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Cell Cycle and Senescence

A Systematic Analysis of Negative Growth Control Implicates the DREAM Complex in Cancer Cell Dormancy

James MacDonald, Yudith Ramos-Valdes, Pirunthan Perampalam, Larissa Litovchick, Gabriel E. DiMattia and Frederick A. Dick
DOI: 10.1158/1541-7786.MCR-16-0323-T Published April 2017

Abstract

Epithelial ovarian cancer (EOC) generates multicellular aggregates called spheroids that detach from the primary tumor and disseminate through ascites. Spheroids possess a number of characteristics of tumor dormancy including withdrawal from the cell cycle and resistance to chemotherapeutics. This report systematically analyzes the effects of RNAi depletion of 21 genes that are known to contribute to negative regulation of the cell cycle in 10 ovarian cancer cell lines. Interestingly, spheroid cell viability was compromised by loss of some cyclin-dependent kinase inhibitors such as p57Kip2, as well as Dyrk1A, Lin52, and E2F5 in most cell lines tested. Many genes essential for EOC spheroid viability are pertinent to the mammalian DREAM repressor complex. Mechanistically, the data demonstrate that DREAM is assembled upon the induction of spheroid formation, which is dependent upon Dyrk1A. Loss of Dyrk1A results in retention of the b-Myb–MuvB complex, elevated expression of DREAM target genes, and increased DNA synthesis that is coincident with cell death. Inhibition of Dyrk1A activity using pharmacologic agents Harmine and INDY compromises viability of spheroids and blocks DREAM assembly. In addition, INDY treatment improves the response to carboplatin, suggesting this is a therapeutic target for EOC treatment.

Implications: Loss of negative growth control mechanisms in cancer dormancy lead to cell death and not proliferation, suggesting they are an attractive therapeutic approach. Mol Cancer Res; 15(4); 371–81. ©2016 AACR.

This article is featured in Highlights of This Issue, p. 359

Introduction

Metastatic dissemination of cancer cells is the major source of cancer-related mortality (1). These cells may also enter dormancy and as a consequence become impervious to standard chemotherapy paradigms dependent on DNA replication and a highly active metabolic state (1, 2). Tumor cell dormancy is particularly relevant to malignancies that generate ascites such as epithelial ovarian cancer (EOC). These tumors shed cells into the ascites which then aggregate to form multicellular clusters, or spheroids. The ascites acts as a conduit for movement of these spheroids throughout the abdomen and pelvis to seed secondary tumor deposits usually leading to carcinomatosis in high-grade ovarian cancer (3). The persistence of drug-resistant EOC cells remains a major hurdle to successful cancer treatment and underscores the necessity to understand biochemical mechanisms critical to the formation and viability of cancer cell spheroids, and pave the way to new treatment options.

Ovarian cancer cell lines, and primary ascites-derived EOC cells, induced to produce spheroids enter G0 (4–6). This is accompanied by increased expression of the RB family member p130 (RBL2), p27kip1, and Dyrk1B (4, 5, 7). In addition, the ubiquitin ligase subunit Skp2, which targets both p130 and p27kip1 for degradation, is downregulated and AKT activity is reduced (5). Increased expression of p27Kip1 and p130 is known to mark the G0 state (8–10). While p27Kip1 is a CDK inhibitor, p130 acts to enforce cell-cycle exit through repression of gene expression as part of the DREAM complex. DREAM consists of DP, an RB-like pocket protein (p130 or p107), a repressor E2F (E2F4 or E2F5) and the Multi-vulval class B (MuvB) core of proteins (Lin9, Lin37, Lin52, Lin54, and RBBP4) (11–13). Outside of G0, the MuvB core also binds b-Myb and FoxM1 to drive relevant cell-cycle–dependent gene expression (14). Upon cell-cycle exit, DREAM assembly is mediated by the inhibition of CDKs leading to dephosphorylation of p107 and p130, allowing them to bind to MuvB (15). Simultaneously, phosphorylation of Lin52 at S28 serves to increase the binding affinity of Lin52 for the dephosphorylated RB-family proteins (15). Once activated, DREAM can repress approximately 800 cell-cycle–regulated genes (11).

