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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
Departments of 1 Gynecologic Oncology, 2 Experimental Therapeutics, 3 Cancer Biology, and 4 Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas
Requests for reprints: Anil K. Sood, Departments of Gynecologic Oncology and Cancer Biology, The University of Texas M.D. Anderson Cancer Center, 1155 Herman Pressler, CPB6.3244, Unit 1362, Houston, TX 77030. Phone: 713-745-5266; Fax: 713-792-7586; E-mail: asood{at}mdanderson.org.
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Abstract |
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Introduction |
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We have used an ovarian cancer xenograft mouse model to examine the efficacy of in vivo gene silencing by siRNA. Ovarian cancer is associated with the highest mortality among all gynecologic malignancies, with an estimated 22,220 cases and 16,210 deaths in the United States in 2005 (11). The majority of ovarian cancer patients respond to initial therapy of tumor cytoreductive surgery and platinum-based chemotherapy, but of these, 
70% will recur and succumb to disease (12). Therefore, novel therapeutic strategies are urgently needed to improve the outcome of women with ovarian cancer. Fortunately, ovarian cancer has a favorable mouse model. I.p. injected ovarian cancer cells form tumors resembling human cancer in growth pattern, and their response to therapy tends to be predictive of response in human patients (13, 14).
EphA2 is a tyrosine kinase receptor in the ephrin family that plays a key role in neuronal development (15, 16). In adults, it is expressed to a low degree, primarily in epithelial cells (17). Several investigators have reported EphA2 overexpression in human cancers (18–21), and we have shown that the high rate of overexpression in ovarian cancer is associated with poor clinical outcome (22). EphA2 can function as an oncoprotein (23), and down-regulation reduces tumorigenicity in preclinical studies of breast and pancreatic cancer (24–26), making it an ideal therapeutic target.
We have previously used liposomes composed of the neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to deliver antisense oligonucleotides in vivo (27). Here, we sought to determine the feasibility and effectiveness of delivering EphA2-targeting siRNA in DOPC. Therapeutic delivery of siRNA directed against EphA2 resulted in decreased protein expression in the tumor and remarkably decreased tumor growth when combined with chemotherapy in an orthotopic mouse model of ovarian cancer.
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Materials and Methods |
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Small interfering RNA constructs and in vitro delivery. siRNA was purchased from Qiagen (Valencia, CA) in three formulations. A nonsilencing siRNA sequence, shown by BLAST search to not share sequence homology with any known human mRNA (target sequence 5'-AATTCTCCGAACGTGTCACGT-3') and tagged with Alexa 555, was used to determine uptake and distribution in various tissues when given in vivo. siRNA with the target sequence 5'-AATGACATGCCGATCTACATG-3', designed and shown (26) to target mRNA of the receptor tyrosine kinase EphA2, was used to down-regulate EphA2 in vitro and in vivo. A nonsilencing siRNA construct (sequence as above without an Alexa 555 tag) was used as control for EphA2-targeting experiments. For in vitro delivery, siRNA (5 µg) was incubated with 30 µL RNAiFect transfection reagent (Qiagen) for 10 minutes at room temperature and added to cells in culture at 80% confluence in 35 mm culture plates. The medium was changed 6 hours later, and cells collected after 48 hours as lysate for Western blot analysis.
Liposomal preparation. siRNA for in vivo delivery was either given naked (without transfection agent), incorporated into N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP; Roche, Indianapolis, IN), or incorporated into DOPC. DOPC and siRNA were mixed in the presence of excess tertiary butanol at a ratio of 1:10 (w/w) siRNA/DOPC. Tween 20 was added to the mixture in a ratio of 1:19 Tween 20:siRNA/DOPC. The mixture was vortexed, frozen in an acetone/dry ice bath, and lyophilized. Before in vivo administration, this preparation was hydrated with normal 0.9% saline at a concentration of 15 µg/mL to achieve the desired dose in 150 to 200 µL per injection. To estimate the quantity of siRNA not taken up by liposomes, free siRNA was separated from liposomes using 30,000 nominal molecular weight limit filter units (Millipore Corp., Billerica, MA). The liposomal suspension was added to the filters and centrifuged at 5,000 x g for 40 minutes at room temperature. Fractions were collected, the material trapped in the filter was reconstituted with 0.9% saline, and siRNA of the collected fraction and the elute were measured by spectrophotometry.
