Genetic inhibition of the atypical kinase Wee1 selectively drives apoptosis of p53 inactive tumor cells

All cell lines were obtained from ATCC (Manassas, VA, U.S.A.). All cell lines were grown according to manufacturer’s conditions in the presence of fetal bovine serum (Invitrogen, Carlsbad, CA, U.S.A.).

Viable cell numbers were measured using CellTiter-Glo reagent (Promega, Madison, WI, U.S.A.) or CyQuant (Invitrogen). Caspase-Glo 3/7 assay (Promega) was used to measure cellular caspase-3/7 activity.

Antibodies used included mouse anti-Wee1, mouse anti-P53 (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), rabbit anti-phospho-CDK1 (Y15), rabbit anti-phospho-H2AX (S139), mouse anti-phospho-Histone H3 (S10), rabbit anti-Histone H3 (Cell Signaling Technology, Danvers, MA, U.S.A.), mouse anti-CDK1 (Millipore, Billerica, MA, U.S.A.), mouse anti-P21 (BD Biosciences, San Diego, CA, U.S.A.), and mouse anti-Actin (Sigma, St. Louis, MO, U.S.A.). Western blots were performed as previously described [13].

The human Wee1 coding sequence was amplified using standard PCR protocols and cloned into the pLVX-puro lentiviral construct (Clontech, Mountain View, CA, U.S.A.). Kinase-altered Wee1 (K328R) and siRNA resistant constructs were introduced using the QuikChange site directed mutagenesis kit (Stratgene, Santa Clara, CA, U.S.A.). Cell lines were generated immediately after infection by mass selection in 2 μg/ml puromycin (Clontech).

Engineered NCI-H1299 cell lines were grown in DMEM containing 10% fetal bovine serum and 2 μg/ml puromycin. Cell lysate samples were collected 48 hr after transfection for Western blot analysis. Five days after siRNA transfection, microscopic cell images were taken and both floating and attached cells were then collected by trypsinization and counted for viable cell number using a Vi-CELL cell viability analyzer (Beckman Coulter, Brea, CA, U.S.A.).

NCI-H1299 and A549 cells were plated and transfected 24 hours later with either control siRNA (NT1) or Wee1 siRNA (#8). Plates were placed in an incubator and images were captured every 30 minutes using IncuCyte™ High Definition (HD) Imaging (Essen Bioscience, Ann Arbor, MI, U.S.A). Movies were extracted for the indicated time frames (3 days of siRNA treatment) at a rate of 10 frames per second.

At varied times after transfection with 5nM siRNA, adherent cells were trypsinized and combined with any floating cells present, then washed with cold PBS. Cells were then stained with 0.5 ml PBS containing 50 μg/ml propidium iodide (PI), 0.1 mg/ml RNase A, 0.1% BSA, and 0.1% Triton-X100 for 20 min and cell cycle distribution was analyzed using a BD LSR-II flow cytometer (BD Biosciences, San Jose, CA, U.S.A.).

H1299 cells were treated with 2 mM thymidine (Sigma) and blocked for 20 hrs. Cells were then released by washing with PBS and feeding with regular culture medium. Four hours after the first release, cells were transfected with 5nM siRNA and incubated for another 4.5 hrs before the second treatment of 2 mM thymidine. Cells were blocked with thymidine for another 14.5 hrs and released again into regular medium. At the second release (t = 0), cells should be synchronized at G1/S phase boundary of the cell cycle. Cell samples were collected at varied time points for cell cycle and Western blot analysis as described above.

NCI-H1299 cells were plated in 6-well plate and transfected with 5 nM siRNA as described above. After 24 or 48 hr incubation, cells were labeled with 10 μM EdU (Invitrogen) for 30 min. Both floating and attached cells were then collected for fixation, Click-iT reaction, and cell cycle staining following manufacturer’s protocol. Cell samples were analyzed for DNA content and EdU level using BD LSR-II flow cytometer.

