David T Dicker

Publications of David T Dicker

• High-resolution imaging and antitumor effects of GFP(+) bone marrow-derived cells homing to syngeneic mouse colon tumors.

  Authors: Niklas K Finnberg, Lori S Hart, Nathan G Dolloff, Zachary B Rodgers, David T Dicker, Wafik S El-Deiry

  The American journal of pathology. 11/2011; 179(5):2169-76.

  Bone marrow-derived cells (BMDCs) participate in the growth and spread of tumors of the breast, brain, lung, and stomach. To date, there are limited reports of bone marrow involvement in colon cancerBone marrow-derived cells (BMDCs) participate in the growth and spread of tumors of the breast, brain, lung, and stomach. To date, there are limited reports of bone marrow involvement in colon cancer pathogenesis, but such findings would have the potential to generate novel treatments for colon cancer patients. We have established a mouse model for imaging BMDCs from whole tumor to single-cell resolution, whereby the bone marrow of lethally irradiated host animals is reconstituted with EGFP-expressing bone marrow cells from matched TgActb(EGFP) donors. The BM transplants yield mice with fluorescently labeled bone marrow, and so BMDCs can subsequently be monitored within a tumor through optical imaging. Successful BM reconstitution was confirmed at 8 weeks after transplantation, when surviving BALB/c mice were injected with CT26 mouse colon cancer cells. We find that up to 45% of cells dissociated from the tumors are GFP(+) and approximately 50% of Lin(+), CD11b(+), and CD3(+) cells express high levels of GFP. Notably, tumor growth is reduced in BM transplanted animals, compared with untransplanted host mice or EGFP-expressing BM donor mice. A needed next step is to separate the molecular and cellular (eg, T cells, NK cells, macrophages) bases of the antitumor effect of the BMDCs from any protumorigenic effect that could be subverted for therapeutic gain.

• Identification and enumeration of circulating tumor cells in the cerebrospinal fluid of breast cancer patients with central nervous system metastases.

  Authors: Akshal S Patel, Joshua E Allen, David T Dicker, Kristi L Peters, Jonas M Sheehan, Michael J Glantz, Wafik S El-Deiry

  Oncotarget. 10/2011; 2(10):752-60.

  The number of circulating tumor cells (CTCs) in the peripheral blood of metastatic breast cancer patients is now an established prognostic marker. While the central nervous system is a common site ofThe number of circulating tumor cells (CTCs) in the peripheral blood of metastatic breast cancer patients is now an established prognostic marker. While the central nervous system is a common site of metastasis in breast cancer, the standard marker for disease progression in this setting is cerebrospinal fluid (CSF) cytology. However, the significance of CSF cytology is unclear, requires large sample size, is insensitive and subjective, and sometimes yields equivocal results. Here, we report the detection of breast cancer cells in CSF using molecular markers by adapting the CellSearch system (Veridex). We used this platform to isolate and enumerate breast cancer cells in CSF of breast cancer patients with central nervous system (CNS) metastases. The number of CSF tumor cells correlated with tumor response to chemotherapy and were dynamically associated with disease burden. This CSF tumor cell detection method provides a semi-automated molecular analysis that vastly improves the sensitivity, reliability, objectivity, and accuracy of detecting CSF tumor cells compared to CSF cytology. CSF tumor cells may serve as a marker of disease progression and early-stage brain metastasis in breast cancer and potentiate further molecular analysis to elucidate the biology and significance of tumor cells in the CSF.

• Hyperspectral imaging: a non-invasive method of imaging melanoma lesions in a patient with stage IV melanoma, being treated with a RAF inhibitor.

  Authors: David T Dicker, Nadia Kahn, Keith T Flaherty, Jeremy Lerner, Wafik S El-Deiry

  Cancer biology & therapy. 08/2011; 12(4):326-34.

