Publications by authors named "Harold I Saavedra"

26 Publications

  • Page 1 of 1

Mitotic kinases as drivers of the epithelial-to-mesenchymal transition and as therapeutic targets against breast cancers.

Exp Biol Med (Maywood) 2021 Feb 18:1535370221991094. Epub 2021 Feb 18.

Department of Basic Sciences, Division of Pharmacology and Cancer Biology, 6650Ponce Health Sciences University/Ponce Research Institute, Ponce, PR 00732, USA.

Biological therapies against breast cancer patients with tumors positive for the estrogen and progesterone hormone receptors and Her2 amplification have greatly improved their survival. However, to date, there are no effective biological therapies against breast cancers that lack these three receptors or triple-negative breast cancers (TNBC). TNBC correlates with poor survival, in part because they relapse following chemo- and radio-therapies. TNBC is intrinsically aggressive since they have high mitotic indexes and tend to metastasize to the central nervous system. TNBCs are more likely to display centrosome amplification, an abnormal phenotype that results in defective mitotic spindles and abnormal cytokinesis, which culminate in aneuploidy and chromosome instability (known causes of tumor initiation and chemo-resistance). Besides their known role in cell cycle control, mitotic kinases have been also studied in different types of cancer including breast, especially in the context of epithelial-to-mesenchymal transition (EMT). EMT is a cellular process characterized by the loss of cell polarity, reorganization of the cytoskeleton, and signaling reprogramming (upregulation of mesenchymal genes and downregulation of epithelial genes). Previously, we and others have shown the effects of mitotic kinases like Nek2 and Mps1 (TTK) on EMT. In this review, we focus on Aurora A, Aurora B, Bub1, and highly expressed in cancer (Hec1) as novel targets for therapeutic interventions in breast cancer and their effects on EMT. We highlight the established relationships and interactions of these and other mitotic kinases, clinical trial studies involving mitotic kinases, and the importance that represents to develop drugs against these proteins as potential targets in the primary care therapy for TNBC.
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http://dx.doi.org/10.1177/1535370221991094DOI Listing
February 2021

Tank Binding Kinase 1 modulates spindle assembly checkpoint components to regulate mitosis in breast and lung cancer cells.

Biochim Biophys Acta Mol Cell Res 2021 Mar 11;1868(3):118929. Epub 2020 Dec 11.

Department of Tumor Biology, H, Lee Moffitt Cancer Center and Research Institute, 12902 USF Magnolia Drive, Tampa, FL 33612, United States of America. Electronic address:

Error-free progression through mitosis is critical for proper cell division and accurate distribution of the genetic material. The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase regulates the progression from metaphase to anaphase and its activation is controlled by the cofactors Cdc20 and Cdh1. Additionally, genome stability is maintained by the spindle assembly checkpoint (SAC), which monitors proper attachment of chromosomes to spindle microtubules prior to cell division. We had shown a role for Tank Binding Kinase 1 (TBK1) in microtubule dynamics and mitosis and here we describe a novel role of TBK1 in regulating SAC in breast and lung cancer cells. TBK1 interacts with and phosphorylates Cdc20 and Cdh1 and depletion of TBK1 elevates SAC components. TBK1 inhibition increases the association of Cdc20 with APC/C and BubR1 indicating inactivation of APC/C; similarly, interaction of Cdh1 with APC/C is also enhanced. TBK1 and TTK inhibition reduces cell viability and enhances centrosome amplification and micronucleation. These results indicate that alterations in TBK1 will impede mitotic progression and combining TBK1 inhibitors with other regulators of mitosis might be effective in eliminating cancer cells.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118929DOI Listing
March 2021

Correction for Adon et al., "Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells".

Mol Cell Biol 2020 Nov 6;40(23). Epub 2020 Nov 6.

Department of Radiation Oncology, Emory University School of Medicine, 1365C Clifton Rd., Room C3084, Atlanta, Georgia 30322

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http://dx.doi.org/10.1128/MCB.00488-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652402PMC
November 2020

Role of E2Fs and mitotic regulators controlled by E2Fs in the epithelial to mesenchymal transition.