Previous published reports on Wee1 inhibition by small molecule intervention or siRNA knockdown provide conflicting results regarding whether the loss of Wee1 kinase activity can result in the inhibition of growth in tumor cell lines [8, 10‐12, 14]. To test the hypothesis that ablation of Wee1 in the absence of cytotoxics would have effects on cellular health we performed siRNA experiments on a panel of human cell lines derived from solid tumors of diverse origin. Four distinct siRNAs targeting Wee1 as well as negative (non-targeting siRNA #1 and #2) and positive (Ran, [15]) controls were added to cells for the duration of the experiments. The negative controls ensure that siRNA transfection has no deleterious effects on cell growth while the positive control provides a reference of maximum cell killing possible and ensures transfection efficiency. The resulting phenotypes of Wee1 siRNA resulted in dramatic inhibition of cellular proliferation as measured by MTS reagent in H1299 non-small cell lung (NSCLC) and Daoy glioma cell lines but did not affect growth of A549 NSCLC, D54MG glioma or A2780 ovarian tumor lines (Figure 1). Similar results were obtained using DNA content (CyQuant assay) as a measure of proliferation (Additional file 1).

The striking differences between responding and non-responding tumor cell lines to Wee1 siRNA could not be easily explained by off-target effects because of the complete lack of efficacy in A549, D54MG and A2780 cells. However, one possible explanation for the mixed phenotypic effects of Wee1 siRNAs was differential loss of the Wee1 protein. To explore this possibility, we performed Western blot analysis of all five cell lines after siRNA transfection to assess the loss of Wee1 protein. We also examined pCDK which served as a biomarker readout for loss of Wee1 activity in these cells. siRNA treatment resulted in a roughly equivalent loss of Wee1 protein from effective siRNAs in all cell lines examined (Figure 2 and Additional file 2). Moreover, the loss of Wee1 kinase activity also appeared to be roughly equivalent in each of the tumor cell lines based on the levels of CDK Y15 phosphorylation (Figure 2 and Additional file 2). The siRNAs targeting Wee1 used in this study produced protein knock-down and downstream effects over a range of low nanomolar concentrations in all cell lines analyzed (data for Daoy cells shown in Additional file 3). It should be noted that the peptide-based pCDK monoclonal antibody that is used in this study and others cannot distinguish between CDK1, CDK2 and CDK5 phosphorylation as each of these kinases have identical peptides in the region surrounding the Y15 phosphorylation site. In total, these results indicate that differential loss of Wee1 protein or kinase activity amongst cell lines is not the cause of the dramatic differences seen in cellular phenotypes after Wee1 siRNA treatment. These data indicate that in a number of cancer cell lines Wee1 is absolutely essential for survival.

We performed siRNA rescue experiments to rule out the possibility of off-target effects leading to the inhibition of proliferation seen after Wee1 siRNA treatment in the subset of cell lines we assayed. To accomplish this goal, stable NCI-H1299 NSCLC cell lines with a vector control or a siRNA-resistant version of Wee1 were isolated and subjected to siRNA treatment for both proliferation and Western blot analyses. The Wee1 siRNA-resistant DNA construct had scrambled seed regions for both siRNAs #6 and #8 but still coded for the WT amino acid sequence of Wee1. Our results indicate that, while the vector control cell line responded phenotypically (proliferation) and molecularly (Western) as expected, the siRNA-resistant Wee1 cells were unaffected by treatment (Figure 3a–j). These results confirm that the siRNAs targeting Wee1 are on-target and that the dramatic cellular changes are solely a result of the loss of Wee1 protein.

