CYR61 mediates HGF-dependent glioma cell survival, migration and AKT activation
First Place
Xin (Linda) Zhou
Department of Neurology, Neuroscience, and Neuro-oncology, Kennedy Krieger Institute, Baltimore, MD
Tumor cell responses to the growth-factor inducible early response gene product CCN1/Cyr61 overlap with those induced by the HGF:c-Met signaling pathway. In this study, we investigate if Cyr61 is a downstream effector of HGF/c-Met pathway activation in human glioma cells. A semi-quantitative immunohistochemical analysis of 112 human glioma and normal brain specimens showed that levels of tumor-associated Cyr61 protein correlated with tumor grade (P< 0.001) and with c-Met protein (R2=0.4791, P<0.0001). Purified HGF rapidly up-regulated Cyr61 mRNA (peak at 30 min) and protein expression (peak at 2 hr) in HGF-/c-Met+ human glioma cell lines via a transcription- and translation-dependent mechanism. Conversely, HGF/c-Met pathway inhibitors reduced Cyr61 expression in HGF+/c-Met+ human glioma cell lines in vitro and in HGF+/c-Met+ glioma xenografts. Targeting Cyr61 expression with siRNA inhibited HGF-induced cell migration (P < 0.01) and cell proliferation (P < 0.001). The effect of Cyr61 on HGF-induced Akt pathway activation was also examined. Cyr61 siRNA had no effect on the early wave of glioma cell HGF-induced Akt activation/phosphorylation (Ser-473) 30 min post-stimulation with HGF. Cyr61 siRNA inhibited a second wave of Akt phosphorylation measured 12 hr after cell stimulation with HGF and also inhibited HGF-induced phosphorylation of the Akt target GSK3. We treated pre-established subcutaneous U87 glioma xenografts with direct intratumorally delivered Cyr61 siRNA or control siRNA. Cyr61 siRNA inhibited xenograft growth by up to 30 percent in a dose-dependent manner (P < 0.05). These results identify a Cyr61-dependent pathway by which c-Met activation mediates cell proliferation, cell migration, and long-lasting signaling events in glioma cell lines and possibly astroglial malignancies.
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Second Place
Parthenolide and structurally related natural products as anti-cancer stem cell agents: A new era in treatment of multiple myeloma.
Huynh, D.T.1, Iannotti, M.J.1, Gunn E.J.1, Han, C.2, Barrios, F.J.3, Colby D.A.2,3,Kirshner, J.1
1Department of Biological Sciences, Purdue University, West Lafayette, IN; 2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN; and 3Department of Chemistry, Purdue University, West Lafayette, IN
Multiple myeloma (MM) is an incurable plasma cell malignancy where even patients in remission succumb to an inevitable relapse. Pre-clinical testing of emerging therapeutics is hindered by the failure of the standard models to sustain the ex vivo growth of the MM clone. We have recently described a 3-D culture system where the extracellular matrix and cellular compartments of the bone marrow (BM) are reconstructed in vitro, recapitulating the native microenvironment of the human BM where cells occupy distinct niches. Cells from the BM aspirates are grown in a fibronectin, laminin, and collagen-rich extracellular matrix designed to reconstruct endosteal and central marrow compartments of the BM (rBM). In the rBM model, the MM undergoes up to 15-fold expansion of malignant cells harboring the clonotypic IgH VDJ. The currently used MM pre-clinical models only target the plasma cells, comprising the bulk of the tumor, leaving the cancer stem cells (CSCs) to repopulate the tumor and trigger the relapse. The localization of non-proliferating, drug resistant CD20+ B cells to the reconstructed endosteum, in contact with N-cadherin and osteocalcin positive osteoblastic cells, suggests the presence of MM-cancer stem cells (MM-CSC) in the endosteal niche of rBM; the niche known to harbor the hematopoietic stem cells. MM-CSCs exhibit tumorigenic and self-renewal potential, measured by their ability to generate clonotypic colonies and differentiate into plasma cells in colony forming unit assays. Thus, the rBM model provides biologically relevant pre-clinical paradigm enabling the evaluation of therapeutic vulnerabilities of all compartments of the MM clone, including the drug-resistant MM-CSC. We have previously demonstrated that melphalan, dexamethasone, vincristine, and bortezomib therapies are not curative because these drugs target CD138+CD56+ MM plasma cells, thus failing to eliminate the MM-CSC. Here we show that parthenolide and structurally related natural product inhibitors of NFkB are potent anti-MM-CSC agents (LC50=5μM), inducing apoptosis in MM-CSCs through the activation of caspases 8 and 3. Combination treatment with conventional chemotherapeutic agents melphalan or bortezomib and parthenolide were cytotoxic and eliminated both the plasma cell burden and the MM-CSC, implying that such combinations may have curative potential. We determined that an analog of parthenolide, 13-(3-trifluoromethyl-phenyl)-parthenolide, has MM-CSC activity (LC50=15?M). The lower cytotoxicity of the fluorinated analog makes it a candidate to be used as an imaging probe. Therefore, we plan to test this compound as a labeling reagent for PET detection of MM-CSCs. This is a first report of an anti-CSC agent in MM, suggesting that parthenolide and its analogs could improve the survival of patients with MM by eliminating the relapse causing MM-CSCs.
