Paraskevi Giannakakou

Cornell University, Итак, New York, United States

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Publications (120)893.79 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Background Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons.Objectives We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype β-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia.Patients/Methods Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H β-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of β-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP β-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization.ResultsWe found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP and 76 in SNP patients (both x 109/L; p<0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of β tubulin; in vitro microtubule recovery assays revealed cells transfected with R307H SNP β-1 had significantly impaired microtubule recovery.Conclusions Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior.This article is protected by copyright. All rights reserved.
    Journal of Thrombosis and Haemostasis 12/2014; 13(4). DOI:10.1111/jth.12824 · 5.55 Impact Factor
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    ABSTRACT: Taxanes are the only chemotherapies used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study, we show that ERG overexpression in in vitro and in vivo models of CRPC is associated with decreased sensitivity to taxanes. ERG affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of docetaxel or cabazitaxel with tubulin. Finally, analysis of a cohort of 34 men with metastatic CRPC treated with docetaxel chemotherapy reveals that ERG-overexpressing prostate cancers have twice the chance of docetaxel resistance than ERG-negative cancers. Our data suggest that ERG plays a role beyond regulating gene expression and functions outside the nucleus to cooperate with tubulin towards taxane insensitivity. Determining ERG rearrangement status may aid in patient selection for docetaxel or cabazitaxel therapy and/or influence co-targeting approaches.
    Nature Communications 11/2014; 5:5548. DOI:10.1038/ncomms6548 · 10.74 Impact Factor
  • Tito Fojo, Paraskevi Giannakakou
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    ABSTRACT: Over the past decade, funding for cancer research by the US government and others has stagnated, while the demand for investment has grown because of the increasing cancer incidence worldwide. We discuss how National Cancer Institute funding efforts have developed during this period, and the contemporary and future impact of these measures on cancer research in the USA.
    Nature Reviews Clinical Oncology 10/2014; 11(11). DOI:10.1038/nrclinonc.2014.173 · 15.70 Impact Factor
  • Prashant K Khade, Paraskevi Giannakakou
    Proceedings of the National Academy of Sciences 07/2014; 111(31). DOI:10.1073/pnas.1410788111 · 9.81 Impact Factor
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    ABSTRACT: Nicotinamide adenine dinucleotide (NAD(+)) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD(+)-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD(+). Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD(+) levels. We find that these effects of NAD(+) are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD(+) on microtubule polymers. Taken together, these data demonstrate that NAD(+) and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.
    Proceedings of the National Academy of Sciences 06/2014; 111(24):E2443-52. DOI:10.1073/pnas.1404269111 · 9.81 Impact Factor
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    ABSTRACT: Circulating tumor cells (CTCs) have emerged as a viable solution to the lack of tumor tissue availability for patients with a variety of solid tumors, including prostate cancer. Different approaches have been used to capture this tumor cell population and several of these techniques have been used to assess the potential role of CTCs as a biological marker to predict treatment efficacy and clinical outcome. CTCs are now considered a strong tool to understand the molecular characteristics of prostate cancer, and to be used and analyzed as a 'liquid biopsy' in the attempt to grasp the biological portrait of the disease in the individual patient.
