Table of Contents

Volume 11, Issue 14

  July 15, 2012

  Pages 2581 - 2761

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 Issue Catalogue (MARC XML)

EDITORIALS: CELL CYCLE FEATURES

Open Access Article

p53 reactivation: The link to zinc

Gabriella D’Orazi and David Givol
Pages 2581 - 2582
http://dx.doi.org/10.4161/cc.21020
Abstract | Full Text | PDF

Comment on: Yu X, et al. Cancer Cell 2012; 21:614-25.

Open Access Article

p53 and cancer stem cells: The mevalonate connexion

Christophe Ginestier, Emmanuelle Charafe-Jauffret and Daniel Birnbaum
Pages 2583 - 2584
http://dx.doi.org/10.4161/cc.21092
Abstract | Full Text | PDF

Comment on: Freed-Pastor WA, et al. Cell 2012; 148:244-58 and Ginestier C, et al. Stem Cells 2012; 1327-37.

Open Access Article

Wnt signaling regulates experience-dependent synaptic plasticity in the adult nervous system

Michael Jensen, Penelope J. Brockie and Andres V. Maricq
Pages 2585 - 2586
http://dx.doi.org/10.4161/cc.21138
Abstract | Full Text | PDF

Comment on: Jensen M, et al. Cell 2012; 149:173-87.

Open Access Article

The transient and constitutive inflammatory signaling in tumorigenesis

Matjaz Rokavec and Jun-Li Luo
Pages 2587 - 2588
http://dx.doi.org/10.4161/cc.21139
Abstract | Full Text | PDF

Comment on: Rokavec M, et al. Mol Cell 2012; 45:777-89.




CELL CYCLE NEWS & VIEWS

Open Access Article

The Herculean task of killing cancer cells: Suppression of FOXO3A in acute leukemia involves a hydra of multiple survival kinases

Peter P. Ruvolo
Page 2589
http://dx.doi.org/10.4161/cc.21233
Abstract | Full Text | PDF

Comment on : Buontempo F, et al. Cell Cycle 2012; 11:2467-75.

Open Access Article

Protein phosphatase PP4: Role in dephosphorylation of KAP1 and DNA strand break repair

Gerd P. Pfeifer
Page 2590
http://dx.doi.org/10.4161/cc.21234
Abstract | Full Text | PDF

Comment on: Liu J, et al. Cell Cycle 2012; 11:2643-9.

Open Access Article

p53-dependent growth arrest and induction of p21: A critical role for PCAF-mediated histone acetylation

Richard Eckner
Page 2591
http://dx.doi.org/10.4161/cc.21235
Abstract | Full Text | PDF

Comment on: Love IM, et al. Cell Cycle 2012; 11:2458-66

Open Access Article

CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis

Varun V. Prabhu, Noel A. Warfel and Wafik S. El-Deiry
Pages 2592 - 2593
http://dx.doi.org/10.4161/cc.21240
Abstract | Full Text | PDF

Comment on: Capparelli C, et al. Cell Cycle 2012; 11:2272-84 and Capparelli C, et al. Cell Cycle 2012; 11:2285-302.




EXTRA VIEWS

RGG motif proteins: Modulators of mRNA functional states

Purusharth Rajyaguru and Roy Parker
Pages 2594 - 2599
http://dx.doi.org/10.4161/cc.20716
Abstract | Full Text | PDF

A recent report¹ demonstrates that a subset of RGG-motif proteins can bind translation initiation factor eIF4G and repress mRNA translation. This adds to the growing number of roles RGG-motif proteins play in modulating transcription, splicing, mRNA export and now translation. Herein, we review the nature and breadth of functions of RGG-motif proteins. In addition, the interaction of some RGG-motif proteins and other translation repressors with eIF4G highlights the role of eIF4G as a general modulator of mRNA function and not solely as a translation initiation factor.

