EpCAM (epithelial cell adhesion molecule) is a cell surface molecule that is known to be highly expressed in colon and other epithelial carcinomas. EpCAM is involved in cell-to-cell adhesion and has been the target of antibody therapy in several clinical trials. To assess the value of EpCAM as a novel target for breast cancer gene therapy, we performed real-time reverse transcription-PCR to quantify the level of EpCAM mRNA expression in normal breast tissue and primary and metastatic breast cancers. We found that EpCAM is overexpressed 100-to 1000-fold in primary and metastatic breast cancer. Silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in a 35-80% decrease in the rate of cell proliferation in four different breast cancer cell lines. EpCAM siRNA treatment decreased cell migration by 91.8% and cell invasion by 96.4% in the breast cancer cell line MDA-MB-231 in vitro. EpCAM siRNA treatment was also associated with an increase in the detergent-insoluble protein fraction of E-cadherin, ␣-catenin, and ␤-catenin, consistent with the known biology of EpCAM as a regulator of cell adhesion. Our hypothesis is that modulation of EpCAM expression can affect cell migration, invasion, and proliferation by enhancing E-cadherinmediated cell-to-cell adhesion. These data provide compelling evidence that EpCAM is a potential novel target for breast cancer gene therapy and offer insights into the mechanisms associated with EpCAM gene silencing.

In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/ mass spectroscopy. Interestingly, only one specific ceramide, C 18:0 -ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C 18 -ceramide was not detected. These data suggested the hypotheses that decreased levels of C 18 -ceramide might impart a growth advantage to HNSCC cells and that increased generation of C 18 -ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C 18 -ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C 18 -ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/ mUOG1 resulted in increased levels of only C 18:0 -ceramide by ϳ2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C 18 -ceramide by mLAG1/mUOG1 inhibited cell growth (ϳ70 -80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C 18 -ceramide in the regulation of growth of HNSCC.

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK1/S1P pathway also plays a critical role in regulation of cyclooxygenase-2 (COX-2), a well-established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK1 and COX-2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK1 staining was found in 21/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK1 staining was observed in normal mucosa. The increase in SK1 and COX-2 expression in AOM-induced rat colon adenocarcinoma was confirmed at the level of mRNA by real-time RT-PCR. In addition, it was found that 1) down-regulation of SK1 in HT-29 human colon cancer cells by small interfering RNA (siRNA) decreases COX-2 expression and PGE2 production; 2) overexpression of SK1 in RIE-1 rat intestinal epithelial cells induces COX-2 expression; and 3) S1P stimulates COX-2 expression and PGE2 production in HT-29 cells. These results suggest that the SK1/S1P pathway may play an important role in colon carcinogenesis, in part, by regulating COX-2 expression and PGE2 production.

Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth