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Investigating a Small Molecule Inhibitor of Aurora Kinase-A in Ras-driven Pancreas Cancer Cells Brian W. Wakefield1,2 ; Amy E. Chang2 ; Vikas K. Goel, Ph.D2 ; Sunil R. Hingorani, M.D., Ph.D. 2 1Whitman College, Walla Walla, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA 2010 Best Poster Presentation Award RalGTPase Pulldown Assay: RalA activity decreases with AKAi-1. Results Cell Proliferation: AKAi-1 inhibits cell growth in KPC cells. AKA P + P+ Fig. 10: Western Blot. KPC cells treated with AKAi-1 or vehicle for 36 hours. Signal intensity normalized to GST loading control. Inhibition of AKA by AKAi-1 causes downregulation of active RalA. P + P+ AKA Conclusions AKAi-1 treatment in primary KPC cell lines: Inhibits cell proliferation with increased G2/M arrest Increases apoptosis These effects occurred in a concentration-dependent manner. Abstract Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancerrelated deaths in the United States and, with a five-year survival rate of less than 5%, is almost uniformly fatal. Current therapies are only able to improve survival by several weeks and new approaches to the study of this disease are therefore imperative. Over 90% of pancreas cancers have an activating mutation in the KRAS proto-oncogene while 75% have an inactivating mutation in the tumor suppressor gene, TP53. We have shown that endogenous expression of KrasG12D and Trp53R172H in the pancreata of mice induces disease that mimics human pancreas cancer from precursor lesions to fully invasive and metastatic PDA. Direct inhibition of oncogenic KRAS in patients has proved challenging, causing a shift in focus toward its downstream effectors. The small GTPase, RalA, is downstream target of Ras signaling, necessary for Ras-mediated oncogenesis, and is also activated by Aurora Kinase A (AKA). We describe the effects of a small molecule inhibitor of AKA (AKAi-1) on primary tumor cells derived from the pancreata of mice expressing oncogenic KrasG12D and mutant Trp53R172H . Standard assay methods for apoptosis, proliferation, and cell cycle progression were used. These experiments were designed to determine whether this drug should proceed to preclinical trials in the autochthonous mouse model. CDC25 No additive or synergistic effects were observed with simultaneous treatment of AKAi-1 and Gemcitabine. Fig. 3: Aurora Kinase A and Kras converge to activate the small GTPase RalA These data suggest that AKAi-1 may be effective in treating Ras-driven carcinomas and justify testing this inhibitor in our preclinical mouse model. AKAi-1 1 2 5 10 - - - 1 2 5 10 1 2 5 10 (µM) GEM - - - - 1 5 10 1 1 1 1 5 5 5 5 (nM) Fig. 5: Growth inhibition by AKAi-1 in KPC cells. Concentration-dependent inhibition was seen in cells treated with AKAi-1 as compared to DMSO control. No additive or synergistic effects were observed from co-administration of AKAi-1 with Gemcitabine. Cell Cycle: AKAi-1 promotes G2/M arrest in KPC cells Vehicle Cell Cycle Progression Background Targeted expression of oncogenic KrasG12D to the pancreas initiates pancreas cancer1 Concomitant expression of mutant Trp53 cooperates to accelerate widely metastatic disease2 Future Directions 157 To gain a better understanding of the molecular interactions between Kras and RalA, we will quantify the effects on RalA activity of silencing KrasG12D using shRNA in an inducible system. Mouse PDA Cells NIH3T3 Cells Control V33 V28 V24 V14 C. V28 V24 V14 DOX - + - + - + - + - + + + + + AKAi-1 0.5 µM AKAi-1 2.5 µM AKAi-1 - 0.5 1 2.5 5 - - - 0.5 0.5 1 1 (µM) GEM - - - - - - 2 5 2 5 2 5 (nM) Fig. 6: Effect of AKAi-1on cell cycle progression in KPC cells. Concentration-dependent increase in G2/M arrest was seen in AKAi-1-treated cells. No additive or synergistic effects were observed from coadministration of AKAi-1 with Gemcitabine. Fig7: Representative plots. Apoptosis: AKAi-1 increases apoptosis in KPC cells Materials and Methods Experiments were conducted in primary cell lines derived from murine pancreatic tumors (KPC): KPC LSL-KrasG12D activating mutation LSL-Trp53R172H mutation Fig. 1: Conditional LSL-KRASG12D and LSL-Trp53R172H (KPC) alleles and generation of functional mutant via Cre recombinase2 Pdx-1-Cre Fig. 11: Western blot. KPC cells (left) infected with a Doxycyclin-inducible TripZ vector (V) carrying KrasG12D shRNA show silencing of oncogenic Kras when exposed to Doxycyclin. NIH3T3 cells (right) Fig. 2b: Specimen from same mouse showing hepatic metastases (black arrows) and a large head-of-the-pancreas primary tumor ( * ) 2 Fig. 2a: Gross pathological photograph of metastatic PDA in a KPC mouse2 expressing wild-type Kras show no effect. Ras activity will be quantified with a RasGTPase pull-down assay RalA activity will be quantified with a RalGTPase pull-down assay We will administer AKAi-1 to KPC animals with established tumors alone and in combination with Gemcitabine. The results of the in vivo study together with the in vitro data will help determine whether this drug should proceed to clinical trials. Vehicle Control AKAi-1 0.5µM AKAi-1 1.0µM Fig. 8: Representative apoptosis flow cytometry data from KPC cells. Double positive (upper right, circled) population represents late apoptotic and dead cells. Annexin V positive population (lower right) represents early apoptotic cells. Fig. 4: Western blot. KPC tumor cells show high levels of AKA expression and expression of RalA Fig. 2d: Welldifferentiated liver metastasis (m) and surrounding normal tissue (h) 2 Fig. 2c: Welldifferentiated, glandular PDA2 Cell Proliferation: cells were plated in 96-well plates and exposed to a spectrum of AKAi-1 and Gemcitabine concentrations alone and in combination. Proliferation was analyzed by WST-1 and absorbance was read at 450nm. References 1.Hingorani, Sunil et al. “Preinvasive and Invasive Ductal Pancreatic Cancer and its Early Detection in the Mouse.” Cancer Cell 4(2003): 437-450. 2.Hingorani, Sunil et al. “Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.” Cancer Cell 7(2005): 469-483 3.Brunner, Thomas et al. “Farnesyltransferase Inhibitors: An Overview of the Results of Preclinical and Clinical Investigations.” Cancer Research 63(2003): 5656-5668. 4.Lim, Kian-Huat et al. “Aurora-A Phosphorylates, Activates, and Relocalizes the Small GTPase RalA.” Molecular and Cellular Biology 30(2010): 508-523 Cell Cycle and Apoptosis: cells were plated in 60mm plates and exposed to a spectrum of AKAi-1 and Gemcitabine concentrations alone and in combination. Cell cycle was analyzed by propidium iodide staining and flow cytometry. Apoptosis was analyzed by PE-Annexin V staining and flow cytometry. Direct inhibition of constitutively active oncogenic Kras or its upstream regulators ineffective or impossible in clinical populations 3 Targeted inhibition of downstream effectors of Kras may reveal new potential therapeutic targets RalA Activity: Active, phosphorylated RalA was quantified using a RalGTP assay (MilliporeTM ). Active RalA was pulled down by RalBP1 as per manufacturer’s instructions and quantified by western blot. Acknowledgements Hypotheses Targeting specific downstream effectors of Kras maybe an effective method of inhibiting Ras-driven PDA This program was supported by a research grant from the Howard Hughes Medical Institute and Whitman College. I would personally like to thank Dr. Sunil Hingorani, Dr. Vikas Goel, Amy Chang, Natalie Wong, Layken Fletcher, Dr. Paolo Provenzano, and Dr. Libing Feng for their help and guidance during this internship. AKAi-1 - 0.5 1 2.5 5 - - - 0.5 0.5 1 1 (µM) GEM - - - - - - 5 10 5 10 5 10 (nM) Fig. 9: Effect of AKAi-1on apoptosis in KPC cells. Concentration-dependent increase in apoptosis was seen in AKAi-1-treated cells. Effect plateaued near IC50 value for AKAi-1 (2.06 µM). Concentrationdependent increase in apoptosis was also observed for Gemcitabine treatments. No additive or synergistic effects were observed from co-administration of AKAi-1 with Gemcitabine. Knockdown of KrasG12D by shRNA: Cells were infected with Doxycyclin-inducible TripZ vectors containing Kras shRNA. Knockdown efficiency was estimated by western blot. Using RalA to link Aurora Kinase A (AKA) and Kras validates AKA as a therapeutic target in treatment of PDA
