Identification and characterization of mechanism of action of P61-E7, a novel phosphine catalysis-based inhibitor of geranylgeranyltransferase-I

Small molecule inhibitors of protein geranylgeranyltransferase-I (GGTase-I) provide a promising type of anticancer drugs. Here, we first report the identification of a novel tetrahydropyridine scaffold compound, P61-E7, and define effects of this compound on pancreatic cancer cells. P61-E7 was identified from a library of allenoate-derived compounds made through phosphine-catalyzed annulation reactions. P61-E7 inhibits protein geranylgeranylation and blocks membrane association of geranylgeranylated proteins. P61-E7 is effective at inhibiting both cell proliferation and cell cycle progression, and it induces high p21(CIP1/WAF1) level in human cancer cells. P61-E7 also increases p27(Kip1) protein level and inhibits phosphorylation of p27(Kip1) on Thr187. We also report that P61-E7 treatment of Panc-1 cells causes cell rounding, disrupts actin cytoskeleton organization, abolishes focal adhesion assembly and inhibits anchorage independent growth. Because the cellular effects observed pointed to the involvement of RhoA, a geranylgeranylated small GTPase protein shown to influence a number of cellular processes including actin stress fiber organization, cell adhesion and cell proliferation, we have evaluated the significance of the inhibition of RhoA geranylgeranylation on the cellular effects of inhibitors of GGTase-I (GGTIs). Stable expression of farnesylated RhoA mutant (RhoA-F) results in partial resistance to the anti-proliferative effect of P61-E7 and prevents induction of p21(CIP1/WAF1) and p27(Kip1) by P61-E7 in Panc-1 cells. Moreover, stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation caused by P61-E7. Taken together, these findings suggest that P61-E7 is a promising GGTI compound and that RhoA is an important target of P61-E7 in Panc-1 pancreatic cancer cells.     

Development of Nanoparticles Incorporating a Novel Liposomal Membrane Destabilization Peptide for Efficient Release of Cargos into Cancer Cells

In anti-cancer therapy mediated by a nanoparticle-based drug delivery system (DDS), overall efficacy depends on the release efficiency of cargos from the nanoparticles in the cancer cells as well as the specificity of delivery to tumor tissue. However, conventional liposome-based DDS have no mechanism for specifically releasing the encapsulated cargos inside the cancer cells. To overcome this barrier, we developed nanoparticles containing a novel liposomal membrane destabilization peptide (LMDP) that can destabilize membranes by cleavage with intramembranous proteases on/in cancer cells. Calcein encapsulated in liposomes modified with LMDP (LMDP-lipo) was effectively released in the presence of a membrane fraction containing an LMDP-cleavable protease. The release was inhibited by a protease inhibitor, suggesting that LMDP-lipo could effectively release its cargo into cells in response to a cancer-specific protease. Moreover, when LMDP-lipo contained fusogenic lipids, the release of cargo was accelerated, suggesting that the fusion of LMDP-lipo with cellular membranes was the initial step in the intracellular delivery. Time-lapse microscopic observations showed that the release of cargo from LMDP-lipo occurred immediately after association of LMDP-lipo with target cells. Consequently, LMDP-lipo could be a useful nanoparticle capable of effective release of cargos specifically into targeted cancer cells.     

Farnesylated RhoB Prevents Cell Cycle Arrest and Actin Cytoskeleton Disruption Caused by the Geranylgeranyltransferase I Inhibitor GGTI-298

Here we demonstrate that the geranylgeranyltransferase-I inhibitor GGTI-298 inhibits the RhoB pathway and disrupts stress fiber and focal adhesion formation in NIH-3T3 cells. Farnesylated V14RhoB-CAIM (resistant to GGTI-298), but not geranylgeranylated V14RhoB (-CLLL), prevented inhibition of actin stress fiber and focal adhesion formation, underlining the critical role of RhoB. In contrast, farnesylated, V14RhoA (-CVLS) was unable to prevent effects of GGTI 298 on cytoskeleton organization. Furthermore, the ability of GGTI-298 to induce p21WAF and to block cells in the G0/G1 phase of the cell cycle was also prevented by farnesylated V14RhoB but not by farnesylated V14RhoA. Moreover, treatment with GGTI-298 of cells expressing farnesylated RhoB results in accumulation of these cells in the G2/M phase. Therefore, the RhoB pathway is a critical target of GGTI-298.     

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics.     

Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism. We previously reported that 5-LOX and 12-LOX are upregulated in human pancreatic cancer cells and that blockade of these enzymes abolishes pancreatic cancer cell growth. The present study was aimed at evaluating the effect of LOX inhibition on differentiation and apoptosis in pancreatic cancer cells in parallel with growth inhibition. Four human pancreatic cancer cell lines, PANC-1, MiaPaca2, Capan2, and HPAF, were used. Apoptosis was evaluated by three separate methods, including DNA propidium iodide staining, DNA fragmentation, and the TUNEL assay. Morphological changes and carbonic anhydrase activity were used to determine the effect of LOX inhibitors on differentiation. The general LOX inhibitor NDGA, the 5-LOX inhibitor Rev5901, and the 12-LOX inhibitor baicalein all induced apoptosis in all four pancreatic cancer cell lines, as confirmed by all three methods, suggesting that both the 5-LOX and 12-LOX pathways are important for survival of these cells. Furthermore, NDGA, Rev5901, or baicalein resulted in marked cellular morphological changes in parallel with increased intracellular carbonic anhydrase activity, indicating that LOX blockade induced a more differentiated phenotype in human pancreatic cancer cells. Together with our previous findings, this study suggests that perturbations of LOX activity affect pancreatic cancer cell proliferation and survival. Blockade of LOX enzymes may be valuable for the treatment of human pancreatic cancer.     

Inhibitors of protein geranylgeranyltransferase-I lead to prelamin A accumulation in cells by inhibiting ZMPSTE24

Protein farnesyltransferase (FTase) inhibitors, generally called "FTIs," block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis-GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl-prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing.     

Farnesylated RhoB prevents cell cycle arrest and actin cytoskeleton disruption caused by the geranylgeranyltransferase I inhibitor GGTI-298

Here we demonstrate that the geranylgeranyltransferase-I inhibitor GGTI-298 inhibits the RhoB pathway and disrupts stress fiber and focal adhesion formation in NIH-3T3 cells. Farnesylated (V14)RhoB-CAIM (resistant to GGTI-298), but not geranylgeranylated (V14)RhoB (-CLLL), prevented inhibition of actin stress fiber and focal adhesion formation, underlining the critical role of RhoB. In contrast, farnesylated, (V14)RhoA (-CVLS) was unable to prevent effects of GGTI 298 on cytoskeleton organization. Furthermore, the ability of GGTI-298 to induce p21(WAF) and to block cells in the G(0)/G(1) phase of the cell cycle was also prevented by farnesylated (V14)RhoB but not by farnesylated (V14)RhoA. Moreover, treatment with GGTI-298 of cells expressing farnesylated RhoB results in accumulation of these cells in the G(2)/M phase. Therefore, the RhoB pathway is a critical target of GGTI-298.     

Inhibitors of protein geranylgeranyltransferase I and Rab geranylgeranyltransferase identified from a library of allenoate-derived compounds

Protein geranylgeranylation is critical for the function of a number of proteins such as RhoA, Rac, and Rab. Protein geranylgeranyltransferase I (GGTase-I) and Rab geranylgeranyltransferase (RabGGTase) catalyze these modifications. In this work, we first describe the identification and characterization of small molecule inhibitors of GGTase-I (GGTI) with two novel scaffolds from a library consisting of allenoate-derived compounds. These compounds exhibit specific inhibition of GGTase-I and act by competing with a substrate protein. Derivatization of a carboxylic acid emanating from the core ring of one of the GGTI compounds dramatically improves their cellular activity. The improved GGTI compounds inhibit proliferation of a variety of human cancer cell lines and cause G(1) cell cycle arrest and induction of p21(CIP1/WAF1). We also report the identification of novel small molecule inhibitors of RabGGTase. These compounds were identified first by screening our GGTI compounds for those that also exhibited RabGGTase inhibition. This led to the discovery of a common structural feature for RabGGTase inhibitors: the presence of a characteristic six-atom aliphatic tail attached to the penta-substituted pyrrolidine core. Further screening led to the identification of compounds with preferential inhibition of RabGGTase. These compounds inhibit RabGGTase activity by competing with the substrate protein. These novel compounds may provide valuable reagents to study protein geranylgeranylation.     

