414VACCINATION WITH RAD5 CARRYING A FUSION CONSTRUCTOF PROSTATE-SPECIFIC ANTIGENS GENERATES EFFECTORT CELLS

Felipe Rosso*, Jeffrey Holzbeierlein, Peter Van Veldhuizen, DevKaran, Kansas City, KS

INTRODUCTION AND OBJECTIVES: Immunotherapy hasemerged as a promising approach in the development of novel thera-pies for prostate cancer. While most currently approved vaccines aretargeted toward a single antigen, the heterogeneity of prostate cancermay create a limitation toward achieving a maximal effector responsewith this approach. Our goal is to expand the range of anti-tumorresponses by creating a multi-antigenic T cell response utilizing asingle immunization with a recombinant viral construct containing afusion of two known prostate cancer antigens.

METHODS: A fusion construct (PSPA) containing prostate-specific antigen (PSA) and prostate stem cell antigen (PSCA) wascreated and combined with a recombinant adenovirus type 5 (rAd5)vector and injected into mice. Following immunization, spleen cellsuspensions were processed to analyze the production of IFN-� in thevaccinated mice compared to control. In vivo cytotoxic T cell assaysand tumor growth challenge studies were compared in the vaccinatedgroup compared to control mice. Student’s t-test was used to determinelevels of significance among groups studied.

RESULTS: Average immune responses of anti-PSA(p�0.037) and anti-PSCA (p�0.026) CD8 T cells following Ad5-PSPA immunization were significantly higher as compared to con-trols. A reduced level of anti-PSA IFN-� was noted in comparison toanti-PSCA following immunization (p�0.056). Immunized miceshowed a strong cytotoxic response, being able to lyse 80% and100% of PSA or PSCA coated target cells, respectively (p�0.034and 0.005, respectively). Immunized mice demonstrated a signifi-cant inhibition of tumor outgrowth derived from PSA-expressingmurine tumor cell lines with a total survival rate of 60%.

CONCLUSIONS: Immunization of mice with rAd5 vector carry-ing a fusion construct of PSA and PSCA simultaneously induces theexpansion of anti-PSA and anti-PSCA CD8 T cells. The developedantigen-specific T cell responses were efficient in eliminating targetcells expressing cognate antigens, and strong anti-tumor immunity waselicited when challenged with prostate tumor cell lines. The effectorresponse caused by the use of multiple antigens may have a syner-gistic effect to significantly reduce tumor burden and have importantimplications for human clinical trials targeted at prostate cancer.

Source of Funding: Hearst Foundation and the VeteransAffairs Medical Center

415ANDROGRAPHOLIDE TARGETS AR PATHWAY INCASTRATION-RESISTANT PROSTATE CANCER

Chengfei Liu*, Meng Sun, Wei Lou, Jaeyeon Chun, NagalakshmiNadiminty, Christopher Evans, Allen Gao, Sacramento, CA

INTRODUCTION AND OBJECTIVES: The Androgen Receptorpathway plays a central role in growth and survival of castration-resistant prostate cancer (CRPC). The first line of treatment of andro-gen-dependent prostate cancer is the use of androgen-deprivationtherapy. However, most of the patients will eventually relapse due todevelopment of CRPC. Bicalutamide is used as a second line treatmentoption for patients who fail androgen&endash;deprivation therapy. Un-fortunately, patients will also develop a resistance to bicalutamidetreatment. Thus develop a strategy to treat bicalutamide resistantprostate cancer is urgently needed.

METHODS: We have previously identified andrographolide cansuppress tumor growth of prostate cancer cells by screening com-pounds from the Natural compound library. LNCaP and C4-2 cells weretreated with either bicalutamide or andrographolide in FBS or charcoal-stripped FBS conditions. Cell growth was determined and the levels of

AR expression were measured by qRT-PCR and Western blots. Theeffects on PSA and NKX3.1 were also examined by ELISA and lu-ciferase reporter assays. AR nuclear translocation was examined byimmunofluorescence assays. The effects of andrographolide on therecruitment of AR to the androgen responsive genes were examined byChromatin Immunoprecipitation (ChIP) Assays.

RESULTS: We identified andrographolide, a diterpenoid lac-tone isolated from a traditional Chinese and Indian medicinal plantAndrographis paniculata which exhibit a wide spectrum of biologicalactivities of therapeutic importance, can significantly inhibit prostatecancer cell growth and induce apoptosis. Andrographolide not onlyinhibits AR activity, such as inhibition of the expression of androgenregulated genes (PSA and NKX3.1), but is also able to down regulateAR expression at both mRNA and protein levels and prevents itsnuclear translocation. Andrographolide prevents the binding of Hsp90to AR, resulting in proteasome-mediated AR degradation. Furthermore,andrographolide inhibits bicalutamide resistant C4-2 cell growth byreducing AR expression and activity. Combination of andrographolidewith bicalutamide achieved synergistic effect in inhibition of C4-2 cellgrowth and AR expression.

CONCLUSIONS: We have characterized and identified an-drographolide as a potential therapeutic agent for prostate cancer byinhibiting androgen receptor signaling. Andrographolide inhibits thegrowth of castration resistant prostate cancer after failure of bicaluta-mide treatment. Andrographolide could be developed as a potentialanti-androgen against castration-resistant prostate cancer.

Source of Funding: R01CA140468

416IMPACT OF COMBINED HDAC AND MTOR INHIBITION ON THEINVASIVE POTENTIAL OF PROSTATE CANCER CELLS

Steffen Wedel*, Lucasz Hudak, Jasmina Makarevic,Jens-Michael Seibel, Eva Juengel, Axel Haferkamp, Roman Blaheta,Frankfurt am Main, Germany

INTRODUCTION AND OBJECTIVES: The concept of molecu-lar tumor targeting has recently become standard in the therapy of renalcell carcinoma and might also provide new hope in the treatment ofadvanced prostate cancer. We evaluated metastasis blocking proper-ties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA)and the mammalian target of rapamycin (mTOR) inhibitor RAD001 onprostate cancer cell lines.

METHODS: RAD001 or VPA were applied to PC-3, DU-145and LNCaP cells, either separately or in combination. Adhesion tovascular endothelium or to immobilized collagen, fibronectin or lamininwas quantified. Migration and invasion were explored by a modifiedBoyden chamber assay. Integrin � and � subtypes were analyzed byflow cytometry, western blotting and RT-PCR. Effects of drug treatmenton integrin related signaling, Akt and p70S6kinase activation, histoneH3 and H4 acetylation were also determined.

RESULTS: Separate application of RAD001 or VPA distinctlyreduced tumor cell adhesion, migration and invasion, accompanied byelevated Akt activation and p70S6kinase deactivation. Integrin subtypeexpression was altered significantly by both compounds (VPA �RAD001). Particularly,�3 coding mRNA was diminished drastically withthe combination being superior then single drug treatment. Further-more, when both drugs were used in concert additive effects wereobserved on the migratory and invasive behavior of the prostate cancercells.

CONCLUSIONS: Separate mTOR or HDAC inhibition slowsprocesses related to tumor metastasis. The RAD001-VPA combinationshowed advantage over VPA application alone with particular respectto migration and invasion. Ongoing studies are required to assess therelevance of VPA monotherapy versus VPA-RAD001 combination ontumor cell motility.

Source of Funding: supported by the ”Jung-Stiftung”

Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011 THE JOURNAL OF UROLOGY� e167