    05/2014; 18(4). DOI:10.1007/s40291-014-0101-8
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    ABSTRACT: Prostate cancer growth depends on androgen receptor (AR) signaling. Androgen ablation therapy induces expression of constitutively active AR splice variants which drive disease progression. Taxanes are a standard of care therapy in castration-resistant prostate cancer (CRPC), however, mechanisms underlying the clinical activity of taxanes are poorly understood. Recent work suggests that the microtubule network of prostate cells is critical for AR nuclear translocation and activity. In this study, we employed a set of AR deletion mutants to identify the microtubule-binding domain of AR, which encompasses the DNA binding domain plus hinge region. We report that two clinically relevant AR splice variants, ARv567 and ARv7, differentially associate with microtubules and dynein motor protein, thereby resulting in differential taxane sensitivity in vitro and in vivo. ARv7, which lacks the hinge region, did not co-sediment with microtubules or co-precipitate with dynein motor protein, unlike ARv567. Mechanistic investigations revealed that the nuclear accumulation and transcriptional activity of ARv7 was unaffected by taxane treatment. In contrast, the microtubule-interacting splice variant ARv567 was sensitive to taxane-induced microtubule stabilization. In ARv567-expressing LuCap86.2 tumor xenografts, docetaxel treatment was highly efficacious, whereas ARv7-expressing LuCap23.1 tumors xenografts displayed docetaxel resistance. Our results suggest that AR variants which accumulate in CRPC cells utilize distinct pathways of nuclear import that affect the antitumor efficacy of taxanes, suggesting a mechanistic rationale to customize treatments for CRPC patients which might improve outcomes.
    Cancer Research 02/2014; 74(8). DOI:10.1158/0008-5472.CAN-13-2876 · 9.28 Impact Factor
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    ABSTRACT: Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm(2). CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLe(x)) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLe(x) expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis.
    PLoS ONE 12/2013; 8(12):e85143. DOI:10.1371/journal.pone.0085143 · 3.53 Impact Factor
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    ABSTRACT: Circulating tumor cells (CTCs) have emerged as a reliable source of tumor cells, and their concentration has prognostic implications. CTC capture offers real-time access to cancer tissue without the need of an invasive biopsy, while their phenotypic and molecular interrogation can provide insight into the biological changes of the tumor that occur during treatment. The majority of the CTC capture methods are based on EpCAM expression as a surface marker of tumor-derived cells. However, EpCAM protein expression levels can be significantly down regulated during cancer progression as a consequence of the process of epithelial to mesenchymal transition. In this paper, we describe a novel HER2 (Human Epidermal Receptor 2)-based microfluidic device for the isolation of CTCs from peripheral blood of patients with HER2-expressing solid tumors. We selected HER2 as an alternative to EpCAM as the receptor is biologically and therapeutically relevant in several solid tumors, like breast cancer (BC), where it is overexpressed in 30% of the patients and expressed in 90%, and gastric cancer (GC), in which HER2 presence is identified in more than 60% of the cases. We tested the performance of various anti HER2 antibodies in a panel of nine different BC cell lines with varying HER2 protein expression levels, using immunoblotting, confocal microscopy, live cells imaging and flow cytometry analyses. The antibody associated with the highest capture efficiency and sensitivity for HER2 expressing cells on the microfluidic device was the one that performed best in live cells imaging and flow cytometry assays as opposed to the fixed cell analyses, suggesting that recognition of the native conformation of the HER2 extracellular epitope on living cells was essential for specificity and sensitivity of CTC capture. Next, we tested the performance of the HER2 microfluidic device using blood from metastatic breast and gastric cancer patients. The HER2 microfluidic device exhibited CTC capture in 9/9 blood samples. Thus, the described HER2-based microfluidic device can be considered as a valid clinically relevant method for CTC capture in HER2 expressing solid cancers.
