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BackgroundRHEB is a unique member of the RAS superfamily of small GTPases expressed in all tissues and conserved from yeast to humans. Early studies on RHEB indicated a possible RHEB-RAF interaction, but this has not been fully explored. Recent work on cancer genome databases has revealed a reoccurring mutation in RHEB at the Tyr35 position, and a recent study points to the oncogenic potential of this mutant that involves activation of RAF/MEK/ERK signaling. These developments prompted us to reassess the significance of RHEB effect on RAF, and to compare mutant and wild type RHEB.MethodsTo study RHEB-RAF interaction, and the effect of the Y35N mutation on this interaction, we used transfection, immunoprecipitation, and Western blotting techniques. We generated cell lines stably expressing RHEB WT, RHEB Y35N, and KRAS G12V, and monitored cellular transforming properties through cell proliferation, anchorage independent growth, cell cycle analysis, and foci formation assays.ResultsWe observe a strong interaction between RHEB and BRAF, but not with CRAF. This interaction is dependent on an intact RHEB effector domain and RHEB-GTP loading status. RHEB overexpression decreases RAF activation of the RAF/MEK/ERK pathway and RHEB knockdown results in an increase in RAF/MEK/ERK activation. RHEB Y35N mutation has decreased interaction with BRAF, and RHEB Y35N cells exhibit greater BRAF/CRAF heterodimerization resulting in increased RAF/MEK/ERK signaling. This leads to cancer transformation of RHEB Y35N stably expressing cell lines, similar to KRAS G12 V expressing cell lines.ConclusionsRHEB interaction with BRAF is crucial for inhibiting RAF/MEK/ERK signaling. The RHEB Y35N mutant sustains RAF/MEK/ERK signaling due to a decreased interaction with BRAF, leading to increased BRAF/CRAF heterodimerization. RHEB Y35N expressing cells undergo cancer transformation due to decreased interaction between RHEB and BRAF resulting in overactive RAF/MEK/ERK signaling. Taken together with the previously established function of RHEB to activate mTORC1 signaling, it appears that RHEB performs a dual function; one is to suppress the RAF/MEK/ERK signaling and the other is to activate mTORC1 signaling.

New therapy development is critically needed for ovarian cancer. We used the chicken egg CAM assay to evaluate efficacy of anticancer drug delivery using recently developed biodegradable PMO (periodic mesoporous organosilica) nanoparticles. Human ovarian cancer cells were transplanted onto the CAM membrane of fertilized eggs, resulting in rapid tumor formation. The tumor closely resembles cancer patient tumor and contains extracellular matrix as well as stromal cells and extensive vasculature. PMO nanoparticles loaded with doxorubicin were injected intravenously into the chicken egg resulting in elimination of the tumor. No significant damage to various organs in the chicken embryo occurred. In contrast, injection of free doxorubicin caused widespread organ damage, even when less amount was administered. The lack of toxic effect of nanoparticle loaded doxorubicin was associated with specific delivery of doxorubicin to the tumor. Furthermore, we observed excellent tumor accumulation of the nanoparticles. Lastly, a tumor could be established in the egg using tumor samples from ovarian cancer patients and that our nanoparticles were effective in eliminating the tumor. These results point to the remarkable efficacy of our nanoparticle based drug delivery system and suggests the value of the chicken egg tumor model for testing novel therapies for ovarian cancer.

Epithelial ovarian cancer (EOC) is the most deadly gynecologic malignancy on account of its late stage at diagnosis and frequency of drug resistant recurrences. Novel therapies to overcome these barriers are urgently needed. TWIST is a developmental transcription factor reactivated in cancers and linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance, making it a promising therapeutic target. In this work, we demonstrate the efficacy of TWIST siRNA (siTWIST) and two nanoparticle delivery platforms to reverse chemoresistance in EOC models. Polyamidoamine dendrimers and mesoporous silica nanoparticles (MSNs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with cisplatin plus MSN-siTWIST exhibited lower tumor burden than mice treated with cisplatin alone, with most of the effect coming from reduction in disseminated tumors. This platform has potential application for overcoming the clinical challenges of metastasis and chemoresistance in EOC and other TWIST overexpressing cancers.