Orthotopic in vivo model of advanced ovarian cancer and tissue processing. Female athymic nude mice (NCr-nu) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD) and housed in specific pathogen-free conditions. They were cared for in accordance with guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and the USPHS "Policy on Human Care and Use of Laboratory Animals," and all studies were approved and supervised by The University of Texas M.D. Anderson Cancer Center Institutional Animal Care and Use Committee. Tumors were established by i.p. injection of cells prepared as above. This model reflects the i.p. growth pattern of advanced ovarian cancer, as intra-abdominal spread is the main mechanism of ovarian cancer metastasis (12–14). Studies to determine uptake of single-dose fluorescent siRNA in tissue or silencing potential of single-dose siRNA against EphA2 were initiated once i.p. tumors reached a size of 0.5 to 1.0 cm3 as assessed by palpation (17 days after injection). Liposomal siRNA (5 µg) was given as a 200 µL i.v. bolus into the tail vein under normal pressure, and tumor and other tissues were harvested at various time points after injection (1 hour, 6 hours, 48 hours, 4 days, 7 days, or 10 days). Tissue specimens were snap frozen for lysate preparation, fixed in formalin for paraffin embedding, or frozen in OCT medium for frozen slide preparation. For long-term experiments to assess tumor growth, therapy began 1 week after tumor cell injection. Paclitaxel (100 µg) or vehicle was injected i.p. once weekly; siRNAs (nonspecific or EphA2 targeting, 150 µg/kg) in liposomes, or empty liposomes, were injected twice weekly i.v. in 150 to 200 µL volume (depending on mouse weight) with normal pressure. Mice (n = 10 per group) were monitored for adverse effects, and tumors were harvested after 4 weeks of therapy or when any of the mice began to appear moribund. Mouse weight, tumor weight, and distribution of tumor were recorded. Vital organs were also harvested and necropsies were done by a board-certified pathologist for evidence of tissue toxicity.
Immunofluorescence and confocal microscopy. Tissue for immunofluorescence was collected from sacrificed mice, immediately placed in OCT medium, and rapidly frozen. Frozen sections were cut at 8 µm sections for conventional microscopy and 30 µm sections for confocal microscopy. Tissue was fixed with acetone and either examined immediately or costained for f4/80 (to detect scavenging macrophages) or CD31 (to detect endothelial cells). For immunofluorescence detection, slides were blocked with 5% normal horse serum and 1% normal goat serum (Invitrogen, Carlsbad, CA) in PBS, exposed to 10 µg/mL anti-f4/80 antibody (Serotec, Oxford, United Kingdom) or 0.625 µg/mL anti-CD31 antibody in blocking solution overnight at 4°C, washed with PBS, and exposed to 4 µg/mL anti-rat antibody-Alexa 488 (Molecular Probes, Eugene, OR) in blocking solution for 1 hour at room temperature. Slides were washed with PBS, exposed to either 1.0 µg/mL Hoescht (Molecular Probes, in PBS) or 10 nmol/L Sytox green (Molecular Probes, in PBS) for 10 minutes to stain nuclei, washed, and covered with propylgallate and coverslips for microscopic evaluation. Conventional microscopy was done with a Zeiss AxioPlan 2 microscope (Carl Zeiss, Inc., Germany), Hamamatsu ORCA-ER Digital camera (Hamamatsu Corp., Japan), and ImagePro software (Media Cybernetics, Silver Spring, MD). Fluorescence in three dimensions within 30 µm sections was examined with a Zeiss LSM 510 confocal microscope and LSM 510 Image Browser software (Carl Zeiss).
