Our RNAi library was made of lentivirus solutions in 384-well plates. Each well contains lentiviruses harboring expression plasmids encoding a single shRNA that is designed to target a single mRNA. The arrayed library targets 1,006 human genes, including 571 kinases; most of them are protein kinases, while other kinases acting on nucleic acids, lipids, and carbohydrates are included (Figure ).
Figure 1 Lentiviral RNAi library screen of human kinases identifies regulators of cancer cell growth. (a) Target genes covered by the shRNA library were classified according to gene function using Gene Ontology groups . (b) Two human sarcoma cell lines, HT1080 (more ...)
We began the screen with two different sarcoma cell lines, with the goal of pre-selecting shRNAs that are lethal to these tumor-derived cell lines. We infected U-2-OS, osteosarcoma-derived cells, and HT1080, fibrosarcoma-derived cells, in triplicate and incubated them for three days. This allows time for the shRNAs to be expressed, to bind to their target mRNAs, and cause a reduction in expression of the encoded protein, as the protein is turned over. Percent growth inhibition was determined by adding alamar blue to the culture, and by measuring fluorescence.
A number of shRNA clones displayed growth inhibitory effects (Figure and Additional data file 1). Statistical analysis of primary screening data revealed 195 genes whose knockdown inhibited growth of either cell lines more than 25% (P < 0.01; Figure and Additional data file 1). Fifty-two genes affected cell growth in both cell lines, while other genes had specific effects on each cell line, which may reflect the different tissue origin of these two sarcomas. Some of the hit genes in common between the two cell lines are well-known regulators of the cell cycle or cell survival, but there were nine genes whose functions have not been described (Additional data file 1). We were most interested in genes whose functions are most critical to the survival of these two cancer cell lines. Accordingly, we calculated the sum of the percent growth inhibition in each cell line and selected seven shRNAs whose summed values were >90% (Figure ). Six out of these seven genes were among the 52 common hits in Figure . One gene, PPM1E, was not statistically significant and, as expected, shRNAs targeting PPM1E were not active in our follow-up analysis (data not shown).
Reducing expression of these six target genes causes growth arrest or cell death in two different sarcoma-derived cancer cell lines; however, we were concerned that knockdown of these six genes may affect normal cells to the same degree. To identify shRNA clones that have cancer cell selectivity, we created fresh batches of lentivirus harboring the six shRNA clones and retested them in a pair of nearly isogenic cell lines, BJ-TERT and BJ-TERT/LT/ST/RASV12
. Both cell lines were derived from primary human BJ foreskin fibroblasts [16
]. These BJ primary cells were engineered successively to express the catalytic subunit of human telomerase (hTERT), the SV40 large T and small T oncoproteins (LT and ST), and an oncogenic allele of HRAS
). We refer to these cells lines as BJ-TERT, BJ-TERT/LT/ST, and BJ-TERT/LT/ST/RASV12
. Only the BJ-TERT/LT/ST/RASV12
cells form tumors in nude mice. Therefore, testing of shRNAs in BJ-TERT and BJ-TERT/LT/ST/RASV12
should enable one to identify genes with a function that is essential in tumor cells, but not normal cells. We measured trypan blue exclusion to evaluate the cytotoxic or growth inhibitory effect of these shRNA clones on normal cells and their isogenic engineered tumor cell counterparts.
Out of these six shRNA clones, five did not show differential activity in the two cell lines; however, the shRNA targeting CSNK1E
) had a tumorigenic-cell-line-specific activity (Figure ). The activity of shCSNK1E
was further tested in four different BJ-derived cell lines, namely, BJ-TERT, BJ-TERT/LT/ST, BJ-TERT/LT/ST/RASV12
, and DRD cells. DRD cells were engineered to express hTERT, ST, HRASG12V
, dominant negative p53, and constitutively active cyclin-dependent kinase (CDK)4/cyclin D, which inactivates the RB protein [17
]. The p53DD/CDK4/cyclin D1 combinations substitute for LT. DRD cells are tumorigenic in nude mice, which is expected from the fact that they are also derived from BJ primary cells and the effects of mutations in both cell lines should be similar. The growth inhibitory potential of shCSNK1E
increased as the cell doubling time decreased, suggesting that the activity is proliferation-rate dependent rather than genotype dependent (Figure ).
Figure 2 CSNK1E is a target for developing anti-cancer drugs with a potentially high therapeutic index. (a) Retesting of six hit shRNA clones in isogenic BJ-derived cell lines. Knocking down CSNK1E induced cancer-cell-specific growth inhibition, whereas knocking (more ...)