  Utilizing a macroscopic Prism and Reflectance Imaging Spectroscopy System (MACRO-PARISS), spectral data are taken from human skin using a fiber optic light probe. The subject was an oncology patientUtilizing a macroscopic Prism and Reflectance Imaging Spectroscopy System (MACRO-PARISS), spectral data are taken from human skin using a fiber optic light probe. The subject was an oncology patient at the University of Pennsylvania, with biopsy proven melanoma, who underwent chemotherapy with a novel small molecule BRAF inhibitor as part of a Phase I clinical trial. The BRAF mutation had been previously identified in this patient. Cutaneous lesions were identified by a clinical oncologist, and lesions were imaged periodically over a 1 month period of time. Spectra are classified using a linearity-independent algorithm over a wavelength range of 450-920 nm. Spectral signatures, or characteristic features, of generated spectra correlate with gross features of lesions that are readily observable. Using PARISS we demonstrated differences between spectral signatures of treated lesions over a treatment course, indicating spectral data correlation with changes in disease. PARISS may be useful to better establish a noninvasive means of following disease progression, and ultimately establish characteristic spectral signatures to define disease presence in the future.

• Prediction of proapoptotic anticancer therapeutic response in vivo based on cell death visualization and TRAIL death ligand-receptor interaction.

  Authors: LanLan Zhou, Wenge Wang, David T Dicker, Robin C Humphreys, Wafik S El-Deiry

  Cancer biology & therapy. 08/2011; 12(4):335-48.

  Tumor growth is often associated with insufficient apoptosis. The Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) and its proapoptotic receptors death receptor 4 (DR4) and DR5Tumor growth is often associated with insufficient apoptosis. The Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) and its proapoptotic receptors death receptor 4 (DR4) and DR5 agonistic monoclonal antibodies are being developed as targeted therapeutics because they kill cancer cells while sparing normal cells. A challenge to targeted therapeutics is the selection of patients who are most likely to benefit from targeted drugs because of the heterogeneity of cancer. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and for predicting therapeutic response. We hypothesized that the cell surface expression level of DR4/5 may predict the proapoptotic targeted therapeutic response if the signaling pathway downstream is intact. The goal of this proof-of-concept study was to develop a molecular imaging strategy to predict proapoptotic anti-cancer therapy response at an early stage of treatment. TRAIL and the DR5 agonistic monoclonal antibody HGS-ETR2 (Lexatumumab, TRM-2) were labeled with a near-infrared dye and these were used to image the TRAIL receptors on cultured TRAIL sensitive and TRAIL resistant human tumor cells as well as tumor xenografts. Imaging of cells and tumor-bearing animals was conducted with near infrared fluorescence imagers and apoptosis in cells was assessed by western blots of PARP-cleavage and flow cytometry of sub-G1 content. Apoptosis in tumors was evaluated by imaging near-infrared dye-labeled Annexin V and tumor tissue activated caspase-3 staining. Both in vitro and in vivo studies showed that imaging of death inducing ligand-receptor interaction was consistent with the apoptosis readout. Thus TRAIL sensitive tumors that express TRAIL receptors underwent cell death following treatment whereas tumors lacking TRAIL receptor expression were shown to be TRAIL resistant. In vivo molecular imaging of TRAIL receptor expression correlated with response to TRAIL therapy and an apoptotic response in vivo.

• Spectral imaging-based methods for quantifying autophagy and apoptosis.

  Authors: Nathan G Dolloff, Xiahong Ma, David T Dicker, Robin C Humphreys, Lin Z Li, Wafik S El-Deiry

  Cancer biology & therapy. 08/2011; 12(4):349-56.

  Spectral imaging systems are capable of detecting and quantifying subtle differences in light quality. In this study we coupled spectral imaging with fluorescence and white light microscopy toSpectral imaging systems are capable of detecting and quantifying subtle differences in light quality. In this study we coupled spectral imaging with fluorescence and white light microscopy to develop new methods for quantifying autophagy and apoptosis. For autophagy, we employed multispectral imaging to examine spectral changes in the fluorescence of LC3-GFP, a chimeric protein commonly used to track autophagosome formation. We found that punctate autophagosome-associated LC3-GFP exhibited a spectral profile that was distinctly different from diffuse cytosolic LC3-GFP. We then exploited this shift in spectral quality to quantify the amount of autophagosome-associated signal in single cells. Hydroxychloroquine (CQ), an anti-malarial agent that increases autophagosomal number, significantly increased the punctate LC3-GFP spectral signature, providing proof-of-principle for this approach. For studying apoptosis, we employed the Prism and Reflector Imaging Spectroscopy System (PARISS) hyperspectral imaging system to identify a spectral signature for active caspase-8 immunostaining in ex vivo tumor samples. This system was then used to rapidly quantify apoptosis induced by lexatumumab, an agonistic TRAIL-R2/DR5 antibody, in histological sections from a preclinical mouse model. We further found that the PARISS could accurately distinguish apoptotic tumor regions in hematoxylin and eosin-stained sections, which allowed us to quantify death receptor-mediated apoptosis in the absence of an apoptotic marker. These spectral imaging systems provide unbiased, quantitative and fast means for studying autophagy and apoptosis and complement the existing methods in their respective fields.

• Quinacrine synergizes with 5-fluorouracil and other therapies in colorectal cancer.

  Authors: Jean-Nicolas Gallant, Joshua E Allen, Charles D Smith, David T Dicker, Wenge Wang, Nathan G Dolloff, Arunasalam Navaraj, Wafik S El-Deiry

  Cancer biology & therapy. 08/2011; 12(3):239-51.

  Although treatments have improved patient prognosis in surgically resectable colorectal cancer, new effective drugs with improved safety profiles are needed to improve the currently poor outcomes ofAlthough treatments have improved patient prognosis in surgically resectable colorectal cancer, new effective drugs with improved safety profiles are needed to improve the currently poor outcomes of patients with recurrent or metastatic colorectal cancer. Quinacrine, a small molecule anti-malarial agent that has activity in giardiasis, lupus, prion disease, and used as a means of non-surgical sterilization, has shown cytotoxic activity across a broad range of cancers. Here, we evaluate the potential of adding quinacrine to anticancer chemotherapeutics and targeted agents as a potential novel combinatorial therapy for advanced colon cancer. We show that quinacrine synergizes with 5-fluorouracil and significantly enhances the cytotoxicity of sorafenib in a panel of 10 human colorectal cancer cell lines, including those with KRAS mutations protein gel blot analysis confirmed that quinacrine's anticancer activity partially arises from its ability to stabilize p53 and lower anti-apoptotic protein levels. In a series of in vivo studies, quinacrine monotherapy lowered the tumor load of nu/nu mice bearing human colorectal cancer xenografts. In combination, quinacrine and 5-Fluorouracil significantly delayed tumor growth of a variety of different xenografts, as compared to each agent administered alone. Our results suggest that the administration of quinacrine in combination with chemotherapeutic agents and targeted agents should be further explored in patients with recurrent, locally advanced, or metastatic colorectal cancer.

• Quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapeutic agents.

  Authors: Wenge Wang, Jean-Nicolas Gallant, Sharyn I Katz, Nathan G Dolloff, Charles D Smith, Junaid Abdulghani, Joshua E Allen, David T Dicker, Bo Hong, Arunasalam Navaraj, Wafik S El-Deiry

  Cancer biology & therapy. 08/2011; 12(3):229-38.

  Quinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. TreatmentQuinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. Treatment by quinacrine alone at concentrations of 10-20 mM for 1-2 d cannot kill hepatocellular carcinoma cells, such as HepG2, Hep3B, Huh7, which are also resistant to TRAIL. However, quinacrine renders these cells sensitive to treatment by TRAIL. Co-treatment of these cells with quinacrine and TRAIL induces overwhelming cell death within 3-4 h. Levels of DR5, a pro-apoptotic death receptor of TRAIL, are increased upon treatment with quinacrine, while levels of Mcl-1, an anti-apoptotic member of the Bcl-2 family, are decreased. While the synergistic effect of quinacrine with TRAIL appears to be in part independent of p53, knockdown of p53 in HepG2 cells by siRNA results in more cell death after treatment by quinacrine and TRAIL. The mechanism by which quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapies, and the potential for clinical application currently are being further explored. Lastly, quinacrine synergizes with chemotherapeutics, such as adriamycin, 5-FU, etoposide, CPT11, sorafenib, and gemcitabine, in killing hepatocellular carcinoma cells in vitro and the drug enhances the activity of sorafenib to delay tumor growth in vivo.

• Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab.

  Authors: Lori S Hart, Nathan G Dolloff, David T Dicker, Constantinos Koumenis, James G Christensen, Adda Grimberg, Wafik S El-Deiry

  Cell cycle (Georgetown, Tex.). 07/2011; 10(14):2331-8.

  Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSCCancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemo-resistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.

• Identifying circulating tumor stem cells that matter: the key to prognostication and therapeutic targeting.

