SIM2 was originally thought to contribute to Down’s syndrome. As a transcription factor, murine SIM2 mediates gene expression through CNS midline enhancer element with its dimerization partner ARNT via ARNT carboxy-terminus. The transcription AZ 960 factor c-myb regulates SIM2 transcription in glioblastoma cells, and a nuclear localization signal mediates nuclear localization of SIM2. A prior in silico bioinformatics approach using the Cancer Genome Anatomy Project database of the National Cancer Institute identified SIM2 as associated with colon, pancreas and prostate carcinomas, while absent in the corresponding normal tissues. Two different spliced isoforms of SIM2 transcript, SIM2- long and SIM2-short, have been reported while their differential function in humans are not known yet. SIM2-s was specifically expressed in early stages of colon cancer. Antisense inhibition of SIM2-s expression by antisense oligos caused growth inhibition and apoptosis in colon cancer cell line RKO and tumor growth in nude mice and also in pancreatic cancer cell line CAPAN1. Apoptosis was induced by SIM2-s inhibition in the RKO colon cancer cell line. SIM2-s was also found to have tumor suppressive activity in breast cancer. The invasion potential of glioblastoma was decreased significantly by SIM2s inhibition, consistent with a decrease in the expression of matrix metalloproteinase 2 at both mRNA and protein levels. We have previously reported SIM2 as a potential biomarker and immunotherapy target for human prostate cancer. Although SIM2-s expression has been associated with aggressive histopathology in prostate cancer, and overexpressing ectopic SIM2s enhanced survival in certain conditions in PC3AR+ cells, the functional role of SIM2 gene in prostate cancer cell is largely unknown. In this study we sought to elucidate the functional role of SIM2 in PCa using a gene silencing approach and characterization of molecular and functional changes by both gene expression profiling and metabolomic profiling. In our previous biomarker identification efforts, we have identified SIM2 as a potential biomarker for PCa. Thanks to its overexpression in prostate tumors and its highly restricted expression in humans, we proposed to use SIM2 as an immunotherapy target and were able to identify 5 HLA-A2.1, SIM2-derived immunogenic epitopes. In the present study we attempted to characterize the role of SIM2 in prostate cancer using a short hairpin RNA-induced gene silencing approach in PC3 cells as a model. We focused on profiling both the transcriptome and metabolome in SIM2low and normal PC3 cells, and evaluated the impact of SIM2 silencing on cell signaling and function. The SIM2s isoform has been reported to be expressed in colon, pancreas, and prostate tumors while absent in the corresponding benign tissues. We found that SIM2 genes are detectable in all these prostate cancer cells by real time PCR. However the expression levels in DU145 and LNCaP are relatively lower than other prostate cancer cells while PC3 cells express moderate level of SIM2 genes which are consistent with other report. The whole spectrum of regulation of gene expression by the transcription factor SIM2 is still poorly defined.