DREAM-associated proteins and their roles in cell-cycle exit are tempting targets for treating cancer cell dormancy. For example, targeted knockdown of Lin52 or Dyrk1A alleviates imatinib induced quiescence leading to apoptosis in gastrointestinal stromal tumor cells (16). Enhanced apoptosis also occurs in ovarian cancer cell line spheroids upon inhibition of Dyrk1B and mTOR (6). In addition, suboptimal culture conditions can compromise viability in some ovarian cancer cells lines and p130-dependent arrest is proposed to participate in this effect (4). However, these effects may also be mediated by reactive oxygen species (17, 18), or by multiple negative growth control pathways, leaving the question of cell-cycle control in spheroids relatively unexplored. In this study, we have used siRNA knockdown of known cell-cycle regulators in a panel of 10 EOC cell lines to systematically investigate which genes are needed for arrest and survival in spheroids. We show that loss of negative growth regulators have little effect on EOC cells under optimal culture conditions. Depletion of some cyclin-dependent kinase inhibitors (CKI) compromises spheroid viability. In addition, loss of p130, Dyrk1A, E2F5, and Lin52 have the most damaging effects among non-CKIs, and these all contribute to DREAM assembly and function. Notably, the role(s) of pRB1, p19INK4D, and Dyrk1B in survival are limited, indicating specificity in spheroid survival. We show that Dyrk1A is essential for DREAM assembly in spheroids, cell-cycle exit, and that DREAM target genes are upregulated as a result. Finally, we show that chemical inhibition of Dyrk1A compromises spheroid viability suggesting it may have therapeutic applications in EOC.

Materials and Methods

Cell lines and culture conditions

Primary cultures of ascites cells were established as published previously (19). The majority of patients were diagnosed with advanced stage (stage II–IV) high-grade serous EOC. All primary iOvCa lines were evaluated by STR and found to be unique, all possess TP53 mutations and further details will be published elsewhere. Information regarding specific gynecologic malignancies and staging for the seven primary cell lines is listed in Table 1. All primary lines were used between passage 40 and 50. Established ovarian cancer cell lines, OVCAR3, OVCAR5, OVCAR8, and Hey were from ATCC and were subject to routine STR analysis. EOC cells were cultured in DMEM/F12 supplemented with 10% FCS and penicillin/streptomycin/glutamine. Primary iOvCa cells such as iOvCa147E2 and stable shRNA–expressing derivatives were cultured approximately 1 month before reverting to fresh, earlier passage stocks. HEK293T cells were cultured in DMEM (Invitrogen) supplemented as above. Adherent EOC cells were maintained on standard tissue culture treated polystyrene plates (Greiner) and nonadherent cells were plated on ultra-low attachment (ULA) tissue culture plates (Corning).

View this table:
Table 1.

Clinical characteristics of cell lines used in this study

Cell viability assays

In adherent culture, viability was assessed after 72 hours by measuring ATP levels using CellTiter-Glo (Promega). Spheroids were generated as described previously (5). Briefly, cells were seeded into ULA dishes and incubated for 72 hours. Aggregates were collected, trypsinized briefly, resuspended in CellTiter-Glo/trypsin (1:1), and luminescence measured using a microplate spectrophotometer. For inhibitor studies, spheroids were incubated for 48 hours in the presence of 30 μmol/L INDY (Sigma), 20 μmol/L Harmine (Sigma), or 100–150 μmol/L carboplatin (Sigma; see figure legend for details). For reattachment assays, suspension cultures of EOC cells were transferred to regular tissue culture dishes after 72 hours in suspension and 24 hours later stained with HEMA3. The data were quantitated by counting reattached aggregates and tabulated.

siRNA transfections and lentiviral shRNA delivery

Dharmacon Smart Pool small interfering RNA (Thermo Scientific) each containing four individual siRNA molecules were transfected into cell lines using Dharmafect. The primary iOvCa cells were freshly thawed from stocks prior to transfection and used between passages 40 and 50. Cells were maintained in antibiotic-free DMEM/F12 containing 10% FBS for three days after which they were replated for spheroid formation or adherent culture. Primary iOvCa147E2 cells stably expressing shRNA's were generated by lentiviral infection, and these were independent sequences from the Smart Pool. Lentivirus particles containing short hairpin RNAs (Mission shRNA, Sigma) were generated in HEK293T cells. Culture media was transferred to iOvCa147E2 cells for 24 hours, followed by selection with 4 μg/mL puromycin for an additional 48 hours.

