Upregulation of the S1P3 receptor in metastatic breast cancer cells increases migration and invasion by induction of PGE2 and EP2/EP4 activation

Abstract

Breast cancer is one of the most common and devastating malignancies among women worldwide. Recent evidence suggests that malignant progression is also driven by processes involving the sphingolipid molecule sphingosine 1-phosphate (S1P) and its binding to cognate receptor subtypes on the cell surface. To investigate the effect of this interaction on the metastatic phenotype, we used the breast cancer cell line MDA-MB-231 and the sublines 4175 and 1833 derived from lung and bone metastases in nude mice, respectively. In both metastatic cell lines expression of the S1P receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca]), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E synthase expression, and consequently with increased PGE synthesis. PGE synthesis was decreased by antagonists and siRNA against S1P and S1P. Moreover, in parental MDA-MB-231 cells overexpression of S1P by cDNA transfection also increased PGE synthesis, but only after treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, indicating reversible silencing of the COX-2 promoter. Functionally, the metastatic sublines showed enhanced migration and Matrigel invasion in adapted Boyden chamber assays, which further increased by S1P stimulation. This response was abrogated by either S1P antagonism, COX-2 inhibition or PGE receptor 2 (EP) and 4 (EP) antagonism, but not by S1P antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE signaling via EP and EP.

Ceramide Kinase Is Upregulated in Metastatic Breast Cancer Cells and Contributes to Migration and Invasion by Activation of PI 3-Kinase and Akt

“…The migratory capacity of cells was measured in an adapted Boyden chamber assay. Both metastatic cell lines showed enhanced migration compared to the parental cells ( Figure 2), which confirms previous findings in this metastases model [26]. As expected, in the presence of the CerK inhibitor NVP-231 [27], migration of the two sublines dose-dependently decreased (Figure 2), reaching maximal inhibition of 30% at 1 μM in 4175 cells and of 70% at 1 μM in 1833 cells.…”

“…As expected, in the presence of the CerK inhibitor NVP-231 [27], migration of the two sublines dose-dependently decreased (Figure 2 Another feature of metastatic cells is invasiveness [28,29]. We previously reported that the 4175 and the 1833 sublines also have an increased capacity of invasion, as detected in a Matrigel assay [26]. Here, we found that this process was also mitigated by the CerK inhibitor NVP-231 ( Figure 3).…”

“…The cPLA 2 is another intracellular target of C1P, which, upon C1P binding, is activated to release arachidonic acid and thereby increases prostaglandin E 2 (PGE 2 ) formation [32]. Chronic inflammation is a well-known risk factor for cancer development and, in view of our previous findings that the metastatic sublines 4175 and 1833 produce much higher amounts of PGE 2 [26] and that cPLA 2 is implicated in breast cancer cell migration [35,36], it is plausible that the pro-migratory effect of CerK/C1P involves cPLA 2 . However, in our breast cancer model, neither inhibition of CerK with NVP-231 nor CerK knockdown could attenuate PGE 2 formation ( Figure S10), thus excluding the notion that high PGE 2 production in metastatic cells is due to enhanced C1P production by CerK.…”

“…Cells were incubated for 24 h in serum-free DMEM prior to stimulation as specified in the figure legends. To stop stimulation, the supernatant was discarded, and the cell monolayer was washed with phosphate-buffered saline (PBS), scraped, and homogenized by sonication in lysis buffer [26]. Samples were centrifuged for 10 min at 13,000× g, and 30 µg of protein from the supernatant was separated by SDS-PAGE, transferred to nitrocellulose membrane, and used for Western blot analysis using the antibodies as indicated in the figure legends.…”

Prostaglandin E2-Induced COX-2 Expressions via EP2 and EP4 Signaling Pathways in Human LoVo Colon Cancer Cells

“…Inflammation is emerging as a new hallmark of cancer metastasis and invasion and cyclooxygenases (COXs) and lipoxygenase (LOX) play major roles in arachidonic acid-related inflammatory cascades [23,24]. Previous studies show that COX-2 as a rate-limiting enzyme involved in the formation of PGE2 from arachidonic acid, and is over-expressed in the colorectal cancer tumor tissues.…”