Correspondence Re S. S. Virtanen et al., Alendronate Inhibits Invasion of PC-3 Prostate Cancer Cells by Affecting the Mevalonate Pathway. Cancer Res 2002;62:2708–14. Re K. Sawada et al., Alendronate Inhibits Lysophosphatidic Acid-Induced Migration of Human Ovarian Cancer Cells by Attenuating the Activation of Rho. Cancer Res 2002;62:6015–20.
Authors: Valentine B. Andela
Journal: Cancer Research (2004)

Virtanen et al. (1) report that the aminobisphosphonate, alendronate, unlike the first generation bisphosphonate, clodronate, inhibits prostate cancer invasiveness by decreasing the cellular pool of farnesyl and geranylgeranyl isoprenoids. Mechanistically, aminobisphosphonates act as nonhydrolyzable analogs of PPi (isoprenoid) intermediates of the mevalonate pathway (Fig. 1), whereas the first generation “simple” structured bisphophonates approximate the structure of ATP and, as such, perturb ATP-dependent processes.As pointed out by Virtanen et al., tumor cell invasiveness is a coordinated process that involves cell adhesion, motility, and secretion of matrix-degrading enzymes. The molecular bases for these cellular processes have been elucidated in significant detail, with the notable implication of geranylgeranylated Rho proteins in the orchestration of actin-cytoskeletal dynamics and cellular processes germane to tumor cell invasiveness. RhoB is a notable exception to the rule, granted it is preferentially farnesylated and distinctly localized in the endosomal compartment in which it is implicated in vesicular transport (2, 3). Of general interest, the overt toxicity of geranylgeranyl transferase inhibitors has not only hindered clinical development but obscured the fundamental role of geranylgeranylated proteins in cancer progression (4). It must be emphasized that the validation of farnesyl transferase inhibitors as anticancer agents rests in part on their exquisite effects on the actin-cytoskeleton, which are due to a shift from farnesylated to geranylgeranylated forms of RhoB and the aberrant relocalization of RhoB to the plasma membrane where it perturbs the function of Rho proteins implicated in actin-cytoskeletal dynamics (5, 6).It is, thus, well documented that geranylgeranyl PPi, unlike farnesyl PPi, rescues the inhibition of tumor cell invasiveness induced by inhibitors of the mevalonate synthesis pathway (7, 8). Discordantly, Virtanen et al. report that farnesol is equally as potent as geranylgeraniol, if not moreso, in reversing the effects of alendronate on PC-3 migration and invasiveness. Their observations are nonetheless corroborated by Sawada et al. (9), who demonstrate partial restoration of Rho protein function at the cellular and molecular level, using one-third less farnesol and three times more alendronate on lysophatidic acid-stimulated ovarian cancer cells. Sawada et al. raise the compelling argument that farnesol has untoward cellular effects, but fall short in suggesting that farnesol is processed to geranylgeranyl PPi and is subsequently used in Rho geranylgeranylation. The very premise for using isoprenols, notably farnesol, over readily usable farnesyl PPi, rests on a “salvage pathway” that converts farnesol to farnesyl PPi without further processing to geranylgeranyl PPi, as would occur in the normal sequence of reactions in de novo synthesis (10). Thus. although farnesol affords specific replenishment of farnesyl PPi after depletion studies, its untoward cellular effects disfavor its use in rescue experiments.As referred by Dr. Andela, we have demonstrated that the aminobisphosphonate alendronate inhibits the in vitro invasion of PC-3 human prostate cancer cells by mechanisms that interfere with the mevalonate pathway (1). In contrast to osteoclasts (2), the primary target cells of bisphosphonates, alendronate inhibition of prostate cancer cell invasion and migration was rescued not only by geranylgeraniol but also by farnesol. We totally agree with Dr. Andela about the importance of geranylgeranylated proteins in cellular processes, which need cytoskeletal reorganization. This is also demonstrated by our results of changes in actin organization after alendronate treatment (1). However, our results clearly showed that providing precursors for farnesylation reactions is also important for prostate cancer cell invasion. This was demonstrated by reversal of mevastatin inhibition of PC-3 cell invasion by adding either farnesol or geranylgeraniol. Also, farnesol as well as geranylgeraniol, was able to protect PC-3 cells against alendronate inhibition of invasion. Both also opposed mevastatin or alendronate-caused inhibition of migration although to a much lesser extent. Different results were obtained with murine lung alveolar carcinoma cell line (Line 1) in the experiments of Dr. Andela et al. (3), in which geranylgeraniol but not farnesol was able to rescue from alendronate-caused decrease of cell proliferation, viability and invasion. These reports together point to obvious, although yet poorly identified, differences in isoprenylation reactions, pathways, and target proteins, which are critical for various functions in various cell types. The salvage pathway that converts farnesol to farnesyl pyrophosphosphate without further processing it to geranylgeranylpyrophosphate (4) and that possibly functions in PC-3 cells also