Extract preparation and immunoprecipitation

To prepare cell lysates, adherent cells were washed three times with ice-cold PBS, collected, and pelleted by centrifugation. Spheroids (5 × 105 cells/mL in nonadherent conditions for 24–48 hours) were collected by centrifugation and washed three times in ice cold PBS. Cell pellets were lysed in RIPA buffer. Clarified lysates were flash-frozen in dry ice/ethanol and stored at −80°C until used. For immunoprecipitations, the cells were collected as above, washed once in ice-cold equilibration buffer (50 mmol/L Tris-Cl, pH 7.5, 100 mmol/L NaCl, 1.5 mmol/L MgCl2, 5% glycerol) and lysed in immunoprecipitation buffer (50 mmol/L Tris-Cl, pH 7.5, 100 mmol/L NaCl, 1.5 mmol/L MgCl2, 5% glycerol, 1% NP-40 5 mmol/L NaF, 0.5 mmol/L Na2VO4, 25 mmol/L β-glycerophosphate, 5 μg/mL aprotinin, 5 μg/mL leupeptin and 1 mmol/L PMSF). To immunoprecipitate DREAM, antibodies against Lin9 or Lin37 (3 μg) were added to at least 1.0-mg lysate protein, mixed, and collected on protein G Dynabeads. SDS-PAGE and Western blotting were performed using standard protocols.

Antibodies

Antibodies to Lin9 and Lin37 were as reported previously (11, 20), while those to p107 (C-18), p130 (C-20, F-8), b-Myb (C-5), p21Cip1(C-19), p27Kip1(C-19), CDC6, CDK1, CyclinA2, and PCNA were from Santa Cruz Biotechnology. The antibodies to Dyrk1A, Dyrk1B, p57Kip2, α-tubulin, and the phosphospecific pRB pS807pS811 were from Cell Signaling Technology. The antibody against β-actin was from Sigma.

Kinase assays

Immunoprecipitations were performed as above and Dyrk1A and Dyrk1B precipitates were resuspended in kinase buffer (50 mmol/L HEPES, pH 7.4, 10 mmol/L MgCl2, 10 mmol/L MnCl2). Assays were performed using 5 μg GST-Lin52, 100 μmol/L γ32P-ATP (3,000 Ci/mmole, PerkinElmer). The enzyme-mediated phosphorylation of GST-Lin52 was determined by SDS-PAGE followed by autoradiography. CDK2 immunoprecipitates were prepared as above. Kinase activity was assessed by in vitro phosphorylation of GST-RBC (aa792-928) and detection was done with a pS807/pS811–specific antibody.

Real-time PCR

RNA was isolated from adherent or spheroid cells using an RNA extraction kit as recommended by the manufacturer (Sigma). Two micrograms was reverse transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen) to create cDNA. Real-time PCR measurements used to evaluate the transcript levels of specific DREAM target genes were made on a CFX Connect real-time PCR system from Bio-Rad using iQ SYBR Green Supermix (Bio-Rad) and gene-specific primers. Each gene was normalized to the expression of β-actin.

Thymidine incorporation

Cells were seeded at a density of 5.0 × 105 cells per well in ULA plates to induce spheroids. 3H-thymidine (20 Ci/mmole, PerkinElmer) was added to each well and incubated for 3 hours. Cells were pelleted, washed twice in ice-cold 1× PBS, followed by ice-cold 10% TCA. Cells were lysed by adding 1N NaOH/0.1% SDS and mixing. Cell lysates were added to an equal volume of 1N HCl and mixed with 5 mL of liquid scintillation fluid. Incorporation was quantitated using a Beckman Coulter Model LS6500 liquid scintillation counter.

Flow cytometry

Cells were seeded in ULA plates to induce spheroids as above. Cells were collected and washed twice in cold 1× PBS. EDTA (3 mmol/L) was added to each pellet and incubated at 37°C for 10 minutes to dissociate aggregates. Cells were pelleted and the supernatant was removed. Cells were suspended in PBS and fixed using 95% ethanol drop-wise while vortexing. Cells were then pelleted, resuspended in propidium iodide, and RNase A and incubated overnight at 4°C. DNA content was measured by flow cytometry on a Beckman Coulter Cytomics FC500 flow cytometer.

Results

Depletion of specific cell-cycle–regulatory proteins reduces EOC spheroid viability

A defining characteristic of EOC is the abdominal localization of primary tumor–derived dormant spheroids. As growth arrest is abrogated at the primary site during tumorigenesis, we sought to understand how these cancer cells arrest growth as spheroids and whether there are common mechanisms. We employed an in vitro method to induce spheroid formation under nonadherent culture conditions using ULA culture dishes (3, 5, 21, 22) and targeted well-known negative growth regulators including components of the RB pathway, the Hippo pathway, Cyclin Dependent Kinase Inhibitors, and DREAM components using siRNA SMARTpools. Because of the heterogenous mutational landscape in EOC (23), knockdowns were performed in 10 cell lines from gynecologic malignancies including seven cell lines derived from the ascites obtained from patients presenting with EOC (see Table 1 for details of iOvCa147E2, iOvCa130, iOvCa185, iOvCa129, iOvCa246, iOvCa256, and 105C patients) and three established EOC cell lines (OVCAR5, OVCAR8, and HEY; ref. 24).