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Third Place
The effect of a novel angiogenesis inhibitor (TM) on the efficacy of oncolytic virus
Azeem Kaka, Jason Pradarelli, Christopher Alvarez-Breckenridge, Ji Young Yoo, Quintin Pan, E. A. Chiocca, Ted Teknos, Balveen Kaur
Oncolytic viruses (OV) are modified viruses specifically designed to lyse cancerous cells while leaving surrounding tissue intact. Although effective tumor killing has been demonstrated in vitro and in animal models, OV therapy has shown limited effectiveness in clinical trials for glioma treatment. The tumor microenvironment is a potent barrier for OV propagation and spread; in particular, the angiogenic and inflammatory responses that follow OV inoculation have been demonstrated to significantly limit OV efficacy. Physiologic levels of copper support angiogenesis and has been shown to inhibit wild-type HSV infection and replication. The goal of our current project is to determine if the novel copper-chelating agent, tetrathiomolybdate (TM), represents a potential co-therapy that can be used to enhance overall OV efficacy in malignant glioma. Based on previous findings, the anti-cancerous effects of OV can be significantly increased by pre-treatment with an angiostatic agent. The angiostatic and immunosuppressive properties of TM make it an ideal candidate to enhance OV therapy. In this study, we are testing if copper inhibits OV-mediated glioma cell killing and if TM can reverse this effect. We found a significant inhibitory effect of copper, at physiologically relevant concentrations (1 mg/L in serum), on the cytotoxic ability of OV against multiple glioma and head and neck squamous cell carcinoma cc cell lines. We demonstrated that TM-mediated copper chelation restores the ability of OV to replicate in vitro and, further, that TM removes the copper-mediated inhibition of glioma killing by OV. These results encourage testing the therapeutic efficacy of combining TM and OV for treating glioma in vivo. Compared with athymic nude mice treated with OV alone, our preliminary data suggests that on day 25 post glioma implantation, athymic nude mice treated with TM and OV combination therapy had significantly smaller tumors (21.51 versus 153.93 mm3).
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Honorable Mention
Manipulation of HuR expression alters the efficacy of PARP-inhibitors in pancreatic and ovarian cancer cells.
