Pim-selective inhibitor DHPCC-9 reveals Pim kinases as potent stimulators of cancer cell migration and invasion.

BACKGROUND: Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9), which we had recently identified as a potent and selective inhibitor for all Pim family members. RESULTS: We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases. CONCLUSIONS: Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.

Inhibition of GGTase-I and FTase disrupts cytoskeletal organization of human PC-3 prostate cancer cells.

The mevalonate synthesis pathway produces intermediates for isoprenylation of small GTPases, which are involved in the regulation of actin cytoskeleton and cell motility. Here, we investigated the role of the prenylation transferases in the regulation of the cytoskeletal organization and motility of PC-3 prostate cancer cells. This was done by using FTI-277, GGTI-298 or NE-10790, the specific inhibitors of FTase (farnesyltransferase), GGTase (geranylgeranyltransferase)-I and -II, respectively. Treatment of PC-3 cells with GGTI-298 and FTI-277 inhibited migration and invasion in a time- and dose-dependent manner. This was associated with disruption of F-actin organization and decreased recovery of GFP-actin. Immunoblot analysis of various cytoskeleton-associated proteins showed that the most striking change in GGTI-298- and FTI-277-treated cells was a markedly decreased level of total and phosphorylated cofilin, whereas the level of cofilin mRNA was not decreased. The treatment of PC-3 cells with GGTI-298 also affected the dynamics of GFP-paxillin and decreased the levels of total and phosphorylated paxillin. The levels of phosphorylated FAK (focal adhesion kinase) and PAK (p-21-associated kinase)-2 were also lowered by GGTI-298, but levels of paxillin or FAK mRNAs were not affected. In addition, GGTI-298 had a minor effect on the activity of MMP-9. RNAi knockdown of GGTase-Ibeta inhibited invasion, disrupted F-actin organization and decreased the level of cofilin in PC-3 cells. NE-10790 did not have any effect on PC-3 prostate cancer cell motility or on the organization of the cytoskeleton. In conclusion, our results demonstrate the involvement of GGTase-I- and FTase-catalysed prenylation reactions in the regulation of cytoskeletal integrity and motility of prostate cancer cells and suggest them as interesting drug targets for development of inhibitors of prostate cancer metastasis.

Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells.

Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.

Alendronate inhibits invasion of PC-3 prostate cancer cells by affecting the mevalonate pathway.

Breast and prostate cancer preferentially metastasize in the skeleton, inducing locally increased bone resorption by osteoclasts. Bisphosphonates (BPs), potent inhibitors of osteoclasts and bone resorption, are able to reduce metastatic bone lesions, but the metastasis-related cellular target molecules for BPs have not yet been identified. In osteoclasts, nitrogen-containing BPs inhibit the function of the mevalonate pathway, impairing the prenylation and activation of small GTPases. In addition, direct effects of BPs on cancer cells have been suggested. In the present study, the effects of two clinically used BPs, the amino-BP alendronate and clodronate, on adhesion, invasion, and migration of human PC-3 prostate cancer cells were examined in vitro. We also studied the possible role of the mevalonate pathway in invasion and migration of PC-3 cells using the beta-hydroxy-beta-methylglutaryl-CoA reductase inhibitor mevastatin and the mevalonate pathway intermediates mevalonate (mevalonic acid lactone), geranylgeraniol, and trans-trans-farnesol. The results demonstrate that alendronate pretreatment very effectively inhibited in vitro invasion of prostate cancer cells in a dose-dependent manner, with an IC50 as low as approximately 1 pM. The inhibition was similar to that of mevastatin. Clodronate also inhibited invasion, but the IC50 was 0.1 microM. Importantly, geranylgeraniol and trans-trans-farnesol reversed the inhibitory effect of alendronate and mevastatin but not the clodronate-induced inhibition of invasion. Alendronate pretreatment also inhibited migration, which was partially reversed by geranylgeraniol and trans-trans-farnesol. Adhesion of PC-3 cells to various matrices was reduced, and their F-actin organization was changed. Alendronate pretreatment also inhibited invasion of human Du-145 prostate and MDA-MB-231 breast cancer cells. As a conclusion, the results demonstrate that the mevalonate pathway leading to protein prenylation is important for cancer cell invasion and migration in vitro. They further suggest that interference with this pathway is involved in inhibition of invasion and migration of prostate cancer cells by the amino-BP alendronate but that the mechanism of clodronate inhibition is different. It is possible that BPs have therapeutic potential in preventing the spread of prostate cancer.

Adenosine inhibits tumor cell invasion via receptor-independent mechanisms.

UNLABELLED: Extracellular adenosine mediates diverse anti-inflammatory, angiogenic, and other signaling effects via binding to adenosine receptors, and it also regulates cell proliferation and death via activation of the intrinsic signaling pathways. Given the emerging role of adenosine and other purines in tumor growth and metastasis, this study evaluated the effects of adenosine on the invasion of metastatic prostate and breast cancer cells. Treatment with low micromolar concentrations of adenosine, but not other nucleosides or adenosine receptor agonists, inhibited subsequent cell invasion and migration through Matrigel- and laminin-coated inserts. These inhibitory effects occurred via intrinsic receptor-independent mechanisms, despite the abundant expression of A2B adenosine receptors (ADORA2B). Extracellular nucleotides and adenosine were shown to be rapidly metabolized on tumor cell surfaces via sequential ecto-5'-nucleotidase (CD73/NT5E) and adenosine deaminase reactions with subsequent cellular uptake of nucleoside metabolites and their intracellular interconversion into ADP/ATP. This was accompanied by concurrent inhibition of AMP-activated protein kinase and other signaling pathways. No differences in the proliferation rates, cytoskeleton assembly, expression of major adhesion molecules [integrin-1ß (ITGB1), CD44, focal adhesion kinase], and secretion of matrix metalloproteinases were detected between the control and treated cells, thus excluding the contribution of these components of invasion cascade to the inhibitory effects of adenosine. These data provide a novel insight into the ability of adenosine to dampen immune responses and prevent tumor invasion via two different, adenosine receptor-dependent and -independent mechanisms. IMPLICATIONS: This study suggests that the combined targeting of adenosine receptors and modulation of intracellular purine levels can affect tumor growth and metastasis phenotypes.