Antibody-Mediated Targeting of pH-Responsive “Smart Polymers” Facilitates siRNA Delivery to Cancer Cells Youcef Ouadah 1 , Maria C. Palanca-Wessels, MD, PhD 1 , Richelle Cutler-Strom, PhD 1 , Anthony J. Convertine, PhD 2 , Patrick S. Stayton, PhD 2 , and Oliver W. Press, MD, PhD 1 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Department of Bioengineering, University of Washington, Seattle, WA 2010 Best Poster Presentation Award Conclusions Transcript Knockdown Assays Performed Abstract • Intracellular delivery of small interfering RNA (siRNA) molecules and target transcript knockdown can both be achieved with the novel polyplex system. Total RNA in SKBR3 Cells under polyplex treatment, 15nM siRNA Flow Cytometry: Cells were plated at 200 thousand cells per well and pulse-labeled for one hour with polyplexes containing 20nM AlexaFluor 647-labeled siRNA on ice. Cells were then washed with PBS and resuspended in DMEM + 5% FBS during incubation at 37°C. After specified incubation periods, cells were washed extensively with acid and PBS prior to flow cytometry analysis on a BD FACS Canto system. • Small Interfering RNA (siRNA) molecules are potent and specific inhibitors of gene expression in eukaryotic systems, including humans. neg. control siRNA anti-GAPD siRNA • siRNA internalization is dramatically increased in the Her2/neu expressing cell population and is dependent on anti-Her2/neu antibody targeting. Quantitative RT-PCR: Cells were plated at 50 thousand cells per well and treated in triplicate with polyplex containing 15nM siRNA for 48 hours prior to RNA extraction with an RNeasy Mini Kit (Qiagen). RNA was converted to cDNA using an ABI Taqman Reverse Transcription kit and PCR was performed using an ABI 7900HT Real-Time PCR system. • Delivery of pro-apoptotic siRNA to cancer cells may reduce tumor size and sensitize malignant cells to chemotherapy, however effective delivery to disseminated tumors remains a major obstacle1,2 . • GAPD transcript knockdown in SKBR3 breast cancer cells is increased under Her2/neu targeted polyplex treatment with low corresponding effects on global mRNA levels. Total RNA quantification data indicates little effect on global mRNA expression due to polyplex treatment. 100 80 60 40 20 0 Percent RNA Relative to Untreated BHV1-SA 10H8-SA Trast-SA GAPD mRNA Expression in SKBR3 Cells under polyplex treatment, 15nM siRNA LDH Enzyme Activity Assay: Lactate dehydrogenase (LDH) activity provided a measure of cell viability and was measured using the Cytotoxicity Detection kitPLUS (Roche). Cells were plated in 96-well tissue-culture treated plates at 15 thousand cells per well and incubated continuously with drug at 37°C. Lysates were transferred to new plates at 1:5 dilution with PBS. The assay was performed as described in the kit manual and spectrophotometric measurements were taken at 492nm. • Monoclonal antibodies (mAb) Trastuzumab and 10H8 bind Her2/neu internalizing receptors overexpressed on ovarian and breast cancer cells allowing a mechanism for in vivo siRNA targeting and intracellular delivery3 . • Trastuzumab-SA targeting conjugate performs slightly better than 10H8-SA in both internalization and knockdown studies. neg. control siRNA anti-GAPD siRNA • Dose-dependent cytotoxicity profiles demonstrate that SKOV3 ovarian cancer cells are sensitive to carboplatin, paclitaxel, and 5fluorouracil. Results siRNA Internalization • An additional barrier to intracellular siRNA delivery is degradation of endosomal contents by fusion with the lysosome, so a pH-sensitive endosomolytic “smart polymer” (b-P7v6) has been developed to allow siRNA escape into the cytosol4 . Future Work 140 120 100 80 60 40 20 0 Percent Expression Relative to Untreated 158 • Pulse labeling studies should be performed to demonstrate a more significant difference in target transcript knockdown between Her2/neu targeting and non-targeting conjugates and to better model in vivo delivery strategies. Significant GAPD transcript knockdown is observed in SKBR3 cells with all tested conjugates but is highest with Trastuzumab-SA conjugate. NIH3T3 p185 (Her2/neu+) NIH3T3 wildtype (Her2/neu–) BHV1-SA 10H8-SA Trast-SA p185 time 0 no treatment 88.5 p185 time 0 no conjugate 90.9 p185 time 0 BHV1 87.9 100 p185 time 0 Trast 88.9 Cytotoxicity Assays 80 • In this study the mAb–”smart polymer”–siRNA polyplex is investigated for its ability to induce siRNA internalization through Her2/neu-mediated receptor endocytosis and to knockdown target mRNA transcripts. 