Theoretically, the length of shRNA involved in base paring with the target mRNA is long enough to ensure specificity of the shRNA clone. However, mismatches between a shRNA and target mRNAs are tolerable; RISC is able to suppress expression of off-target mRNAs whose sequences do not perfectly complement the guide strand of the shRNA [7
]. In order to confirm our hypothesis that knocking down expression of CSNK1E
is responsible for the observed growth inhibition, we tested multiple shRNA clones targeting the CSNK1E
gene; each shRNA clone binds to different regions of the CSNK1E
mRNA. If more than a single shRNA clone induces growth inhibition, CSNK1E
is likely to be the relevant target, because the probability of a common off-target effect of multiple shRNA clones with unrelated sequences is low. We found that four shRNAs targeting CSNK1E
induced strong growth inhibition in HT1080 cells (Figure ). The level of CSNK1E
mRNA decreased upon expression of these shRNAs, as assessed by real-time quantitative PCR analysis (Figure ). Note that one of these shRNAs, clone 1838, did not display stronger growth inhibition effects even though the mRNA level was decreased significantly. This is likely to reflect an off-target effect of this particular shRNA.
gene encodes the CK1ε protein, whose main function is to regulate the circadian rhythm by phosphorylating other clock gene products [18
]. The role of CK1ε in cancer has been speculated upon, because CK1ε was shown to phosphorylate key proteins in cancer signaling pathways, such as p53 [19
] and β-catenin [20
]. However, the significance of these phosphorylation events in carcinogenesis is not known, and the possibility of using CK1ε as a pharmacological target for cancer treatment has not been considered. Therefore, we analyzed the expression level of CSNK1E
in human tumor samples to obtain support for its involvement in human cancer. Some genes that are specifically required for tumor maintenance are overexpressed in cancer cells over normal cells. We analyzed the gene-expression database Oncomine for differential expression patterns in normal versus tumor in different tissue types [21
]. The Oncomine database contained microarray expression data for CSNK1E
from ten different tissues, including brain, head and neck, renal, bladder, leukemia, lung, melanoma, prostate, salivary gland, and seminoma. Interestingly, all tumor tissues in the database showed upregulated CSNK1E
expression compared to normal tissues, suggesting a positive role of CK1ε in cancer maintenance or neogenesis (Figure ).
Figure 3 Gene expression studies comparing normal and cancer tissues were analyzed for CSNK1E using Oncomine . CSNK1E was found to be over-expressed in cancer samples over normal samples regardless of tissue origin. The graph shows representative results of (more ...)
The proliferation-rate-dependent action of shCSNK1E (Figure ) raises the possibility that shRNA treatment induces cell cycle arrest; thus, fast growing cells have greater growth inhibition. To test this hypothesis, we stained the DNA of shRNA-treated cells with propidium-iodide and analyzed the cell cycle distribution by flow cytometry. The cell cycle distribution profile indicates that, after expression of shCSNK1E, HT1080 cells were arrested in the second gap (G2) phase of the cell cycle, with a concomitant increase in the population of cells harboring less than the normal diploid DNA content (that is, sub-G1), implying apoptosis had occurred (Figure ).
Figure 4 Knocking down CSNK1E induces G2/M cell cycle arrest and caspase-mediated apoptosis. (a) Two days after non-targeting shRNA or shCSNK1E treatment, HT1080 cells were fixed in methanol and stained with propidium iodide (Materials and methods). Flow cytometry (more ...)
The cell cycle is primarily regulated by the activity of cyclins and CDKs. Among CDK/cyclin complexes, CDK1-cyclin A promotes the transition from G2 to mitosis (M), while CDK1-cyclin B governs maturation of M phase [22
]. We examined whether shCSNK1E
treatment affected expression of cyclin A2 and cyclin B1 in HT1080 cells. Real-time PCR analysis revealed that shCSNK1E
decreased mRNA levels of cyclin B1 and cyclin A2 (Figure ). In contrast, mRNA levels of cyclin D1, whose function is important for the G1 to S transition, were slightly increased (Figure ). These data are consistent with the cell cycle distribution pattern after shCSNK1E
treatment observed by flow cytometry; knocking down CSNK1E
expression caused down-regulation of cyclin B1 and cyclin A2, which results in cell cycle arrest at the G2/M phase.
In addition to G2/M phase cell cycle arrest, shCSNK1E treatment induced apoptotic cell death, as evidenced by the appearance of small, fragmented cells and a sub-G1 population (Figure ). To confirm the apoptotic phenotype of shCSNK1E-treated cells, we examined cleavage of poly(ADP-ribose)polymerase-1 (PARP1), which is cleaved by caspases during apoptosis. Western blot analysis with antibodies specific to PARP1 showed that cells treated with a non-targeting shRNA contained only full length PARP1, whereas those treated with shCSNK1E or staurosporine, a known inducer of caspase-dependent apoptosis, contained a diagnostic PARP1 fragment, indicating that apoptotic caspases were activated by these treatments (Figure ). Activation of apoptotic caspases was further confirmed by western blot, which detected the active form of caspase-3 only in shCSNK1E or staurosporine-treated samples (Figure ). Thus, shCSNK1E induces caspase-mediated apoptosis in sensitive cancer cells.
