MUC1 siRNA SILENCING IMPACT IN GASTRIC CANCER CELLS PHENOTYPE

MUC1 siRNA SILENCING IMPACT IN GASTRIC CANCER
CELLS PHENOTYPE
N. Costa, P. Paulo, R. Barbosa, H. Tsutsumida, T. Caffrey,
M.A. Hollingsworth, F. Santos-Silva*
Eppley Cancer Institute, UNMC, Omaha, NE, USA
IPATIMUP, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal
*[email protected]
INTRODUCTION. MUC1 is a trans-membrane glycoprotein expressed on
epithelial surfaces that was been associated with protection, cell signaling, cellcell interactions and cell migration (1). Over-expression and abnormal
glycosylation of MUC1 has been reported in several cancer models, including
gastric cancer (2). We evaluated the relevance of MUC1 for gastric cancer
tumorigenicity using a siRNA silencing strategy.
METHOD. We used small interfering RNAs (siRNA) to knock down MUC1
expression in MKN45 gastric cancer cell line. MUC1 expression levels in
silenced clones were confirmed by immunofluorescence and western-blot using
HMFG1 MAb and by real-time PCR. MUC1 silenced clones were evaluated for
proliferation (MTT assay), apoptosis (TUNEL assay) and invasion (Matrigel
invasion assay). Signaling pathways were evaluated with KinetworksTM protein
kinase multiblot analysis.
RESULTS. Using the RNAi strategy we stably down–regulated MUC1
expression. The MUC1 silenced clones (C1, C2) had significantly increased rates
of growth (proliferation), apoptosis and invasion activity when compared with
scramble control (SC) (figure 1). Furthermore these clones also showed
alterations in phosphorylation of proteins involved in oncogenic signaling
pathways: Erk1, decreased phosphorylation of T202 and Y204; Erk2, increased
phosphorylation of T185 and Y187; and cell cycle CDK1/2, increased
phosphorylation of T14, Y15, and T160/T161.
Figure 1
DISCUSSION. MKN45 is a gastric cell line with high levels of MUC1
expression. This study showed that down-regulation of MUC1 in MKN45 cells
promotes oncogenesis-associated events (proliferation, apoptosis and invasion).
Alterations in phosporylation of signaling targets implicate the involvement of
proliferation related cascades (CDK1/2). These findings are opposite from those
described in breast and pancreatic models (3, 4), which suggests the need to
identify MUC1 signaling partners in gastric cancer cells.
Our RNAi model is the first to show that MUC1 affects the transformed status of
gastric cancer cells, through effects on oncogenic signaling pathways.
ACKNOWLEDGMENT. This work was supported by Fundação para a Ciência
e Tecnologia (grants: POCTI/CBO/44812/2002, POCI/SAU-IMI/ 56895/2004 and
SFRH/BD/27669/2006) and Fundação Luso-Americana para o Desenvolvimento.
REFERENCES
1. Hollingsworth, M.A., Swanson, B.J. (2004) Nat. Rev. Cancer. 4:45-60.
2. Silva, F. et al (2001) Eur. J. Hum. Genet. 9:548-52.
3. Tsutsumida, H. et al (2006) Clin. Cancer Res. 12:2976-87.
4. Hattrup, C.L., Gendler, S.J. (2006) Breast Cancer Res. 8(4):R37.