Costantino CL, Richards NG, Rittenhouse DW, Cozzitorto JA, Witkiewicz AK, Yeo CJ, Brody JR
PARP-inhibitors (PARPi) are a novel class of agents that target cancer cells deficient in DNA repair. Early phase trials show promising success with PARP-inhibitors, yet drug resistance mechanisms and off-target pathways affected by drug exposure have not been explored. HuR (ELAV1) is a protein that binds and stabilizes mRNA transcripts upon certain stress stimuli. HuR targeting of mRNAs result in an increase in protein expression; and thus, most likely protein function. Recently, we demonstrated that HuR binds and regulates deoxycytidine kinase (dCK), the gemcitabine metabolizing enzyme. Thus, HuR expression enhances gemcitabine efficacy against pancreatic cancer cells. We subsequently screened various chemotherapeutics and found that PARPi, specifically ABT-888 (Abbott Labs) and to a lesser extent DPQ (3,4-Dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone), and BYK204165 (4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione) stimulate the HuR stress response pathway. ABT-888 is a novel, safe, orally bioavailable small molecule potent PARP inhibitor that delays the repair of DNA damage induced by chemotherapeutics and increases sensitivity of tumor cells to damaging agents, in vitro and demonstrated PARP inhibition in murine tumors, in vivo, and human peripheral blood mononuclear cells and tumors, ex vivo. In both pancreatic and ovarian cancer cell lines, siRNA knockdown of >90% of HuR mRNA expression, detected by qPCR, rendered cells more sensitive to ABT-888 compared to a control siRNA. Other PARPis, DPQ and BYK showed no real differences between HuR siRNA knockdown and control siRNA knockdown lines. Immunofluorescence studies indicate that a three hour treatment with 100 µM ABT 888 directly caused HuR to transport from the nucleus to the cytoplasm, presumably regulating a select set of ARE-rich mRNA cargoes after drug exposure. Accordingly, immunoblot analysis indicated increased HuR accumulation in cytosolic extracts as early as one hour after 75 µM ABT-888 treatment. Focused gene expression analysis on HuR bound, immunoprecipitated RNA defined established and novel HuR mRNA targets including dCK, VEGF, HuR and death receptor 5 (DR5). Current studies will reveal and validate the importance of these downstream mRNA targets in PARP-inhibitor metabolism and efficacy. We hypothesize that HuR may be central to PARPi effectiveness (particularly ABT-888) and may be useful in understanding PARPi de novo drug resistance mechanisms. Future work will reveal whether 1) HuR status can be utilized as a predictive marker for PARP-inhibitor-based therapy; 2) to what extent HuR is important to PARPi efficacy; and 3) if all clinically available PARP-inhibitors engage HuR’s regulation of unique mRNA cargoes in cancer cells upon treatment.
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Honorable Mention
Effect of inflammation on apoptosis in myeloid-derived suppressor cells (MDSC) in breast cancer
Lydia Grmai, Olesya Chornoguz
Suzanne Ostrand-Rosenberg, Professor, Department of Biological Sciences
Department of Biological Sciences, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
Myeloid-derived suppressor cells (MDSC) are a major cause of immune suppression in cancer patients by inhibition of T-lymphocyte activation. Transfection of 4T1 mammary carcinoma cells with interleukin-1β (an inflammatory cytokine) leads to induction of more aggressive and higher levels of MDSC. Pathway analyses of the proteins expressed by MDSC induced by these two tumors highlighted numerous pathways that differ between 4T1 and 4T1/IL-1β-induced MDSC, including the extrinsic apoptotic pathway activated through Fas. To determine if this pathway contributed to the enhanced survival of 4T1/IL-1β-induced MDSC, we treated MDSC with Jo2, an agonist antibody for Fas. Apoptosis of the treated cells was measured by flow cytometry using antibodies to activated caspase-3, a molecule that is upregulated in apoptotic cells. 4T1/IL-1β-induced MDSC contained more activated caspase-3 than 4T1-induced MDSC, although the two MDSC populations expressed similar levels of Fas. These results suggest that 4T1/IL-1β-induced tumors may attribute their aggressiveness to the resistance of MDSC to Fas-mediated apoptosis. Future studies will explore the possible role of suppressors of apoptosis in 4T1/IL-1β-induced MDSC. Upregulating apoptosis in MDSC may reduce their accumulation and promote immunotherapy in cancer patients.
This work was funded in part by NIH/HIGMS MARC U*STAR T34 08663 National Research Service Award to UMBC and NIH, National Cancer Institute RO1CA84232.