Western blot. Cultured cell lysates were prepared by washing cells with PBS followed by incubation in modified radioimmunoprecipitation assay buffer (RIPA) lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton, 0.5% deoxycholate plus 25 µg/mL leupeptin, 10 µg/mL aprotinin, 2 mmol/L EDTA, 1 mmol/L sodium orthovanadate; Sigma Chemical Co., St. Louis, MO) for 10 minutes at 4°C. Cells were scraped from plates and centrifuged at 13,000 rpm for 20 minutes at 4°C and the supernatant was stored at –80°C. To prepare lysate from snap-frozen tissue, 30 mm3 cuts of tissue were incubated on ice in RIPA for 3 hours, mortar and pestle disrupted and homogenized, and centrifuged, and the supernatant was stored at –80°C. Samples from three regions of the tumor were collected and tested individually. Protein concentrations were determined using a BCA Protein Assay Reagent kit (Pierce Biotechnology, Rockford, IL) and subjected to 10% SDS-PAGE separation. Samples transferred to a nitrocellulose membrane by semidry electrophoresis (Bio-Rad Laboratories, Hercules, CA) were incubated with 0.625 µg/mL anti-EphA2 antibody (Upstate, Lake Placid, NY) overnight at 4°C, detected with 1 µg/mL horseradish peroxidase (HRP)–conjugated anti-mouse IgG (Amersham, Piscataway, NJ), and developed using enhanced chemiluminescence detection kit (Pierce Biotechnology). Membranes were tested for ß-actin (0.1 µg/mL anti-ß-actin primary antibody; Sigma Chemical) to confirm equal loading.
Immunohistochemistry. Formalin-fixed, paraffin-embedded sections were deparaffinized by sequential washing with xylene, 100% ethanol, 95% ethanol, 80% ethanol, and PBS. Antigen retrieval was done by heating in steam cooker in 0.2 mol/L Tris-HCl (pH 9.0) for 20 minutes. After cooling and PBS wash, endogenous peroxide was blocked with 3% H2O2 in methanol for 5 minutes. Nonspecific proteins and exposed endogenous mouse IgG antibodies were blocked with 0.13 µg/mL mouse IgG Fc blocker (The Jackson Laboratory, Bar Harbor, ME) in 0.5% blocking agent (TSA Biotin System kit, Perkin-Elmer, Boston, MA) overnight at 4°C. Slides were incubated in primary antibody (5 µg/mL mouse anti-EphA2 clone EA5, a kind gift of Dr. Michael Kinch, MedImmune, Inc., Gaithersburg, MD) for 4 hours at 4°C and washed followed by 1.5 µg/mL biotinylated horse anti-mouse (Vector Labs, Burlingame, CA) for 1 hour at room temperature. The secondary antibody signal was enhanced with 0.75 µg/mL streptavidin-HRP (DakoCytomation, Carpinteria, CA) for 30 minutes, detected with 3,3'-diaminobenzidine (DAB; Phoenix Biotechnologies, Huntsville, AL) substrate for 7 minutes, and counterstained with Gill no. 3 hematoxylin (Sigma Chemical) for 20 seconds.
Microvessel density. To determine microvessel density (MVD), collected tissue was frozen in OCT with liquid nitrogen. Eight-micron sections were fixed in acetone for 10 minutes, washed in PBS, and blocked with 10% fish gelatin for 10 minutes. Slides were exposed to rat anti-mouse CD31 for 2 hours at room temperature, diluted in blocking solution, and exposed, after three PBS washes (5 minutes each), to anti-rat IgG conjugated to HRP. HRP was detected with DAB for 10 minutes and counterstained with Gill no. 3 hematoxylin for 20 seconds. To calculate MVD, five representative photographs were taken of each slide (1 slide per mouse, 5 slides per group), and the number of vessels per field (final magnification, x100) was counted by an examiner blinded to treatment group. A vessel was defined as an open lumen with at least one adjacent CD31-positive cell. Multiple positive cells beside a single lumen are counted as one vessel.
Statistical considerations. For in vivo therapy experiments, 10 mice in each group were used as directed by a power analysis to detect a 50% reduction in tumor size (ß error = 0.2). Continuous variables (tumor size and MVD) were analyzed for statistical significance (achieved if P < 0.05) with Student's t test for two-group comparisons and ANOVA for multiple-group comparisons. If values were not normally distributed, the Mann-Whitney rank sum test was used using Stata 8 software (College Station, TX).
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Results |
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65% of the siRNA are incorporated into liposomes. In our experience, the liposomal-nucleic acid complexes are stable for at least 4 weeks when stored at –20°C.
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To confirm that the siRNA was present in tumor cells and not simply scavenged by macrophages, separate slides were stained for f4/80 to identify scavenging macrophages. These macrophages are seen to surround nests of tumor cells that contain perinuclear siRNA and have about the same rate of siRNA uptake as tumor cells (Fig. 1F), suggesting that siRNA is delivered directly into the tumor cells. To confirm that the fluorescent signal was not a contaminating secondary antibody or an artifact of processing, 30 µm sections were examined with confocal microscopy. This technique permitted signal detection within the middle of tissue rather than from surface emissions alone. After evaluating emissions at multiple depths, a three-dimensional cross-section was created. Lateral views clearly show the presence of fluorescently tagged siRNA within tissue parenchyma (Fig. 1G). In this view, fluorescent emission from macrophage staining (green) was noted only at the surface, because the detecting antibody is too large to penetrate tissue. Emission from nuclear staining (blue) is the result of a dye (Hoescht) small enough to penetrate tissue. Tumors collected at 4, 7, and 10 days after a single injection were also noted to retain siRNA fluorescence. However, because this fluorescently tagged siRNA should be a nonsilencing construct, longevity after administration of a mRNA-targeting construct is likely to be of shorter duration.
Tissue distribution of small interfering RNA after delivery by conventional methods. To compare tumor delivery of siRNA with other methods, we injected siRNA i.v. either without a transfection agent (naked) or complexed in DOTAP. After administration of naked fluorescent siRNA, fluorescence was rarely observed (2% of x40 fields, <1% of cells; Fig. 2A) although notably present in the desired perinuclear location in positive cells. Administration of siRNA complexed with DOTAP showed sporadic presence of fluorescence within tumor tissue (7% of all fields examined). However, the observed fluorescence was primarily adjacent to CD31-positive endothelial cells (shown green in Fig. 2B), bringing into question whether the liposomal contents were released or trapped in the vasculature. Tissue obtained after siRNA delivery in DOPC and stained with CD31 showed that delivery was not restricted to the vasculature and is efficiently delivered deep into the tumor parenchyma. Our DOPC liposome preparation was associated with an estimated 10-fold improvement in delivery of siRNA compared with DOTAP and 30-fold improvement over naked siRNA.
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Down-regulation of EphA2 with liposomal small interfering RNA. We have shown previously that EphA2 is overexpressed by a large percentage of patients with ovarian cancer and that overexpression is predictive of poor outcome (22). Furthermore, this protein has low relative expression in the adult and so is an attractive tumor selective target. Therefore, we used EphA2 as a model to test the efficacy of siRNA therapy. In vitro, both HeyA8 and SKOV3ip1 ovarian cancer cell lines transfected with EphA2 siRNA showed a 95% decrease in EphA2 expression compared with transfection with control siRNA as determined by Western blot analysis (data not shown). Subsequently, we tested the ability of DOPC liposomal siRNA to silence EphA2 in an orthotopic in vivo model. EphA2-targeting siRNA-DOPC was given to tumor-bearing mice and tumor collected at various time points. Measurement of EphA2 by Western blot of tumor lysate (Fig. 4A) and by immunohistochemistry (Fig. 4D) showed that tumor collected 48 hours following administration of single-dose anti-EphA2 siRNA had significantly decreased EphA2 expression compared with treatment with a nonspecific siRNA (Fig. 4B) or naked siRNA (Fig. 4C). Expression of EphA2 remained suppressed at 4 days, was recovering after 7 days, and had returned to normal levels by 10 days. Therefore, we used twice-weekly dosing of anti-EphA2 siRNA for subsequent therapy experiments.
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Effects of EphA2-targeted therapy on vascular density. To explore the mechanisms involved in the reduced tumor formation with this therapy, we first confirmed that EphA2 levels remained low with long-term therapy. Tumors harvested at the conclusion of therapy trials were subjected to immunohistochemistry for EphA2 (Fig. 6A). Both groups treated with EphA2-targeting siRNA showed 50% decrease in distribution of expression compared with control siRNA or control siRNA plus paclitaxel. The intensity of staining was not significantly different among the groups, suggesting that gene silencing is effective if delivery is achieved.
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No toxicities were observed by behavioral changes, such as eating habits and mobility in animals treated with liposomal siRNA preparations, both those that are nonsilencing and those targeting EphA2. Mouse weights were not significantly different among the five groups of animals, suggesting that eating and drinking habits were not affected. Organ sections were reviewed by a board-certified pathologist, and after 5 weeks of therapy, no histologic toxicities were detected in the liver, kidney, heart, lung, or brain. A slight increase in the size of the white pulp of the spleen was noted in all four siRNA groups, which may be indicative of a general inflammatory response.
