METHODS: Western blotting was used to determine the expressionof eIF4E, 4E-BP1, and phospho-4E-BP1 in breast cancer cell lines.eIF4E knockdown was achieved with two separate siRNA sequencesand compared to control siRNA. Cell growth was assessed with sul-forhodamine B assay and colony formation assay.

RESULTS: In breast cancer cell lines with a spectrum of genomic/signaling aberrations, although the expression and phosphorylationof 4E-BP1 differed, eIF4E was ubiquitously expressed. eIF4E siRNAachieved upwards of 80% decrease in eIF4E levels. In ER/PR/HER2(�) MDA-MB-468 cells, eIF4E knockdown decreased cellgrowth by 73% at 5 days (p�.0035). Significant growth inhibitionwith eIF4E knockdown was also observed in MCF7 (ER�,HER2�), BT474 (ER�, HER2�) and SKBR3 (ER�,HER2�)cells. Further, eIF4E knockdown led to an 86% decline (p�.0001) inanchorage-dependent colony formation ability.

CONCLUSIONS: eIF4E siRNA produces successful eIF4E knock-down. eIF4E knockdown leads to growth inhibition in a variety ofgenetic backgrounds, including triple negative cells, for which thereare currently no targeted therapies. Studies to better characterize themechanism of eIF4E-mediated growth and its effect in vivo are on-going.

Novel role of tyrosine kinase inhibition in theprevention of immune tolerance with immunemodulated therapy in advanced malignancyJunko J Ozao, MD, Ping-Ying Pan, PhD, George Wang, DVM, PhD,BingJiao Yin, PhD, Celia M Divino, MD, Shu-Hsia Chen, PhDMount Sinai School of Medicine, New York, NY

INTRODUCTION: Immune tolerance is associated with the accu-mulation of myeloid derived suppressor cells (MDSCs) in mice withlarge tumor burdens. MDSC induces Foxp3� T regulatory cells(Tregs). Blocking the receptor for SCF, ckit, can prevent MDSCaccumulation and Treg development.

METHODS: C-kit blocking antibody was compared to sunitinibmalate,a tyrosine kinase inhibitor for efficacy of ckit-mediated rever-sion of MDSC immune tolerance. The long-term survival effect ofblocking c-kit function on our combination immune therapy (IL-12and 4-1BB activation) for treatment of large MCA26, murine coloncancer cell line tumors was also examined.

RESULTS: Sunitinib malate, similar to anti-ckit, can decreaseGr1�CD115� MDSC accumulation in a dose-dependent fashion.More importantly, sunitinib malate and anti-ckit can decrease tumorinduced Treg development in vivo in our MCA-26 murine coloncancer model. Sunitinib malate and anti-ckit were able to reverse Tcell anergy of sorted Thy1.2� CD4 HA-specific TCR-transgenic Tcells when stimulated with HA in vitro. Anti-ckit in combinationwith our immune therapy protocol was able to improve the long-term survival rate of MCA-26 advanced stage, large tumor-bearingmice (P�0.001, log rank test).

CONCLUSIONS: Modulation of c-kit function can prevent Tregdevelopment induced by MDSC and can enhance the efficacy ofimmune modulation therapy in treating immune tolerance in large

tumor-bearing animals. Since sunitimib malate is already an FDAapproved drug, it offers a potentially useful orally administered drugfor enhancing the efficacy of immune modulation therapy.

Gene silencing of apoptosis-inducing factor blocksthe chemosensitizing effects of the nitric oxidedonor DETA/NONOate in metastatic colon cancercellsSergio Huerta, MD, Sapna Chilka, MD, Alejandro Vega, PhD,Fernando Rivera, MD, Thomas Anthony, MD,Edward Livingston, MDUniversity of Texas Southwestern-Dallas VA Medical Center,Dallas, TX

INTRODUCTION: Metastatic colon cancer cells SW620 are resis-tant to cisplatin (CDDP)-induced apoptosis. DETA/NONOate(DETA) is a nitric oxide donor which chemosensitizes these cells toapoptosis. Because SW620 cells have altered levels of Apaf-1, wehypothesized that a caspase-independent pathway of apoptosis mightbe responsible for the chemosensitizing effects of DETA. The follow-ing study was undertaken to assess the role of AIF in DETA-mediatedchemosensitization.

METHODS: SW620 cells were pre-treated with 0.5 and 1 mMDETA for 24-h followed by 5 mg/mL CDDP. Cells were treated withsiRNA specific for AIF. Apoptosis was assessed by TUNEL. Proteinlevels were evaluated by Western blot analysis with anti-AIF antibod-ies. RT-PCR assessed AIF expression.

RESULTS: Pre-treatment with 0.5 �M DETA resulted in a 3.6-foldincrease in apoptosis and a 4.6-fold increase with 1.0 mM DETA(both p’s�0.05). Western blot analysis with anti-AIF antibodies re-vealed an increase of AIF levels in a dose dependent fashion. Thisincrease correlated with an augmentation of AIF mRNA. AIF spe-cific siRNA showed a 55% decrease in apoptosis in cells pre-treatedwith 1.0 �M DETA followed by CDDP (p�0.01). There was nodecrease in apoptosis in siRNA treated-cells receiving CDDP only.

CONCLUSIONS: The chemosensitizing effects of the DETA aremediated in part by an increase in AIF transcription. Understandingthe transcription factors affected by DETA, may result in a potentialrole for nitric oxide in chemosensitization of metastatic colon cancerresistant to conventional chemotherapeutic agents.

Activation of Ras/MAPK and mTOR pathways inhepatocellular carcinoma: Tumor growth inhibitionwith Ras/mTOR pathway abrogation by a novelcombination of Sorafenib and rapamycinPhilippa H Newell, MD, Augusto Villanueva, MD, Judit Peix, BA,Isabel Fiel, MD, Swan Thung, MD, Steven Yea, BA,Sasan Roayaie, MD, Myron Schwartz, MD, Scott Friedman, MD,Josep Llovet, MDMount Sinai School of Medicine, New York, NY

INTRODUCTION: Resection and transplantation are available onlyto patients with early hepatocellular carcinoma (HCC). Sorafenib, a

S93Vol. 205, No. 3S, September 2007 Surgical Forum Abstracts