Cyclin D1 Degradation Is Sufficient to Induce G1 Cell Cycle Arrest Despite Constitutive Expression of Cyclin E2 in Ovarian Cancer Cells

Home , Cyclin D, Cyclin D1, Cyclin E, G1 phase

Published OnlineFirst July 28, 2009; DOI: 10.1158/0008-5472.CAN-09-0913

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Cyclin D1 Degradation Is Sufficient to Induce G1 Cell Cycle Arrest despite Constitutive Expression of Cyclin E2 in Ovarian Cancer Cells

Chioniso Patience Masamha1 and Doris Mangiaracina Benbrook1,2

Departments of 1Biochemistry and Molecular Biology and 2Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Abstract All cancers are characterized by abnormalities in apoptosis and differentiation and altered cell proliferation (4). Cancer cells often D- and E-type cyclins mediate G1-S phase cell cycle progression through activation of specific cyclin-dependent kinases have a selective growth advantage due to deregulation of cell cycle (cdk) that phosphorylate the retinoblastoma protein (pRb), proteins, causing aberrant growth signaling that drives tumor thereby alleviating repression of E2F-DP transactivation of development (1, 5). Exit of cells from quiescence and cell cycle S-phase genes. Cyclin D1 is often overexpressed in a variety of progression is induced by sequential activation of cyclin-dependent cancers and is associated with tumorigenesis and metastasis. kinases (cdk) by cyclins. Once the cell progresses through late G1 into the Sphase, it is irrevocably committed to DNA replication Loss of cyclin D can cause G1 arrest in some cells, but in other cellular contexts, the downstream cyclin E protein can and cell division (6). Deregulation of G1 to S-phase transition is implicated in the pathogenesis of most human cancers, including substitute for cyclin D and facilitate G1-S progression. The objective of this study was to determine if a flexible ovarian cancer (7). D- and E-type cyclins act as positive regulators heteroarotinoid anticancer compound, SHetA2, regulates cell of this critical transition. Cyclin D expression is the converging cycle proteins and cell cycle progression in ovarian cancer point at which diverse mitogenic and transformation signaling cells. SHetA2induced cyclin D1 phosphorylation, ubiquitina- cascades integrate to mediate engagement of the cell cycle machinery (8). Three cyclin D isoforms (D1, D2, and D3) are tion, and proteasomal degradation, causing G1 arrest in ovarian cancer cells despite continued cyclin E2expression differentially expressed in various tissues (9). Of these, cyclin D1 and independently of p53 and glycogen synthase kinase-3B. overexpression is most frequently associated with human cancer Cyclin D1 loss inhibited pRb S780 phosphorylation by cyclin (10). Cyclin D1 is rarely mutated, but its overexpression confers a D1-cdk4/6 and released p21 from cyclin D1-cdk4/6-p21 selective growth advantage and hence acts as a driver of neoplastic protein complexes to form cyclin E2-cdk2-p21 complexes, growth in various cancers (11). In epithelial ovarian cancer, which repressed phosphorylation of pRb S612by cyclin E2- overexpression of cyclin D1 has been associated with decreased survival in patients (12). An estimated 26% of sporadic epithelial cdk2and ultimately E2F-DPtranscriptional activity. G 1 arrest was prevented by overexpression or preventing degradation of ovarian cancers overexpress cyclin D1 (13). Proteasomal degrada- cyclin D1 but not by restoration of pRb S612phosphorylation tion has emerged as an important regulator of cyclin D1 levels in through p21 knockdown. In conclusion, we show that loss of cancer cells (14). In addition to cyclin D, both human E-type cyclins cyclin D1 in ovarian cancer cells treated with SHetA2is (E/E1 and E2) are also involved in G1- to S-phase progression and sufficient to induce G1 cell cycle arrest and this strategy is not in some cases can compensate for cyclin D knockout (15–17). impeded by the presence of cyclin E2. Therefore, cyclin D1 is a Overexpression of either E-type cyclin has been associated with cell sufficient therapeutic target in ovarian cancer cells. [Cancer proliferation and metastasis (18). The activities of both G1 cyclins Res 2009;69(16):6565–72] D and E converge on the retinoblastoma protein (pRb). Unphosphorylated pRb binds to E2F-DP transcription factor heterodimers, thus masking their transactivation domain (6). In Introduction this state, pRb also recruits histone deacetylases (HDAC), SWI/SNF, Ovarian cancer is the most lethal of gynecologic malignancies and histone methylases, which all serve to maintain the repressed with more than 70% of cases diagnosed at an advanced stage that state of chromatin located at E2F-responsive genes required for cell has only a 30% average 5-year survival rate. Unlike other cancers, cycle progression (6, 19). In healthy cells, increased cyclin D levels the molecular mechanisms involved in ovarian cancer etiology are in response to mitogenic stimuli lead to formation of active cyclin poorly understood, mainly due to difficulties in detecting and D-cdk4/6 complexes that hypophosphorylate pRb and disrupt its obtaining early-stage ovarian cancer lesions for study (1–3). Thus, association with HDAC at E2F-DP binding sites. This results in there is a great need for low-toxicity drugs that can prevent and partial activation of E2F-DP, allowing transcription from the cyclin treat ovarian cancer. E promoter only. Ongoing pRb-SWI/SNF interactions on other E2F- DP promoters maintains E2F-DP in the repressed state (20). The newly synthesized cyclin E binds and activates cdk2, causing further pRb hyperphosphorylation resulting in additional weaken- Note: Supplementary data for this article are available at Cancer Research Online ing of pRb affinity for E2F-DP. The released E2F-DP transcription (http://cancerres.aacrjournals.org/). Requests for reprints: Doris Mangiaracina Benbrook, Department of Obstetrics factor complex is now free to drive transcription of S-phase genes, and Gynecology, Oklahoma University Health Science Center, Oklahoma City, OK hence facilitating cell cycle progression (6, 20). Disruption of the 73104. Phone: 405-271-5523; Fax: 405-271-3874; E-mail: [email protected]. I2009 American Association for Cancer Research. pRb pathway in cancer is not surprising given its complex control doi:10.1158/0008-5472.CAN-09-0913 of cell cycle progression (21). www.aacrjournals.org 6565 Cancer Res 2009; 69: (16). August 15, 2009