Exp Biol Med (Maywood) 2019 11 1;244(16):1419-1429. Epub 2019 Oct 1.

Basic Sciences Department, Division of Pharmacology and Toxicology, Ponce Research Institute, Ponce Health Sciences University, Ponce PR 00732, USA.

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http://dx.doi.org/10.1177/1535370219881360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900705PMC
November 2019

Centrosome aberrations and chromosome instability contribute to tumorigenesis and intra-tumor heterogeneity.

J Cancer Metastasis Treat 2018 7;4. Epub 2018 Aug 7.

Basic Sciences Department, Division of Pharmacology and Toxicology, Ponce Health Sciences University, Ponce Research Institute, Ponce, PR 00732, USA.

Centrosomes serve as the major microtubule organizing centers in cells and thereby contribute to cell shape, polarity, and motility. Also, centrosomes ensure equal chromosome segregation during mitosis. Centrosome aberrations arise when the centrosome cycle is deregulated, or as a result of cytokinesis failure. A long-standing postulate is that centrosome aberrations are involved in the initiation and progression of cancer. However, this notion has been a subject of controversy because until recently the relationship has been correlative. Recently, it was shown that numerical or structural centrosome aberrations can initiate tumors in certain tissues in mice, as well as invasion. Particularly, we will focus on centrosome amplification and chromosome instability as drivers of intra-tumor heterogeneity and their consequences in cancer. We will also discuss briefly the controversies surrounding this theory to highlight the fact that the role of both centrosome amplification and chromosome instability in cancer is highly context-dependent. Further, we will discuss single-cell sequencing as a novel technique to understand intra-tumor heterogeneity and some therapeutic approaches to target chromosome instability.
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http://dx.doi.org/10.20517/2394-4722.2018.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205736PMC
August 2018

TTK promotes mesenchymal signaling via multiple mechanisms in triple negative breast cancer.

Oncogenesis 2018 Sep 12;7(9):69. Epub 2018 Sep 12.

Winship Cancer Institute, Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA, USA.

Abnormal expression of TTK kinase has been associated with the initiation, progression, and therapeutic resistance of breast and other cancers, but its roles remain to be clarified. In this study, we examined the role of TTK in triple negative breast cancer (TNBC), and found that higher TTK expression correlated with mesenchymal and proliferative phenotypes in TNBC cells. Pharmacologic inhibition and genomic silencing of TTK not only reversed the epithelial-to-mesenchymal transition (EMT) in TNBC cells, but also increased the expression of KLF5, an effector of TGF-β signaling and inhibitor of EMT. In addition, TTK inhibition decreased the expression of EMT-associated micro-RNA miR-21 but increased the expression of miR-200 family members and suppressed TGF-β signaling. To test if upregulation of KLF5 plays a role in TTK-induced EMT, TTK and KLF5 were silenced simultaneously, which reversed the decreased EMT caused by loss of TTK. Consistently, the decrease in miR-21 expression and increase in miR-200 expression caused by TTK silencing were rescued by loss of KLF5. Altogether, this study highlights a novel role and signaling pathway for TTK in regulating EMT of TN breast cancer cells through TGF-β and KLF5 signaling, highlighting targetable signaling pathways for TTK inhibitors in aggressive breast cancer.
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http://dx.doi.org/10.1038/s41389-018-0077-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133923PMC
September 2018

Detection of E2F-DNA Complexes Using Chromatin Immunoprecipitation Assays.

Methods Mol Biol 2018 ;1726:143-151

Pharmacology Division, Department of Basic Sciences, Ponce Health Sciences University, Ponce, Puerto Rico.

