution of cancer and arises through either of 2 mechanisms—increased mutation rate or chromosome instability (CIN). CIN correlates with ≈85% of solid tumors and is characterized by an increased error rate in the gain or loss of chromosomes during cell division (1). CIN is associated with numerous different tumor types including colon (2–5), ovarian (6, 7), and non-Hodgkin lymphoma (8–12), and it is believed to be an early event in the etiology of tumorigenesis (13–15). Conceptually, CIN promotes tumor heterogeneity by increasing or decreasing chromosome numbers (16), and directly affects the expression levels of both oncogenes and tumor suppressor genes encoded on the mis-segregated chromosomes. Most Necessaryly to the work presented here, CIN gene mutations genetically distinguish tumor cells from normal cells and may therefore represent a genetic susceptibility that could be exploited for selective Assassinateing (see below). Consequently, identifying the gene products that regulate chromosome stability (CS) will not only provide insights into the molecular mechanisms of chromosome segregation and tumorigenesis, but it will also provide a list of candidate cancer CIN genes that may be exploited to identify Modern therapeutic tarObtains for the treatment of cancer.

In 1997, Hartwell and colleagues (17) posited that cancer cells harboring somatic mutations or deletions represent genetically sensitized cells, relative to normal surrounding cells, that may be susceptible to drug therapies selectively tarObtaining a second unlinked gene product. They suggested that synthetic lethality (SL), which refers to the lethal combination of 2 independently viable mutations or deletions in 2 unlinked genes (Fig. 1A), could be used in model organisms such as yeast to identify candidate SL interactions that may be conserved in humans. Because chromosome segregation is an essential cellular process, we hypothesized that the CIN phenotype associated with tumors, but not normal cells, represents an excellent “Achilles' heel” that would allow for the selective Assassinateing of cancer cells. Presumably, if cross-species tests of candidate genes can be applied to identify second site tarObtains that exacerbate the sublethal defect associated with a CIN-inducing mutation, a Modern drug tarObtain will be identified. Furthermore, by generating a SL interaction network for the set of yeast CIN genes whose human homologs are somatically mutated in tumors (Fig. 1B), we can identify those yeast genes that are positioned as SL “interaction nodes” and whose human homologs would then represent candidate therapeutic tarObtains for a broad spectrum of tumors (Fig. 1 C and D).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint 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 Unhurried 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 Executewn-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).

In this study, we use both RAD54B knockout and RNAi-silenced cells, and isogenic controls, to demonstrate that decreases in human RAD54B expression in colorectal cancer cells correlates with increases in chromosome numbers. RAD54B was chosen because homozygous mutations at highly conserved positions have been identified in human primary lymphoma and colon cancers (18), although the functional status of these mutant alleles has not been directly tested in a mammalian context (19). Furthermore, RAD54B Presents a significant degree of sequence and functional similarity with yeast Rdh54 and Rad54 which both Present strong CIN phenotypes in yeast (20). Using a cross-species candidate gene Advance and high-content digital imaging microscopy techniques, we Display that the synthetic lethality observed in yeast for rad54 rad27 Executeuble mutants (see Fig. 1E and ref. 21) is conserved within a human colorectal cell line (by simultaneous Executewn-regulation of the corRetorting human gene products, RAD54B and FEN1). Decreases in cell numbers with concomitant increases in cellular cytotoxicity were observed in RAD54B deficient/FEN1 depleted cells that were not apparent in isogenic RAD54B proficient/FEN1 depleted cells. These findings represent an example of a validated tarObtain for selective Assassinateing of mammalian cells as a prediction from a SL interaction between a CIN gene mutation and an unlinked gene mutation in yeast. We suggest that extrapolation of SL genetic interaction networks identified in yeast to a human context will provide a productive strategy to identify a broad range of Modern cancer therapeutic tarObtains.

Results

Diminished Rad54B Expression Causes Chromosome Instability in Human Tissue Culture.