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Figure 1. SHetA2 induces G1-phase cell cycle arrest and cyclin D1 loss in ovarian cancer cell lines. A, cell cycle profiles of cultures treated with the indicated concentrations of SHetA2 for 24 h (top graphs) or with 5 Amol/L SHetA2 or the delivery vehicle DMSO for the indicated number of hours (bottom bar graphs) were evaluated using PI staining and flow cytometry. Data are representative of at least two independent experiments. Points and columns, mean (n z 3); bars, SE. P values are based on a two-tailed Student’s t test between the cells in G1 DMSO and G1 SHetA2 for the same time point. B, Western blots of cyclin D1 expression in cell extracts from cultures treated with 5 Amol/L SHetA2 over a 24-h time period, including a 24-h DMSO solvent control (24C; left) or with different concentrations of SHetA2 for 24 h (right). C, left, average total cell numbers in cultures treated with 5 Amol/L SHetA2 (T) or DMSO (C) for 24 h fromtwo independent experiments; right, representative data of the percentage of apoptotic cells measured by two independent experiments.

Flexible heteroarotinoids (Flex-Het) are small molecules that cytostatic (28). The objective of this study was to determine if offer promise in ovarian cancer because they induce differentiation, alterations in cell cycle proteins and cell cycle progression apoptosis, and growth inhibition without evidence of toxicity contribute to the mechanism of growth inhibition induced by (22–27). The lead Flex-Het, SHetA2, is currently in preclinical SHetA2 in ovarian cancer cell lines. development and was chosen because it exhibited the greatest In this study, we report that targeting cyclin D1 for proteasomal efficacy in inducing apoptosis in multiple cancer cell lines without degradation is sufficient to induce G1 cell cycle arrest in ovarian killing the normal cells. The mechanism of this differential cancer cell lines. The downstream consequences on pRb phos- apoptosis occurs through direct effects on mitochondria and Bcl- phorylation, E2F activity, and cyclin A expression could not be 2 family proteins (22, 24). Because apoptosis and the cell cycle are compensated for by continued expression of endogenous cyclin E2 closely coupled, most compounds that are cytotoxic are also or by relief of p21 repression of cyclin E2-cdk2 activity in ovarian

