guanosine-5--o-(3-thiotriphosphate) and Neoplasm-Metastasis

Increased circulating levels of L-alpha-lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G-protein-coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.. Quantitative RT-PCR was used to measure GPR55 expression in breast cancer cell lines. Cell migration and invasion were measured using a Boyden chamber chemotaxis assay and Cultrex invasion assay, respectively. Cell polarization and orientation in response to the microenvironment were measured using slides containing nanometric grooves.. GPR55 expression was detected in the highly metastatic MDA-MB-231 breast cancer cell line. In these cells, LPI stimulated binding of [(35)S]GTPgammaS to cell membranes (pEC(50) 6.47 +/- 0.45) and significantly enhanced cell chemotaxis towards serum. MCF-7 cells expressed low levels of GPR55 and did not migrate or invade towards serum factors. When GPR55 was over-expressed in MCF-7 cells, serum induced a robust migratory and invasive response, which was further enhanced by LPI and prevented by siRNA to GPR55. The physical microenvironment has been identified as a key factor in determining breast tumour cell metastatic fate. LPI endowed MDA-MB-231 cells with the capacity to detect shallow (40 nm deep) grooved slides and induced marked cancer cell polarization on both flat and grooved surfaces.. LPI and GPR55 play a role in the modulation of migration, orientation and polarization of breast cancer cells in response to the tumour microenvironment.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Polarity; Chemotaxis; Female; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Lysophospholipids; Neoplasm Invasiveness; Neoplasm Metastasis; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; RNA, Small Interfering

The two major forms of lung carcinoma, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), are clinically distinct, and are also differentiated by morphology and behavior in culture. SCLC cells have a greater metastatic potential than NSCLC cells in vivo, and exhibit a unique spherical morphology in culture due to their inability to adhere and spread on the substratum. Because the small GTPase RhoA affects metastatic properties and regulates cell morphology, we examined whether differences in RhoA expression and activity contribute to the distinct SCLC and NSCLC phenotypes. We found that the expression and GTPgammaS-dependent activation of RhoA are generally greater in SCLC cell lines (SCC-9, NCI-H69, NCI-H146, and NCI-H345) than in NSCLC cell lines (NCI-H23, NCI-H157, NCI-H520, and NCI-H522). The effects of inhibiting Rho-mediated signaling in these cells were investigated by transfecting the cells with cDNA coding for C3 exoenzyme, which ADP-ribosylates and inactivates Rho. Expression of C3 exoenzyme in SCLC cells induces cell-cell compaction, and causes NCI-H345 cells to adhere and spread on collagen IV. In contrast, expression of C3 exoenzyme in NSCLC cells does not induce detectable compaction, but induces cell spreading of NCI-H23 and NCI-H157 cells. Cell proliferation is diminished by Rho inactivation in the majority of the NSCLC cell lines, but not the SCLC cell lines. Expression of p21Cip1/WAF1 is also diminished by Rho inactivation in two of the SCLC cell lines, but is not significantly altered in the NSCLC lines. These results indicate that Rho-mediated signaling may regulate different events in SCLC and NSCLC cells, including adhesion of SCLC cells and proliferation of NSCLC cells.

Topics: Adenocarcinoma; Adenosine Diphosphate Ribose; ADP Ribose Transferases; Botulinum Toxins; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Cell Size; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA, Complementary; Gene Expression Regulation, Neoplastic; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Proteins; rhoA GTP-Binding Protein; Signal Transduction

Phospholipase D (PLD) is a highly regulated enzyme involved in lipid-mediated signal transduction processes affecting vesicular trafficking and cytoskeletal reorganization. It is regulated by protein kinase C, adenosine diphosphate (ADP)-ribosylation factors and Rho family proteins, and both protein kinase C and Rho family proteins have been implicated in the metastatic potential of melanoma. We analysed PLD in four human melanoma cell lines and in primary human melanocytes. Melanoma cell lines showed phosphatidylcholine-hydrolysing, phosphatidylinositol 4,5-bisphosphate-dependent PLD activity, which was activated by phorbol ester and a non-hydrolysable guanosine triphosphate (GTP) analogue in a dose-dependent and synergistic manner, whereas primary melanocytes exhibited only low PLD activity compared with the melanoma cell lines. As determined by reverse transcription polymerase chain reaction, both splicing variants of PLD1, PLD1a and PLD1b, and the isoenzyme PLD2, are expressed in melanoma cells and melanocytes. Western blot analysis showed that PLD1 expression was low in primary melanocytes in contrast to melanoma cells, which is in agreement with our finding of low activity. Interestingly, Rho protein mRNA was elevated in all melanoma cell lines. We conclude that in human melanoma cells, the PLD activity that is stimulated by phorbol ester requires ADP-ribosylation factor, protein kinase C and Rho proteins for full activity, and most probably represents the isoenzyme PLD1.

Topics: Adenosine Diphosphate; ADP-Ribosylation Factors; Alternative Splicing; Blotting, Western; Cell Division; Cell Line, Tumor; Cells, Cultured; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation, Enzymologic; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoblotting; Melanocytes; Melanoma; Neoplasm Metastasis; Oleic Acid; Phorbol Esters; Phospholipase D; Protein Isoforms; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA; RNA, Messenger

Rho-like GTPases have been implicated in the regulation of the actin cytoskeleton which controls the morphology, adhesion and motility of cells. Like Ras proteins, they become activated when bound GDP is exchanged for GTP, a process catalysed by GDP-dissociation stimulator (GDS) proteins. Several GDS proteins specific for Rho-like GTPases have been identified. Most of these contain a conserved catalytic domain, the DBL-homology (DH) domain, and activate Cdc42 or Rho but not Rac. We have isolated the invasion-inducing Tiam1 gene, which also encodes a protein with a DH domain. Here we show that Tiam1 is a GDS protein for Rho-like GTPases in vitro. In fibroblasts, Tiam1 induces a similar phenotype as constitutively activated (V12)Rac1, including membrane ruffling, and this is inhibited by dominant negative (N17)Rac1. Moreover, T-lymphoma cells expressing V12Rac1 become invasive, indicating that the Tiam1-Rac signalling pathway could be operating in the invasion and metastasis of tumour cells.

Topics: 3T3 Cells; Animals; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Fibroblasts; Glutathione Transferase; GTP Phosphohydrolases; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Proteins; rac GTP-Binding Proteins; rap GTP-Binding Proteins; Recombinant Fusion Proteins; rhoA GTP-Binding Protein; T-Lymphoma Invasion and Metastasis-inducing Protein 1; Transfection; Tumor Cells, Cultured