Cells transfected with siRNA were split onto regular tissue culture plasticware, and ULA dishes to allow aggregation into spheroids over a 3-day period. Under adherent conditions, only minimal effects on viability were observed, irrespective of siRNA pool or cell line (Fig. 1A). However, under nonadherent conditions, some knockdowns exerted severe effects on cell viability (Fig. 1A). Importantly, the siRNA control exerted little effect on viability and clustered closest with nontransfected cells (Fig. 1A). Despite variability between cell lines for individual knockdowns, we observed that depletion of some proteins disrupted survival across most cell lines. Cumulative effects on survival were quantitated by comparing viability for each gene across all 10 cell lines under nonadherent conditions with the average cumulative viability of all knockdowns under adherent conditions (z-score, Fig. 1B; Supplementary Fig. S1). While depletion of CDK inhibitors such as p57Kip2 have strong effects, the presence of E2F5, Dyrk1A, p130 and Lin52 near the bottom of this chart, and pRB among others near the top (Fig. 1B; Supplementary Fig. S1) suggests that DREAM assembly specifically contributes to survival under nonadherent culture conditions.

Figure 1.
Figure 1.

Differential effects of negative growth-regulatory genes on EOC cell viability in a culture model of dormancy. A, siRNA knockdown was used for the indicated genes in 10 EOC cell lines. Cells were assayed for viability relative to a control siRNA 72 hours following plating in adherent culture conditions. siRNA-transfected cells were also transferred to nonadherent culture conditions to form dormant spheroids and assayed for viability after 72 hours. B, Cumulative viability of each gene depletion across all 10 cell lines was determined and compared to the cumulative viability for these same genes under adherent conditions. Differences from mean viability are expressed as a Z-score with genes plotted in descending order.

Images of cells depleted of Dyrk1A, E2F5, Lin9, p57Kip2, p107, and p130 in suspension culture indicate aggregation is reduced in low viability genotypes (Supplementary Fig. S2). We extended this viability analysis by determining whether spheroids could reattach to culture dishes and resume proliferation akin to metastatic dissemination in the abdomen (Fig. 2A). Reattachment of HEY, iOvCa147E2, iOvCa129, and iOvCa185 cells depleted of Dyrk1A, E2F5, p130, and p57Kip2 was assessed after 72 hours under nonadherent conditions. We found that spheroids depleted of these proteins contained fewer viable cells as evidenced by fewer colonies (Fig. 2B; Supplementary Fig. S3). Together, the data in Figs. 1 and 2 indicate that rather than causing a gain in proliferation, loss of specific negative cell-cycle regulators results in an irreversible loss in spheroid viability.

Figure 2.
Figure 2.

Depletion of cell-cycle–regulatory proteins reduces reattachment of EOC spheroids. A, EOC cell lines were transfected with siRNAs to deplete expression of the indicated proteins. Cells were transferred to nonadherent conditions for 72 hours before replating and culture under adherent conditions for 24 hours. B, Reattached spheroid-derived colonies from the indicated cell lines were stained with HEMA3 and quantitated. Errors bars, 1 SD from the mean and an asterisk represents a significant difference (ANOVA, *, P < 0.05).

Dyrk1A is activated during spheroid formation

Previous work has shown that Dyrk1B plays a key role in EOC cell-cycle arrest (6, 7, 25, 26), and it is amplified in approximately 10% of high-grade EOC cases (23). Thus, we were surprised by the observation that EOC spheroid viability was more sensitive to loss of Dyrk1A than Dyrk1B (Fig. 1B). We evaluated Dyrk1A and Dyrk1B expression under adherent and nonadherent conditions in our 10 selected EOC cell lines. OVCAR3 cells, which overexpress Dyrk1B, served as a positive control (6, 27). We found that, although there was variation between the cell lines, all the EOC cells in our study expressed Dyrk1A (Fig. 3C; Supplementary Fig. S4A). In contrast, appreciable levels of Dyrk1B were observed only in OVCAR3 and iOvCa246 cells, although low but detectable levels were observed in other cell lines (Fig. 3C; Supplementary Fig. S4B).

Figure 3.
Figure 3.

Differential effects on EOC spheroid viability by knockdown of Dyrk1A and Dyrk1B. A, The indicated EOC cell lines were transfected with siRNAs to deplete Dyrk1A, Dyrk1B, or both. Representative knockdown of Dyrk1A, Dyrk1B, and in combination is shown for adherent iOvCa147E2 cells. B, Viability was measured following 72 hours of culture as before. C, Western blots displaying relative expression levels of Dyrk1A and Dyrk1B are labeled, and Actin blots are shown as a loading control. Dyrk1A and Dyrk1B were immunoprecipitated from adherent (A) and spheroid (S) cell cultures as indicated and detected by Western blotting. D, The immunoprecipitated Dyrk1 kinases were used to measure in vitro enzyme activity with GST-LIN52 as a substrate. The panel depicts an autoradiograph showing 32P incorporation into GST-LIN52. Relative substrate input for each reaction was determined by Coomassie staining and is shown at the bottom.