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Cyclin E2 Does Not Compensate for Cyclin D1 Loss cancer cell lines. Thus, cyclin D1 is a sufficient target for inducing after trypsinization. The total number of cells was determined with a Beckman Coulter counter. G1 arrest of ovarian cancer cells in chemoprevention and therapeutic strategies for ovarian cancer. For the apoptosis assay, cells were pelleted and resuspended in Annexin V-FITC-PI stain (100 AL Annexin binding buffer, 5 AL Annexin V-FITC conjugate, and 2 AL of 100 Ag/mL PI; Invitrogen) for 10 min at room Materials and Methods temperature. An additional 400 AL of the Annexin binding buffer was then Cell lines. Ovarian cancer cell lines SK-OV-3 and Caov-3 (American Type added and the samples were run through a FACSCalibur. Summit for MoFlo Culture Collection) were maintained in McCoy’s 5A and DMEM (with Acquisition and Sort Control Software (Cytomation, Inc.) were used to L-glutamine) media, respectively. A2780 cells were cultured in RPMI 1640 quantify apoptosis. A with L-glutamine (supplemented with 1 mmol/L sodium pyruvate and Transient expression of cyclin D1. Cells were transfected with 5 g 1 mmol/L HEPESbuffer). All media were supplemented with 10% fetal bovine human pcDNA3 cyclin D1 T286 mutant (Mt) and pcDNA3 cyclin D1 wild- serum and antibiotic/antimycotic. SHetA2 was synthesized by type (Wt) using Metafectene Pro transfection reagent (Biontex) according to K. Darrell Berlin (Oklahoma State University, Stillwater, OK; ref. 25), dissolved the manufacturer’s protocol. Plasmids were a kind gift from Dr. Osamu in DMSO, and manipulated under reduced lighting conditions. All cells were Tetsu (University of California San Francisco, San Francisco, CA) and were treated for 24 h with 5 Amol/L SHetA2 or DMSO unless stated otherwise. previously described (29). After 24 h, the cells were replated and cultured for Cell cycle analysis. Cells were seeded onto six-well plates (5 Â 105 per a further 24 h before treatment. well). After treatment, cells were harvested and fixed in 70% ethanol and Immunoprecipitation. Cells transfected with 10 Ag Wt cyclin D1 were stored overnight at 4jC. For analysis, 1 mL of freshly prepared PBSstaining treated with 5 Amol/L SHetA2 for 8 h in the presence of 10 Amol/L MG132, solution [200 Ag/mL RNase A, 20 mg/mL propidium iodide (PI), and 0.1% a proteasomal degradation inhibitor. Protein extracts (200 Ag) were Triton X-100] was added to the cells. DNA content was analyzed the same immunoprecipitated with 8 Ag of cyclin D1 or p21 antibody using day by flow cytometry (FACSCalibur from Becton Dickinson). ModFit Cell Dynabeads Protein G (Invitrogen) according to the manufacturer’s protocol Cycle Analysis software was used to determine the cell cycle phase. with the addition of a 1-h Dynabead preclearance step to reduce nonspecific Total cell number determination and apoptosis assay. Cells were binding. plated in six-well plates (5 Â 105 per well) overnight before treatment. After Western blotting. For whole-cell extraction, cells were lysed with M-PER treatment, the medium and PBSused to wash the cells were harvested to mammalian protein extraction reagent (Pierce) containing protease collect any detached cells and pooled with attached cells that were collected inhibitor cocktail (Sigma). Cytoplasmic and nuclear proteins were extracted

Figure 2. SHetA2 induces cyclin D1 T286 phosphorylation, ubiquitination, and proteasomal degradation independent of GSK-3h. A, Western blots of enriched cytoplasmic protein extracts from cells treated with 5 Amol/L SHetA2 or DMSO were hybridized with antibodies recognizing phosphorylated cyclin D1 and total cyclin D1. B, cancer cells transfected with Wt cyclin D1 were treated with SHetA2 (T) or DMSO (C) in the presence of 10 Amol/L MG132 for 8 h. Cell lysates were immunoprecipitated with cyclin D1 antibodies and the Western blot was probed with ubiquitin antibody. C, cell cycle profile of cells treated for 24 h with 5 Amol/L SHetA2 in the presence of different concentrations of MG132. Columns, mean (n z 3); bars, SE. D, cell cycle profiles of cultures treated with 5 Amol/L SHetA2 in the presence or absence of 10 mmol/L LiCl or SB216763 (10 Amol/L) for 24 h. Columns, mean (n z 3); bars, SE.