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Honorable Mention
Thioredoxin and thioredoxin reductase 1 regulate CLIC4 nuclear translocation in squamous cell carcinoma cells
Kasey Jividen1, Mariam Malik1, Kwang Suh1 and Stuart H. Yuspa1
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, MD
Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its roles in stress-induced apoptosis, modulating TGFβ-signaling and Ca2+-induced differentiation in keratinocytes. Moreover, CLIC4 protein is reduced and excluded from the nucleus in tumor cells of multiple human cancers. Our laboratory has established that CLIC4 nuclear translocation is regulated by cellular redox, specifically through direct modification of a cysteine residue by nitric oxide; S-nitrosylation enhances its association with nuclear import proteins by inducing a conformational change in the protein. Interestingly, CLIC4 is a structural homolog of glutathione S-transferase ω (GSTω) with thioredoxin folds at the amino and carboxy termini. We hypothesize that the altered distribution of CLIC4 observed in tumor cells is due to an altered redox environment, namely through the reducing activity of the thioredoxin (Trx)/thioredoxin reductase 1 (TR1) pair, the major denitrosylating enzyme pair. To test this hypothesis, we examined the level of these enzymes and modulated their activity in tumor cells to monitor effects on CLIC4.
Inhibition of TR1 by the chemical inhibitor auranofin, enhances S-nitrosylation and nuclear translocation of CLIC4 in normal cells underlining the importance of the Trx/TR1 pathway in determining CLIC4 subcellular distribution. CLIC4 nuclear translocation is delayed in response to TNFα in mouse squamous cell carcinoma cells and to etoposide in human squamous cell carcinoma cells as well as in H-Ras-transformed mouse keratinocytes. TR1 protein and enzyme activity are elevated in murine squamous papilloma and carcinoma cells compared to normal cells. A similar increase in TR1 protein and activity is detected in human squamous carcinoma cells. Chemical inhibition of TR1 by auranofin enhances nuclear translocation of CLIC4 in both Ras-transformed mouse keratinocytes and human squamous carcinoma cells. Furthermore, CLIC4 nuclear translocation can be restored by the genetic silencing of either Trx or TR1 in oncogenic Ras-transformed cells. These results support the hypothesis that the redox environment of cancer cells modifies CLIC4 subcellular localization and function. We are currently investigating the biological consequences of restoring CLIC4 nuclear translocation and whether this could have therapeutic efficacy.
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Honorable Mention
Role of miR-182/miR-96 cluster in prostatic zinc homeostasis
Ekaterina Khramtsova, Avani Vaishnav, Nicoleta Arva, Angeline Antonio, Andre Kajdacsy-Balla, and Larisa Nonn
Zinc is an essential element that is required for the activity of more than 300 enzymes, structure of proteins, and control of genetic expression. It plays an important role in cellular processes such as cell division, growth, differentiation, development, aging, and synthesis and repair of DNA, RNA, and protein. Prostatic secretary epithelial cells accumulate large amounts of zinc and have a high expression of human Zinc transporter 1 (hZIP1). However, prostate cancer (PCa) tissue exhibits lower levels of zinc and hZIP1 compared with surrounding normal-appearing areas. Previously, we examined several miRNAs in formalin-fixed paraffin-embedded prostate tissue. Based on those studies, we hypothesize that miR-182/96 cluster is responsible for regulation of hZIP1 levels in PCa. In this study, we examined the relationship between the levels of miR-182/96 and hZIP1 using cell culture models. We observed an inverse correlation between hZIP1 and miR-182 levels in several prostate cell cultures: primary normal epithelial cells, normal stromal cells, and prostate cancer cells (PC3 and LNCaP). miR-182 is transcribed in a cluster with miR-96 and the miRNAs have an overlapping mRNA target sequence. As expected, miR-96 expression was identical to that of miR-182, thus also inversely correlated with hZIP1 in prostate cell cultures. Overexpression of miR-182 in both normal and LNCaP cells decreased hZIP1 mRNA levels, further implicating miR-182 as a regulator of zinc transport. Ongoing experiments focus on validating the two putative miR-182/96 binding sites in the 3’UTR of hZIP1 and the effect of miR-182 and miR-96 overexpression on zinc transport. Our results may establish a role for miR-182 in prostatic zinc homeostasis. Because zinc levels are linked to PCa risk, these miRNAs may be useful as a prognostic or diagnostic marker in prostate biopsies to identify patients with high risk of PCa recurrence and for identifying individuals for zinc replacement.
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Honorable Mention
Investigation of SPAG5 gene expression as a molecular marker for breast cancer prognosis
Jennifer Y. Y. Kwan1, Dong-Yu Wang2, Thomas R. Cawthorn3, and Susan J. DoneL 2,3.
1Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada; 2Department of Pathology, University Health Network, Toronto, ON, Canada; 3Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada.
Breast cancer is a clinically heterogeneous disease, making it imperative to use biomarkers, such as the estrogen receptor (ER) and human epidermal growth factor 2 (HER2), to stratify patients into specific prognostic groups. Biomarker stratification provides patients with more accurate diagnoses, predictions of clinical outcomes, and offers targeted therapeutic options.
SPAG5 (Sperm Associated Antigen 5) is involved in the maintenance of spindle-pole integrity, efficient chromosomal alignment, and cell proliferation. Its role in cell division may be a cancer susceptibility factor. Low expression of SPAG5 has been correlated with good prognosis and absence of metastases in ER+ breast cancer. However, its relationship to other molecular subtypes of breast cancer and its functional role in the disease are relatively unknown. The purpose of this study was to investigate the role of SPAG5 in breast cancer through (i) microarray analysis and (ii) correlating its expression with invasiveness, a factor involved in disease progression.
In part (i), SPAG5 expression in breast cancers was evaluated by using five Affymetrix, three Agilent and one Illumina microarray datasets. The associations between SPAG5 expression and various tumor pathologies were analyzed via log2 expression ratios. Pathologies that were investigated included the ER+ subtype, HER2 overexpression, lymph node metastasis, and breast cancer stem cell-like/undifferentiated tumors.
In part (ii), SPAG5 expression across five breast cancer epithelial cell lines of varying invasive ability (MDA-MB-231, MDA-MB-157, BT549, MCF-7, and CAMA1) was investigated. The cells were cultured at normal conditions and lysed using the CytoBusterTM protein extraction reagent. The supernatants were collected by microcentrifugation at 16,000 rcf at 4ºC for 5 minutes. Western blotting was completed using standard techniques.
From the microarray analysis, overexpression of SPAG5 showed strong associations with HER2 overexpression, ER-negative and triple-negative cancers, high grade tumors, metastasis, and poor clinical outcomes in a total of 1595 breast cancers. Via immunoblotting, it was shown that higher levels of SPAG5 accompanied highly invasive cell lines (MDA-MB-231, MDA-MB-157, and BT549) and lower levels occurred in weakly invasive cells (MCF-7 and CAMA1). These results suggest that SPAG5 is a biomarker of malignancy and invasiveness and may be a novel therapeutic target for the prevention of metastasis. Stratification into specific prognostic groups using SPAG5 as a biomarker could lead to early detection and intervention of breast cancers likely to metastasize. It is possible that treatments targeting the modulation of SPAG5 expression levels may also aid in improving clinical outcomes. Ongoing functional analysis of SPAG5 will elucidate its role in breast cancer and metastasis and aid in further characterization of SPAG5.
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Honorable Mention
Targeted nanoparticle drug delivery to radiation inducible GRP78 in tumor vasculature
Ralph J. Passarella1, Daniel Spratt1, Alice Van Der Ende1, John G. Phillips1, Hongmei Wu1, Dennis E. Hallahan2, Eva Harth1, Roberto Diaz3. 1Vanderbilt University, Nashville, TN; 2Washington University in St. Louis, St. Louis, MO; 3Emory University, Atlanta, GA
Purpose/Objectives: GRP78 is a major endoplasmic reticulum chaperone that suppresses stress-induced apoptosis. High GRP78 levels have been shown to correlate with a worse pathologic grade and poor patient survival in various cancer types. GRP78 is also known to confer chemoresistance to tumors and tumor-associated endothelial cells. The goal of this study is to elucidate the mechanism responsible for radiation-mediated neoantigen expression in tumor microvasculature. GIRLRG, a peptide already shown to bind specifically to radiation (XRT) treated, responding tumors, was used in tumor protein extraction to identify its possible cellular targets. GRP78 was identified from the protein extraction as a possible receptor for GIRLRG. We tested the hypothesis that GRP78 is upregulated in response to radiation and can be used as a radiation-inducible drug delivery target.