Given the disparity in spheroid cell survival upon targeted loss of Dyrk1A versus Dyrk1B, we examined these kinases in more detail. We investigated siRNA-mediated loss of Dyrk1A or Dyrk1B individually and together in viability assays of adherent and nonadherent cultures in a selection of EOC cells that express low levels of Dyrk1B (iOvCa147E2 and HEY) and that overexpress Dyrk1B (iOvCa246 and OVCAR3). Fig. 3A illustrates the effectiveness and specificity of knockdown in these experiments. Fig. 3B shows a heatmap depicting relative viability in both culture conditions for all knock downs. Knockdown of Dyrk1A, but not Dyrk1B, had a negative effect on spheroid viability in iOvCa147E2 and HEY cells (Fig. 3B), consistent with the results in Fig. 1. Knockdown of Dyrk1A or Dyrk1B in iOvCa246 cells also had little effect on spheroid cell survival (Fig. 3B). Surprisingly, the combined depletion of Dyrk1A and Dyrk1B also had no effect on viability of iOvCa246 spheroids (Fig. 3B). OVCAR3 cells were sensitive to loss of either Dyrk1A or Dyrk1B under both culture conditions (Fig. 3B).

We directly assayed Dyrk1 kinase activity in immunoprecipitates from adherent and spheroid cells (Fig. 3C), employing GST-Lin52 as a substrate. The amount of Dyrk1 kinase in the immunoprecipitates mirrored those observed in the input (Fig. 3C; Supplementary Fig. S4A). These kinase assays showed that Dyrk1A activity is elevated in spheroids relative to proliferating cells in OVCAR3, iOvCa147E2, and iOvCa246 cells, and is already present at high levels in HEY (Fig. 3D). Dyrk1B kinase activity towards Lin52 was detectable only in OVCAR3 immunoprecipitates, under either culture condition (Fig. 3D). These findings point to Dyrk1A as a key survival kinase in EOC spheroids and suggest that Dyrk1B overexpression does not necessarily replace Dyrk1A function in spheroid survival.

DREAM complex assembly in EOC cells is dependent on Dyrk1A and CDK inhibition

The spheroid cells in our study showed greatest sensitivity to loss of E2F5, Lin52, p130, Dyrk1A, and CKIs such as p57Kip2 (Fig. 1). With the exception of p57Kip2, these are either components of, or assembly factors for the mammalian DREAM complex (28). However, because Dyrk1A and CKIs can also inhibit phosphorylation of p130 and contribute to DREAM assembly through CDK regulation (15, 29), we investigated the possibility that the DREAM assembly is central to spheroid arrest and survival.

The dynamics of Myb-MuvB and DREAM complexes between proliferative and nonproliferative states are illustrated in Fig. 4A (12, 14, 28). Fig. 4B shows that the levels of p130 and the MuvB core proteins Lin37 and Lin9 are modestly increased in spheroids in iOvCa147E2 cells. The shift in p130 migration also suggests a change from a hyper- to hypo-phosphorylated state (pp130 to p130). Furthermore, p107 levels decrease under nonadherent conditions, also consistent with cell-cycle exit (30). Lin9 was precipitated from both adherent and 24-hour spheroid cultures, and detection of Lin37 in both confirms the integrity of the MuvB core (Fig. 4B). DREAM assembly was detected only in spheroids by coimmunoprecipitation of p130 with Lin9. We generated iOvCa147E2 cells stably depleted of p130 using shRNA expression delivered by lentiviral transduction (Fig. 4C). Western blotting for p107 revealed only a modest increase in expression, suggesting that there is little compensation for p130 loss. Indeed, immunoprecipitation of Lin37 shows only a small increase in p107 assembly into DREAM in p130-depleted spheroids, indicating that in these cells, DREAM is largely unassembled. Depletion of Dyrk1A (Fig. 4D) also resulted in a loss of Lin37–p130 interactions in spheroids. However, similar to adherent, proliferating iOvCa147E2 cells, b-Myb remains bound to the MuvB core in Dyrk1A-deficient spheroids (Fig. 4E). Together these data show Dyrk1A-dependent DREAM assembly in EOC spheroids.

Figure 4.
Figure 4.