Why minimal is not optimal: Driving the mammalian cell cycle—and drug discovery—with a physiologic CDK control network

Karl A. Merrick and Robert P. Fisher
Pages 2600 - 2605
http://dx.doi.org/10.4161/cc.20758
Abstract | Full Text | PDF

Progression through the eukaryotic cell division cycle is governed by the activity of cyclin-dependent kinases (CDKs). For a CDK to become active it must (1) bind a positive regulatory subunit (cyclin) and (2) be phosphorylated on its activation (T) loop. In metazoans, multiple CDK catalytic subunits, each with a distinct set of preferred cyclin partners, regulate the cell cycle, but it has been difficult to assign functions to individual CDKs in vivo. Biochemical analyses and experiments with dominant-negative alleles suggested that specific CDK/cyclin complexes regulate different events, but genetic loss of interphase CDKs (Cdk2, -4 and -6), alone or in combination, did not block proliferation of cells in culture. These knockout and knockdown studies suggested redundancy or plasticity built into the CDK network but did not address whether there was true redundancy in normal cells with a full complement of CDKs. Here, we discuss recent work that took a chemical-genetic approach to reveal that the activity of a genetically non-essential CDK, Cdk2, is required for cell proliferation when normal cyclin pairing is maintained. These results have implications for the systems-level organization of the cell cycle, for regulation of the restriction point and G₁/S transition and for efforts to target Cdk2 therapeutically in human cancers.

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ

Teodora Nikolova, Frank Hennekes, Anita Bhatti and Bernd Kaina
Pages 2606 - 2619
http://dx.doi.org/10.4161/cc.20862
Abstract | Full Text | PDF

Chloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto- and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci and displayed co-staining with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G₁ but increased significantly in S phase and remained at a high level in G₂, where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O⁶-chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O⁶-chloroethylguanine-triggered cross-links, which give rise to DSBs in the treatment cell cycle, and that HR, but not NHEJ, is the major route of protection against this group of anticancer drugs. Base excision repair had no significant impact on ACNU-induced cytotoxicity.




PERSPECTIVES

The importance of being modified: PCNA modification and DNA damage response

Inbal Gazy and Martin Kupiec
Pages 2620 - 2623
http://dx.doi.org/10.4161/cc.20626
Abstract | Full Text | PDF

PCNA (proliferating cell nuclear antigen) is a sliding clamp that plays important roles during DNA replication and repair. In yeast, PCNA can be modified by either mono- or poly-ubiquitin or by addition of SUMO moieties. These different modifications direct the activity of PCNA toward alternative DNA transactions. In mammals, PCNA ubiquitination was reported, and it seems to have similar effects to those observed in yeast. However, for a long time, no SUMOylation of PCNA could be detected. Two recent papers report the detection of SUMOylated PCNA in mammalian cells. Here, we summarize similarities and differences between the various biological systems and present the current view of the way by which PCNA modification can affect DNA replication and repair pathways.

MiR-1246: A new link of the p53 family with cancer and Down syndrome

Jun-Ming Liao, Xiang Zhou, Yu Zhang and Hua Lu
Pages 2624 - 2630
http://dx.doi.org/10.4161/cc.20809
Abstract | Full Text | PDF

Since the discovery of miRNAs, a number of miRNAs have been identified as p53’s transcriptional targets. Most of them are involved in regulation of the known p53 functions, such as cell cycle, apoptosis and senescence. Our recent study revealed miR-1246 as a novel target of p53 and its analogs p63 and p73 to suppress the expression of DYRK1A and consequently activate NFAT, both of which are associated with Down syndrome and possibly with tumorigenesis. This finding suggests that miR-1246 might serve as a likely link of the p53 family with Down syndrome. Here, we provide some prospective views on the potential role of the p53 family in Down syndrome via miR-1246 and propose a new p53-miR-1246-DYRK1A-NFAT pathway in cancer.




REVIEW

Shugoshins function as a guardian for chromosomal stability in nuclear division

Yixin Yao and Wei Dai
Pages 2631 - 2642
http://dx.doi.org/10.4161/cc.20633
Abstract | Full Text | PDF

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.