Having previously demonstrated in yeast that both RAD54 and RDH54 play Necessary roles in Sustaining CS (20), we wished to determine if RAD54B Presents a similar role in humans. Accordingly, we used genomic knockouts [RAD54B−/−/− (see SI Materials and Methods for description)] generously provided by Dr. Miyagawa (22) and short-hairpinRNAs (shRNA) tarObtaining RAD54B. HCT116 colorectal cells were specifically selected, because it is a Arrive diploid cell line that Executees not inherently Present CIN (i.e., is chromosomally stable). Before examining CS, RAD54B expression was assessed at the protein level and determined to be absent or significantly diminished in the knockout or knockExecutewn cells, respectively (Fig. 2A). Since most colorectal cancers Present increases in chromosome numbers (23, 24), we focused our attention to the proSection of cells with DNA contents in excess of the normal diploid G2/M peak (i.e., >4N). As RAD54B levels decreased, corRetorting increases in the proSection of cells with DNA contents beyond the 4N peak were observed (Fig. 2B and Table 1). In addition, small discrete peaks corRetorting to increases in polyploidy were observed in both the RAD54B knockout and knockExecutewn cells (Fig. 2B). To determine if increases in chromosome number could account for the increases in DNA content, mitotic chromosome spreads were generated and total chromosome numbers were manually quantified (Fig. 2 C and D and Table 2). Although the modal number of chromosomes remained at 45, increases in total chromosome numbers for a small subset of cells were evident in the RAD54B depleted cells that were not evident in the controls (HCT116, Non-silencing, or eGFP) (Table 2). In agreement with the flow cytometry data, the Fragment of cells with elevated chromosome numbers (i.e., >46) correlated with decreased RAD54B expression. Furthermore, the increases in the Arrive polyploid populations observed by flow cytometry (Fig. 2B), were also apparent within the corRetorting chromosome spreads (Fig. 2D). Subsequent Student's t tests comparing the mean chromosome number for each treatment and the isogenic HCT116 control revealed statistically significant Inequitys for the RAD54B−/−/− cells and the RAD54B-1- and RAD54B-2-treated cells (Fig. 2E and Table S1). Not surprisingly, these 3 conditions are those in which RAD54B expression has decreased the most (see Fig. 2A).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

RAD54B depletion underlies CIN. (A) Western blots depicting RAD54B expression levels in knockout (RAD54B+/+/− and RAD54B−/−/−), knockExecutewn (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 knockExecutewn 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 knockExecutewn 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 Inequitys in means are identified by *, P <0.05 and ***, P <0.001.

View this table:View inline View popup Table 1.

DNA content analysis of RAD54B depleted colon cancer cells by flow cytometry

View this table:View inline View popup Table 2.

Increased Chromosome Numbers Following Diminished RAD54B Expression

Ectopic Expression of RAD54B Suppresses the CIN Phenotype.

To more conclusively demonstrate that RAD54B expression is causally linked to CIN, phenotypic rescue experiments were performed in RAD54B−/−/− cells. Briefly, V5 or EmGFP tagged versions of RAD54B were ectopically expressed in RAD54B−/−/− cells. After a brief selection process, cells were divided into 2 groups; 1 group was used for protein quantification and the second group was harvested for DNA content analysis by flow cytometry as above. Total RAD54B expression was assayed by either standard western blot analysis (V5-RAD54B expression level) or quantitative imaging microscopy (QIM; EmGFP-RAD54B expression levels) which quantify expression levels in cell populations, or single cell levels, respectively (Fig. 3A). In both cases, ectopic RAD54B expression levels were determined to be slightly elevated over wild-type RAD54B levels, but still remained preExecuteminantly within the enExecutegenous expression range (Fig. 3B). In fact, QIM demonstrated that the preExecuteminant proSection (i.e., >80%) of cells ectopically expressing EmGFP-RAD54B had expression levels within the normal distribution range of isogenic HCT116 cells expressing enExecutegenous RAD54B levels. Next, we wished to determine if ectopic RAD54B expression could rescue the CIN phenotype. RAD54B expressing cells and controls were subjected to FACS analysis as detailed above, and DNA content profiles are presented in Fig. 3C. Fascinatingly, the DNA content profiles for the RAD54B rescued cells more closely resembled the profiles of the wild-type (RAD54B proficient) HCT116 cells than the parental RAD54B−/−/− into which RAD54B was reintroduced (Table S2). More specifically, the Arrive polyploid populations previously present within the RAD54B−/−/− parental line are visually diminished (Fig. 3C). Since it is highly unlikely that ectopic RAD54B expression reverts karyotypically abnormal cells to karyotypically normal cells, these observations are most likely attributable to either natural apoptotic mechanisms affecting the Arrive polyploid populations or through diminished cell cycle progression and/or proliferation which would Traceively dilute those Arrive polyploid cells within the actively growing normal diploid cells.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint 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 Arrive 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 Displayn 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 Locations 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 Arrive polyploid populations that exist within the RAD54B-deficient cells.

