ADAM9 silencing inhibits breast tumor cell invasion in vitro

Abstract

ADAM9 (A Disintegrin And Metalloproteinase 9) is a member of the ADAM protein family which contains a disintegrin domain. This protein family plays key roles in many physiological processes, including fertilization, migration, and cell survival. The ADAM proteins have also been implicated in various diseases, including cancer. Specifically, ADAM9 has been suggested to be involved in metastasis. To address this question, we generated ADAM9 knockdown clones of MDA-MB-231 breast tumor cells using silencing RNAs that were tested for cell adhesion, proliferation, migration and invasion assays. In RNAi-mediated ADAM9 silenced MDA-MB-231 cells, the expression of ADAM9 was lower from the third to the sixth day after silencing and inhibited tumor cell invasion in matrigel by approximately 72% when compared to control cells, without affecting cell adhesion, proliferation or migration. In conclusion, the generation of MDA-MB-231 knockdown clones lacking ADAM9 expression inhibited tumor cell invasion in vitro, suggesting that ADAM9 is an important molecule in the processes of invasion and metastasis.

ADAM9 silencing inhibits breast tumor cells transmigration through blood and lymphatic endothelial cells

“…Furthermore, the deficiency of some ADAMs, such as ADAM9, ADAM15 and ADAM17 resulted in decreased tumor cell growth in mice models of metastasis [11,12]. In addition, a recent study from our group showed that ADAM9 silencing inhibited matrigel invasion of MDA-MB-231 metastatic breast tumor cells (about 70%) [16].…”

“…These results strongly suggest a key role for ADAM9 in the regulation of MMP2 expression. However, despite the increase in MMP2 expression, siADAM9 inhibited MDA-MB-231 cells invasion into matrigel by approximately 70% [16].…”

“…Previous studies from our laboratory have shown that ADAM9 has an important role in the invasion of MDA-MB-231 breast tumor cells [16]. Fry and Toker [17] using RNAi technique applied to different breast tumor cells demonstrated that ADAM9-S (secreted) and ADAM9-L (transmembrane) act in opposite ways in cell migration regulation, thus ADAM9-S promotes migration and ADAM9-L attenuates it.…”

“…The Silencer ® siRNA Starter Kit (Ambion) was used and the protocol was performed as described by Micocci and coworkers [16]. After 72 h of transfection, the cells were washed with phosphatebuffered saline (PBS) (1Â) and removed from the plates for gene expression assays (using TRIzol reagent -Life Technologies).…”

“…The primer sequences were: sense (5'-rCrCrArGrArGrUrArCrUrGrCrArArUrGrGrUrUrCrUrUrCTC-3 0 ) and anti-sense (5'-rGrArGrArArGrArArCrCrArUrUrGrCrArGrUrArCrUrCrUrGrGrArA-3 0 ). The scrambled primer used was the Silencer Select Negative Control N o 1 siRNA (Ambion) [16]. The esiRNA human ADAM9 silencing primer (Sigma-Aldrich, catalog number: EHU020151) and esiRNA targeting EGFP (Sigma-Aldrich, catalog number: EHUEGFP) were also used.…”