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E2F luciferase reporter assay. Cultures were transfected with 5 Ag pTA-E2F-luc and pTA-luc plasmids (Clontech) using Metafectene Pro as described earlier. The next day, cells were replated and incubated overnight. Cultures were then treated for 24 h and luciferase activity was determined using a luciferase assay (Promega). Each luciferase value was normalized to the protein concentration and then to the control sample transfected with pTA-luc and treated with DMSO solvent. Statistics. Results shown are representative of at least two independent experiments performed in triplicate. The data are presented as the mean F SE. The Student’s independent t test was used to determine the statistical

significance between the G1-phase cells in the DMSO control and SHetA2 treatment group. For cyclin D1 overexpression, one-way ANOVA was used

to analyze the statistical significance of the percentage of cells in the G1 phase after SHetA2 treatment followed by Tukey’s multiple comparison test. In each case, P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Results

SHetA2induces G 1-phase cell cycle arrest and cyclin D1 loss independent of p53 and apoptosis in ovarian cancer cells. The effects of SHetA2 on cell cycle profiles were measured by flow cytometric analysis of three ovarian cancer cell lines that differed by p53 status and origin. A2780, established from an ovarian adenocarcinoma of an untreated patient, expresses Wt p53 (30); SK-OV-3, established from the ascitic fluid of an ovarian cancer patient, has a p53 gene deletion and does not express any p53 mRNA or protein (30); whereas Caov-3, established from an ovarian adenocarcinoma, expresses Mt p53 (31). Despite these differences, all three ovarian cancer cell lines exhibited a dose-dependent increase in the percentage of cells in the G1 phase of the cell cycle (Fig. 1A for A2780 and SK-OV-3; data not shown for Caov-3), indicating that p53 is not required for the mechanism of G1 arrest. Maximal G1 arrest was induced by 5 Amol/L SHetA2, and this concentration was used to treat cells in subsequent experiments. G1 arrest increased in a time-dependent manner (Fig. 1A). A2780 cells responded more rapidly to SHetA2, exhibiting statistically significant G1 arrest after 4 hours (P = 0.0257). Both SK-OV-3 and A2780 cells exhibited significant G1-phase arrest at 16 hours (A2780, P = 0.000712; SK-OV-3, P = 0.00409) and at 24 hours (A2780, P = 0.000346; SK-OV-3, P = 0.00759). This G1 arrest was associated Figure 3. Overexpression of Wt and degradation-resistant cyclin D1 partially with both a time- and dose-dependent decrease of cyclin D1 attenuated G1 arrest. A, cell cycle profiles of cultures transfected with Wt or T286 Mt cyclin D1 plasmids and treated for 24 h with DMSO (C)or5Amol/L protein (Fig. 1B) in SHetA2-treated cultures compared with SHetA2 (T) were determined by flow cytometry. Data are representative of two untreated controls. experiments performed in triplicate. Columns, mean (n z 3); bars, SE. Flow cytometric analysis of cells stained with Annexin V and PI B, Western blot verification of cyclin D1 overexpression. was performed to determine if apoptosis was occurring at the 5 Amol/L dose and 24-hour time point that induced maximal G using the Nuclear Extract kit (Active Motif). Protein concentrations were 1 arrest. Although this treatment induced a statistically significant determined by the Bicinchoninic Acid kit (Pierce) to ensure equal loading. Proteins were separated on 10% SDS-PAGE gels and transferred to reduction in total cell number of cells (A2780, P = 0.0157; SK-OV-3, polyvinylidene difluoride membranes as previously described (24). Blots P = 0.0378; Fig. 1C, left), there was no significant increase in the were incubated with the following antibodies: cyclin D1 (BD Pharmingen); number of cells undergoing apoptosis (Fig. 1C, right). S9 phospho–glycogen synthase kinase-3h (GSK-3h), GSK-3h, T286 phos- The mechanism of cyclin D1 loss involves T286 phosphor- pho–cyclin D1, cyclin A, p21, p42 phospho-pRb (S780, S795), and total pRb ylation, ubiquitination, and proteasomal degradation inde- (Cell Signaling); ubiquitin, cyclin E2, p21, h-actin, and glyceraldehyde-3- pendent of GSK-3B. Cyclin D1 levels are frequently up-regulated in phosphate dehydrogenase (Santa Cruz Biotechnology); and phospho-pRb cancer and this may be partly due to deregulated proteasomal S612 (Invitrogen). The blots were incubated with the appropriate secondary degradation (32). Western blot analysis of enriched nuclear and antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology). cytoplasmic extracts showed that SHetA2 induced phosphorylation RNA interference. Cells were transfected with human-specific p21 small of cyclin D1 on T286, which is known to target this protein for interfering RNA (siRNA) SignalSilence p21Waf1/Cip1 siRNA kit (Santa Cruz Biotechnology). Then, 48 h after transfection, the cultures were treated with ubiquitination (10). This occurred in a time-dependent manner either DMSO or 5 Amol/L SHetA2 for 24 h. Western blots of protein extracts in both fractions with a concomitant decrease in total cyclin D1 from these cultures were probed with anti-p21 and anti-p42 mitogen- (Fig. 2A; results for nuclear extracts not shown). Subsequent activated protein kinase (MAPK) antibodies (provided with kit to confirm increases in cyclin D1 ubiquitination were confirmed after 8 hours knockout specificity). of SHetA2 treatment in both cell lines (Fig. 2B). An inhibitor of