suggests that farnesylated proteins have a role in the invasion of at least these cancer cells. Compartmental separation of branches of the isoprenoid pathway, as well as of target proteins for isoprenoid transferases in different cell types, may be a critical step in the outcome of rescue experiments.Although the rescue experiments in our work (1) were carried out using 10 μm alendronate, it should be noted that invasion of human PC-3 prostate cancer cells was almost totally inhibited at much lower alendronate concentration (50% at 1 pm and &gt;80% inhibition at 1 nm concentration) than inhibition of proliferation/viability in our experiments (at &gt;10 μm) and a decrease in responses (proliferation, viability, invasion; at 3–30 μm) observed in the experiments with murine alveolar lung carcinoma cells referred to by Andela et al. (3). Therefore, it is possible that the mechanisms involved in alendronate inhibition of invasion at very low concentrations and in inhibition of proliferation and viability at very much higher concentrations are different, although the interference of the mevalonate pathway would be involved in both cases. It is also possible that there are, thus far, unknown steps in the salvage pathways of the externally added precursors (farnesyl and geranylgeraniol) to phosphorylated isoprenoids (farnesylpyrophosphate and geranylgeranylpyrophosphate) as also suggested by Crick et al., (4).All of these results emphasize the importance of the isoprenylated small GTPases in malignant behavior of cancer cells and the multiple mechanisms, which may differentially regulate various features of malignant growth (such as invasion and proliferation). The final picture will not be clear until we get detailed information about the targets of various isoprenylation reactions and the levels and cellular localization of the prenylated proteins after various treatments.Dr. Andela commented that geranylgeranyl PPi, unlike farnesyl PPi, rescued the inhibition of tumor cell invasiveness induced by inhibitors of the mevalonate pathway. But, in our article (1), farnesol (FOH) appears to be effective in restoring the activation of Rho, and the phosphorylation of the myosin light chain (MLC) to some extent and therefore migration activity is partially restored to a lesser extent than geranylgeraniol. Despite Dr. Andela’s comments, we believe that FOH might have the possibility of restoring the Rho-activation induced by inhibitors of the mevalonate pathway. This discrepancy could be interpreted as follows: lysophosphatidic acid activates not only Rho but also Ras in mammalian cells, and activated Ras might modulate Rho-activation, followed by the stimulated migration of cancer cells. In fact, in our unpublished data,1 alendronate inhibits Ras-activation induced by lysophosphatidic acid in human ovarian cancer cells, and the addition of FOH restores the inhibition of Ras-activation. Much evidence indicates that Rho-GTPases are key downstream targets in Ras-mediated signaling (234). In addition to their well-characterized roles as inducers of cell proliferation, Ras proteins have rapid and profound effects on the actin cytoskeleton (567). When activated Ras is microinjected into fibroblasts, there is induction of membrane ruffling and assembly of actin stress fibers (6). Microinjection experiments reveal that Rho proteins (Cdc42, Rac, and Rho) act in concert and function in a cascade when Ras activates Rac, which results in membrane ruffling followed by activation of Rho-mediated actin stress fiber development (5, 6, 8). Thus, by controlling the activation state of Rac and Rho, Ras can regulate the cell cytoskeleton as well as stimulate cell growth. In the absence of Rac or Rho activation (e.g., in cells treated with alendronate), Ras-activation induced by FOH could still stimulate cell spreading and actin filament assembly through the activation of Rho. Besides, it has been previously reported that Ras activates phosphatidylinositol 3 kinase (PI3K) the products of which, phosphatidylinositol 4,5 bisphosphate [PI(3,4)P2] and phosphatidylinositol 3,4,5 trisphosphate [PI(3,4,5)P3], induce Rho-mediated F-actin assembly (7, 9, 10).In addition, according to the previous report using the macrophage-like cell line, although both FOH and geranylgeraniol were effective in preventing caspase activation and apoptosis after 24 h of treatment with aminobisphosphonates, only geranylgeraniol was effective after 48 h of treatment (11). This suggests that geranylgeranylated proteins (such as Rho and Rac) rather than farnesylated proteins (such as Ras) may be particularly important for preventing apoptosis. In this report, the authors comment that the protective effect of FOH after 24 h may result from the conversion of some FOH to geranylgeranyl PPi via farnesyl PPi, although Crick et al. (12) concluded that FOH could not be converted to geranylgeranyl PPi in rat glial cells and African green monkey kidney cells (13, 14). Therefore, we discussed the possibility of the conversion from FOH to geranylgeranyl PPi in our article, although this speculation has not been examined in ovarian cancer cells.Requests for reprints: Ken-ichirou Morishige, Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3351; Fax: 81-6-6879-3359; E-mail: mken@gyne.med.osaka-u.ac.jp1Unpublished observations.