    Lab on a Chip 11/2013; 14(1). DOI:10.1039/c3lc51039e · 5.75 Impact Factor
  • G. Galletti, B. Kirby, L. T. Vahdat, P. Giannakakou
    Cancer Research 08/2013; 73(8 Supplement):1467-1467. DOI:10.1158/1538-7445.AM2013-1467 · 9.28 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):2888-2888. DOI:10.1158/1538-7445.AM2013-2888 · 9.28 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):5162-5162. DOI:10.1158/1538-7445.AM2013-5162 · 9.28 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):LB-59-LB-59. DOI:10.1158/1538-7445.AM2013-LB-59 · 9.28 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):3492-3492. DOI:10.1158/1538-7445.AM2013-3492 · 9.28 Impact Factor
  • M. Carbonaro, D. Hassane, P. Giannakakou
    Cancer Research 08/2013; 73(8 Supplement):2318-2318. DOI:10.1158/1538-7445.AM2013-2318 · 9.28 Impact Factor
  • M Sung, P Giannakakou
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    ABSTRACT: The taxanes are effective microtubule-stabilizing chemotherapy drugs used in the treatment of various solid tumors. However, the emergence of drug resistance hampers their clinical efficacy. The molecular basis of clinical taxane resistance remains poorly understood. Breast cancer 1, early onset gene, BRCA1, is a tumor-suppressor gene, whose expression has been correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer. However, the molecular mechanism underlying the relationship between BRCA1 (B1) expression and taxane activity remains unclear. To this end, we created a stable B1 knockdown A549 cell line (B1-KD) to investigate B1's role in microtubule biology and response to taxane treatment. We show that B1-KD rendered A549 cells resistant to paclitaxel (PTX), phenocopying clinical studies showing that low B1 expression correlated with taxane resistance. As previously reported, we show that loss of B1 enhanced centrosomal γ-tubulin localization and microtubule nucleation. Interestingly, we found that the B1-KD cells exhibited increased microtubule dynamics as compared with parental A549 cells, as assessed by live-cell confocal microscopy using enhanced green fluorescent protein-tagged α-tubulin or EB1 protein. In addition, we showed that loss of B1 impairs the ability of PTX to induce microtubule polymerization using immunofluorescence microscopy and a cell-based tubulin polymerization assay. Furthermore, B1-KD cells exhibited significantly lower intracellular binding of a fluorescently labeled PTX to microtubules. Recent studies have shown that PTX-stabilized microtubules serves as a scaffold for pro-caspase-8 binding and induction of apoptosis downstream of induced-proximity activation of caspase-8. Here we show that loss of B1 reduces the association of pro-caspase-8 with microtubules and subsequently leads to impaired PTX-induced activation of apoptosis. Taken together, our data show that B1 regulates indirectly endogenous microtubule dynamics and stability while its loss leads to microtubules that are more dynamic and less susceptible to PTX-induced stabilization conferring taxane resistance.Oncogene advance online publication, 25 March 2013; doi:10.1038/onc.2013.85.
    Oncogene 03/2013; DOI:10.1038/onc.2013.85 · 8.56 Impact Factor
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    ABSTRACT: Predictive biomarkers are needed in immune thrombocytopenia (ITP). Single nucleotide polymorphisms (SNPs) in beta 1 tubulin are potential candidates, as beta 1 tubulin is integral for platelet production and function, and SNPs in beta 1 tubulin have been associated with distinct phenotypes in platelets. We investigated the most prevalent beta 1 tubulin SNP (R307H) as a biomarker in patients with ITP via a retrospective chart review. Allelic frequencies between a group of 191 ITP patients and a healthy control group showed no difference, suggesting no direct aetiological role for the SNP in ITP. However, over similar periods of follow-up, both heterozygote and homozygote minor allele ITP patients were treated with significantly more treatment modalities and had significantly higher risk of failure to immune-modulatory therapies [relative risk (RR) = 1·5, 95% confidence interval (CI) = 1·1-2·1; P = 0·01]; with rituximab, in particular, ITP patients with the SNP experienced a 58% failure rate (RR = 1·6, 95%CI = 1·03-2·5; P = 0·04). Analysis of the absolute immature platelet fraction (A-IPF) as a marker of platelet production showed that SNP patients had significantly higher median A-IPFs compared to non-SNP patients when complete responses were achieved using immune modulatory therapies. The data suggest that the beta 1 tubulin R307H SNP has potential for use as a biomarker in ITP and may affect platelet turnover.