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Cyclin E2 Does Not Compensate for Cyclin D1 Loss proteasomal degradation, MG132, was used to test if SHetA2- SHetA2-induced cyclin D1 phosphorylation and subsequent events. induced cyclin D1 loss and G1 arrest are due to enhanced In contrast, Western blot analysis of both nuclear and cytoplasmic proteasomal degradation. Treatment with SHetA2 in the presence extracts showed that SHetA2 induced phosphorylation of GSK-3h of MG132 totally abrogated the G1 arrest observed in the presence at S9, which inhibits its kinase activity (Supplementary Fig. S1C). of SHetA2 alone (Fig. 2C). MG132 also partially restored the cyclin Cyclin D1 overexpression attenuated SHetA2-induced G1 D1 in SHetA2-treated extracts (Supplementary Fig. S1A) to levels arrest. To directly test the role of cyclin D1 in the mechanism of comparable with treatment with MG132 alone, implicating cyclin SHetA2-induced G1 arrest, the effects of expression of Wt and D1 degradation in the mechanism of G1 arrest. Simultaneous T286A, a degradation-resistant Mt, of cyclin D1 on SHetA2 G1 arrest treatment with LiCl and SB216763 to inhibit GSK-3h, a kinase were evaluated. Transfection with either Wt or Mt cyclin D1 caused known to phosphorylate cyclin D1 at T286 (32, 33), did not prevent partial reversal of G1 loss after SHetA2 treatment in comparison SHetA2-induced cyclin D1 degradation (Supplementary Fig. S1B)or with mock-transfected A2780 cells (Fig. 3A). Western blots verified G1 arrest (Fig. 2D), suggesting that GSK-3h does not mediate the the transfection and cyclin D1 expression (Fig. 3B). The lack of

Figure 4. Role of p21 in ovarian cancer cell cycle progression. A, Western blots of whole-cell extracts fromcultures treated with DMSO solvent control (C)or5Amol/L SHetA2 (T) for 24 h. B, whole-cell extracts were immunoprecipitated with p21 antibody and Western blotted with cyclin D1 and cyclin E2 antibodies. C, cultures were treated with 5 Amol/L SHetA2 over a 24-h time period with a 24-h–treated DMSO control (24C). The immunoblots were probed for phosphorylated pRb. D, right, Western blots of cell extracts from cultures transfected with p21 siRNA and treated with DMSO or SHetA2 for 24 h to verify p21 knockdown. p42 MAPK was the loading control. Left, after 24-h treatment with 5 Amol/L SHetA2 or DMSO, flow cytometry was used to determine the DNA content. Data are representative of two experiments. Columns, mean (n = 3); bars, SE. Western blots of cell extracts fromp21 RNA interference cells treated with SHetA2 (T) or DMSO (C) hybridized with Rb phospho-specific antibodies.

