Tivantinib (ARQ 197) affects the apoptotic and proliferative machinery downstream of c-MET: role of Mcl-1, Bcl-xl and Cyclin B1
Shuai LüHelga‐Paula TörökEike GallmeierFrank T. KolligsAntonia RizzaniSabrina ArenaBurkhard GökeAlexander L. GerbesEnrico N. De Toni
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// Shuai Lu 1 , Helga-Paula Török 1 , Eike Gallmeier 2 , Frank T. Kolligs 1, 3 , Antonia Rizzani 1 , Sabrina Arena 4, 5 , Burkhard Göke 1 , Alexander L. Gerbes 1 , Enrico N. De Toni 1 1 Medizinische Klinik und Poliklinik 2, Klinikum der Universität München, Campus Grosshadern, Munich, Germany 2 Department of Gastroenterology, Endocrinology and Metabolism, University Hospital of Marburg, Philipps-University of Marburg, Marburg, Germany 3 Department of Internal Medicine and Gastroenterology, HELIOS Klinikum Berlin-Buch, Berlin, Germany 4 Department of Oncology, University of Torino, Candiolo, Torino, Italy 5 Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy Correspondence to: Enrico De Toni, e-mail: enrico.detoni@med.uni-muenchen.de Keywords: HCC, targeted therapies, c-MET, apoptosis Received: January 28, 2015 Accepted: May 28, 2015 Published: June 10, 2015 ABSTRACT Tivantinib, a c-MET inhibitor, is investigated as a second-line treatment of HCC. It was shown that c-MET overexpression predicts its efficacy. Therefore, a phase-3 trial of tivantinib has been initiated to recruit "c-MET-high"patients only. However, recent evidence indicates that the anticancer activity of tivantinib is not due to c-MET inhibition, suggesting that c-MET is a predictor of response to this compound rather than its actual target. By assessing the mechanisms underlying the anticancer properties of tivantinib we showed that this agent causes apoptosis and cell cycle arrest by inhibiting the anti-apoptotic molecules Mcl-1 and Bcl-xl, and by increasing Cyclin B1 expression regardless of c-MET status. However, we found that tivantinib might antagonize the antiapoptotic effects of c-MET activation since HGF enhanced the expression of Mcl-1 and Bcl-xl. In summary, we show that the activity of tivantinib is independent of c-MET and describe Mcl-1, Bcl-xl and Cyclin B1 as effectors of its antineoplastic effects in HCC cells. We suggest that the predictive effect of c-MET expression in part reflects the c-MET-driven overexpression of Mcl-1 and Bcl-xl in c-MET-high patients and that these molecules are considered as possible response predictors.Keywords:
Cyclin B1
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Progression through the cell cycle in somatic eukaryotic cells is regulated by variations in the levels of cyclin proteins. These protein levels are in turn regulated by cyclical oscillations in their mRNA levels. We show here that regulation of RNA stability plays a role in the mechanisms underlying cell cycle progression. Both cyclin A and B1 messages are expressed at high levels in G2-M and at low levels in early G1. The half-lives of their messages mirror this pattern, long in G2-M (>8 h) and short in early G1 (1-2 h). However, there is evidence of specificity to these changes, because the cyclin A message becomes stable at the G1-S boundary, whereas the cyclin B1 message is unstable until later in S phase. Furthermore, although cyclin B1 mRNA levels are lowered after irradiation because of enhanced instability, cyclin A mRNA levels and message stability are unaffected by irradiation. Additional evidence of specificity was found in an analysis of cyclin E mRNA stability, which remains constant through the cell cycle, although the cyclin E message displays cell cycle-dependent changes in expression. These studies suggest that specific alterations in RNA stability are an important component in regulating the expression of cyclins A and B1 and hence in controlling the cell cycle.
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The cyclins are tightly regulated elements governing eukaryotic cell cycle progression by means of sequential activation-inactivation of cyclin-dependent kinases. In one manifestation of this regulation, the mRNA levels of several cyclin genes oscillate during the cycle in mammalian cells. Such cycle-dependent fluctuations in transcript levels could result from changes not only in rates of transcription, but also in mRNA stability. Here we used a new, minimally-disturbing method for producing multi-cycle synchronous growth of human MOLT-4 cells, in combination with quantitative real-time RT-PCR, to compare cell cycle-dependent transcript levels and half-lives of cyclin A2, B1, D3, E and PCNA mRNAs. While all mRNA levels except cyclin D3 varied in the cycle, there were no apparent variations in message half-lives. This differs from several previous reports of dramatic fluctuations in the stabilities of cyclin mRNAs, and infers that fluctuations in cyclin mRNA transcript levels during the MOLT-4 cell cycle are not due to variations in half-lives. The discrepancy in mRNA stability determinations could be due to differences in cell types or synchronization methods, but our findings may be representative of mRNA processing in the cycle of cells in unstressed steady-state growth.
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B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary.
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Cyclin B, a regulatory subunit of maturation/M‐phase promoting factor (MPF), has several subtypes in many vertebrate species. However, it is not known whether the different B‐type cyclins have any different functions in vertebrate cells, although their subcellular localizations seem to differ largely from each other. To examine the roles of two major B‐type cyclins, B1 and B2, in spindle formation in M phase, we overexpressed their N‐termini in Xenopus oocytes; the N‐termini of cyclins B1 and B2 contained a cytoplasmic retention signal (CRS), and hence their overexpressions were expected to competitively inhibit the subcellular localizations of the endogenous cyclins B1 and B2, respectively. Upon entry into meiosis I, oocytes overexpressing the cyclin B1 N‐terminus formed an apparently normal bipolar spindle, but those oocytes overexpressing the cyclin B1 N‐terminus formed a monopolar (or monoastral) spindle. This defect in bipolar spindle formation was observed only when the cyclin B2 N‐terminus contained its own CRS sequence, and was able to be rescued by overexpression of full‐length cyclin B2. These results suggest, for the first time, that the correct subcellular localization of cyclin B2, but not of cyclin B1, is essential for (the initiation of) bipolar spindle formation in Xenopus oocytes.