Chromatin immunoprecipitation (ChIP), originally developed by John T. Lis and David Gilmour in 1984, has been useful to detect DNA sequences where protein(s) of interest bind. ChIP is comprised of several steps: (1) cross-linking of proteins to target DNA sequences, (2) breaking genomic DNA into 300-1000 bp pieces by sonication or nuclease digestion, (3) immunoprecipitation of protein bound to target DNA with an antibody, (4) reverse cross-linking between target DNA and the bound protein to liberate the DNA fragments, and (5) amplification of target DNA fragment by PCR. Since then, the technology has evolved significantly to allow not only amplifying target sequences by PCR, but also sequencing all DNA fragment bound to a target protein, using a variant of the approach called the ChIP-seq technique (1). Another variation, the ChIP-on-ChIP, allows the detection of protein complexes bound to specific DNA sequences (2).
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http://dx.doi.org/10.1007/978-1-4939-7565-5_13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070307PMC
January 2019

The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1.

Oncotarget 2017 Sep 8;8(44):77649-77672. Epub 2017 Sep 8.

Department of Basic Sciences, Program of Pharmacology, Ponce Health Sciences University-School of Medicine/Ponce Research Institute, Ponce, 00716-2348 Puerto Rico.

The E2F1, E2F2, and E2F3a transcriptional activators control proliferation. However, how the E2F activators regulate mitosis to maintain genomic integrity is unclear. Centrosome amplification (CA) and unregulated spindle assembly checkpoint (SAC) are major generators of aneuploidy and chromosome instability (CIN) in cancer. Previously, we showed that overexpression of single E2F activators induced CA and CIN in mammary epithelial cells, and here we show that combined overexpression of E2F activators did not enhance CA. Instead, the E2F activators elevated expression of multiple mitotic regulators, including Sgo1, Nek2, Hec1, BubR1, and Mps1/TTK. cBioPortal analyses of the TCGA database showed that E2F overexpression in lobular invasive breast tumors correlates with overexpression of multiple regulators of chromosome segregation, centrosome homeostasis, and the SAC. Kaplan-Meier plots identified correlations between individual or combined overexpression of E2F1, E2F3a, Mps1/TTK, Nek2, BubR1, or Hec1 and poor overall and relapse-free survival of breast cancer patients. In MCF10A normal mammary epithelial cells co-overexpressing E2Fs, transient Sgo1 knockdown induced CA, high percentages of premature sister chromatid separation, chromosome losses, increased apoptosis, and decreased cell clonogenicity. BubR1 silencing resulted in chromosome losses without CA, demonstrating that Sgo1 and BubR1 maintain genomic integrity through two distinct mechanisms. Our results suggest that deregulated activation of the E2Fs in mammary epithelial cells is counteracted by activation of a Sgo1-dependent mitotic checkpoint.
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http://dx.doi.org/10.18632/oncotarget.20765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652806PMC
September 2017

Centrosome - a promising anti-cancer target.

Biologics 2016 13;10:167-176. Epub 2016 Dec 13.

Department of Pharmacology, Ponce Health Sciences University-School of Medicine, Ponce Research Institute, Ponce, Puerto Rico.

The centrosome, an organelle discovered >100 years ago, is the main microtubule-organizing center in mammalian organisms. The centrosome is composed of a pair of centrioles surrounded by the pericentriolar material (PMC) and plays a major role in the regulation of cell cycle transitions (G1-S, G2-M, and metaphase-anaphase), ensuring the normality of cell division. Hundreds of proteins found in the centrosome exert a variety of roles, including microtubule dynamics, nucleation, and kinetochore-microtubule attachments that allow correct chromosome alignment and segregation. Errors in these processes lead to structural (shape, size, number, position, and composition), functional (abnormal microtubule nucleation and disorganized spindles), and numerical (centrosome amplification [CA]) centrosome aberrations causing aneuploidy and genomic instability. Compelling data demonstrate that centrosomes are implicated in cancer, because there are important oncogenic and tumor suppressor proteins that are localized in this organelle and drive centrosome aberrations. Centrosome defects have been found in pre-neoplasias and tumors from breast, ovaries, prostate, head and neck, lung, liver, and bladder among many others. Several drugs/compounds against centrosomal proteins have shown promising results. Other drugs have higher toxicity with modest or no benefits, and there are more recently developed agents being tested in clinical trials. All of this emerging evidence suggests that targeting centrosome aberrations may be a future avenue for therapeutic intervention in cancer research.
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http://dx.doi.org/10.2147/BTT.S87396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167523PMC
December 2016

Silencing of E2F3 suppresses tumor growth of Her2+ breast cancer cells by restricting mitosis.