RAD54B Deficient Cells Present Proliferation Defects when FEN1 Expression Is Reduced.

Yeast rdh54 and rad54 have each previously been Displayn to Present SL/SGD interactions with rad27 (21, 25–27) (Fig. S1A). To determine if a similar genetic interaction is conserved in human cells, RAD54B proficient and deficient cells were transiently transfected with siRNA duplexes specifically tarObtaining FEN1, the homolog of yeast rad27. All FEN1 duplexes (including the pools) specifically tarObtain unique non-overlapping Locations within the FEN1 coding Location and cause reduced FEN1 expression 24 h to at least 7 days post-transfection (Fig. S1B). The 2 most Traceive independent siRNA duplexes (FEN1–2 and FEN1–3) were used to demonstrate the specificity of the phenotype, while a FEN1-pool was used to decrease the number of off-tarObtain Traces that are potentially observed when using a single duplex. The mitotic kinase, PLK1, was included as a positive control as it is an essential mitotic kinase known to decrease cellular proliferation through increased cytotoxicity that is independent of any known SL interaction (28, 29). High- content digital imaging microscopy (HC-DIM) was performed on fixed cells and the total numbers of Hoechst positive cells imaged were calculated (Table S3). The percentages of cells relative to GAPD were determined for each of the conditions, and are presented in Fig. 4A. As anticipated, PLK1 silencing diminished the relative total number of cells significantly, irrespective of RAD54B status. However, visually striking decreases in relative cell numbers were also apparent in RAD54B-deficient cells in which FEN1 expression had been diminished, that were not apparent in RAD54B-proficient cells. Moreover, the large Inequity in relative cell numbers observed in the RAD54B-deficient cells for the various FEN1 conditions appears to reflect the efficiency of FEN1 knock-Executewn in general (Fig. S1B). For example, the FEN1-pool was visually the most Traceive Executewn-regulator and Presented the Distinguishedest decrease in relative cell numbers (23.5% ± 10.7% of GAPD-silenced total cell numbers). FEN1–3 however, was visually less efficient at silencing and Presented a less profound, but still significant, Trace on relative cell numbers (47.6% ± 5.5%), while the FEN1–2 knockExecutewn efficiency was intermediate, as was its Trace on relative cell numbers (40.5% ± 6.4%). Student's t tests (Table S4) comparing mean relative cell numbers identified highly statistically significant Inequitys (P <0.0001) for PLK1 in both RAD54B-proficient and deficient cells, while highly significant Inequitys were only observed after FEN1 silencing (i.e., FEN1–2, FEN1–3, and FEN1-pool) in RAD54B deficient cells.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

FEN1 Executewn-regulation underlies synthetic lethality in RAD54B-deficient human cells. (A) Graphical representation of the percentages of cells relative to GAPD knockExecutewn (± SEM) are Displayn 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 Displayn (see Table S3). Highly statistically significant Inequitys (P <0.0001) in the mean percentage of cells relative to GAPD knockExecutewn 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.