Methods: Proteins from untreated and XRT treated GL261 gliomas were extracted and incubated with agarose beads coated with GIRLRG and then analyzed by gel electrophoresis and mass spectrometry. A fluorescently labeled antibody to GRP78 was used to assess location of GRP78 expression post-XRT in vitro. WB analysis, immunohistochemistry (IHC), and in vivo imaging of GRP78 expression levels was performed in XRT treated and untreated GL261 and MDA-MB-231 breast tumors 48 hours post-XRT. In vivo blocking experiments were performed to assess GRP78 as the radiation inducible receptor for the GIRLRG peptide. Finally, GIRLRG was conjugated to a slow release nanoparticle drug delivery system encapsulating paclitaxel to target GRP78 in vivo.
Results: Mass spectrometry analysis revealed that GIRLRG specifically extracted a 78k-dalton protein, GRP78 from XRT treated tumor samples. Similarly, WB, IHC, and in vivo imaging analysis showed an upregulation of GRP78 expression in XRT treated GL261 and MDA-MB-231 samples. In vitro imaging showed GRP78 to be upregulated at the cell surface in response to XRT. A blocking antibody to GRP78 was able to significantly abrogate GIRLRG binding post-XRT (p < 0.05). The GIRLRG-nanoparticle drug delivery system produced a significant tumor growth delay relative to all control groups (p < 0.05).
Conclusions: GRP78 has been identified as GIRLRG’s binding partner. We found that GRP78 is induced by XRT. This finding correlates with GRP78’s known stress induced anti-apoptotic function, as XRT is a known cellular stressor. Furthermore, we utilized upregulation of GRP78 post-XRT by combining chemotherapeutic agents to a peptide ligand, GIRLRG, that recognizes the XRT-induced receptor, providing a targeting system that increases chemotherapeutic efficacy for irradiated tumors in vivo. In the future we plan to investigate if blocking GRP78 could potentially radiosensitize tumors by promoting an apoptotic state. Overall the regulation of GRP78 has the potential to provide exciting new targets for anti-cancer strategies.
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Honorable Mention
An improved model for constitutive mTOR inhibition and implications for compromised immune system function
Margaret R. Pruitt1, Shuling Zhang1*, Julie Readinger2*, Mirkka Janka-Junttila3, Richard Robinson1, Wendy DuBois1, Val Bliskovsky1, Julie Wu2, Kaori Sakakibara1, Carol Parent3, Lino Tessarollo4, Pam Schwartzberg2, and Beverly Mock1
1Laboratory of Cancer Biology and Genetics, CCR/NCI, 2Genetic Disease Research Branch, NHGRI, and 3Laboratory of Cellular and Molecular Biology, CCR/NCI, 4Mouse Cancer Genetics Program, CCR/NCI
*These authors contributed equally.
Mammalian Target of Rapamycin (mTOR) contributes to cell metabolism through its incorporation in either of two protein complexes. The first complex, mTORC1, is involved in cell growth and protein synthesis, while the second complex, mTORC2, plays a role in controlling actin cytoskeleton and cell spreading. Rapamycin and its analogs specifically target mTOR and are being evaluated in clinical trials for cancer treatment, making mTOR a particularly important gene to investigate. Still, current in vivo models that block mTOR function have been limited to conditional knock-outs in specific cell types. Because rapamycin mainly influences mTORC1 function, systems using this form of mTOR inhibition are limited in their study of mTORC2. Additionally, knock-out of mTOR and genetic inactivation of the kinase domain of mTOR in mice both resulted in embryonic lethality. The model created by our lab group addresses these obstacles. Neo insertion into the mTOR gene, between exons 12 and 13, interrupted transcription, and thereby substantially reduced mTOR expression in both mTORC1 and mTORC2, while maintaining mouse viability. The effects of reduced mTOR on cells of the immune system are particularly important since the immune system provides a physiological defense against cancer cells. The mTOR inhibitor, rapamycin has frequently been used as an immunosuppressant drug. Homozygous knock-in (KI) mice containing the neo insertion displayed small body, organ, and cell sizes compared to wild-type (WT). Reduced mTOR affected T lymphocyte activation, differentiation, migration and proliferation. Low mTOR also affected B lymphocyte population size, development in the bone marrow (BM), differentiation in the spleen (SPL), and proliferation of resting B cells: 1) the percentages of B220+ B cells were fewer in the spleens and bone marrow of KI mice and the percentages of CD138+ plasma cells were lower in KI spleens; 2) KI mice exhibited a partial block in B cell development in BM; 3) in SPL, the KI mice had more mature B cells than the WT; and 4) CD43- resting B cells from KI mice did not proliferate in response to signaling through the B cell receptor (BCR). Collectively this information demonstrates that the neo-knock-in mouse provides a good model for the long-term consequences of reduced mTOR activity, which may include compromised immune cell activation, differentiation, migration, and proliferation. Responses in these mice may phenocopy effects of treatment with the newer dual kinase inhibitors affecting both mTORC1 and mTORC2 function.