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Discussion |
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Because delivery in this study was efficient to other vital organs, most notably in the liver and kidney, this method may be used in noncancerous conditions shown to be amenable to siRNA therapy in preclinical models, such as viral hepatitis (9, 29) and HIV (30). However, this mode of delivery is not tissue specific, so it will be important that the gene chosen to down-regulate with siRNA is not crucial to function by normal cells. Alternatively, further modifications of the liposome may allow tumor-selective delivery (31, 32).
The first demonstration that siRNA had activity in vivo was in the hydrodynamic injection of naked siRNA that effectively decreased luciferase expression in the livers of mice (29). Along with confirmatory reports of high-pressure i.v. injection (33, 34), others have shown that siRNA has activity in vivo using delivery in viral vectors (8, 9), retinal electroporation (35), and direct intracellular (36), intratumoral (37), intravitreal (38), intranasal (39), and intrathecal (40) administration. Although these methods are useful in a preclinical setting, their delivery methods and the climate of viral gene therapy make clinical applicability limited.
Sorensen et al. effectively reduced tumor necrosis factor-
expression in the liver and spleen by delivering siRNA packaged in cationic liposomes (DOTAP), protecting mice from a lethal dose of lipopolysaccharide (41). We have found that DOTAP accumulates near the vasculature and is preferentially taken up by the liver and spleen, limiting its effectiveness in systemic or antitumor therapy. Soutschek et al. have reported that siRNA conjugated with cholesterol improved delivery to multiple organs and that down-regulation of ApoB was achieved in liver and jejunum (10). However, the effects of cholesterol conjugation on siRNA activity and duration of effect, efficiency of uptake in tumors, and toxicities are not known. Duxbury et al. have shown that systemic delivery of naked siRNA-targeting FAK (42), EphA2 (26), or CEACAM6 (43) down-regulated protein expression and decreased growth of a single s.c. injected malignant pancreatic cell line. It is possible that naked siRNA may be effectively delivered to s.c. sites, but not to orthotopic sites, as supported by our results. To the best of our knowledge, others have not reported successful therapy with naked unaltered siRNA in other cancer models.
Recent studies suggest that the specificity of siRNA may not be as absolute as initially hoped. An analysis of gene expression profiling suggested that RNA down-regulation might occur with as few as 11 complementary base pairs within the 21-bp siRNA sequence (44). A recent study of commonly used siRNA sequences found that 75% of these sequences had nonspecific targeting (45). Therefore, in siRNA design, a BLAST search for cross-reactive 21-bp sequences is insufficient to have confidence that the mRNA of interest is the only target. Furthermore, siRNAs may bind mRNA of only near-perfect complementarity and prevent translation without degradation (46). This is the mechanism used by endogenously produced microRNAs (miRNA), believed to be another method of natural regulation of gene expression (47). Crossover of siRNA into the miRNA pathway or down-regulation by partial homology seem to be minimal and require participation of several siRNA sequences, but this potential should caution conclusions made regarding the specificity of gene down-regulation. It is difficult at this time to speculate which particular proteins could be "off-site" targets of nonspecific siRNA silencing. Studies with microarray analysis or reporter arrays may allow such projections to be made in the future (44, 48). Another level of questionable specificity of siRNA introduction lies in activation of the innate immune system. siRNA therapy has, in some circumstances, been shown to activate IFN (49, 50). Of course, in the treatment of cancer, IFN induction may be of additional benefit, as long as toxicities are limited. This is supported by our finding that therapy with a nonspecific siRNA construct results in some reduction in tumor growth compared with empty liposomes.
Toxicities of liposomes are believed to be limited. Liposomal chemotherapy is routinely used in treatment of ovarian and other cancers (51). In a phase I trial with cationic liposomes carrying a plasmid encoding the E1A gene, fever and pain 3 hours after treatment were the dose-limiting toxicities (52). Although this is the best estimation of side effects we can currently predict, delivery of siRNA is less likely to be recognized as foreign, and host response will almost certainly differ.