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complete abrogation of G1 arrest is likely due to low transfection efficiencies in these cell lines. A similar trend was seen in SK-OV-3 cells, although G1 attenuation was less than in the A2780 cells (Fig. 3A). When the percentage of A2780 cells in G1 was compared by one-way ANOVA, the P value was <0.00001, and Tukey’s multiple comparison test for each set of pairs gave the following values: mock versus Wt, P < 0.05; mock versus Mt, P < 0.001; and Wt versus Mt, P < 0.01. In SK-OV-3, the ANOVA P value was <0.001, and for pairwise comparisons, the ANOVA P value was as follows: mock versus Wt, P < 0.05; mock versus Mt, P < 0.001; and Wt versus Mt, P > 0.05. In A2780 cells when compared with the mock-transfected cells, there was greater G1 arrest attenuation in the degradation- resistant cyclin D1 Mt–transfected cells when compared with Wt cyclin D1–transfected cells after treatment with SHetA2. This further supports the role of proteasomal degradation in the SHetA2-induced mechanism. SHetA2alters cyclin E2levels and p21interactions. The effects of SHetA2 on the expression and interaction of additional G1 phase cell regulatory molecules that have been implicated in ovarian cancer, cyclin E2 and p21 (1), were evaluated. Both cyclin E2 and p21 proteins were elevated at the 24-hour SHetA2 treatment time point at protein level (Fig. 4A). Cyclin E2 mRNA levels were also elevated after treatment with SHetA2 in all three ovarian cancer cell lines (Supplementary Fig. S2). Both cyclin D1 and cyclin E2 coimmunoprecipitated with p21 in the untreated control cells (Fig. 4B). In SHetA2-treated cells, cyclin D1 did not coimmuno- precipitate with p21, which was expected because cyclin D1 protein is absent at this treatment time point as shown earlier (Fig. 1B). In Figure 5. E2F transactivation and cyclin A expression are repressed addition, greater amounts of cyclin E2 coimmunoprecipitated with downstreamof cyclin D1 loss. A, cells transiently transfected with the empty pTA-luc plasmid vector or pE2F-TA-luc plasmid were treated for 24 h with DMSO p21 in treated cells compared with the untreated control. or 5 Amol/L SHetA2. Luciferase activity was normalized to protein concentration p21 siRNA restores pRb S612 phosphorylation but not G1-S and then normalized to pTA-luc–transfected, DMSO-treated control cultures. Results are representative of two independent experiments. Columns, mean; progression. Both cyclin D1–stimulated and cyclin E2–stimulated bars, SE. B, Western blots of whole-cell extracts fromcultures treated for cdk activity converges on hyperphosphorylation of pRb; hence, the 24 h with 5 Amol/L SHetA2 (T) or DMSO (C) and probed for cyclin A. effect of SHetA2 on the phosphorylation status of pRb was investigated by Western blot (Fig. 4C). SHetA2 caused a reduction in pRb phosphorylation at S780 and S612 but not S795. Because p21 complexed with both cyclin D1 and cyclin E2 in our studies, p21 that degradation of cyclin D1 through treatment with SHetA2 is was predicted to be involved in the mechanism of SHetA2- sufficient to induce G1 cell cycle arrest. This conclusion is supported mediated G1 arrest. Specific siRNA knockdown of p21 did not affect by the demonstration that G1 arrest can be inhibited by overriding SHetA2-induced G1 arrest in either cell line (Fig. 4D). Knockdown the cyclin D1 loss through overexpression of Wt or a nondegradable of p21 in SHetA2-treated cells did not prevent SHetA2-mediated Mt of cyclin D1 or by inhibiting proteasomal degradation. Whereas loss of phosphorylation of pRb S780 but did partially restore pRb cyclin E has been shown to compensate for knockout of cyclin D S612 phosphorylation, a site phosphorylated by cyclin E2-cdk2 during fetal development and in other studies (15–17), our studies (Fig. 4D, bottom; ref. 34). show that cyclin E2 expression is insufficient to facilitate G1 to S SHetA2inhibits E2Ftransactivation and cyclin A expression. progression in the absence of cyclin D1. Under normal conditions, Decreased phosphorylation of pRb has been shown to inhibit expression of cyclin E is controlled by cyclin D through activation of uncoupling of E2F-DP from pRb and to repress E2F transactivation cdk4/6 hypophosphorylation of pRb (Fig. 6), causing the partial (20). To test this, the effects of SHetA2 on luciferase activity were release of E2F-DP at the cyclin E promoter from repression by pRb measured in ovarian cancer cell lines cotransfected with a and HDAC (6, 9, 20). In SHetA2-treated ovarian cancer cell lines, luciferase reporter construct driven by E2F DNA binding sites. however, cyclin E2 continued to be expressed at the 24-hour time SHetA2 inhibited E2F promoter activity f3-fold (Fig. 5A). To verify point despite complete loss of cyclin D1 at an earlier (8 hours) that the observed decrease in E2F promoter activity altered treatment time point. This suggests that in the ovarian cancer cells expression of E2F target genes, the effects of SHetA2 on studied, cyclin E2 expression may be independent of cyclin D1. endogenous cyclin A levels were determined using Western blot. Although both cyclin E2 mRNA and protein levels were increased in As expected, cyclin A levels were decreased in SHetA2-treated cells SHetA2-treated cultures, it cannot be concluded that the effect is a in comparison with control (Fig. 5B). direct effect of SHetA2 on transcription because the increased cyclin E2 levels could be due to the higher percentage of G1-phase cells in the SHetA2-treated cultures that may have contained cyclin E2 Discussion protein before treatment. The results of this study show that cyclin D1 plays a dominant The inability of cyclin E2 to compensate for cyclin D1 loss can be role in regulating cell cycle progression in ovarian cancer cells and explained by the molecular events that occur downstream of cyclin