Using these experimental methods we could also determine if the siRNA effects were a result of loss of a scaffolding function or kinase activity of Wee1. To answer this question, we generated a siRNA-resistant/kinase-altered version of Wee1 in which altering the critical active site lysine of Wee1 to an arginine residue (K328R) results in a decrease of Wee1 kinase activity [16]. NCI-H1299 cells over-expressing the siRNA-resistant/kinase-altered version of Wee1 were not rescued to a similar extent as the kinase-active version (Figure 3f–o). The siRNA-resistant/kinase altered Wee1 construct does produce a slight amount of phenotypic rescue (~35%) for both siRNA #6 and #8, but this can be explained by one of two likely possibilities: the K328R change may not produce a fully kinase-dead Wee1 or the over-expressed siRNA-resistant Wee1 constructs may cause some degree of siRNA protection to the endogenous Wee1 mRNA present in the cells. These effects can be seen on the molecular level when relative levels of phospho- to total-CDK (p/t CDK) are taken into account. The level of p/t CDK is essentially absent in the vector control NCI-H1299 cells (Figure 3e), restored in the siRNA-resistant cells (Figure 3j) and partial when the siRNA-resistant/kinase altered Wee1 is over-expressed (Figure 3o). These experimental results imply that the Wee1 siRNAs are on-target and that the loss of kinase activity and the subsequent downstream signaling effects are responsible for the dramatic effects of Wee1 siRNA treatment on cellular proliferation seen in a specific subset of solid tumor cell lines.

Having ruled out several plausible explanations for the drastically different phenotypic response to Wee1 siRNA treatment in our initial 5 cell lines we decided to expand the number of cell lines in our study. We added 15 more tumor cell lines for a total of 20 from various tissue origins and found that, in general, response to Wee1 siRNA treatment correlated well with inactive p53 status (Table 1). The trend in sensitivity upon Wee1 knockdown towards inactive p53 status can be observed when comparing wild-type p53 tumor cell lines (top 7 cell lines with an average 76% viability) to tumor cell lines with deletion/nonsense mutations and the E6 virus that result in cell lines with recognized inactive p53 status (bottom 6 cell lines with an average of 12.5% viability).

The observed effects of Wee1 inhibition and their apparent relation to p53 status presented herein and elsewhere [8, 12] suggested that abrogation of p53 in a normally p53 wild-type setting would generate sensitivity to Wee1 inhibition. To directly test this hypothesis we performed experiments in isogenic pairs of the colorectal carcinoma cell line RKO over-expressing either a vector control or the human papillomavirus protein E6. E6 expression is associated with a substantial decrease in p53 protein levels in these cells resulting in a clonal cell line with inactive p53 [18]. We performed siRNA experiments and found dramatic inhibition of proliferation in the RKO-E6 cells treated with Wee1 siRNAs while the RKO-vector cells were unperturbed (Figure 4a). Western blot analyses revealed that Wee1 protein knockdown and subsequent loss of cellular activity as shown by pCDK levels were roughly equivalent in both cell lines although there was a marked difference in the levels of p53 protein and its transcriptional target p21 in the two cell lines (Figure 4b).

In an attempt to decipher the potential mechanisms by which Wee1 ablation affects cell growth, we performed caspase-activity assays after Wee1 siRNA treatment. Wee1 siRNAs resulted in an induction of Caspase 3/7 activity in a time-dependent fashion in NCI-H1299 and Daoy cells but did not affect A549, D54MG and A2780 cells (Figure 5). These effects correspond well to the cellular proliferation data seen in Figure 1 and suggest that these cells are dying from caspase-dependent apoptosis rather than inhibition of proliferation from cytostatic effects. This caspase-dependent apoptosis is rescued in cells over-expressing the siRNA-resistant Wee1 construct (data not shown). Additionally, the likely cell-cycle related effects of Wee1 knockdown resulted in activation of caspase activity at a slower rate than the positive control Ran siRNA. For example, when comparing the Caspase-3/7 activity at 48 and 72 hours it should be noted that effects of Ran siRNA in cells peaks at 48 hours and decreases in most cell lines by 72 hours, presumably because most cells have already undergone apoptosis. However, Wee1 knockdown has a slower rate of caspase induction that begins at ~ 48 hours and remains steady at 72 hours (Figure 5 and data not shown).