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Honorable Mention
Nuclear expression of the aryl hydrocarbon receptor elicits resistance to aminoflavone prodrug
Karri Stark, Binh Nguyen, Phillip Shelton, Lisa Polin, Patricial M. LoRusso, Angelika M. Burger
Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI; Tigris Pharmaceuticals, Bonita Springs, FL, USA.
Aminoflavone Prodrug (AFP464) is a novel anticancer agent currently in Phase I clinical trials in the US and Europe. Its target is the aryl hydrocarbon receptor (AhR). Upon binding to AhR in the cytoplasm, the receptor/AFP464 complex dimerizes with the AhR nuclear translocator and is shuttled into the nucleus where it induces xenobiotic response via the cytochrome P (CYP) system. CYP1A1 is critical to the activity of AFP464 in cells as it converts the drug into metabolites that cause DNA double strand breaks and cell death. Previous screening of AFP464 in tumor cell lines in vitro has revealed differential cytotoxicity with some lines being sensitive at nanomolar concentrations, while others were resistant even at >100μM. Here, we have investigated the cause of intrinsic resistance of tumor cells to AFP464. We used a panel of 15 breast, ovarian and pancreas cancer cell lines and compared their sensitivity to AFP464 with the cellular localization of the AhR. Five-day MTT assays were used to determine in vitro antiproliferative activity and nude mouse xenografts to assess in vivo efficacy. Immunofluorescence and immunoperoxidase staining of fixed tumor cells and archival tissue were employed to analyze AhR expression. Five breast cancer cell lines (MCF-7, T47D, MDA-MB-468, MCF-7TAM1, MCF-7Her2-18), two ovarian (OVCAR-3, IGROV-1), and two pancreatic (ASPC-1 and Hs766T) lines exhibited cytoplasmic AhR; their inhibitory concentration 50 percent (IC50) ranged from 20nM (MDA-MB-468) to 10μM (Hs766T). The IC50s for breast (MDA-MB-231, Hs578T), ovarian (OVCAR-8) and pancreatic (PANC-1, MIAPaCa2, Capan-1) cell lines with nuclear AhR were between 13μM (OVCAR-8) and >100μM (pancreatic lines). These in vitro results translated into in vivo efficacy: MDA-MB-468 xenografts responded to single agent AFP464 at a dose of 35 mg/kg (given i.v. 4 cycles [q2dx3]) with 62 percent growth inhibition compared to vehicle control, while MDA-MB-231 xenografts were resistant to AFP464, even at the maximal tolerated dose of 70mg/kg. To test whether primary human tumors would also show nuclear or cytoplasmic AhR and to assess the extent to which AhR was expressed, we analyzed 165 archival human tissues comprising breast, pancreas, ovarian and renal cell cancers. We found the 59 percent of all cases had detectable AhR, amongst those 78 percent exhibited cytoplasmic AhR and 22 percent nuclear AhR. Pancreatic (70%) and breast cancers (46%) showed the highest percentage of cytoplasmic AhR.
Together our data indicate that AhR has a distinct distribution pattern in tumor cells. Cytoplasmic AhR expression elicits sensitivity to the AhR ligand AFP464. If AhR is located in the nucleus, xenobiotic response is impaired and AFP464 cannot be activated. Thus, immunohistological analysis of AhR should be considered as a tool in the upcoming Phase II trials in breast and pancreatic cancer to select patients that are most likely to benefit from AFP464 treatment.
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