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Cyclin E2 Does Not Compensate for Cyclin D1 Loss

D1 loss in ovarian cancer cells. The SHetA2-induced decrease in The SHetA2-induced reduction in pRb S612 and S795 phosphor- phosphorylation of pRb at S780 is consistent with cyclin D1 loss ylation was expected to inhibit release of E2F from pRb. The and the report that this site is exclusively phosphorylated by cyclin consequence of decreased release of E2F was confirmed by the D-cdk4/6 (34). Decreased phosphorylation of pRb on the S612 site observed reduction in E2F transactivation activity and transcrip- reported to be exclusively phosphorylated by cyclin E-cdk2, tion of an E2F endogenous gene, cyclin A. Cyclin A regulates however, is not consistent with the continued expression of cyclin S-phase progression and mitosis because it binds to and activates E2 observed after SHetA2 treatment (34) but can be explained by both cdk2 and cdk1 (37). Thus, the downstream mechanism of p21 repression of cdk2 kinase activity. There has been controversy SHetA2-induced G1 arrest through cyclin D1 degradation involves over the role of p21 in cdk4/6 inhibition; however, the general both decreased expression of cyclin A and release of p21, which consensus is that p21 is a positive regulator of cdk4/6 but inhibits serves to repress cdk2 activity (Fig. 6). cdk2 when complexed with either cyclin E or cyclin A (20, 35, 36). GSK-3h was the first kinase shown to phosphorylate cyclin D1, In this study, coimmunoprecipitation experiments showed that p21 thus targeting it for nuclear export by CRM1, ubiquitination, and binds both cyclin D1 and cyclin E2. As cyclin D1 is lost in SHetA2- proteasomal degradation (33). In the ovarian cell lines used in this treated cells, and cyclin E2 levels are increased, the increased cyclin study, however, cyclin D1 T286 phosphorylation and degradation is E2 is bound by p21 protein (Fig. 6). Repression of cdk2 activity by independent of GSK-3h because two separate GSK-3h inhibitors p21 has been previously reported (35, 36) and is consistent with the failed to abrogate cyclin D1 degradation. Instead, SHetA2 induced ability of p21 siRNA to partially reverse the reduction in pRb S612 phosphorylation of GSK-3h at S9, an event known to inhibit the phosphorylation caused by SHetA2-induced transfer of p21 from kinase activity (33, 38). These observations suggest the existence of cyclin D1 to cyclin E2. Thus, in untreated ovarian cancer cells, another redundant kinase for which cyclin D1 (T286) acts as a cyclin D1 facilitates cdk2 activity by titrating p21 from cyclin substrate. E2-cdk2. In SHetA2-treated cells, increased levels of cyclin E2 are The ability to inhibit the deregulated cell cycle progression of bound and inhibited by increased levels of total p21 and may be ovarian cancer cell lines through a specific molecular mechanism supplemented with p21 released from degraded cyclin D1 supports the application of SHetA2 in ovarian cancer chemo- complexes. prevention. SHetA2 offers promise in this regard in that it has not The inability of p21 siRNA to attenuate SHetA2-induced G1 exhibited toxicities in multiple animal models, including models of arrest further exemplifies the dominant role of cyclin D1 activity ovarian cancer xenograft growth, skin irritancy, and teratogenicity over cyclin E2 activity in ovarian cancer cell lines. As anticipated, (22, 26, 27). Further evidence for SHetA2 chemoprevention activity p21 siRNA released repression of cyclin E2-cdk2 activity and includes the reversal of carcinogen-induced transformation in an allowed pRb S612 phosphorylation, but due to the absence of cyclin in vitro organotypic model and in vivo inhibition of angiogenesis D1 in SHetA2-treated cells, pRb S780, a cyclin D-cdk4/6 and xenograft growth (23, 27). The demonstration in this study that phosphorylation site, remained unphosphorylated in the presence SHetA2 induces G1 arrest in all cell lines tested regardless of the of p21 siRNA. Thus, pRb S612 phosphorylation by cyclin E2-cdk2 is status of p53 suggests that it will be effective against a broad insufficient to overcome loss of cyclin D1 and the resulting spectrum of tumor types, including tumors that have lost p53 reduction of pRb S780 phosphorylation. This dominant role of function. The tumor suppressor gene p53 is the most commonly cyclin D1-cdk4 over cyclin E2-cdk2 in driving cell cycle progression mutated gene in all cancers. The efficacy of SHetA2 is not limited indicates that cyclin D1 is a relevant target for the development of to ovarian cancer because it can inhibit growth of a wide variety of drugs for ovarian cancer. cancers (22).