  Authors: Bishoy Faltas, Amer Zeidan, Kristi Peters, Avisnata Das, Jamal Joudeh, Arunasalam Navaraj, Nathan G Dolloff, Harold A Harvey, Yixing Jiang, Joshua E Allen, David T Dicker, Wafik S El Deiry

  Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 06/2011; 29(21):2946-7; author reply 2947-8.

• Off-target lapatinib activity sensitizes colon cancer cells through TRAIL death receptor up-regulation.

  Authors: Nathan G Dolloff, Patrick A Mayes, Lori S Hart, David T Dicker, Robin Humphreys, Wafik S El-Deiry

  Science translational medicine. 06/2011; 3(86):86ra50.

  Lapatinib, a dual HER2/EGFR (human epidermal growth factor receptor 2/epidermal growth factor receptor) inhibitor, is a recently approved targeted therapy for metastatic breast cancer. BecauseLapatinib, a dual HER2/EGFR (human epidermal growth factor receptor 2/epidermal growth factor receptor) inhibitor, is a recently approved targeted therapy for metastatic breast cancer. Because lapatinib enhances the efficacy of the chemotherapeutic agent capecitabine in breast cancer patients, we tested whether lapatinib also enhances the activity of anticancer agents in colorectal cancer. We found that lapatinib improved the proapoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two TRAIL receptor agonists, the antibodies mapatumumab and lexatumumab. Tumors from mice treated with a combination of lapatinib and TRAIL exhibited more immunostaining for cleaved caspase-8, a marker of the extrinsic cell death pathway, than did tumors from mice treated with lapatinib or TRAIL alone. Furthermore, combination therapy suppressed tumor growth more effectively than either agent alone. Lapatinib up-regulated the proapoptotic TRAIL death receptors DR4 and DR5, leading to more efficient induction of apoptosis in the presence of TRAIL receptor agonists. This activity of lapatinib was independent of EGFR and HER2. The off-target induction of DR5 by lapatinib resulted from activation of the c-Jun amino-terminal kinase (JNK)/c-Jun signaling axis. This activity of lapatinib on TRAIL death receptor expression and signaling may confer therapeutic benefit when increased doses of lapatinib are used in combination with TRAIL receptor-activating agents.

• Overcoming hypoxia-induced apoptotic resistance through combinatorial inhibition of GSK-3β and CDK1.

  Authors: Patrick A Mayes, Nathan G Dolloff, Colin J Daniel, J Judy Liu, Lori S Hart, Kageaki Kuribayashi, Joshua E Allen, David I H Jee, Jay F Dorsey, Yingqiu Y Liu, David T Dicker, J Martin Brown, Emma E Furth, Peter S Klein, Rosalie C Sears, Wafik S El-Deiry

  Cancer research. 06/2011; 71(15):5265-75.

  Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that couldTumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and cyclin-dependent kinase (CDK) 1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator hypoxia-inducible factor (HIF) 1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.

• The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling, tumor growth and chemosensitivity under hypoxic conditions.

  Authors: Elizabeth M Matthew, Lori S Hart, Aristotelis Astrinidis, Arunasalam Navaraj, Nathan G Dolloff, David T Dicker, Elizabeth P Henske, Wafik S El-Deiry

  Cell cycle (Georgetown, Tex.). 12/2009; 8(24):4168-75.

  Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to asTuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently overexpressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT -11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently overexpressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT -11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT -11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.

• Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and paclitaxel have cooperative in vivo effects against glioblastoma multiforme cells.

  Authors: Jay F Dorsey, Akiva Mintz, Xiaobing Tian, Melissa L Dowling, John P Plastaras, David T Dicker, Gary D Kao, Wafik S El-Deiry

  Molecular cancer therapeutics. 12/2009;

  Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in conjunction with microtubule-targeting agents may be a promising novel anticancer treatment strategy. In vitro studies haveTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in conjunction with microtubule-targeting agents may be a promising novel anticancer treatment strategy. In vitro studies have suggested that relatively low concentrations of TRAIL enhance the lethality of paclitaxel (Taxol) against human cancer cells. The increased efficacy may be due to the triggering of caspase activation, resulting in mitotic checkpoint abrogation and catastrophe. We show here that wild-type p53 protects cells from caspase-dependent death induced by this therapeutic combination in vitro. We have now also developed an imaging-based model system to test the in vivo efficacy of combined TRAIL and Taxol, in which tumor growth and treatment response can be monitored noninvasively and in real-time. We further utilize bioluminescence, F(18)-fluorodeoxyglucose-positron emission tomography, and microscale computed tomography imaging to confirm the effects of combined treatment on tumors. These studies together provide the first in vivo confirmation that combined TRAIL plus paclitaxel results in better tumor control compared with either TRAIL or paclitaxel alone, and with no discernable increased normal tissue toxicity in the mouse. Interestingly, the in vivo antitumor response elicited by combined treatment was not affected by the p53 status of the tumor cells. These preclinical observations together suggest the therapeutic potential of combining TRAIL plus paclitaxel in cancer treatment, and support further preclinical and future clinical testing. [Mol Cancer Ther 2009;8(12):3285-95].

• Sorafenib inhibits ERK1/2 and MCL-1(L) phosphorylation levels resulting in caspase-independent cell death in malignant pleural mesothelioma.

  Authors: Sharyn I Katz, Lanlan Zhou, Grace Chao, Charles D Smith, Thomas Ferrara, Wenge Wang, David T Dicker, Wafik S El-Deiry

  Cancer biology & therapy. 12/2009; 8(24):2406-16.

  Malignant pleural mesothelioma (MPM) is an aggressive, rapidly progressive malignancy without effective therapy. We evaluate sorafenib efficacy and impact on the cellular pro-survival machinery inMalignant pleural mesothelioma (MPM) is an aggressive, rapidly progressive malignancy without effective therapy. We evaluate sorafenib efficacy and impact on the cellular pro-survival machinery in vitro, efficacy of sorafenib as monotherapy and in combination with the naturally occurring death receptor agonist, TRAIL using human MPM cell lines, MSTO-211H, M30, REN, H28, H2052 and H2452. In vitro studies of the six MPM lines demonstrated single agent sensitivity to the multikinase inhibitor sorafenib and resistance to TRAIL. H28 and H2452 demonstrated augmented apoptosis with the addition of TRAIL to sorafenib in vitro. Treated cell lines demonstrated sorafenib-induced rapid dephosphorylation of AKT followed shortly by near complete dephosphorylation of the constitutively phosphorylated ERK1/2. Sorafenib therapy also decreased phosphorylation of B-Raf and mTOR in several cell lines. Within 3 h of sorafenib treatment, a number of known pro-survival molecules were dephosphorylated and/or downregulated in expression including MCL-1(L), c-FLIP(L), survivin and cIAP(1). These changes and eventual cell death did not elicit significant caspase-3 activation or PARP cleavage and pretreatment with the pan-caspase inhibitor, Z-VAD-FMK, did not block sorafenib efficacy but did block the effect of TRAIL monotherapy. Pre-treatment with Z-VAD-FMK did not block the synergistic effect of TRAIL and sorafenib in H28. In summary, single agent treatment with sorafenib results in widespread inhibition of the pro-survival machinery in vitro leading to cell death via a primarily caspase-independent mechanism. Combining sorafenib therapy with TRAIL, may be useful in order to provide a more efficient death signal and this synergistic effect appears to be caspase-independent. Pilot in vivo data demonstrates promising evidence of therapeutic efficacy in human tumor bearing xenograft nu/nu mice. We document single agent activity of sorafenib against MPM, unravel novel effects of sorafenib on anti-apoptotic signaling mediators, and suggest the combination of sorafenib plus TRAIL as possible therapy for clinical testing in MPM.

• Reduced cell death, invasive and angiogenic features conferred by BRCA1-deficiency in mammary epithelial cells transformed with H-Ras.

  Authors: Arunasalam Navaraj, Niklas Finnberg, David T Dicker, Wensheng Yang, Elizabeth M Matthew, Wafik S El-Deiry

  Cancer biology & therapy. 12/2009; 8(24):2417-44.