    British Journal of Haematology 11/2012; DOI:10.1111/bjh.12124 · 4.96 Impact Factor
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    ABSTRACT: Circulating tumor cells (CTCs) are tumor cells found in the peripheral blood that putatively originate from established sites of malignancy and likely have metastatic potential. Analysis of CTCs has demonstrated promise as a prognostic marker as well as a source of identifying potential targets for novel therapeutics. Isolation and characterization of these cells for study, however, remain challenging owing to their rarity in comparison with other cellular components of the peripheral blood. Several techniques that exploit the unique biochemical properties of CTCs have been developed to facilitate their isolation. Positive selection of CTCs has been achieved using microfluidic surfaces coated with antibodies against epithelial cell markers or tumor-specific antigens such as EpCAM or prostate-specific membrane antigen (PSMA). Following isolation, characterization of CTCs may help guide clinical decision making. For instance, molecular and genetic characterization may shed light on the development of chemotherapy resistance and mechanisms of metastasis without the need for a tissue biopsy. This paper will review novel isolation techniques to capture CTCs from patients with advanced prostate cancer, as well as efforts to characterize the CTCs. We will also review how these analyzes can assist in clinical decision making. Conclusion: The study of CTCs provides insight into the molecular biology of tumors of prostate origin that will eventually guide the development of tailored therapeutics. These advances are predicated on high yield and accurate isolation techniques that exploit the unique biochemical features of these cells.
    Frontiers in Oncology 10/2012; 2:131. DOI:10.3389/fonc.2012.00131
  • Maria Thadani-Mulero, David M Nanus, Paraskevi Giannakakou
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    ABSTRACT: Recent studies have shown that the microtubule-stabilizing drug paclitaxel, which is commonly used for the treatment of prostate cancer, inhibits signaling from the androgen receptor by inhibiting its nuclear accumulation downstream of microtubule stabilization. This mechanism is independent of paclitaxel-induced mitotic arrest and could provide an alternative mechanism of drug action that can explain its clinical activity. In this review, we highlight the importance of signaling and trafficking pathways that depend on intact and dynamic microtubules, and, as such, they represent downstream targets of microtubule inhibitors. We showcase prostate cancer, which is driven by the activity of the androgen receptor, as recent reports have revealed a connection between the microtubule-dependent trafficking of the androgen receptor and the clinical efficacy of taxanes. Identification and further elucidation of microtubule-dependent tumor-specific pathways will help us better understand the molecular basis of clinical taxane resistance as well as to identify individual patients more likely to respond to treatment. Cancer Res; 72(18); 4611-5. ©2012 AACR.
    Cancer Research 09/2012; 72(18):4611-5. DOI:10.1158/0008-5472.CAN-12-0783 · 9.28 Impact Factor
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    ABSTRACT: Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45- cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2-400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.
    PLoS ONE 07/2012; 7(4):e35976. DOI:10.1371/journal.pone.0035976 · 3.53 Impact Factor

Publication Stats

6k Citations
893.79 Total Impact Points


  • 2014
    • Cornell University
      Итак, New York, United States
  • 2007–2014
    • Weill Cornell Medical College
      • • Department of Medicine
      • • Division of Hematology/Medical Oncology
      • • Department of Pharmacology
      New York, New York, United States
  • 2011
    • Victoria University of Wellington
      • School of Biological Sciences
      Wellington, Wellington, New Zealand
  • 2001–2009
    • Emory University
      • • Winship Cancer Institute
      • • Department of Pharmacology
      Atlanta, GA, United States
    • Mario Negri Institute for Pharmacological Research
      • Department of Oncology
      Milano, Lombardy, Italy
  • 2005–2008
    • Georgia Institute of Technology
      • School of Electrical & Computer Engineering
      Atlanta, GA, United States
    • The Rockefeller University
      • Laboratory of Chemistry and Cell Biology
      New York, New York, United States
  • 1997–2004
    • The Scripps Research Institute
      • • Skaggs Institute for Chemical Biology
      • • Department of Chemistry
      La Jolla, CA, United States
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 1997–2000
    • National Cancer Institute (USA)
      • Developmental Therapeutics Program
      베서스다, Maryland, United States
  • 1998–1999
    • National Institutes of Health
      • Branch of Medical Genetics
      Bethesda, MD, United States