REPORTS

Open Access Article

Protein phosphatase PP4 is involved in NHEJ-mediated repair of DNA double-strand breaks

Jinping Liu, Linli Xu, Jianing Zhong, Ji Liao, Jing Li and Xingzhi Xu
Pages 2643 - 2649
http://dx.doi.org/10.4161/cc.20957
Abstract | Full Text | PDF

Reversible phosphorylation is an essential posttranslational modification to turn on/off a protein function and to regulate many cellular activities, including DNA repair. A DNA double-strand break (DSB) is the most lethal form of DNA damage and is mainly fixed by the error-prone nonhomologous end joining (NHEJ)-mediated repair and by the high-fidelity homology recombination (HR)-mediated repair. We found previously that protein phosphatase PP4 is required for HR-mediated DSB repair. In this report, we showed that depletion of PP4C by siRNA compromised NHEJ-mediated repair of DSBs induced by the nuclease I-SceI. Both PP4C and its regulatory subunit PP4R2 physically interacted with the chromatin condensation factor KAP1 (KRAB-associated protein 1). Depletion of PP4C led to sustained phosphorylation of KAP1 at Ser824. Conversely, overexpression of PP4C resulted in a decrease of KAP1 phosphorylation. PP4 dephosphorylated pKAP1 in vitro. Inhibition of KAP1 expression resulted in a defect on NHEJ-mediated DSB repair, and co-depletion of PP4c and KAP1 did not have significant synergistic effect on NHEJ-mediated DSB repair. Taken together, our results suggest that PP4C and KAP1 are in the same epistasis group, and PP4 is involved in NHEJ-mediated DSB repair, possibly through regulating the phosphorylation status of KAP1.

Rapid proliferation of daughter cells lacking particular chromosomes due to multipolar mitosis promotes clonal evolution in colorectal cancer cells

Chao Yang, Xiaoyun Shi, Yun Huang, Zhen Zhang, Howard J. Cooke, Mingrong Wang and Qinghua Shi
Pages 2650 - 2659
http://dx.doi.org/10.4161/cc.20976
Abstract | Full Text | PDF

Aneuploidy and chromosome instability (CIN) are hallmarks of the vast majority of solid tumors. However, the origins of aneuploid cells are unknown. The aim of this study is to improve our understanding of how aneuploidy and/or CIN arise and of karyotype evolution in cancer cells. By using fluorescence in situ hybridization (FISH) on cells after long-term live cell imaging, we demonstrated that most (> 90%) of the newly generated aneuploid cells resulted from multipolar divisions. Multipolar division occurred in mononucleated and binucleated parental cells, resulting in variation of chromosome compositions in daughter cells. These karyotypes can have the same chromosome number as their mother clone or lack a copy of certain chromosomes. Interestingly, daughter cells that lost a chromosome were observed to survive and form clones with shorter cell cycle duration. In our model of cancer cell evolution, the rapid proliferation of daughter cells from multipolar mitosis promotes colonal evolution in colorectal cancer cells.

Fenofibrate-induced nuclear translocation of FoxO3A triggers Bim-mediated apoptosis in glioblastoma cells in vitro

Anna Wilk, Katarzyna Urbanska, Maja Grabacka, Jennifer Mullinax, Cezary Marcinkiewicz, David Impastato, John J. Estrada and Krzysztof Reiss
Pages 2660 - 2671
http://dx.doi.org/10.4161/cc.21015
Abstract | Full Text | PDF

Anti-neoplastic potential of calorie restriction or ligand-induced activation of peroxisome proliferator activated receptors (PPARs) has been demonstrated in multiple studies; however, mechanism(s) by which tumor cells respond to these stimuli remain to be elucidated. One of the potent agonists of PPARα, fenofibrate, is a commonly used lipid-lowering drug with low systemic toxicity. Fenofibrate-induced PPARα transcriptional activity is expected to shift energy metabolism from glycolysis to fatty acid β-oxidation, which in the long-term, could target weak metabolic points of glycolysis-dependent glioblastoma cells. The results of this study demonstrate that 25 μM fenofibrate can effectively repress malignant growth of primary glial tumor cells and glioblastoma cell lines. This cytostatic action involves G₁ arrest accompanied by only a marginal level of apoptotic cell death. Although the cells treated with 25 μM fenofibrate remain arrested, the cells treated with 50 μM fenofibrate undergo massive apoptosis, which starts after 72 h of the treatment. This delayed apoptotic event was preceded by FoxO3A nuclear accumulation, FoxO3A phosphorylation on serine residue 413, its elevated transcriptional activity and expression of FoxO-dependent apoptotic protein, Bim. siRNA-mediated inhibition of FoxO3A attenuated fenofibrate-induced apoptosis, indicating a direct involvement of this transcription factor in the fenofibrate action against glioblastoma. These properties of fenofibrate, coupled with its low systemic toxicity, make it a good candidate in support of conventional therapies against glial tumors.

Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes

Hua Shao, Chunqi Ma, Xuan Zhang, Ruizhen Li, Ann L. Miller, William M. Bement and X. Johné Liu
Pages 2672 - 2680
http://dx.doi.org/10.4161/cc.21016
Abstract | Full Text | PDF

Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine¹²² is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.

Open Access Article

Cyclin D1 inhibits hepatic lipogenesis via repression of carbohydrate response element binding protein and hepatocyte nuclear factor 4α

Eric A. Hanse, Douglas G. Mashek, Jennifer R. Becker, Ashley D. Solmonson, Lisa K. Mullany, Mara T. Mashek, Howard C. Towle, Anhtung T. Chau and Jeffrey H. Albrecht
Pages 2681 - 2690
http://dx.doi.org/10.4161/cc.21019
Abstract | Full Text | PDF

Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.

Induction of cancer cell stemness by chemotherapy

Xingwang Hu, Laura Ghisolfi, Andrew C. Keates, Jian Zhang, Shuanglin Xiang, Dong-ki Lee and Chiang J. Li
Pages 2691 - 2698
http://dx.doi.org/10.4161/cc.21021
Abstract | Full Text | PDF

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.

Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances Cyclin D1 expression and promotes human endometrial cancer cell proliferation

Ivan Casaburi, Paola Avena, Marilena Lanzino, Diego Sisci, Francesca Giordano, Pamela Maris, Stefania Catalano, Catia Morelli and Sebastiano Andò
Pages 2699 - 2710
http://dx.doi.org/10.4161/cc.21029
Abstract | Full Text | PDF

Endometrial cancer exhibits a strong incidence in western developed countries mainly due to fat-rich diet and obesity. Processing of dietary lipids is triggered by bile acids, amphipathic detergents that are synthesized in the liver and stored in the gallbladder. In addition to their well-recognized role in dietary lipid absorption and cholesterol homeostasis, bile acids can also act as signaling molecules with systemic endocrine functions.

In the present study we investigated the biological effects of the primary bile chenodeoxycholic acid (CDCA) on a human endometrial cancer cell line, Ishikawa. Low concentrations of CDCA are able to stimulate Ishikawa cell growth by inducing a significant increase in Cyclin D1 protein and mRNA expression through the activation of the membrane G protein-coupled receptor (TGR5)-dependent pathway. Dissecting the molecular mechanism underlying this effect by mutagenesis, EMSA and ChIP analysis revealed that CDCA-induced Cyclin D1 expression requires the enhanced recruitment of the transcription factor CREB on the cyclic AMP-responsive element motif within the Cyclin D1 gene proximal promoter.

Our results suggest a novel molecular mechanism explaining the potential contribution of high-fat diet and obesity to endometrial cancer growth and progression opening the rationale for strategies to prevent the risk of this obesity-related cancer in women.

The conserved C terminus of Claspin interacts with Rad9 and promotes rapid activation of Chk1

Shizhou Liu, Na Song and Lee Zou
Pages 2711 - 2716
http://dx.doi.org/10.4161/cc.21041
Abstract | Full Text | PDF

Claspin is a key mediator of the ATR-Chk1 checkpoint pathway. In response to DNA damage, Claspin interacts with Rad17 and Chk1 in a phosphorylation-dependent manner, enabling ATR to phosphorylate Chk1 efficiently. Claspin also interacts with Rad9, but how they interact and whether the interaction is functional remains unknown. Unexpectedly, our analysis of two splicing isoforms of Claspin provided an answer to these questions. The Claspin¹³³⁹ isoform contains an evolutionarily conserved C terminus, but the Claspin¹³³² isoform does not. Although the transcripts encoding both Claspin isoforms coexist in HCT116 cells, Claspin¹³³⁹ is the predominant form responsible for Chk1 activation. When expressed in cells depleted of endogenous Claspin, both Claspin¹³³⁹ and Claspin¹³³² are able to mediate Chk1 activation. However, the activation of Chk1 is delayed in Claspin¹³³²-expressing cells compared with Claspin¹³³⁹-expressing cells. Furthermore, only Claspin¹³³⁹ but not Claspin¹³³² interacts with Rad9 efficiently. Together, these results suggest that the conserved C terminus of Claspin interacts with Rad9 and ensures timely activation of the ATR-Chk1 pathway.