The timing of caspase activity induction and the known functions of Wee1 at both the mitotic and DNA synthesis checkpoints suggested that cell death in sensitive lines was related to cell cycle control. To interrogate this possibility, we performed propidium iodide (PI) DNA staining of cells and performed flow cytometry at time points after Wee1 siRNA treatment. A549, D54MG and A2780 cells had unperturbed cell cycles after Wee1 knockdown that were indistinguishable from a non-targeting siRNA control at 24, 48 and 72 hours after treatment (Figure 6 and data not shown). However, two cell lines with impaired p53 activity, NCI-H1299 and Daoy, had significant cell cycle effects after Wee1 knockdown as compared to the non-targeting siRNA control at all time points analyzed (Figure 6 and data not shown). Loss of Wee1 kinase activity in NCI-H1299 cells resulted in a substantial accumulation of DNA content greater than 2 N that corresponded to a mixture of S-phase, G2/M and >4 N DNA content in cells that ultimately led to a significant fraction of sub-G1 DNA content corresponding with cell death that peaks at 48 hours post-transfection (Figure 6). We also visualized NCI-H1299 and A549 cells by live cell imaging after transfection with Wee1 and controls siRNAs (Additional files 4, 5, 6 and 7) and found that the A549 cells treated with control and Wee1 siRNAs were indistinguishable in phenotype and growth rates. However, NCI-H1299 cells treated with Wee1 siRNA appear to start and abort mitosis at more rapid time periods than their respective non-targeting siRNA control counterparts as visualized by cellular shape changes from flat to round [19]. The cumulative results shown in Figure 6 and the time-lapse light microscopy movies suggest that NCI-H1299 and Daoy cellular DNA content may be undergoing concomitant attempts to perform DNA synthesis and mitosis leading to aneuploidy and incomplete DNA synthesis and this ultimately leads to apoptotic cell death after treatment with Wee1 siRNA.

Cell populations can be synchronized at specific stages of the cell cycle through various means including serum starvation and chemical methods. However, several of these methods are harsh and not amenable to co-treatment with siRNA because of the length of time required for turnover of endogenous protein levels to decrease. We found that Wee1 siRNA treatment resulted in optimal loss of Wee1 protein at 16 hours post-transfection (data not shown). With this timing limitation in mind, we decided upon double thymidine block to synchronize cells in late G1/early S-phase because of its relative lack of toxicity coupled with the length of treatment. NCI-H1299 cells treated with non-targeting or Wee1 siRNAs were also subjected to synchronization and analyzed for DNA content and Western blot at time points following thymidine block release. The cells treated with control siRNA proceeded through the cell cycle from the S-phase block to G2/M and back to G1 as expected, but the cells without Wee1 activity never make it past S-phase and eventually die (Figure 7a). Of particular interest is that cells treated with Wee1 siRNA already have the mitotic marker phospho-Histone H3 (pHH3) present at the release point (t = 0 h) as seen by Western blot analysis (Figure 7b). This demonstrates that despite the lack of G2 DNA content in these cells that they still enter M-phase. This results in DNA damage that is again visible at the release from thymidine block in Wee1 knockdown cells as seen by the increased signal of the rapid response DNA damage marker γ-H2AX (Figure 7b). The gross phenotype of NCI-H1299 cells without functional Wee1 at 24 hours after thymidine release also suggests an abnormal percentage of cells in a “rounded” mitotic state (Figure 7c).

To look more closely at the non-mitotic effects resulting from the loss of Wee1 kinase activity, we performed EdU incorporation assays in both A549 and NCI-H1299 cells. We transfected Wee1 siRNA and a non-targeting siRNA control and performed dual Edu and DNA content flow cytometry on cells at 24 and 48 hours after treatment (Figure 8). In this assay, A549 cells treated with either siRNA had G1 DNA content that could incorporate EdU until a DNA content of 4 N is reached corresponding to the G2/M stage of the cell cycle indicating the lack of premature mitosis in these cells. NCI-H1299 cells with Wee1 knocked down continued to accumulate DNA content (>4 N) while still trying to add more DNA as seen by incorporation of EdU (Figure 8). These results are reflective of the recently reported effects of Wee1 siRNA treatment in U2OS cells resulting in increased activity of both CDK1 and CDK2 kinase activity [14].