SCC-S2/GG2-1/NDED (approved gene symbol TNFAIP8) is a transcription factor NF-kappaB-inducible, antiapoptotic, and oncogenic molecule. In this study, we examined the role of SCC-S2 in invasion and experimental metastasis. We demonstrate that expression of SCC-S2 cDNA in MDA-MB 435 human breast cancer cells is associated with enhanced invasion in vitro and increased frequency of pulmonary colonization of tumor cells in athymic mice. Systemic treatment of athymic mice with a cationic liposomal formulation of SCC-S2 antisense oligo led to decreased incidence of pulmonary metastasis and inhibition of SCC-S2 expression in vivo. Antisense inhibition of endogenous SCC-S2 expression correlated with decreased expression of VEGF receptor-2 in tumor cells and human lung microvascular endothelial cells and loss of endothelial cell viability. In addition, downregulation of SCC-S2 expression in tumor cells was associated with decreased expression of known metastasis-related molecules MMP-1 and MMP-9. These results demonstrate a novel role for SCC-S2 in tumor progression, involving multiple effectors, and provide a basis for SCC-S2-targeted cancer gene therapy.

TNFAIP8 is a NF-κB-inducible, oncogenic molecule. Previous “promoter array” studies have identified differential methylation and regulation of TNFAIP8 in prostate epithelial and cancer cell lines. Here we demonstrate that TNFAIP8 expression is induced by androgen in hormone-responsive LNCaP prostate cancer cells. In athymic mice bearing hormone-refractory PC-3 prostate tumor xenografts, intravenous treatment with a liposomal formulation of TNFAIP8 antisense oligonucleotide (LE-AS5) caused reduced expression of TNFAIP8 in tumor tissues, and a combination of LE-AS5 and radiation or docetaxel treatment resulted in significant inhibition of PC-3 tumor growth as compared to single agents. The immunohistochemical evaluation of TNFAIP8 expression revealed correlation of both cytoplasmic and nuclear TNFAIP8 overexpression with high grade prostatic adenocarcinomas, while nuclear overexpression was found to be an independent predictor of disease recurrence controlling for tumor grade. Increased nuclear TNFAIP8 expression was statistically significantly associated with a 2.44 fold (95 % confidence interval: 1.01–5.91) higher risk of prostate cancer recurrence. Mechanistically, TNFAIP8 seems to function as a scaffold (or adaptor) protein. In the antibody microarray analysis of proteins associated with the TNFAIP8 immune-complex, we have identified Karyopherin alpha2 as a novel binding partner of nuclear TNFAIP8 in PC-3 cells. The Ingenuity Pathway Analysis of the TNFAIP8 interacting proteins suggested that TNFAIP8 influences cancer progression pathways and networks involving integrins and matrix metalloproteinases. Taken together, present studies demonstrate that TNFAIP8 is a novel therapeutic target in prostate cancer, and indicate a potential relationship of the nuclear trafficking of TNFAIP8 with adverse outcomes in a subset of prostate cancer patients.

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Raf-1 protein serine/threonine kinase plays an important role in ERK signal transduction pathway of cell survival and proliferation. Raf-induced transcriptional changes are dependent on phosphorylation/activation of ERK. However, regulation of phospho-ERK (p-ERK) via Raf transcriptome is as yet unknown. We report the initial characterization of BRCC3, a novel gene discovered previously by mRNA expression profiling in MDA-MB 231 human breast cancer cells treated with Raf antisense oligonucleotide. BRCC3 is localized at human chromosome 5q12.1. BRCC3 open reading frame consists of 529 amino acids, coding for an approximate 60-kDa predominantly membrane-associated protein. Expression levels of BRCC3 mRNA and protein are high during G2/M phase of the cell cycle in breast cancer cells. Treatment of MDA-MB 231 cells with Raf-1 siRNA resulted in decreased expression of Raf-1, BRCC3 and pERK , but not B-Raf. Transient or stable expression of the epitope-tagged BRCC3 cDNA was associated with increased pERK in three different cell lines. Consistently, BRCC3 siRNA treatment of MDA-MB 231 cells caused decreased expression of BRCC3 and pERK. Furthermore, exogenous BRCC3 expression was associated with a delay in etoposide-induced cell death and an increase in cell proliferation. These findings demonstrate that BRCC3 is a novel effector of Raf-1, and implicate a role of BRCC3 in modulation of pERK , cell survival and proliferation.

By virtue of their potential to selectively silence oncogenic molecules in cancer cells, antisense oligonucleotides (ASO) and small interfering RNAs (siRNAs) are powerful tools for development of tailored anti-cancer drugs. The clinical benefit of ASO/siRNA therapeutic is, however, hampered due to poor pharmacokinetics and biodistribution, and suboptimal suppression of the target in tumor tissues. Raf-1 protein serine/threonine kinase is a druggable signaling molecule in cancer therapy. Our laboratory has developed cationic liposomes for systemic delivery of raf ASO (LErafAON) and raf siRNA (LErafsiRNA) to human tumor xenografts grown in athymic mice. LErafAON is also the first ASO containing liposomal drug tested in humans. In this article, we primarily focus on a modified formulation of systemically delivered cationic liposomes containing raf antisense oligonucleotide (md-LErafAON). The cationic liposomes were prepared using dimyristoyl 1,2-diacyl-3-trimethylammonium-propane (DMTAP), phosphatidylcholine (PC), and cholesterol (CHOL). The toxicology, pharmacokinetics, biodistribution, target selectivity, and anti-tumor efficacy studies of md-LErafAON were conducted in mice. We demonstrate that md-LErafAON is the next generation of systemically delivered and well-tolerated antisense therapeutic suitable for clinical evaluation.