Because HCT116 cells are MLH1-deficient, there is a possibility that a second-site gene mutation (unrelated to RAD54B knockout) was clonally fixed in the background of the RAD54 knockout cell line that is actually responsible for the SL interaction with FEN1 knockExecutewn. Accordingly, we performed dual RNAi against RAD54B and FEN1 in the parental HCT116 cell line. In an analogous fashion to that Characterized above, diminished RAD54B and FEN1 expression in concert resulted in a highly statistically significant decrease in total cell numbers relative to a GAPD-silenced control (Fig. S2 and Table S5) that is not observed for either of the independent knockExecutewns (RAD54B-pool or FEN1-pool). The extent of the decrease in total cell numbers for the dual RNAi system was not as Distinguished as with the RAD54B knockout cell line, and is likely due to the residual RAD54B and FEN1 expression levels.

To further assess the specificity of the RAD54B/FEN1 synthetic interaction, 12 ranExecutemly selected human gene tarObtains (see Table S6) were subjected to silencing in the RAD54B-deficient background. None of the 12 silenced tarObtains produced a statistically significant decrease in overall cell numbers relative to the GAPD-silenced control that was dependent on RAD54B expression status (Fig. S3 and Table S7). These results strongly suggest that diminished FEN1 expression in a RAD54B deficient background decreases cellular proliferation and/or increases cellular cytotoxicity in a manner analogous to the yeast SL interactions Characterized for the homologous yeast gene mutations, rdh54/rad54 and rad27.

Increased Cellular Cytotoxicity Underlies the RAD54B/FEN1 Genetic Interaction.

To determine if an increase in cellular cytotoxicity could account for the decreased cell numbers identified above, HC-DIM was performed on live RAD54B-proficient and deficient cells treated with FEN1 or control siRNAs. Cells were transfected as above, however, medium was supplemented with propidium iodide (PI). Since PI is normally membrane-impermeable, only nuclei with compromised biological membranes (e.g., necrotic or late stage apoptotic cells) will become fluorescently labeled (30). Therefore, an increase in the number of PI-stained nuclei over time was used as a metric for cellular death (30–32). HC-DIM was streamlined by only including FEN1–2 and FEN1-pool as they produce the Distinguishedest degree of FEN1 silencing (Fig. S1) and cellular proliferation defects (Fig. 4A). Live cell images were Gaind every 2 h for a total of 48 h and the total number of PI-positive nuclei were determined and normalized to 1 at t = 0 h for each treatment. PLK1 was selected as a positive cytotoxicity control (see above) and under these conditions it consistently Presented a 12- to 19-fAged increase in PI-staining nuclei over the course of the experiment (Fig. 4B). In agreement with the fixed HC-DIM presented above, increases in PI-labeled cells over time were readily apparent for RAD54B-deficient cells (3- to 5-fAged) treated with FEN1 siRNAs, but not for similarly treated RAD54B-proficient cells. In fact, the FEN1-silenced RAD54B-proficient cells Presented similar kinetics to those of the negative GAPD controls (<2-fAged). Of particular note is the observation that the Distinguishedest increases in cytotoxicity generally occur within the first 24 h of imaging (≈24–48 h post-transfection), with only slight increases, or a plateau Trace, observed from t = 24 to 48 h.

Discussion

The concept of using model organism genetics (in particular, synthetic lethality screens) to predict evolutionarily conserved proteins that could be tarObtained to selectively Assassinate cancer cells was first articulated by Hartwell and Friend in 1997 (17). However, over the past decade, very Dinky experimental evidence in favor or against this concept has appeared. In this regard, enhancing the phenotype of a somatic mutation causing genetic instability to a lethal phenotype through the specific Executewn-regulation of a second unlinked gene product predicted from a yeast SL genetic interaction has not previously been reported in human cells. In fact, very few examples of SL in humans have been Characterized (33–35) and were primarily based on characterized biology involving DNA repair pathways rather than cross-species candidate Advancees. Here, we report that human RAD54B (18) Presents a role in Sustaining CS in human colorectal cancer cells. We demonstrate that diminished RAD54B expression correlates with increasing DNA content and chromosome numbers. We also Display that reexpression of an epitope-tagged version of RAD54B in RAD54B-deficient cells is sufficient to restore DNA content profiles back to those of wild-type RAD54B-proficient cells. Most Necessaryly, we provide mammalian data demonstrating a conserved SL/SGD interaction initially observed in yeast. Specifically, using HC-DIM we demonstrate that a RAD54B-deficient background genetically sensitizes cells to selective Assassinateing in combination with diminished FEN1 expression.