60 % of Max • siRNA molecules targeting oncogene expression, such as antiapoptotic protein Bcl2, should be investigated to demonstrate knockdown-induced cytotoxicity. 40 • In addition, cytotoxicity profiles for relevant chemotherapeutics are generated in ovarian cancer cells as a foundation for future studies to demonstrate siRNA-induced enhanced chemosensitivity. 20 Targeting Conjugates no treatment (neg. control) no conjugate BHV1-SA 10H8-SA Trastuzumab-SA Time: 0 hr Cells become sensitive to carboplatin at ~10µM SKOV3 Cell Viability at 72 hours under carboplatin treatment 120 100 80 60 40 10 3 APC-A 0 10 5 10 4 0 10 2 Materials and Methods • Due to the modular design of the polyplex, targeting conjugates can be easily exchanged to investigate other cancer models, such as targeting CD22 surface antigens expressed in B cell lymphomas. p185 no treatment 1hr 97 p185 no conjugate 1hr 96 20 p185 BHV1 1hr 96.3 p185 10H8 1hr 0 80.9 p185 Trast 1hr 91.8 Percent LDH Activity Relative to Untreated wt no treatment 1hr 90.8 wt no conjugate 1hr 81.6 wt BHV1 1hr 86 wt 10H8 1hr 79.3 wt Trast 1hr 77.2 100 100 Time: 1 hr 80 80 siRNA Condensing Cationic Block Polyplex formation 60 • Cytotoxicity assays should be performed in conjunction with polyplex treatment to demonstrate siRNA-induced enhanced sensitivity and to determine optimal chemotherapeutic dosages. % of Max 60 % of Max mAb-SA 40 40 DMAEMA 20 References Paclitaxel’s mechanism of cytotoxicity changes at ~9nM5 . SKOV3 Cell Viability at 72 hours under paclitaxel treatment 20 10 3 10 4 APC-A 0 10 5 10 3 10 4 APC-A 0 10 5 0 10 2 0 10 2 biotin 1. Ozpolat B, Sood AK, Lopez-Berestein G. Nanomedicine based approaches for the delivery of siRNA in cancer. J Internal Medicine 2009; 267: 44-53. 2. De Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nature Reviews Drug Discovery 2007; 6: 443-453. 3. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, Press MJ. Studies of the Her2/neu proto-oncogene in human breast and ovarian cancer. Science 1989; 244: 707-712. 4. Convertine AJ, Benoit DS, Duvall CL, Hoffman AS, Stayton PS. Development of a novel endosomolytic diblock copolymer for siRNA delivery. J Control Release 2009; 133: 221-9. 5. Torres K, Horwitz SB. Mechanisms of taxol-induced cell death are concentration dependent. Cancer Research 1998; 58: 3620-3626. 120 100 80 60 40 20 0 Percent LDH Activity Relative to Untreated siRNA Relative Fluorescence Intensity Under Polyplex Treatment, 20nM siRNA-AF p185 (Her2/neu+) PPAA+DMAEMA+BMA mAb-targeted siRNA containing polyplex pH-Responsive “Smart” Block 0 hour 1 hour 300 250 200 150 100 50 0 Median RFI Relative to Untreated b-P7v6 polymer composition: • PPAA: poly(polypropyl acrylic acid) • DMAEMA: dimethylaminoethyl methacrylate • BMA: butyl methacrylate wildtype (Her2/neu–) Monoclonal antibodies are chemically conjugated to streptavidin (mAb-SA) Her2/neu targeted: Trastuzumab-SA; 10H8-SA Non-targeted: Bovine herpes virus (BHV1-SA) Acknowledgements Dose-dependent cytotoxicity curves demonstrate SKOV3 sensitivity to three commonly prescribed chemotherapeutics in breast and ovarian cancer. SKOV3 Cell Viability at 72 hours under 5-fluorouracil treatment 100 80 60 40 20 0 Percent LDH Activity Relative to Untreated Supported by: NIBIB R01 EB002991; Life Sciences Discovery Fund 08-02; NCI 2K12CA076930-11; Lymphoma Research Foundation FHCRC Summer Undergraduate Research Program (SURP) Michael Press for supplying 10H8 monoclonal antibody and NIH3T3 cell lines Don Hamlin for supplying mAb-streptavidin conjugates Flow cytometry indicates that siRNA internalization is dependent on Her2/neu targeting and is optimized with Trastuzumab-SA conjugate. Cell lines used: SKOV3: Her2/neu expressing ovarian carcinoma SKBR3: Her2/neu expressing breast carcinoma NIH3T3 wt: non-Her2/neu expressing mouse fibroblast NIH 3T3 p185: Her2/neu expressing mouse fibroblast
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