PIASy-mediated Tip60 sumoylation regulates p53-induced autophagy

Samisubbu R. Naidu, Alexander J. Lakhter and Elliot J. Androphy
Pages 2717 - 2728
http://dx.doi.org/10.4161/cc.21091
Abstract | Full Text | PDF

Posttranslational modifications of p53 integrate diverse stress signals and regulate its activity, but their combinatorial contribution to overall p53 function is not clear. We investigated the roles of lysine (K) acetylation and sumoylation on p53 and their relation to apoptosis and autophagy. Here we describe the collaborative role of the SUMO E3 ligase PIASy and the lysine acetyltransferase Tip60 in p53-mediated autophagy. PIASy binding to p53 and PIASy-activated Tip60 lead to K386 sumoylation and K120 acetylation of p53, respectively. Even though these two modifications are not dependent on each other, together they act as a “binary death signal” to promote cytoplasmic accumulation of p53 and execution of PUMA-independent autophagy. PIASy-induced Tip60 sumoylation augments p53 K120 acetylation and apoptosis. In addition to p14^ARF inactivation, impairment in this intricate signaling may explain why p53 mutations are not found in nearly 50% of malignancies.

Coordinated effects of microRNA-494 induce G₂/M arrest in human cholangiocarcinoma

Sumitaka Yamanaka, Nathaniel R. Campbell, Fangmei An, Scot C. Kuo, James J. Potter, Esteban Mezey, Anirban Maitra and Florin M. Selaru
Pages 2729 - 2738
http://dx.doi.org/10.4161/cc.21105
Abstract | Full Text | PDF

MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G₂) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G₂/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G₂/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G₁) to synthesis (S), as described previously, as well as G₂ to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.

C/EBPα bypasses cell cycle-dependency during immune cell transdifferentiation

Alessandro Di Tullio and Thomas Graf
Pages 2739 - 2746
http://dx.doi.org/10.4161/cc.21119
Abstract | Full Text | PDF

Our earlier work has shown that pre-B cells can be converted into macrophage-like cells by overexpression of the transcription factor C/EBPα or C/EBPβ with high efficiency. Using inducible pre-B cell lines, we have now investigated the role of cell division during C/EBP-induced reprogramming. The majority of cells reprogrammed by C/EBPα incorporated BrdU before arresting at G₀, and all C/EBPβ-induced cells incorporated the compound. This contrasts with reports from other systems where transdifferentiating cells essentially do not divide. Although inhibition of DNA synthesis led to an impairment of C/EBPα-induced transdifferentiation, sorted G₀/G₁ and G₂/M fractions showed no significant differences in their reprogramming kinetics. In addition, knocking-down p53 did not accelerate the transdifferentiation frequency, as it has been described for reprogramming of induced pluripotent (iPS) cells. Time-lapse experiments showed that, after C/EBPα induction, approximately 90% of cells divide once or twice, while 8% do not divide at all before acquiring a macrophage phenotype, supporting our BrdU incorporation results. Importantly, the non-dividing cell subset expressed the highest levels of C/EBPα and was the fastest in differentiating, suggesting that high levels of C/EBPα accelerate both the switching process and the cells’ growth arrest. Our data show that traversing the cell cycle is not strictly required for pre-B cell to macrophage conversion and provides new evidence for the notion that the mechanisms of transcription factor induced transdifferentiation and iPS cell reprogramming differ.

CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy

A. Kathleen McClendon, Jeffry L. Dean, Dayana B. Rivadeneira, Justine E. Yu, Christopher A. Reed, Erhe Gao, John L. Farber, Thomas Force, Walter J. Koch and Erik S. Knudsen
Pages 2747 - 2755
http://dx.doi.org/10.4161/cc.21127
Abstract | Full Text | PDF

Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.

Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors

Jeffry L. Dean, A. Kathleen McClendon, Theresa E. Hickey, Lisa M. Butler, Wayne D. Tilley, Agnieszka K. Witkiewicz and Erik S. Knudsen
Pages 2756 - 2761
http://dx.doi.org/10.4161/cc.21195
Abstract | Full Text | PDF

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.