Oncotarget 2015 Nov;6(35):37316-34

Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA.

The E2F transcriptional activators E2F1, E2F2 and E2F3a regulate many important cellular processes, including DNA replication, apoptosis and centrosome duplication. Previously, we demonstrated that silencing E2F1 or E2F3 suppresses centrosome amplification (CA) and chromosome instability (CIN) in Her2+ breast cancer cells without markedly altering proliferation. However, it is unknown whether and how silencing a single E2F activator, E2F3, affects malignancy of human breast cancer cells. Thus, we injected HCC1954 Her2+ breast cancer cells silenced for E2F3 into mammary fat pads of immunodeficient mice and demonstrated that loss of E2F3 retards tumor growth. Surprisingly, silencing of E2F3 led to significant reductions in mitotic indices relative to vector controls, while the percentage of cells undergoing S phase were not affected. Nek2 is a mitotic kinase commonly upregulated in breast cancers and a critical regulator of Cdk4- or E2F-mediated CA. In this report, we found that Nek2 overexpression rescued back the CA caused by silencing of shE2F3. However, the effects of Nek2 overexpression in affecting tumor growth rates of shE2F3 and shE2F3; GFP cells were inconclusive. Taken together, our results indicate that E2F3 silencing decreases mammary tumor growth by reducing percentage of cells undergoing mitosis.
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http://dx.doi.org/10.18632/oncotarget.5686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741932PMC
November 2015

Chromosome instability in diffuse large B cell lymphomas is suppressed by activation of the noncanonical NF-κB pathway.

Int J Cancer 2015 May 11;136(10):2341-51. Epub 2014 Nov 11.

Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA.

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center-like (GCB) and activated-B-cell-like DLBCL (ABC). The most aggressive type, ABC-DLBCL, displays dysregulation of both canonical and noncanonical NF-κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF-kB pathway, the precise role of the noncanonical NF-kB pathway remains unknown. Here we show that activation of the noncanonical NF-κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF-κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin-induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF-κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53-independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF-κB sites to their promoter region. Overall, these results indicate that the noncanonical NF-κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF-kB pathway should be considered as an important component in DLBCL therapeutic approach.
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http://dx.doi.org/10.1002/ijc.29301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880031PMC
May 2015

Differential expression of centrosome regulators in Her2+ breast cancer cells versus non-tumorigenic MCF10A cells.

Cell Div 2014 25;9. Epub 2014 Sep 25.

Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, C3084, 1365C Clifton Road NE, Atlanta, GA 30322, USA.

Centrosome amplification (CA) amongst particular breast cancer subtypes (Her2+ subtype) is associated with genomic instability and aggressive tumor phenotypes. However, changes in signaling pathways associated with centrosome biology have not been fully explored in subtype specific models. Novel centrosome regulatory genes that are selectively altered in Her2+ breast cancer cells are of interest in discerning why CA is more prevalent in this subtype. To determine centrosome/cell cycle genes that are altered in Her2+ cells that display CA (HCC1954) versus non-tumorigenic cells (MCF10A), we carried out a gene microarray. Expression differences were validated by real-time PCR and Western blotting. After the microarray validation, we pursued a panel of upregulated and downregulated genes based on novelty/relevance to centrosome duplication. Functional experiments measuring CA and BrdU incorporation were completed after genetic manipulation of targets (TTK, SGOL1, MDM2 and SFRP1). Amongst genes that were downregulated in HCC1954 cells, knockdown of MDM2 and SFRP1 in MCF10A cells did not consistently induce CA or impaired BrdU incorporation. Conversely, amongst upregulated genes in HCC1954 cells, knockdown of SGOL1 and TTK decreased CA in breast cancer cells, while BrdU incorporation was only altered by SGOL1 knockdown. We also explored the Kaplan Meier Plot resource and noted that MDM2 and SFRP1 are positively associated with relapse free survival in all breast cancer subtypes, while TTK is negatively correlated with overall survival of Luminal A patients. Based on this functional screen, we conclude that SGOL1 and TTK are important modulators of centrosome function in a breast cancer specific model.
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http://dx.doi.org/10.1186/1747-1028-9-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181616PMC
October 2014

