Enhanced migration is an important feature of tumor cells and is also thought to contribute to invasion and metastasis. Migration and invasion in cancer cells results from highly organized sequential modules triggered by external stimuli. Upon stimulation, the leading edge of the cell membrane protrudes in the intended direction and subsequently stabilizes inducing cell body movement followed by tail detachment and retraction. In this study, we provide evidence that the LPA1 receptor but not the LPA2 or LPA3 receptor is required for LPA-induced migration in the two breast cancer cell lines, 4T1, and MDA- MB-231 cells. In 4T1 cells, the RLCs associated with both NMHC II-A and II-B are phosphorylated by LPA1 receptor-dependent ROCK activation.
Although there is considerable information concerning the functional role of NM IIs in mammary cell migration, the role of each of the NM II isoforms remains controversial. For example, the MCF-10A human normal mammary epithelial cell line, which expresses NM II-A and II-B, continues to migrate when treated with the NM II inhibitor, blebbistatin (Even-Ram et al. 2007
). On the other hand, MA-MB-231 human breast cancer cells, which also express NM II-A and II-B, attenuated their migration when NMHC II-A or II-B is depleted using siRNA (Betapudi et al. 2006
). Our findings which are similar to those of Betapudi et al. are that LPA-induced migration is impaired by either blebbistatin, or by depletion of NMHC II-A or II-B in 4T1 mouse breast cancer cells which express both isoforms.
Interestingly, a recent paper demonstrated that the ectopic expression of LPA1
receptors in MCF-10A cells, which are known as slow moving cells, caused these cells to acquire invasive characteristics (Li et al. 2009
). Consistent with this, MDA-MB-231 cells, which are known as highly migratory cells, expressed more LPA1
receptor than any other breast cancer cell line (Bandoh et al. 1999
; Ham et al. 2004
; Shida et al. 2008
; Supple. Fig. 1A
). 4T1 cells, which we employ in this study, also express large numbers of the LPA1
receptor and have enhanced migration characteristics (, Supple. Fig. 1A
). These observations suggest that LPA1
receptors appear to play a significant role in mammary cell migration.
LPA receptors are well known heterotrimeric-G protein coupled receptors (Lee et al. 2008
; Stähle et al. 2003
). The expression profiles and functional roles of the three LPA receptors isotypes have been characterized in a variety of cell types. Specifically, the LPA1
receptor was highly expressed in human breast cancer cells including MDA-MB-231, BT549, and Hs578T (Chen et al. 2007
). In an in vivo
model, the LPA1
receptor rather than either the LPA2
receptor or LPA3
receptor was required for breast cancer cells to metastasize to bone (Boucharaba et al. 2006
receptors appeared to be dominantly expressed in 4T1 cells as shown in Supple. Fig. 1A
. Both LPA1
receptor siRNA and LPA1,3
antagonists significantly inhibited LPA-induced migration (, , and ) in these cells. So, we suggest that LPA1
receptors are required for LPA-induced migration in 4T1 cells.
Our finding that 4T1 cell are relatively insensitive to PTX for LPA-induced migration even at high concentrations (1 μg/ml) is of interest (), since, PTX is usually known as an inhibitor of LPA-induced migration in a wide variety of cancer cells such as U87, PC3, PANC-1, and MDA-MB-231 cells (Anliker and Chun 2004
, Ishii et al. 2004
). These results imply that the LPA1
receptor in 4T1 cells is mainly coupled to the PTX-insensitive Gα subunit and that these cells appear to have distinct signaling pathways bypassing the PTX-sensitive pathway. Previous studies have reported that G12/13
regulates RhoA activity and LPA stimulates RhoA through G12/13
to induce cell migration (Bian et al. 2006
; Hart et al. 1998
). Here, we show that the LPA1
receptor activated RhoA but not Rac1 or Cdc42 and stimulated the RhoA dependent ROCK pathway. These results suggest that LPA enhances migration through LPA1
-PTX insensitive G12/13
-RhoA-ROCK pathway in 4T1 cells.
In view of the fact that the silencing of either NMHC II-A or II-B attenuated LPA-induced migration (), we can set up a hypothesis in which the disruption of either isoform by silencing of NMHC II-A or II-B expression results in impaired migration. Both NMHC II-A and II-B appear to play unique cellular roles in LPA-induced migration because reduction of one isoform couldn’t be compensated for by transfection of the second isoform with respect to LPA-induced migration (). Furthermore, NM II-A appears to be the major isoform in 4T1 cells since it is expressed at a protein level 6–7 times more than NM II-B as determined by mass spectrometry, but we failed to detect any difference in LPA-induced migration by silencing NM II-A or NM II-B and we couldn’t observe any difference in phosphorylation between RLCs associated with NM II-A or NM II-B following LPA treatment.
Previous work has shown that phosphorylation of the RLC regulates cell migration and induces morphological changes. Sandquist et al. showed that the ROCK-induced phosphorylation of the RLC regulates cell migration in the lung carcinoma A 549 cell line. Generally, both ROCK and MLCK are candidates for kinases phosphorylating the RLC in cell migration. Their specific activity and localization have been delineated in a variety of cell types (Totsukawa et al. 2004
). They suggested that ROCK predominantly activates NM II contractility in the central cell region whereas MLCK plays a role at the cell margins. Here, we determine that ROCK rather than MLCK is the main kinase for LPA-induced migration in 4T1 cells (). Based on our studies with MYPT-1, we suggest that ROCK is more likely to phosphorylate and directly activate myosin, than to phosphorylate and inactivate myosin phosphatase ().
Although a number of investigators have utilized 4T1 breast cancer cells as a positive control for the study of metastasis in vivo, the signaling mechanism triggering the induction of migration or invasion was largely unknown. Here, we provide evidence about the mechanism of in vitro migration, and also describe how NM II can contribute to LPA-induced migration in 4T1 cells.