Role of the vacuolar-ATPase in Sindbis virus infection

Bafilomycin A(1) is a specific inhibitor of the vacuolar-ATPase (V-ATPase), which is responsible for pH homeostasis of the cell and for the acidification of endosomes. Bafilomycin A(1) has been commonly used as a method of inhibition of infection by viruses known or suspected to follow the path of receptor-mediated endocytosis and low-pH-mediated membrane fusion. The exact method of entry for Sindbis virus, the prototype alphavirus, remains undetermined. To further investigate the role of the V-ATPase in Sindbis virus infection, the effects of bafilomycin A(1) on the infection of BHK and insect cells by Sindbis virus were studied. Bafilomycin A(1) was found to block the expression of a virus-encoded reporter gene in both infection and transfection of BHK cells. The inhibitory effects of bafilomycin A(1) were found to be reversible. The results suggest that in BHK cells in the presence of bafilomycin A(1), virus RNA enters the cell and is translated, but replication and proper folding of the product proteins requires the function of the V-ATPase. Bafilomycin A(1) had no significant effect on the outcome of infection in insect cells.     

Impact of oncogenic driver mutations on feedback between the PI3K and MEK pathways in cancer cells

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.     

Bafilomycin A1 inhibits Helicobacter pylori-induced vacuolization of HeLa cells

Bafilomycin A1, a specific inhibitor of the vacuolar-type H⁺-ATPase, responsible for acidification of intra-cellular compartments, prevents the vacuolization of Hela cells induced by H. pylori, with an inhibitory concentration giving 50% of maximal (ID₅₀) of 4 nM. Bafilomycin A1 is also very efficient in restoring vacuolated cells to a normal appearance. The vacuolating activity of Helicobacter pylori is not inhibited by a series of specific inhibitors of vacuolar H⁺-ATPases. These findings indicate that a transmembrane pH gradient is needed for the formation and growth of vacuoles caused by the bacterium and that this pH gradient is due to the activity of a vacuolar ATPase proton pump of HeLa cells.     

2-Arylindole-3-acetamides: FPP-competitive inhibitors of farnesyl protein transferase

A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.     

Lipoxygenase inhibitors abolish proliferation of human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism, but their role in pancreatic cancer growth is unknown. The expression of 5-LOX and 12-LOX as well as their effects on cell proliferation was investigated in four human pancreatic cancer cell lines (PANC-1, MiaPaca2, Capan2, and ASPC-1). Expression of 5-LOX and 12-LOX mRNA was measured by nested RT-PCR. Effects of LOX inhibitors and specific LOX antisense oligonucleotides on pancreatic cancer cell proliferation were measured by (3)H-thymidine incorporation. Our results showed that (1) 5-LOX and 12-LOX were expressed in all pancreatic cancer cell lines tested, while they were not detectable in normal human pancreatic ductal cells; (2) both LOX inhibitors and LOX antisense markedly inhibited cell proliferation in a concentration-dependent and time-dependent manner; (3) the 5-LOX and 12-LOX metabolites 5-HETE and 12-HETE as well as arachidonic and linoleic acids directly stimulated pancreatic cancer cell proliferation; (4) LOX inhibitor-induced growth inhibition was reversed by 5-HETE and 12-HETE. The current studies indicate that both 5-LOX and 12-LOX expression is upregulated in human pancreatic cancer cells and LOX plays a critical role in pancreatic cancer cell proliferation. LOX inhibitors may be valuable for the treatment of pancreatic cancer.     