DREAM complex assembly in EOC spheroids is dependent on Dyrk1A. A, The transition between DREAM and MYB-MuvB is used to illustrate the protein interactions that are being studied in these experiments. B, The expression of Lin37, Lin9, p107, and p130 in iOvCa147E2 cells under proliferating, adherent (A) conditions and arrested spheroids (S) was determined by Western blotting. Lin9 was immunoprecipitated from adherent and spheroid-derived extracts and associated Lin37 and p130 was detected by Western blotting. C, iOvCa147E2 cells were stably depleted of p130 using lentiviral transduction of shRNA and cultured in adherent or spheroid conditions. Extracts were blotted to detect p130 and p107 expression. Lin37 was immunoprecipitated and associated p107 and p130 was detected by Western blotting. D, iOvCa147E2 cells were depleted of Dyrk1A by lentiviral shRNA and expression of Dyrk1A was determined by Western blotting extracts from adherent cultures. Adherent and spheroid cells were collected, lysed, and immunoprecipitated for Lin37 and associated p130 was detected by Western blotting. E, Control and Dyrk1A-depleted adherent and spheroid cell lysates were immunoprecipitated for B-Myb and Western blots were performed to detect associated Lin37. F, iOvCa147E2 cells were depleted of p27Kip1 and p57Kip2 by lentiviral shRNA. Left, p27Kip1 and p57Kip2 expression. Spheroid lysates were immunoprecipitated for Lin37 and Western blots were probed for p130 to detect DREAM assembly.

We were surprised to observe a pronounced loss of viability in EOC spheroids upon loss of p57Kip2 as this CKI is known to be downregulated in many epithelial and nonepithelial cancers (31, 32), including EOC (33, 34). We noted increased expression of p57Kip2 in five of our ten cell lines under nonadherent conditions (OVCAR5, OVCAR8, iOvCa147E2, 105C, and iOvCa246), a decrease in expression in three (iOvCa185, iOvCa130 and iOvCa256), and in one (HEY) it remained constant (Supplementary Fig. S5A). Coincidentally, loss of p57Kip2 in iOvCa130 and iOvCa256 spheroids correlates with only a modest loss in spheroid cell viability (Fig. 1A). Expression of p27Kip1, unlike that of p57Kip2, increased in all our cell lines under nonadherent conditions (Supplementary Fig. S5B), consistent with previous observations (5). We determined the effects of p57Kip2 and p27Kip1 knockdown on DREAM assembly in iOvCa147E2 spheroids and surprisingly found a requirement for p27Kip1 but none for p57Kip2 in DREAM assembly (Fig. 4F). While this suggests a role for CDK inhibition in DREAM assembly in spheroids, it also points to functional differences between these CKIs in spheroid viability.

Misregulation of DREAM target genes in knockdown cells

A major consequence of defective DREAM assembly is the loss of transcriptional repression of cell-cycle genes. Accordingly, we analyzed spheroids for expression of the DREAM target genes CDK1, CCNA2, and MYBL2 (11, 13). We observed increased mRNA levels for these DREAM targets at 6- and 12-hour time points in cells depleted of Dyrk1A or p130 relative to control cells at the same time point (Fig. 5), consistent with compromised DREAM assembly and an inability on the part of the cells to transition into G0. Spheroid cells depleted of p57Kip2 maintained repression, indicative of an intact DREAM complex, and consistent with Fig. 4F (Fig. 5). Notably, the mRNA levels for these DREAM targets do not differ significantly from the controls at 24 hours (Fig. 5), suggesting that the initial effect on transcriptional control is most important following the transfer of cells to suspension culture.

Figure 5.
Figure 5.

Misregulation of DREAM target genes in iOvCa147E2 spheroid cells depleted of Dyrk1A or p130. iOvCa147E2 cells carrying shRNAs for the indicated genes were transferred to nonadherent culture conditions for the indicated length of time. RNA was extracted, used for cDNA synthesis and real-time PCR to determine relative message abundance. All reactions were normalized to actin levels. Errors bars, 1 SD from the mean and an asterisk is a significant difference (ANOVA, *, P < 0.05).

Loss of p130 and Dyrk1A deregulates CDK2 activity and DNA synthesis

To further characterize the effects of p130 and Dyrk1A depletion on cell-cycle control, we used 3H-thymidine labeling to measure DNA synthesis following transfer to nonadherent conditions. Dyrk1A- and p130-depleted spheroid cells showed a significantly greater level of DNA synthesis 6 hours following transfer to nonadherent conditions (Fig. 6A) and this trend continued through 12 hours in Dyrk1A knockdowns (Fig. 6A). Knockdown of p57Kip2 had no effect on 3H-thymidine incorporation (Fig. 6A). In addition, we also observed elevated CDK2 activity in Dyrk1A and p130-depleted spheroids, but not in p57Kip2 knockdown cells (Fig. 6B).