The charge of the liposome affects the tissue specificity of liposomal uptake. Macrophages seem to preferentially take up negatively charged liposomes (53). Different malignant cell lines have varying uptake patterns regarding positive, neutral, or negative charges, and in vivo uptake patterns may differ further (53). Liposomal makeup also influences cellular toxicity, with siRNA delivery using a liposome with a higher proportion of neutral lipids leading to less cellular toxicity without compromising ability to down-regulate gene expression in vitro (54). Clearly, a complete understanding of the best liposomal makeup for delivery of therapeutic substances is still evolving. It is possible that with siRNA delivery the use of a neutral lipid, such as DOPC, allows a balance among efficient uptake of the siRNA into a liposome at preparation, uptake of the liposome into a cell, and breakdown of the intracellular liposome with release of siRNA contents into the cytoplasm.
EphA2 is an attractive target for antitumor therapies. It is minimally expressed in adults, being limited to some epithelial tissues (55), and the EphA2 knockout mouse is phenotypically normal (56). However, EphA2 is overexpressed by several cancers (21), including ovarian, in which it is associated with poorer survival (22). Furthermore, the receptor primarily exhibits carcinogenic properties through high levels of the unphosphorylated form. Therefore, decreasing total EphA2 levels are more likely to be effective than attempts to block its activation. We have shown that EphA2-targeting siRNA therapy leads to a decrease in MVD. Others have seen an antiangiogenic effect with EphA2 down-regulation (25), and we have seen this with antibody-based approaches to decrease EphA2 expression in vivo.5 Delineation of biological pathways dependent on EphA2 is difficult, because EphA2 modulation has little effect on monolayer cell culture properties (21). Further studies of in vivo–treated tissues may help to define other mechanisms affected by EphA2 overexpression.
In vivo delivery of siRNA in experimental models has been shown to provide feasibility for use in humans. Liposomal delivery of drugs is established and safe, and their use for siRNA delivery may make this therapeutic modality clinically attractive. We have shown that using DOPC-complexed siRNA allows delivery to tumor and other tissues, with corresponding gene targeting and reduced tumor growth. With further study and a cautious approach, this is a model that can be taken into a clinical setting for cancer therapy as well as for other conditions amenable to specific gene down-regulation.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Footnotes |
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Received 2/17/05. Revised 5/ 2/05. Accepted 5/18/05.
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 W. M. Merritt, Y. G. Lin, W. A. Spannuth, M. S. Fletcher, A. A. Kamat, L. Y. Han, C. N. Landen, N. Jennings, K. De Geest, R. R. Langley, et al. Effect of Interleukin-8 Gene Silencing With Liposome-Encapsulated Small Interfering RNA on Ovarian Cancer Cell Growth J Natl Cancer Inst, March 5, 2008; 100(5): 359 - 372. [Abstract] [Full Text] [PDF]
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 C. N. Landen Jr, M. J. Birrer, and A. K. Sood Early Events in the Pathogenesis of Epithelial Ovarian Cancer J. Clin. Oncol., February 20, 2008; 26(6): 995 - 1005. [Abstract] [Full Text] [PDF]
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M. J. Gray, G. Van Buren, N. A. Dallas, L. Xia, X. Wang, A. D. Yang, R. J. Somcio, Y. G. Lin, S. Lim, F. Fan, et al. Therapeutic Targeting of Neuropilin-2 on Colorectal Carcinoma Cells Implanted in the Murine Liver J Natl Cancer Inst, January 16, 2008; 100(2): 109 - 120. [Abstract] [Full Text] [PDF]
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J. Halder, Y. G. Lin, W. M. Merritt, W. A. Spannuth, A. M. Nick, T. Honda, A. A. Kamat, L. Y. Han, T. J. Kim, C. Lu, et al. Therapeutic Efficacy of a Novel Focal Adhesion Kinase Inhibitor TAE226 in Ovarian Carcinoma Cancer Res., November 15, 2007; 67(22): 10976 - 10983. [Abstract] [Full Text] [PDF]