Poldip2 knockout results in perinatal lethality, reduced cellular growth and increased autophagy of mouse embryonic fibroblasts.

PLoS One 2014 5;9(5):e96657. Epub 2014 May 5.

Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication/repair and ROS production via Nox4. In this study, we have identified a novel role for Poldip2 in regulating the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2-/- embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2-/- mouse embryonic fibroblasts (MEFs) exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to apoptosis or senescence; however, Poldip2-/- MEFs have higher levels of the autophagy marker LC3b. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2-/- cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Increases in p53 S20 and Sirt1 were observed in passage 2 Poldip2-/- MEFs. In passage 4/5 MEFs, Cdk1 and CyclinA2 are downregulated in Poldip2-/- cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21CIP1 is increased in passage 4/5 Poldip2-/- MEFs and its expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative conditions such as cancer, atherosclerosis and restenosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096657PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010529PMC
January 2015

E2F activators signal and maintain centrosome amplification in breast cancer cells.

Mol Cell Biol 2014 Jul;34(14):2581-99

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2(+) cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2(+) cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2(+) breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2.
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http://dx.doi.org/10.1128/MCB.01688-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097658PMC
July 2014

Nek2 and Plk4: prognostic markers, drivers of breast tumorigenesis and drug resistance.

Front Biosci (Landmark Ed) 2014 Jan 1;19:352-65. Epub 2014 Jan 1.

Department of Radiation Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, 30322.

The Nek2 and Plk4 kinases serve as crucial regulators of mitotic processes such as the centrosome duplication cycle and spindle assembly. Deregulation of these processes can trigger chromosome instability and aneuploidy, which are hallmarks of many solid tumors, including breast cancer. Emerging data from the literature illustrated various functions of Nek2 in breast cancer models, with compelling evidence of its prognostic value in breast tumors. The two kinases control distinct steps in the centrosome-centriole cycle and their dysregulation lead to centrosome amplification, marked by the presence of more than two centrosomes within the cell. We found single or composite overexpression of these kinases in breast tumor samples, regardless of subtype, which strongly associated with poor prognosis. Interestingly, in a panel of established cell lines, both kinases are highly expressed in Her2-positive breast cancer cells exhibiting centrosome amplification and trastuzumab resistance. In summary, it appears that Nek2 and Plk4 might synergize to promote breast tumorigenesis and may also be involved in tamoxifen and trastuzumab resistance.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999971PMC
http://dx.doi.org/10.2741/4212DOI Listing
January 2014

Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis.

Cell Div 2013 Jul 25;8(1):10. Epub 2013 Jul 25.

Department of Radiation Oncology, Emory University School of Medicine, Atlanta, USA.

Background: The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery.

Methods: To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks.

Results: Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis.

Conclusion: Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.
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http://dx.doi.org/10.1186/1747-1028-8-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733890PMC
July 2013

Cdk4 and nek2 signal binucleation and centrosome amplification in a her2+ breast cancer model.

PLoS One 2013 11;8(6):e65971. Epub 2013 Jun 11.

Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

Centrosome amplification (CA) is a contributor to carcinogenesis, generating aneuploidy, and chromosome instability. Previous work shows that breast adenocarcinomas have a higher frequency of centrosome defects compared to normal breast tissues. Abnormal centrosome phenotypes are found in pre-malignant lesions, suggesting an early role in breast carcinogenesis. However, the role of CA in breast cancers remains elusive. Identification of pathways and regulatory molecules involved in the generation of CA is essential to understanding its role in breast tumorigenesis. We established a breast cancer model of CA using Her2-positive cells. Our goal was to identify centrosome cycle molecules that are deregulated by aberrant Her2 signaling and the mechanisms driving CA. Our results show some Her2+ breast cancer cell lines harbor both CA and binucleation. Abolishing the expression of Cdk4 abrogated both CA and binucleation in these cells. We also found the source of binucleation in these cells to be defective cytokinesis that is normalized by downregulation of Cdk4. Protein levels of Nek2 diminish upon Cdk4 knockdown and vice versa, suggesting a molecular connection between Cdk4 and Nek2. Knockdown of Nek2 reduces CA and binucleation in this model while its overexpression further enhances centrosome amplification. We conclude that CA is modulated through Cdk4 and Nek2 signaling and that binucleation is a likely source of CA in Her2+ breast cancer cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065971PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679029PMC
January 2014

The G1 phase Cdks regulate the centrosome cycle and mediate oncogene-dependent centrosome amplification.

Cell Div 2011 Jan 27;6. Epub 2011 Jan 27.

Emory University, Department of Radiation Oncology, Winship Cancer Institute, 1701 Uppergate Drive, Atlanta, Georgia, 30322, USA.

Because centrosome amplification generates aneuploidy and since centrosome amplification is ubiquitous in human tumors, a strong case is made for centrosome amplification being a major force in tumor biogenesis. Various evidence showing that oncogenes and altered tumor suppressors lead to centrosome amplification and aneuploidy suggests that oncogenes and altered tumor suppressors are a major source of genomic instability in tumors, and that they generate those abnormal processes to initiate and sustain tumorigenesis. We discuss how altered tumor suppressors and oncogenes utilize the cell cycle regulatory machinery to signal centrosome amplification and aneuploidy.
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http://dx.doi.org/10.1186/1747-1028-6-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038874PMC
January 2011

Cdk2 and Cdk4 regulate the centrosome cycle and are critical mediators of centrosome amplification in p53-null cells.

Mol Cell Biol 2010 Feb 23;30(3):694-710. Epub 2009 Nov 23.

Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA.

The two mitotic centrosomes direct spindle bipolarity to maintain euploidy. Centrosome amplification-the acquisition of > or =3 centrosomes-generates multipolar mitoses, aneuploidy, and chromosome instability to promote cancer biogenesis. While much evidence suggests that Cdk2 is the major conductor of the centrosome cycle and that it mediates centrosome amplification induced by various altered tumor suppressors, the role played by Cdk4 in a normal or deregulated centrosome cycle is unknown. Using a gene knockout approach, we report that Cdk2 and Cdk4 are critical to the centrosome cycle, since centrosome separation and duplication are premature in Cdk2(-)(/)(-) mouse embryonic fibroblasts (MEFs) and are compromised in Cdk4(-)(/)(-) MEFs. Additionally, ablation of Cdk4 or Cdk2 abrogates centrosome amplification and chromosome instability in p53-null MEFs. Absence of Cdk2 or Cdk4 prevents centrosome amplification by abrogating excessive centriole duplication. Furthermore, hyperactive Cdk2 and Cdk4 deregulate the licensing of the centrosome duplication cycle in p53-null cells by hyperphosphorylating nucleophosmin (NPM) at Thr199, as evidenced by observations that ablation of Cdk2, Cdk4, or both Cdk2 and Cdk4 abrogates that excessive phosphorylation. Since a mutant form of NPM lacking the G(1) Cdk phosphorylation site (NPM(T199A)) prevents centrosome amplification to the same extent as ablation of Cdk2 or Cdk4, we conclude that the Cdk2/Cdk4/NPM pathway is a major guardian of centrosome dysfunction and genomic integrity.
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http://dx.doi.org/10.1128/MCB.00253-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812235PMC
February 2010

E2f1, E2f2, and E2f3 control E2F target expression and cellular proliferation via a p53-dependent negative feedback loop.