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Cyclin B1, an important cell cycle regulator, was up-regulated in lymphocytes of human immunodeficiency virus (HlV)-infected patients. However, the mechanism of cyclin B1 up-regulation and the effects of the up-regulation on the host cells remain unclear. Here, we show that HIV-encoded Tat protein regulates cyclin B1 levels in two different ways: first, Tat stimulates the transcription of cyclin B1, which increases cyclin B1 levels and promotes the cells apoptosis; and second, Tat stimulates polyubiquitination-mediated degradation of cyclin B1 through binding to the N-terminal of cyclin B1 (aa 61–129) that is just downstream of the D box, which prevents excessive levels of cyclin B1 in the cells. These results suggest that Tat-regulating cyclin B1 affects the status of HIV: Tat stimulates cyclin B1 expression to slow down the host cell cycle progress and to promote the host cell apoptosis, which might facilitate HIV release;Tat stimulates cyclin B1 degradation to prevent overaccumulation of cyclin B1, which might facilitate HIV replication. Taken together, our results reveal for the first time how HIV-Tat regulates cyclin B1 and keeps its balance in the cells.—Zhang, S.-M., Sun, Y., Fan, R., Xu, Q.-Z., Liu, X.-D., Zhang, X., Wang, Y., Zhou, P.-K HIV-1 Tat regulates cyclin B1 by promoting both expression and degradation. FASEB J. 24, 495–503 (2010). www.fasebj.org
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Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34 cdc2 . To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34 cdc2 kinase to the essential substrates of cyclin B2.
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Cyclin B, a regulatory subunit of maturation/M-phase promoting factor (MPF), has several subtypes in many vertebrate species. However, it is not known whether the different B-type cyclins have any different functions in vertebrate cells, although their subcellular localizations seem to differ largely from each other. To examine the roles of two major B-type cyclins, B1 and B2, in spindle formation in M phase, we overexpressed their N-termini in Xenopus oocytes; the N-termini of cyclins B1 and B2 contained a cytoplasmic retention signal (CRS), and hence their overexpressions were expected to competitively inhibit the subcellular localizations of the endogenous cyclins B1 and B2, respectively. Upon entry into meiosis I, oocytes overexpressing the cyclin B1 N-terminus formed an apparently normal bipolar spindle, but those oocytes overexpressing the cyclin B1 N-terminus formed a monopolar (or monoastral) spindle. This defect in bipolar spindle formation was observed only when the cyclin B2 N-terminus contained its own CRS sequence, and was able to be rescued by overexpression of full-length cyclin B2. These results suggest, for the first time, that the correct subcellular localization of cyclin B2, but not of cyclin B1, is essential for (the initiation of) bipolar spindle formation in Xenopus oocytes.
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Cyclin A functions in both the S and G2-M phases of the cell cycle. The expression of cyclin A during liver regeneration was compared with that of cyclin B1 and p34cdc2. Liver regeneration was followed at 2-h intervals from 12 to 48 h after partial hepatectomy (PH). Immunohistochemical staining using proliferating cell nuclear antigen revealed DNA synthesis peaks at 18 h after PH. The most intense nuclear staining of hepatocytes with cyclins A and B1 and p34cdc2 antibodies occurred at 26 h post-PH, which corresponds with the onset of mitosis. Quantitative mRNA expression of cyclins A and B1 and p34cdc2 was determined by competitive reverse transcription-PCR. Construction of mRNA internal standards and coamplification during reverse transcription-PCR allowed quantitation of all three cell cycle genes. At 24 h post-PH, cyclin A mRNA levels were approximately 5 fg/100 ng total RNA. In contrast, cyclin B1 and p34cdc2 levels were 20-fold higher, 100 fg/100 ng total RNA. Cyclin B1 and p34cdc2 mRNA levels showed two peaks, at 26 and 38-44 h post-PH, whereas the levels of cyclin A were constant during this interval. Immunoblots revealed the presence of cyclin A in normal liver, and significant amounts were present as early as 12 h post-PH. At 26 h post-PH, tyrosine-phosphorylated forms of cyclin A were detected. Cyclin B1 and p34cdc2 protein were not present until 22-24 h post-PH, and two peaks were observed, at 26 and 38-44 h, coinciding with the mRNA pattern. Histone H1 kinase activity was associated with the two peaks of cyclin B1 and p34cdc2 expression. The unique pattern of cyclin A expression and detection of tyrosine-phosphorylated forms suggest a different mechanism for the regulation of cyclin A during liver regeneration.
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We have investigated the relationship between Xenopus laevis c-mos (mosXe) and the cyclin B component of maturation-promoting factor. Microinjection of Xenopus oocytes with in vitro-synthesized RNAs encoding Xenopus cyclin B1 or cyclin B2 induces the progression of meiosis, characterized by germinal vesicle breakdown (GVBD). By preinjecting oocytes with a mosXe-specific antisense oligonucleotide, we show that GVBD induced by cyclin B does not require expression of the mosXe protein. GVBD induced by cyclin B proceeds significantly faster than GVBD induced by progesterone or MosXe. However, coinjection of RNAs encoding cyclin B1 or cyclin B2 with mosXe RNA results in a 2.5- to 3-fold acceleration in GVBD relative to that induced by cyclin B alone. This acceleration of GVBD does not correlate with changes in the level of cyclin B1 and cyclin B2 phosphorylation.
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