  To investigate the role of tumor suppressors BRCA1 and p53 proteins in human breast tumorigenesis, we transformed immortalized human mammary epithelial cells, MCF10A, with or without BRCA1/p53To investigate the role of tumor suppressors BRCA1 and p53 proteins in human breast tumorigenesis, we transformed immortalized human mammary epithelial cells, MCF10A, with or without BRCA1/p53 gene-specific knockdowns. Stable knockdown of BRCA1 alone in MCF10A cells led to centrosome amplification, impaired p53 protein stability, increased sensitivity towards DNA-damaging agents, defective chromosomal condensation at mitosis and elevated protein levels of cyclin D1 and c-myc. While over-expression of mutant H-Ras transformed MCF10A cells, depletion of BRCA1 dramatically enhanced the in vivo tumorigenesis that was associated with higher levels of VEGF, enhanced vascularization and less apoptosis in the BRCA1-deficient Ras-transformed tumors. The Ras-transformed BRCA1-deficient tumors exhibited features of the epithelial-to-mesenchymal transition, appeared to secrete matrix metalloproteases as visualized by in vivo bio-imaging of tumors using fluorescent probe MMP680, and were locally metastatic to lymph nodes. Our results suggest that loss of BRCA1 function may contribute to the aggressiveness of Ras-MAPK driven human breast cancer with associated increase in levels of cyclin D1 and c-myc, enhanced MAPK activity, angiogenic potential & invasiveness. This mammary xenograft tumor model may be useful as a tool to understand human breast tumor angiogenesis and metastasis, as well as to test candidate therapeutics.

• CARPs enhance p53 turnover by degrading 14-3-3sigma and stabilizing MDM2.

  Authors: Wensheng Yang, David T Dicker, Jiandong Chen, Wafik S El-Deiry

  Cell cycle (Georgetown, Tex.). 04/2008; 7(5):670-82.

  CARP1 and CARP2 proteins (CARPs) are E3 ligases that target p53 as well as phospho-p53 for degradation. Because MDM2 is a critical regulator of p53 turnover, we investigated and found that CARPsCARP1 and CARP2 proteins (CARPs) are E3 ligases that target p53 as well as phospho-p53 for degradation. Because MDM2 is a critical regulator of p53 turnover, we investigated and found that CARPs associate with MDM2. We provide evidence that CARPs stabilize MDM2 by inhibiting MDM2 self-ubiquitination. CARPs together with MDM2 enhance p53 degradation, thereby inhibiting p53-mediated cell death. CARP protein levels correlate with MDM2 levels including under hypoxia where both are reduced. CARP2 was found to target 14-3-3sigma for degradation, leading to MDM2 stabilization. MDMX, a homolog of MDM2, is not absolutely required for MDM2 stabilization by CARPs, although overexpression of CARP2 enhances MDM2/MDMX interaction. Taken together, our study identifies novel mechanisms by which CARP proteins regulate the p53 signaling pathway.

• Cell cycle dependent and schedule-dependent antitumor effects of sorafenib combined with radiation.

  Authors: John P Plastaras, Seok Hyun Kim, Yingqiu Y Liu, David T Dicker, Jay F Dorsey, James McDonough, George Cerniglia, Ramji R Rajendran, Anjali Gupta, Anil K Rustgi, J Alan Diehl, Charles D Smith, Keith T Flaherty, Wafik S El-Deiry

  Cancer research. 11/2007; 67(19):9443-54.

  The antineoplastic drug sorafenib (BAY 43-9006) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets ofThe antineoplastic drug sorafenib (BAY 43-9006) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, there are several potential anticancer mechanisms of action, including induction of apoptosis, cytostasis, and antiangiogenesis. We observed that sorafenib has broad activity in viability assays in several human tumor cell lines but selectively induces apoptosis in only some lines. Sorafenib was found to decrease Mcl-1 levels in most cell lines tested, but this decrease did not correlate with apoptotic sensitivity. Sorafenib slows cell cycle progression and prevents irradiated cells from reaching and accumulating at G2-M. In synchronized cells, sorafenib causes a reversible G1 delay, which is associated with decreased levels of cyclin D1, Rb, and phosphorylation of Rb. Although sorafenib does not affect intrinsic radiosensitivity using in vitro colony formation assays, it significantly reduces colony size. In HCT116 xenograft tumor growth delay experiments in mice, sorafenib alters radiation response in a schedule-dependent manner. Radiation treatment followed sequentially by sorafenib was found to be associated with the greatest tumor growth delay. This study establishes a foundation for clinical testing of sequential fractionated radiation followed by sorafenib in gastrointestinal and other malignancies.

• Hyperspectral image analysis of live cells in various cell cycle stages.