These results suggest that chemotherapeutic reagents targeting CK1ε may induce growth arrest and apoptosis with some degree of cancer cell selectivity. To test this hypothesis, we examined the effect of IC261, a kinase inhibitor of CK1ε, in cell culture. IC261 was reported to selectively inhibit casein kinase 1 compared to other protein kinases, by an ATP-competitive mechanism. Moreover, it showed an order of magnitude greater selectivity for CK1δ and CK1ε over other casein kinase 1 isoforms [23
]. Treatment with IC261 started to inhibit the growth of HT1080 cells at submicromolar concentrations (Figure ). When we tested IC261 in BJ-TERT and BJ-TERT/LT/ST/RASV12
cells, the sensitivity of BJ-TERT/LT/ST/RASV12
cells was greater than that of BJ-TERT cells, which was consistent with the results obtained with shCSNK1E
(Figures and ). These data suggest that inhibition of the kinase activity of CK1ε is crucial for the observed growth arrest and apoptosis, as opposed to other functions of this protein, such as those mediated by protein-protein interactions. Moreover, as shRNAs targeting CK1δ were not effective in suppressing cell growth during primary screening, the cancer-cell-selective activity of IC261 can likely be attributed to its inhibition of CK1ε (Additional data file 1).
Figure 5 PERIOD2 is a key substrate of CK1ε that mediates IC261-induced growth inhibition. (a) IC261, a kinase inhibitor of CK1ε, induces growth inhibition in HT1080 cells. (b) IC261 treatment in BJ-derived cell lines showed a similar degree of (more ...)
CK1ε is known to control the circadian rhythm by phosphorylating clock proteins, such as PERIOD and CRYPTOCHROME [24
]. These clock proteins are also reported to regulate the cell cycle, suggesting they have a role in linking the circadian system and the cell cycle machinery [25
]. Mammalian cells have three isoforms of PERIOD proteins and two isoforms of CRYPTOCHROME proteins, which are encoded by PER1
genes, respectively. In order to define the role of each isoform in CK1ε-mediated growth regulation, we conducted counter-screening with shRNAs targeting these genes to identify suppressors of IC261-induced growth inhibition in HT1080 cells. Knocking down expression of PER1
, or CRY2
did not affect growth inhibition by IC261 (Additional data file 2). However, four different shRNA clones targeting PER2
suppressed IC261-induced growth inhibition, implying that PERIOD2 is the most crucial substrate of CK1ε in controlling cell proliferation (Figure ). Note that the maximum growth inhibition by IC261 in Figure is smaller than that in Figure , though they have similar EC50 values of 0.1 μg/ml. This is because cells have been growing for three days before being treated with IC261 in order to express shRNAs targeting PER2
, whereas in Figure , IC261 was added to culture at the time of cell seeding.
As we showed that the proliferation rate of target cells is an important determinant of growth inhibition by CSNK1E
knockdown (Figure ), we measured the proliferation rate of HT1080 cells upon PER2
knockdown using the alamar blue assay (Figure ). None of the shRNA clones targeting PER2
changed the proliferation rate of HT1080 cells, indicating that the protective effect of PER2
knockdown on IC261-induced growth inhibition is not caused by slowing cell growth. It has been shown that a major function of CK1ε in the circadian rhythm is to phosphorylate PERIOD2, which drives proteosome-mediated degradation of PERIOD2 [27
]. In several independent reports, overexpression of PERIOD2 has been shown to exert anti-tumor effects in both cell culture and mouse models [26
]. Therefore, treatment with IC261 is likely to stabilize PERIOD2, which activates the PERIOD2-mediated tumor suppression pathway.
Here, we report the identification of CK1ε as a potential target for developing anticancer reagents. The mammalian CK1 family consists of at least seven isoforms (α, β, γ1, γ2, γ3, δ and ε), as well as additional splice variants [18
]. They share highly conserved kinase domains, but differ significantly in the length and primary structure of their amino- and carboxy-terminal non-catalytic domains, implying that each isoform may play a specific role in regulating biological processes [18
]. Defining isoform-specific functions will aid us in developing agents with enhanced specificity and reduced off-target effects. As the specificity of RNAi agents is potentially high, it allows us to differentiate among these isoforms, which is challenging for some chemical inhibitors.
In our screening, knocking down other isoforms of CK1 was not effective at inducing growth arrest, implying that CK1ε has a unique function in promoting the integrity and proliferation of tumor cells. The nature of the signaling pathway that CK1ε uses to control cell growth remains elusive, but several lines of evidence support a positive role of this kinase in oncogenesis. First, in our gene expression analysis, cancer cells have a high level of CK1ε compared to normal cells, regardless of the tissue origin, implying that a high level of CK1ε causes a growth or survival advantage during tumorigenesis (Figure ). Second, in a recent report, enforced expression of myristoylated-CK1ε, but not other isoforms, induced colony formation in soft-agar-growing engineered human epithelial cells [30
]. Third, deletion of PERIOD2 in mice caused increased tumor development upon gamma-radiation, suggesting a tumor suppressive role of PERIOD2 [26
]. CK1ε is a major kinase that phosphorylates and degrades the PERIOD2 protein through the proteasome [31
]; therefore, it is likely that CK1ε exerts its oncogenic effect by inhibiting the tumor suppressive function of PERIOD2. In accordance with this model, we showed that knocking down PER2
abrogated the growth inhibitory effect of IC261, a kinase inhibitor of CK1ε (Figure ).