Figure 6.
Figure 6.

Spheroids depleted of Dyrk1A and p130 fail to arrest DNA synthesis. A, Suspension cultures of the indicated iOvCa147E2 cells were pulse labeled with 3H-thymidine and processed for liquid scintillation to determine incorporation into DNA. Errors bars, 1 SD from the mean and an asterisk represents a significant difference (ANOVA, *, P < 0.05). B, CDK2 was immunoprecipitated from iOvCa147E2 cells grown under adherent and spheroid conditions and assayed for activity using GST-RBC. Phosphorylation was measured by Western blotting using an antibody to S807/S811. Input for substrate and CDK2 are indicated. C, Suspension cultures of iOvCa147E2 cells with the indicated knockdowns were dissociated, labeled with propidium iodide, and DNA content determined by flow cytometry. Percentage of debris and cell-cycle phases are shown for each.

Because the above experiments indicated that p130- and Dyrk1A-depleted spheroids do not exit the cell cycle, we analyzed cell-cycle kinetics in these cells by flow cytometry Fig. 6C). In general, the number of cells in S-phase decreased over time, although Dyrk1A and p130 knockdowns started at much higher levels, consistent with the elevated 3H-thymidine incorporation described above. Loss of p57Kip2 again demonstrated no increase in S-phase relative to controls. Quantification of sub-2N cellular debris confirmed extensive cell death in Dyrk1A and p57Kip2 spheroids by 24 hours that is lacking in the controls (Fig. 6C). For unknown reasons, p130 knockdown spheroids do not display extensive cell death until 48 hours (Supplementary Fig. S6).

Taken together, these data underscore the central role of Dyrk1A and p130 in regulating cell-cycle exit and survival of EOC spheroid cells as they occur coincidentally in the first 48 hours of suspension culture. Moreover, the data also indicates that loss of viability in p57Kip2-deficient spheroids occurs by a mechanism independent of cell-cycle exit.

Chemical inhibition of Dyrk1A compromises spheroid cell viability and is augmented by carboplatin

Our data suggest that the inhibition of DREAM assembly may be an effective way to compromise EOC spheroid cell viability. To test this possibility, we incubated iOvCa147E2 spheroids with the Dyrk1A inhibitors Harmine and INDY (35–37) and assessed cell viability after 48 hours. In both cases, these agents decreased spheroid cell viability relative to control cells (Fig. 7A). An analysis of the effect of Harmine and INDY on DREAM assembly in spheroids revealed a reduction in the amount of p130 that coimmunoprecipitated with Lin37 (Fig. 7B) suggesting that pharmacologic inhibition of Dyrk1A and its effects on DREAM assembly compromises viability in dormant ovarian cancer cells.

Figure 7.
Figure 7.

EOC spheroid viability is compromised by chemical inhibitors of Dyrk1A. A, iOvCa147E2 spheroids were incubated with 30 μmol/L INDY, 20 μmol/L Harmine, vehicle (DMSO), or left untreated for 48 hours after which spheroids were collected and viability was assessed. Error bars, 1 SD from the mean and an asterisk is a significant difference (ANOVA, *, P < 0.05). B, Harmine, INDY, and vehicle-treated iOcVa147E2 spheroids were collected, lysed, and immunoprecipitated for Lin37. Western blots were probed with the indicated antibodies to determine input levels of proteins and p130 interaction with Lin37. C, Spheroids were treated with vehicle (DMSO), INDY (30 μmol/L), carboplatin (iOvCa147E2, iOvca185, OVCAR8: 100 μmol/L, HEY: 150 μmol/L) or INDY plus carboplatin for 48 hours after which they were collected and assessed for viability. Error bars, 1 SD from the mean and an asterisk indicates a significant difference (ANOVA, *, P < 0.05).

While pharmacologic inhibition of Dyrk1A exerted a negative effect on spheroid cell survival, we were interested to see whether the effects of chemotherapeutic drugs that typically target proliferating cells would be augmented by inhibition of Dyrk1A. In this case, we treated OVCAR8, HEY, iOvCa147E2, and iOvCa185 spheroids with INDY and carboplatin and assessed their survival. We found cell viability to be additionally compromised in spheroids treated with INDY and Carboplatin than when treated with each alone (Fig. 7C). Because carboplatin targets proliferating cells (38), these data provide further evidence that EOC spheroid cells deprived of Dyrk1A have difficulty exiting the cell cycle.