Nanoparticles that respond to internal and external stimuli to carry out controlled release of anticancer drugs have been developed. In this review, we focus on the development of mesoporous silica based nanoparticles, as this type of materials provides a relatively stable material that is amenable to various chemical modifications. We first provide an overview of various designs employed to construct MSN-based controlled release systems. These systems respond to internal stimuli such as pH, redox state and the presence of biomolecules as well as to external stimuli such as light and magnetic field. They are at a different stage of development; depending on the system, their operation has been demonstrated in aqueous solution, in cancer cells or in animal models. Efforts to develop MSNs with multi-functionality will be discussed. Safety and biodegradation of MSNs, issues that need to be overcome for clinical development of MSNs, will be discussed. Advances in the synthesis of mechanized theranostic nanoparticles open up the possibility to start envisioning future needs for medical equipment.

BackgroundKey effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).MethodsWe used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.ResultsImmunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.ConclusionsOur data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer.

BackgroundGlioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has been associated with a high level of the mechanistic target of rapamycin complex 2 (mTORC2) activity. We aimed to observe roles of mTORC2 in GBM cells especially on actin cytoskeleton reorganization, cell migration and invasion, and further determine new important players involved in the regulation of these cellular processes.MethodsTo further investigate the significance of mTORC2 in GBM, we treated GBM cells with PP242, an ATP-competitive inhibitor of mTOR, and used RICTOR siRNA to knock down mTORC2 activity. Effects on actin cytoskeleton, focal adhesion, migration, and invasion of GBM cells were examined. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal arrangement and motility/invasion, we affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Characterization of the protein of interest was performed.ResultsIn addition to the inhibition of mTORC2 activity, we demonstrated significant alteration of actin distribution as revealed by the use of phalloidin staining. Furthermore, vinculin staining was altered which suggests changes in focal adhesion. Inhibition of cell migration and invasion was observed with PP242. Two major proteins that are associated with this mTORC2 multiprotein complex were found. Mass spectrometry identified one of them as Filamin A (FLNA). Association of FLNA with RICTOR but not mTOR was demonstrated. Moreover, in vitro, purified mTORC2 can phosphorylate FLNA likewise its known substrate, AKT. In GBM cells, colocalization of FLNA with RICTOR was observed, and the overall amounts of FLNA protein as well as phosphorylated FLNA are high. Upon treatments of RICTOR siRNA or PP242, phosphorylated FLNA levels at the regulatory residue (Ser2152) decreased. This treatment also disrupted colocalization of Actin filaments and FLNA.ConclusionsOur results support FLNA as a new downstream effector of mTORC2 controlling GBM cell motility. This new mTORC2-FLNA signaling pathway plays important roles in motility and invasion of glioblastoma cells.

Growth and progression of solid tumors depend on the integration of multiple pro-growth and survival signals, including the induction of angiogenesis. TWIST1 is a transcription factor whose reactivation in tumors leads to epithelial to mesenchymal transition (EMT), including increased cancer cell stemness, survival, and invasiveness. Additionally, TWIST1 drives angiogenesis via activation of IL-8 and CCL2, independent of VEGF signaling. In this work, results suggest that chemically modified siRNA against TWIST1 reverses EMT both in vitro and in vivo. siRNA delivery with a polyethyleneimine-coated mesoporous silica nanoparticle (MSN) led to reduction of TWIST1 target genes and migratory potential in vitro. In mice bearing xenograft tumors, weekly intravenous injections of the siRNA-nanoparticle complexes resulted in decreased tumor burden together with a loss of CCL2 suggesting a possible anti-angiogenic response. Therapeutic use of TWIST1 siRNA delivered via MSNs has the potential to inhibit tumor growth and progression in many solid tumor types.From the clinical editorTumor progression and metastasis eventually lead to patient mortality in the clinical setting. In other studies, it has been found that TWIST1, a transcription factor, if reactivated in tumors, would lead to downstream events including angiogenesis and result in poor prognosis in cancer patients. In this article, the authors were able to show that when siRNA against TWIST1 was delivered via mesoporous silica nanoparticle, there was tumor reduction in an in-vivo model. The results have opened up a new avenue for further research in this field.