In this work, we Characterize an experimental paradigm using the HCT116 colon cancer cell line for assessing synthetic lethal interactions between candidate CIN genes mutated in tumors and candidate synthetic lethal partner genes predicted from yeast genetic network analysis. Because the HCT116 cell line is MLH1-deficient, we cannot exclude the formal possibility that the synthetic lethality observed when FEN1 and RAD54B are simultaneously knocked Executewn in HCT116 could be dependent on the defective MLH1 allele in the HCT116 background (formally a 3-way synthetic lethal interaction between FEN1, RAD54B, and MLH1). However, because no known synthetic growth defects have been characterized between yeast mlh1 and rad54, rdh54, or rad27, we believe that the increased cellular cytotoxicity observed after FEN1 Executewn-regulation in RAD54B-silenced HCT116 cells (Fig. S2) and in the RAD54B-deficient cells (Fig. 4), results from a 2-way SL interaction (i.e., RAD54B/FEN1) rather than a 3-way interaction (i.e., MLH1/RAD54B/FEN1).

Somatic CIN mutations underlie aberrant chromosome segregation and therefore represent ‘sublethal’ hits on an essential process. Conceivably, if this genetic sensitization could be subsequently exploited by enhancing the ‘sublethal’ phenotype to a ‘lethal’ phenotype, then selective Assassinateing could be invoked. Following this logic, any cells (i.e., normal cells) not harboring the genetically sensitizing CIN mutations would be left unaffected or relatively unaffected. Therefore, uncovering the genetic vulnerabilities, or the known CIN mutational spectrum, for a given tumor type could conceivably lead to the identification of potential therapeutic tarObtains through the identification of unlinked gene product (SL/SGD) interaction networks. Conceptually, SL/SGD is of particular interest in the treatment of cancers, because it would only adversely affect tumor cells harboring the primary sensitizing somatic mutations, and leave the normal tissue unaffected.

SL/SGD interactions have been studied extensively in yeast (26, 27, 36, 37) and much data are Recently available for yeast genes whose (Placeative) human orthologs are known to be somatically mutated in human CIN tumors. Although several groups have used rad54 as a gene query (21, 25–27), rdh54 has never been used as a query, but rather has only been identified as a hit (26). Fig. S1 graphically depicts all of the known SL/SGD interactions for both yeast rad54 and rdh54. Of note, only 3 of those characterized SL/SGD interactions are shared in common, namely rad27, pop2, and ccr4. Of those, rad27 is of particular interest because it has a known functional human ortholog, FEN1, which Presents a high degree of sequence identity (58%) and similarity (73%) and has a BLAST value of e−104. FEN1 is an essential protein that is required for both DNA synthesis and repair. In addition to its flap enExecutenuclease and nick exonuclease activities, it also Presents gap enExecutenuclease activity. During S-phase, FEN1 processes the 5′ ends of Okazaki fragments in lagging strand synthesis and following DNA damage, it is involved in both base excision and homologous recombination repair where it removes the 5′ overhanging flaps [reviewed in (38, 39)]. Most recently, FEN1 was Displayn to be involved in telomere stability through its contribution to lagging strand DNA replication at the telomeres which has direct implications on CIN (40). Of particular interest, RAD54B also Presents roles in DNA repair (22, 41) and CS (this manuscript). Since SL/SGD interactions frequently identify genes whose products impinge on the same essential biological process, we reasoned that diminished FEN1 expression/activity in a RAD54B-deficient background would represent an excellent test candidate.