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C. N. Landen Jr., Y. G. Lin, G. N. Armaiz Pena, P. D. Das, J. M. Arevalo, A. A. Kamat, L. Y. Han, N. B. Jennings, W. A. Spannuth, P. H. Thaker, et al. Neuroendocrine Modulation of Signal Transducer and Activator of Transcription-3 in Ovarian Cancer Cancer Res., November 1, 2007; 67(21): 10389 - 10396. [Abstract] [Full Text] [PDF]
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 M. B. Nilsson, G. Armaiz-Pena, R. Takahashi, Y. G. Lin, J. Trevino, Y. Li, N. Jennings, J. Arevalo, S. K. Lutgendorf, G. E. Gallick, et al. Stress Hormones Regulate Interleukin-6 Expression by Human Ovarian Carcinoma Cells through a Src-dependent Mechanism J. Biol. Chem., October 12, 2007; 282(41): 29919 - 29926. [Abstract] [Full Text] [PDF]
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C. N. Landen Jr., Y. G. Lin, A. Immaneni, M. T. Deavers, W. M. Merritt, W. A. Spannuth, D. C. Bodurka, D. M. Gershenson, W. R. Brinkley, and A. K. Sood Overexpression of the Centrosomal Protein Aurora-A Kinase is Associated with Poor Prognosis in Epithelial Ovarian Cancer Patients Clin. Cancer Res., July 15, 2007; 13(14): 4098 - 4104. [Abstract] [Full Text] [PDF]
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Y. G. Lin, A. B. Kunnumakkara, A. Nair, W. M. Merritt, L. Y. Han, G. N. Armaiz-Pena, A. A. Kamat, W. A. Spannuth, D. M. Gershenson, S. K. Lutgendorf, et al. Curcumin Inhibits Tumor Growth and Angiogenesis in Ovarian Carcinoma by Targeting the Nuclear Factor-{kappa}B Pathway Clin. Cancer Res., June 1, 2007; 13(11): 3423 - 3430. [Abstract] [Full Text] [PDF]
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S. A. Hammond, R. Lutterbuese, S. Roff, P. Lutterbuese, B. Schlereth, E. Bruckheimer, M. S. Kinch, S. Coats, P. A. Baeuerle, P. Kufer, et al. Selective Targeting and Potent Control of Tumor Growth Using an EphA2/CD3-Bispecific Single-Chain Antibody Construct Cancer Res., April 15, 2007; 67(8): 3927 - 3935. [Abstract] [Full Text] [PDF]
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 J. D. Wright and D. G. Mutch Getting the Message Out: RNA Interference of Human Papillomavirus Reproductive Sciences, January 1, 2007; 14(1): 6 - 7. [PDF]
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C. N. Landen Jr., C. Lu, L. Y. Han, K. T. Coffman, E. Bruckheimer, J. Halder, L. S. Mangala, W. M. Merritt, Y. G. Lin, C. Gao, et al. Efficacy and Antivascular Effects of EphA2 Reduction With an Agonistic Antibody in Ovarian Cancer. J Natl Cancer Inst, November 1, 2006; 98(21): 1558 - 1570. [Abstract] [Full Text] [PDF]
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J. Halder, A. A. Kamat, C. N. Landen Jr., L. Y. Han, S. K. Lutgendorf, Y. G. Lin, W. M. Merritt, N. B. Jennings, A. Chavez-Reyes, R. L. Coleman, et al. Focal Adhesion Kinase Targeting Using In vivo Short Interfering RNA Delivery in Neutral Liposomes for Ovarian Carcinoma Therapy. Clin. Cancer Res., August 15, 2006; 12(16): 4916 - 4924. [Abstract] [Full Text] [PDF]
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 A. K. Sood, C. N. Landen, A. Sanguino, and G. Lopez-Berestein Therapeutic gene silencing in vivo using RNA interference Am. Assoc. Cancer Res. Educ. Book, April 1, 2006; 2006(1): 353 - 358. [Full Text] [PDF]
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A. A. Kamat, M. Fletcher, L. M. Gruman, P. Mueller, A. Lopez, C. N. Landen Jr., L. Han, D. M. Gershenson, and A. K. Sood The Clinical Relevance of Stromal Matrix Metalloproteinase Expression in Ovarian Cancer Clin. Cancer Res., March 15, 2006; 12(6): 1707 - 1714. [Abstract] [Full Text] [PDF]
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J. A. Hickson, D. Huo, D. J. Vander Griend, A. Lin, C. W. Rinker-Schaeffer, and S. D. Yamada The p38 Kinases MKK4 and MKK6 Suppress Metastatic Colonization in Human Ovarian Carcinoma Cancer Res., February 15, 2006; 66(4): 2264 - 2270. [Abstract] [Full Text] [PDF]
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