Figure 6. Model. In normal cells, mitogenic stimuli induce cyclin D expression. Cyclin D binds to cdk4/6, forming a complex, and titrates p21 fromthe cell, facilitating pRb hypophosphorylation. This results in partial derepression of the E2F transcription factor, allowing expression of cyclin E. Cyclin E forms a complex with and activates cdk2, which further hyperphosphorylates pRb, resulting in total derepression of the E2F promoter. This allows transcription of S-phase genes that are under the control of the E2F promoter. In cancer cells, there is aberrant cyclin D1 and cyclin E2 expression. When SHetA2 is added to the cells, cyclin D1 is lost through proteasomal degradation. This loss results in recruitment of p21 to cyclin E2-cdk2 complexes, inhibiting cdk2 activity. This prevents pRb hyperphosphorylation and the E2F promoters remain repressed by the bound pRb complex, resulting in G1 arrest and inhibition of cyclin A expression.

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In summary, this study shows that cyclin D1 is a dominant A are secondary biochemical end points. Some of these regulatory protein of cell cycle progression and a potential downstream effects of SHetA2 may be useful surrogate biomarkers chemopreventive and chemotherapeutic target of ovarian cancer. of treatment responsiveness and efficacy before clinical evidence of Hence, the low-toxicity SHetA2 compound shows promise as a treatment response. chemoprevention agent for ovarian cancer and other types of cancers. This is significant because several molecules that inhibit cyclin D1 or the cognate kinase cdk4/6 are in clinical development Disclosure of Potential Conflicts of Interest and agents that down-regulated cyclin D1 resulted in positive No potential conflicts of interest were disclosed. outcomes (39–41). In addition, cyclin D1 seems to be clinically predictive of cancer risk (39), suggesting that cyclin D1 levels can be used as an end point biomarker for both chemoprevention and Acknowledgments chemotherapy. Whereas cyclin D1 proteasomal degradation is the Received 3/9/09; revised 5/20/09; accepted 6/8/09; published OnlineFirst 7/28/09. primary biochemical end point of SHetA2-induced G1 arrest, Grant support: R01 CA106713. increased cyclin E2 and p21 levels, alterations in cyclin D1-cdk4/6- 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 p21 and cyclin E2-cdk2-p21 protein complexes, decreases in pRb with 18 U.S.C. Section 1734 solely to indicate this fact. S780 and S612 phosphorylation, and decreased expression of cyclin We thank Dr. Tongzu Liu for his help with the cell cycle protocol.

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Cancer Res 2009; 69: (16). August 15, 2009 6572 www.aacrjournals.org

Cyclin D1 Degradation Is Sufficient to Induce G1 Cell Cycle Arrest despite Constitutive Expression of Cyclin E2 in Ovarian Cancer Cells

Chioniso Patience Masamha and Doris Mangiaracina Benbrook

Cancer Res 2009;69:6565-6572. Published OnlineFirst July 28, 2009.

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