Our study provides novel insights into how the RhoA pathway controls the cancer phenotype of cell invasion. In melanoma cell lines, MRDI represents a novel target of Rho signaling, functioned in cell invasion, and was necessary for autophosphorylation of FAK, a key mediator of focal adhesion turnover. MRDI showed targeting to membranes in advanced human melanoma specimens and localizes at the leading edge of cells in culture. A role in regulating membrane events was also suggested in cultured cells by live cell imaging and findings that link MRDI to FAK phosphorylation and stress fiber destabilization consistent with its role in promoting cell invasion. Taken together, our results suggest that MRDI promotes cell invasion under conditions of RhoA pathway dysregulation by regulating molecular events key to the control of membrane dynamics and cell adhesion. A working model is presented in supplemental Fig. S12
Our proteomics screening strategy involved profiling protein responses to RhoA activation in cells in which the pathway was normally “off” and then carrying out a second screen to filter for targets uniformly regulated across cell lines in which RhoA was constitutively “on.” Only one of the five RhoA targets identified initially in premetastatic cells turned out to be highly correlated with constitutive RhoA activation across many cell lines. The fact that MRDI was validated as a regulator of RhoA-dependent cellular responses and correlated with melanoma stage illustrates the utility of prioritizing functional targets by filtering out bystanders that vary between cells because they have no regulatory function.
Our study also showed that the activation state of RhoA is differentially elevated in melanoma cell lines derived from metastatic tumors, revealing a role for RhoA signaling that is distinct from the previous characterization of RhoC in promoting melanoma metastases. The results underscore the importance of monitoring Rho-GTP levels given that the pathway activation in metastatic cell lines was not reflected by Rho protein expression. Rho activity assays are not routinely measured in cancer samples because of difficulties in performing GTP binding assays on tissue specimens. Future studies are needed to examine whether MRDI is a useful marker for Rho activation in clinical samples.
A completely unexpected finding was that MRDI has promiscuous function, acting both as a metabolic enzyme and a regulator of signal transduction. Enzymatic pathways involved in catabolism of MTA are ancient, having been delineated in bacteria and Archaea where they convert MTRu-1-P to 2-keto-4-methylthiobutyrate as a precursor of methionine (40
). In contrast, pathways for methionine salvage in humans are incompletely defined, and many of the enzymes that make up these pathways have not been mapped in mammalian genomes. Thus, metabolic conversion of MTR-1-P to MTRu-1-P has been demonstrated in rat cell extracts (31
), but the isomerase responsible for this conversion has not been identified until now. By showing that MRDI catalyzes the conversion of MTR-1-P to MTRu-1-P and demonstrating the dependence of activity on residues Cys168
, we identified the human MTR-1-P isomerase as well as confirmed residues in the enzyme active site. By showing that MRDI is required for growth in MTA in methionine-free medium and that growth depends on the Cys and Asp residues, we confirmed that MRDI functions as a necessary component in the pathway for methionine salvage from decarboxylated S
-AdoMet. Thus, MRDI joins a handful of metabolic enzymes with “moonlighting” function in signal transduction (46
MTR-1-P isomerase is not the only methionine salvage enzyme to be implicated in cancer. MTA phosphorylase, which catalyzes the conversion of MTA + Pi
→ MTR-1-P + adenine, is deficient in many cancer types including melanoma, often accompanying co-deletion in the chromosome 9q21 region with p19INK4/CDKN2A (46
). MTA phosphorylase is a target for development of inhibitors, which induce apoptosis in head and neck cancer cells, and loss of enzyme enhances cellular sensitivity to chemotherapeutic compounds (47
). The mechanisms for cell toxicity have been variously ascribed to suppression of MTA recycling to S
-AdoMet via methionine or inhibition of purine salvage initiated by the adenine product of MTA phosphorylase. MTA phosphorylase also has tumor suppressor function in certain cancer cells ascribed to autocrine effects of MTA buildup on growth factor and matrix metalloproteinase induction (50
). Our results are in contrast to the example of MTA phosphorylase because we showed unequivocally that MRDI controls cancer cell behavior in a manner that is independent of its catalytic function. We speculate that MRDI elevates cell invasion via protein-protein interactions mediated by surface residues distal to the enzyme active site. Thus, MRDI provides unique evidence that some of the processes regulating control of cell adhesion and motility by signal transduction pathways have evolved from protein scaffolds with ancient metabolic function.