Mol Cell Biol 2007 Jan;27(1):65-78

Human Cancer Genetics program, Department of Molecular Virology, Immunology and Medical Genetics, and Comprehensive Cancer Center, The Ohio State University, 410 W. 12th Avenue, Columbus, OH 43210, USA.

E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21(CIP1). Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21(CIP1) and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.
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http://dx.doi.org/10.1128/MCB.02147-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1800646PMC
January 2007

Control of the p53-p21CIP1 Axis by E2f1, E2f2, and E2f3 is essential for G1/S progression and cellular transformation.

J Biol Chem 2006 Nov 27;281(47):36124-31. Epub 2006 Sep 27.

Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.

The E2F family of transcription factors is believed to have an essential role in the control of cellular proliferation by regulating the transcription of genes involved in cell cycle progression. Previous work has demonstrated that the targeted inactivation of E2f1, E2f2, and E2f3 results in elevated p21(CIP1) protein levels, loss of E2F target gene expression, and cell cycle arrest at G1/S and G2/M, suggesting a strict requirement for these E2Fs in the control of normal cellular proliferation. We now demonstrate that E2f1, E2f2, and E2f3 are also required for oncogene-mediated transformation of mouse embryonic fibroblasts. Analysis of synchronized populations of mouse embryonic fibroblasts revealed that the inactivation of p21(CIP1) restores the ability of E2f1-3-deficient cells to enter and transit through G1/S (but not G2/M). In contrast, loss of p53 restored the ability of these cells to progress through both G1/S and mitosis, leading to their continued proliferation. The inactivation of p53 (but not p21(CIP1)) rendered E2f1-3-deficient cells sensitive to transformation and tumorigenesis. These results suggest that the negative regulation of the p53-p21(CIP1) axis by the E2F1-3 factors is critical for cell cycle progression and cellular transformation.
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http://dx.doi.org/10.1074/jbc.M604152200DOI Listing
November 2006

Aberrant regulation of survivin by the RB/E2F family of proteins.

J Biol Chem 2004 Sep 22;279(39):40511-20. Epub 2004 Jul 22.

Columbus Children's Research Institute, College of Medicine and Public Health, Ohio State University, 43210, USA.

Survivin is a putative oncogene that is aberrantly expressed in cancer cells. It has been hypothesized to play a central role in cancer progression and resistance to therapy in diverse tumor types. Although some of the transcriptional processes regulating its expression have been established, the diversity of genes that may be controlling the levels of its expression in both normal cells as well as in cancer cells has not been fully explored. The most common genetically mutated pathways in human malignancies are the p53 tumor suppressor pathway and the RB/E2F pathway. Both of these pathways, when intact, provide essential checkpoints in the maintenance of normal cell growth and protect the cell from DNA damage. Using non-transformed embryonic fibroblasts, we provide evidence of a molecular link between the regulation of survivin transcription and the RB/E2F family of proteins. We demonstrate that both pRB and p130 can interact with the survivin promoter and can repress survivin transcription. We also show that the E2F activators (E2F1, E2F2, and E2F3) can bind to the survivin promoter and induce survivin transcription. Genetically modified cells that harbor deletions in various members of the RB/E2F family confirm our data from the wild-type cells. Our findings implicate several members of the RB/E2F pathway in an intricate mechanism of survivin gene regulation that, when genetically altered during the process of tumorigenesis, may function within cancer cells to aberrantly alter survivin levels and enhance tumor progression.
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http://dx.doi.org/10.1074/jbc.M404496200DOI Listing
September 2004

Rb function in extraembryonic lineages suppresses apoptosis in the CNS of Rb-deficient mice.

Proc Natl Acad Sci U S A 2003 May 5;100(11):6546-51. Epub 2003 May 5.

Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus 43210, USA.