Discussion

An important aspect of the pathogenesis of high-grade ovarian cancer is the dissemination of multicellular spheroids contributing to metastasis (3). Central to the metastatic nature of these aggregates of cells is their dormant nature, which is exemplified by their resistance to therapeutic treatments that primarily target rapidly growing cells (39–41). Our data demonstrate that components and positive regulators of DREAM play a critical role in cell-cycle withdrawal and survival of ovarian cancer spheroids in a model system of cellular dormancy. In addition, our experiments suggest that additional pathways to viability mediated by p57Kip2 also play an important role in spheroid dormancy.

We were surprised that knockdown of DREAM assembly factors such as Dyrk1A or components such as the Lin proteins had such varying effects on EOC spheroid viability, even within individual cell lines, as they contribute to the same molecular mechanism. In particular, Lin9 is essential for early embryogenesis (42), and cells deficient in Lin9 have been demonstrated to undergo mitotic arrest (42, 43). So its knockdown might be expected to have dramatic effects on viability under all culture conditions. For this reason, it is noteworthy that our assay system for viability in suspension culture does not require long-term proliferation of cells prior to seeding into low attachment dishes. We suggest that our scoring methods for siRNA-transfected EOC cells and spheroid formation may reflect a compromise between viability in suspension and cellular effects of knockdown prior to seeding in suspension. In this way, knockdown of components such as Lin9 may lower proliferation through mitotic arrest resulting in better survivability, thus appearing to have similar modest effects in adherent and suspension viability assays. Ultimately, these types of effects can obscure a particular gene products contribution to DREAM in spheroid dormancy.

Genomic analysis of high-grade EOC suggests that 10% of cases contain Dyrk1B amplifications (23). In addition, cellular studies have suggested that Dyrk1B is expressed in approximately 75% of ovarian cancers and a majority of EOC cell lines (4, 7, 44) and overexpressed in a significant proportion (4, 44). It is difficult to reconcile the overexpression of Dyrk1B, that can send negative growth signals through DREAM assembly, with selection for overexpression during progression to malignancy. For this reason, we interpret data from our comparison of Dyrk1A and Dyrk1B to suggest that Dyrk1A is primarily the kinase responsible for DREAM assembly in spheroid dormancy. Intriguingly, iOvCa246 cells display high expression of Dyrk1B, but we were not able to detect Dyrk1B activity toward GST-Lin52, suggesting elevated Dyrk1B expression may have other functions. It is possible our panel of 10 cell lines does not fully recapitulate what would be found in a broader collection of EOC cells and so the question of differences between Dyrk1A and Dyrk1B will require further investigation. Regardless, our data provide a strong rationale for collectively targeting these kinases in EOC treatment.

Assembly of DREAM has previously been identified as a mediator of quiescence in gastrointestinal stromal tumor (GIST) cells (16, 45). In this instance, DREAM assembly was linked to quiescent imatinib-resistant GIST cells, which also display elevated p27Kip1 and p130 expression levels (16, 45). Knockdown of Dyrk1A or Lin52, or inhibition of Dyrk1A with Harmine, abrogated quiescence and restored imatinib sensitivity (16). Taken together with our study in which sensitivity to carboplatin is increased by Dyrk1A inhibition, it suggests a paradigm where DREAM plays a central role in survival under rare circumstances where growth arrest ironically benefits cancer cells by protecting them from chemotherapy. We also suggest that this role for DREAM in cell-cycle arrest during dormancy may be a common feature of cancers that are characterized by spread through ascites to other abdominal locations.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: J. MacDonald, P. Perampalam, G.E. DiMattia, F.A. Dick

Development of methodology: J. MacDonald, L. Litovchick, G.E. DiMattia, F.A. Dick

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Ramos-Valdes, P. Perampalam, L. Litovchick, G.E. DiMattia

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. MacDonald, P. Perampalam, G.E. DiMattia, F.A. Dick

Writing, review, and/or revision of the manuscript: J. MacDonald, P. Perampalam, L. Litovchick, G.E. DiMattia, F.A. Dick

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. MacDonald, G.E. DiMattia

Study supervision: G.E. DiMattia, F.A. Dick

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

  • Received September 22, 2016.
  • Revision received December 6, 2016.
  • Accepted December 7, 2016.

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A Systematic Analysis of Negative Growth Control Implicates the DREAM Complex in Cancer Cell Dormancy
James MacDonald, Yudith Ramos-Valdes, Pirunthan Perampalam, Larissa Litovchick, Gabriel E. DiMattia and Frederick A. Dick
Mol Cancer Res April 1 2017 (15) (4) 371-381; DOI: 10.1158/1541-7786.MCR-16-0323-T
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