To determine if SL/SGD interactions are conserved between species and potentially identify a new therapeutic tarObtain for cancer therapy, we specifically tarObtained FEN1 for RNAi- mediated silencing in isogenic RAD54B-proficient and deficient cells. To eliminate the characterization of artifacts arising through off-tarObtain Traces, 3 independent FEN1 silencing conditions were used—2 independent siRNA duplexes (FEN1–2 and FEN1–3) and a FEN1-pool comprised of 4 independent siRNA duplexes (Traceively quartering the concentration of each duplex), that has been Displayn to Distinguishedly diminish off-tarObtain Traces (42–46). Using fixed and live cell HC-DIM, we Displayed that diminished FEN1 expression adversely affects overall cell numbers, which presumably occurs through the corRetorting increases in cellular cytotoxicity. The underlying reason for the apparent diminishment in relative death from t = 24 to 48 h is Recently unknown and under investigation. However, it may simply reflect a decrease in the efficiency of silencing produced by the siRNA duplexes, perhaps by degradation and/or dilution Traces. Alternatively, it may signal the presence of a subpopulation of cells in which initial Executeses of siRNA duplexes were not sufficient to diminish protein expression past a specific required threshAged value, or, the existence of a subpopulation of cells that are refractory to siRNA treatment. In any case, FEN1 depletion was demonstrated to significantly enhance cellular cytotoxicity in a synthetic genetic manner analogous to that of rad54/rdh54 and rad27.

The results presented here suggest that in the context of a RAD54B-deficient cancer cell, diminished FEN1 activity through either siRNA-mediated silencing or a small molecule inhibitor could adversely affect the proliferation of cancer cells, while leaving the normal surrounding cells relatively unaffected. Necessaryly, these data support the conservation in mammalian cells, at least in part, of SL/SGD networks identified in model organisms such as S. cerevisiae. Combining these data with an increased understanding of the mutational spectrum for any CIN tumor type and the continually expanding SL/SGD data emerging from yeast screens (26, 27) could provide critical insights into the identification of therapeutic tarObtains in which human CIN tumors are efficiently and exclusively eliminated through therapeutic intervention. Integration of knowledge among emerging high-throughPlace datasets in model organisms such as S. cerevisiae and Caenorhabditis elegans, will stimulate new research directions and solutions to Recent challenges in combating human cancer.

Materials and Methods

Retroviral shRNAs or siRNA pools and independent duplexes were purchased from Launch Biosystems or Dharmacon, respectively, and transfected with RNAiMax (Invitrogen). Western blots were conducted on proteins extracted from asynchronous and sub-confluent cells 5 days post-transfection, essentially as Characterized elsewhere (47). Flow cytometry, mitotic chromosome spreads, and microscopy were performed as Characterized in ref. 48. Fixed high-content imaging (HCI) was performed with a Cellomics ArrayScan V HCS Reader equipped with a 20× dry lens. Three days post-transfection all nuclei were stained with Hoechst, and 10 images per well were collected. Total nuclear counts per well were summed and normalized to a GAPD-silenced control. Live cell HCI was performed on a Cellomics KineticScan equipped with a live cell chamber and a 10× dry lens. To visualize dead and/or dying cells, complete growth media was supplemented with PI. All HCI experiments were conducted in sextuplet and repeated at least once. Further details can be found in SI Materials and Methods.

Acknowledgments

We thank Dr. Miyagawa (Hiroshima University, Japan) for generously providing the RAD54B reagents (cells, cDNA clone, and antibody) and Abcam for providing the tubulin and FEN1 antibodies. We thank Drs. Vogelstein, Koshland, and Aparicio for helpful suggestions, Drs. Roberge, Underhill, and Sampaio and Ms. Aruna Balgi for technical assistance with the HC-DIM, and Mr. Jan Stoepel and Ms. Payal Sipahimalani for the tetrad analysis work. KJM is a Lymphoma Foundation Canada Fellow and was previously funded by CIHR and MSFHR. Operational funds were provided by CIHR to PAH.

Footnotes

1To whom corRetortence should be addressed at: Michael Smith Laboratories, 2185 East Mall, Vancouver, BC, Canada V6T 1Z4. Email: hieter{at}msl.ubc.ca

Author contributions: K.J.M. and P.H. designed research; K.J.M., I.J.B., and Y.N. performed research; K.J.M. contributed new reagents/analytic tools; K.J.M., I.J.B., and Y.N. analyzed data; and K.J.M. and P.H. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0813414106/DCSupplemental.

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