Retinoblastoma (Rb)-deficient embryos show severe defects in neurogenesis, erythropoiesis, and lens development and die at embryonic day 14.5. Our recent results demonstrated a drastic disorganization of the labyrinth layer in the placenta of Rb-deficient embryos, accompanied by reduced placental transport function. When these Rb-/- embryos were supplied with a wild-type placenta by using either tetraploid aggregation or genetic approaches, animals survived until birth. Here we analyze the role of extraembryonic Rb in regulating proliferation, apoptosis, and differentiation in the rescued animals at different developmental stages. Many of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-/- animals, including the ectopic apoptosis in the CNS, were virtually absent in rescued Rb-/- pups. However, rescued animals died at birth with severe skeletal muscle defects. Like in Rb knockout embryos, rescued animals showed a marked increase in DNA replication and cell division in the CNS. In sharp contrast, the typical widespread neuronal apoptosis was absent in Rb-deficient embryos reconstituted with a normal placenta. In lens fiber cells, however, the inappropriate proliferation and apoptosis that is normally observed in Rb-/- embryos continued unabated in rescued animals. These results demonstrate that Rb function in extraembryonic lineages plays an important role in the survival of neuronal cells and in the differentiation of the erythroid lineage, providing mechanistic insight into the cell autonomous and nonautonomous functions of Rb during development.
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http://dx.doi.org/10.1073/pnas.1031853100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC164483PMC
May 2003

Inactivation of E2F3 results in centrosome amplification.

Cancer Cell 2003 Apr;3(4):333-46

Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, Children's Research Institute, The Ohio State University, Columbus, OH 43210, USA.

The E2F family of transcription factors is critical for the control of cell cycle progression. We now show that the specific inactivation of E2F3 in mouse embryo fibroblasts (MEFs) results in a disruption of the centrosome duplication cycle. Loss of E2F3, but not E2F1, E2F2, E2F4, or E2F5 results in unregulated cyclin E-dependent kinase activity, defects in nucleophosmin B association with centrosomes, and premature centriole separation and duplication. Consequently, this defect leads to centrosome amplification, mitotic spindle defects, and aneuploidy. Our findings implicate the E2F3 transcription factor as an important link that orchestrates DNA and centrosome duplication cycles, ensuring the faithful transmission of genetic material to daughter cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033013PMC
http://dx.doi.org/10.1016/s1535-6108(03)00083-7DOI Listing
April 2003

Extra-embryonic function of Rb is essential for embryonic development and viability.

Nature 2003 Feb;421(6926):942-7

Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, Ohio 43210, USA.

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5 (refs 2-4). However, the actual cause of the embryonic lethality has not been fully investigated. Here we show that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. We used two complementary techniques-tetraploid aggregation and conditional knockout strategies-to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals were virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development.
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http://dx.doi.org/10.1038/nature01417DOI Listing
February 2003

Specificity of E2F1, E2F2, and E2F3 in mediating phenotypes induced by loss of Rb.

Cell Growth Differ 2002 May;13(5):215-25

Human Cancer Genetics Program, Department of Molecular Virology, Immunology and Medical Genetics and Department of Molecular Genetics, The Ohio State University, Columbus 43210, USA.

The Rb/E2F pathway plays a critical role in the control ofcellular proliferation. Here, we report that E2F1, E2F2, and E2F3 make major individual contributions toward the in vivo phenotypic consequences of Rb deficiency. In the developing lens of Rb(-/-) embryos, loss of E2F1, E2F2, or E2F3 reduces the unscheduled proliferation of fiber cells, with the loss of E2F3 having the most pronounced effect. In Rb-deficient retinas, all three E2Fs contribute equally to the ectopic proliferation of postmitotic neuronal cells. In contrast, E2F1 is unique in mediating apoptosis in both Rb(-/-) lenses and retinas. In the central nervous system, loss of E2F1 or E2F3 can almost completely eliminate the ectopic DNA replication and apoptosis observed in Rb(-/-) embryos, and loss of E2F2 partially reduces the unscheduled DNA replication and has no effect on apoptosis. These results provide clear evidence for functional specificity among E2Fs in the control of Rb-dependent proliferation and apoptosis in a tissue-specific manner.
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May 2002