Vacuolar H+-ATPase inhibitor induces apoptosis via lysosomal dysfunction in the human gastric cancer cell line MKN-1

We investigated the mechanism of apoptosis induced by bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase. Bafilomycin A(1) significantly inhibited the growth of MKN-1 human gastric cancer cells. Bafilomycin A(1) induced apoptosis as demonstrated by DNA ladder formation and the TUNEL method. We designed a flow cytometric assay to detect the alteration in lysosomal pH using a fluorescent probe, fluorescein isothiocyanate-conjugated dextran. This assay revealed that bafilomycin A(1) dramatically increased lysosomal pH. However, bafilomycin A(1) induced neither significant decrease in mitochondrial transmembrane potential nor the release of mitochondrial cytochrome c into the cytoplasm. Western blotting showed that cathepsin D, but not cathepsin L, was released into the cytoplasm. The activity of caspase-3 was significantly increased by bafilomycin A(1). However, cathepsin D did not directly cleave procaspase-3. These findings suggest that bafilomycin A(1)-induced apoptosis in MKN-1 cells is mediated by other proteases released after lysosomal dysfunction followed by activation of caspase-3 in a cytochrome c-independent manner. The present study showed that flow cytometric analysis of lysosomal pH can be useful to evaluate lysosomal protease-mediated apoptosis.     

The small GTPase RhoA is crucial for MC3T3‐E1 osteoblastic cell survival

Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. RhoA is activated by geranylgeranylation, which promotes its membrane anchoring. In serum‐starved MC3T3‐E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase‐3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase‐3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl tranferase I inhibitor GGTI‐2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1‐34, a potent anti‐apoptotic agent, completely antagonized the stimulatory effects of GGTI‐2166, dominant negative RhoA, and Y27632, on caspase‐3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1‐34 are independent of this pathway. J. Cell. Biochem. 106: 896–902, 2009. © 2009 Wiley‐Liss, Inc.     

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.     

Class I and Class II Histone Deacetylases Are Potential Therapeutic Targets for Treating Pancreatic Cancer

Background

Pancreatic cancer is a highly malignant disease with an extremely poor prognosis. Histone deacetylase inhibitors (HDACIs) have shown promising antitumor activities against preclinical models of pancreatic cancer, either alone or in combination with chemotherapeutic agents. In this study, we sought to identify clinically relevant histone deacetylases (HDACs) to guide the selection of HDAC inhibitors (HDACIs) tailored to the treatment of pancreatic cancer.

Methodology

HDAC expression in seven pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was determined by Western blotting. Antitumor interactions between class I- and class II-selective HDACIs were determined by MTT assays and standard isobologram/CompuSyn software analyses. The effects of HDACIs on cell death, apoptosis and cell cycle progression, and histone H4, alpha-tubulin, p21, and γH2AX levels were determined by colony formation assays, flow cytometry analysis, and Western blotting, respectively.

Results

The majority of classes I and II HDACs were detected in the pancreatic cancer cell lines, albeit at variable levels. Treatments with MGCD0103 (a class I-selective HDACI) resulted in dose-dependent growth arrest, cell death/apoptosis, and cell cycle arrest in G2/M phase, accompanied by induction of p21 and DNA double-strand breaks (DSBs). In contrast, MC1568 (a class IIa-selective HDACI) or Tubastatin A (a HDAC6-selective inhibitor) showed minimal effects. When combined simultaneously, MC1568 significantly enhanced MGCD0103-induced growth arrest, cell death/apoptosis, and G2/M cell cycle arrest, while Tubastatin A only synergistically enhanced MGCD0103-induced growth arrest. Although MC1568 or Tubastatin A alone had no obvious effects on DNA DSBs and p21 expression, their combination with MGCD0103 resulted in cooperative induction of p21 in the cells.

Conclusion

Our results suggest that classes I and II HDACs are potential therapeutic targets for treating pancreatic cancer. Accordingly, treating pancreatic cancer with pan-HDACIs may be more beneficial than class- or isoform-selective inhibitors.     

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‐Iβ 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.     

Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer

Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b, and miR-155, and down-regulation of miR-216a, miR-216b, miR-217, and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP, and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.