  Authors: David T Dicker, Jeremy M Lerner, Wafik S El-Deiry

  Cell cycle (Georgetown, Tex.). 11/2007; 6(20):2563-70.

  In this study we have explored the use of hyperspectral imaging (HSI) to determine the cell cycle status of live cells in culture. Live cancer cell lines in culture were either synchronized byIn this study we have explored the use of hyperspectral imaging (HSI) to determine the cell cycle status of live cells in culture. Live cancer cell lines in culture were either synchronized by release from nocodazole or arrested in various cell-cycle phases with serum starvation (G1), aphidicolin (S), or nocodazole (G2/M). The live cells were then stained with the fluorescent DNA binding dyes Heochst 33342 or DCO along with propidium iodide or MTR. Samples were examined using fluorescence microscopy and entire spectral emission profiles were acquired for each sample using a PARISS HSI system. Classified spectra were incorporated into spectral libraries. All spectra acquired from each sample were correlated with library spectra to a user-determined confidence threshold, generating unique spectral signatures for each sample. Examination of these spectral signatures revealed that all cell cycle phases could be objectively differentiated. Ongoing studies employing other viable cell fluorescent dyes, and dyes in combination may provide more robust spectral signatures defining the status and condition of living cells.

• Effects of low confluency, serum starvation and hypoxia on the side population of cancer cell lines.

  Authors: Raluca T Tavaluc, Lori S Hart, David T Dicker, Wafik S El-Deiry

  Cell cycle (Georgetown, Tex.). 11/2007; 6(20):2554-62.

  The cancer stem cell theory describes a small subset of cancer cells that have the ability to initiate and drive the growth of a tumor. The niche refers to the environmental factors and theThe cancer stem cell theory describes a small subset of cancer cells that have the ability to initiate and drive the growth of a tumor. The niche refers to the environmental factors and the surrounding cells within which the tumor develops. The exact relationship between cancer stem cells and the tumor niche is not known. However, using side population analysis by flow cytometry, it is possible to analyze the relationship between environmental stresses and putative cancer stem cells. The side population is a subpopulation of cells that efflux Hoechst 33342 and has been previously shown to be enriched for cancer stem cells. Using this technique, we characterized the response of side population cells to low confluency, serum starvation and hypoxia using three different human cancer cell lines. We found that these stresses, characteristic of the tumor niche enrich the side population of DLD1, SW480 and MCF7 cancer cell lines, thus possibly predisposing the tumor to a more malignant phenotype.

• Replication stress, defective S-phase checkpoint and increased death in Plk2-deficient human cancer cells.

  Authors: Elizabeth M Matthew, Timothy J Yen, David T Dicker, Jay F Dorsey, Wensheng Yang, Arunasalam Navaraj, Wafik S El-Deiry

  Cell cycle (Georgetown, Tex.). 11/2007; 6(20):2571-8.

  We previously reported that the Polo-like Kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to Taxol-induced apoptosis.We previously reported that the Polo-like Kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to Taxol-induced apoptosis. Our goals have been to better understand why Plk2 is regulated by p53 and how Plk2 signals protection from cell death through checkpoint activation. We found that following knock-down of Plk2 in wild-type p53 expressing H460 human non-small cell lung cancer cells there was a significant increase in cell death observed in aphidicolin-treated cells and a further increase after release from aphidicolin-block. The highest levels of cell death were observed when Plk2-deficient cells were released from both aphidicolin and etoposide treatment. These results suggested that a defective S-phase checkpoint may contribute to enhanced sensitivity of Plk2-deficient cells to replication stress. Consistent with this hypothesis, we observed higher levels of Serine 139 H2AX phosphorylation in Plk2-deficient as compared to control cells before and after aphidicolin treatment indicating that there is more DNA damage when Plk2 is depleted. We also observed higher levels of Chk1 protein in Plk2-deficient cells that were associated with reduced levels of Serine 317-phosphorylated Chk1. In aphidicolin-treated cells, there were lower levels of Serine 317-phosphorylated Chk1 when Plk2 was knocked-down. Plk2 was demonstrated to interact with Chk2, Chk1, Serine 317-phosphorylated Chk1 and p53. Thus, increased cell death observed after aphidicolin treatment and release in Plk2-deficient cells may result from both higher levels of replication stress-induced DNA damage and a dysfunctional S-phase checkpoint.