In this review we highlight recent progress in the study of Rheb family GTPases. Structural studies using X-ray crystallography and NMR have given us insight into unique features of this GTPase. Combined with mutagenesis studies, these works have expanded our understanding of residues that affect Rheb GTP/GDP bound ratios, effector protein interactions, and stimulation of mTORC1 signaling. Analysis of cancer genome databases has revealed that several human carcinomas contain activating mutations of the protein. Rheb's role in activating mTORC1 signaling at the lysosome in response to stimuli has been further elucidated. Rheb has also been suggested to play roles in other cellular pathways including mitophagy and peroxisomal ROS response. A number of studies in mice have demonstrated the importance of Rheb in development, as well as in a variety of functions including cardiac protection and myelination. We conclude with a discussion of future prospects in the study of Rheb family GTPases.

Abstract Background Lung cancer is the leading cause of cancer-related mortality. Therapies against non-small cell lung cancer (NSCLC) are particularly needed, as this type of cancer is relatively insensitive to chemotherapy and radiation therapy. We recently identified GGTI compounds that are designed to block geranylgeranylation and membrane association of signaling proteins including the Rho family G-proteins. One of the GGTIs is P61A6 which inhibits proliferation of human cancer cells, causes cell cycle effects with G1 accumulation and exhibits tumor-suppressing effects with human pancreatic cancer xenografts. In this paper, we investigated effects of P61A6 on non-small cell lung cancer (NSCLC) cells in vitro and in vivo. Methods Three non-small cell lung cancer cell lines were used to test the ability of P61A6 to inhibit cell proliferation. Further characterization involved analyses of geranylgeranylation, membrane association and activation of RhoA, and anchorage-dependent and –independent growth, as well as cell cycle effects and examination of cell cycle regulators. We also generated stable cells expressing RhoA-F, which bypasses the geranylgeranylation requirement of wild type RhoA, and examined whether the proliferation inhibition by P61A6 is suppressed in these cells. Tumor xenografts of NSCLC cells growing in nude mice were also used to test P61A6’s tumor-suppressing ability. Results P61A6 was shown to inhibit proliferation of NSCLC lines H358, H23 and H1507. Detailed analysis of P61A6 effects on H358 cells showed that P61A6 inhibited geranylgeranylation, membrane association of RhoA and caused G1 accumulation associated with decreased cyclin D1/2. The effects of P61A6 to inhibit proliferation could mainly be ascribed to RhoA, as expression of the RhoA-F geranylgeranylation bypass mutant rendered the cells resistant to inhibition by P61A6. We also found that P61A6 treatment of H358 tumor xenografts growing in nude mice reduced their growth as well as the membrane association of RhoA in the tumors. Conclusion Thus, P61A6 inhibits proliferation of NSCLC cells and causes G1 accumulation associated with decreased cyclin D1/2. The result with the RhoA-F mutant suggests that the effect of P61A6 to inhibit proliferation is mainly through the inhibition of RhoA. P61A6 also shows efficacy to inhibit growth of xenograft tumor.

The role of c-Crk (CRK) in promoting metastasis is well described however the role of CRK phosphorylation and the corresponding signaling events are not well explained. We have observed CRK-II serine 41 phosphorylation is inversely correlated with p120-catenin and E-cadherin expressions in non-small cell lung cancer (NSCLC) cells. Therefore, we investigated the role of CRK-II serine 41 phosphorylation in the down-regulation of p120-catenin, cell motility and cell invasiveness in NSCLC cells. For this purpose, we expressed phosphomimetic and phosphodeficient CRK-II serine 41 mutants in NSCLC cells. NSCLC cells expressing phosphomimetic CRK-II seine 41 mutant showed lower p120-catenin level while CRK-II seine 41 phosphodeficient mutant expression resulted in higher p120-catenin. In addition, A549 cells expressing CRK-II serine 41 phosphomimetic mutant demonstrated more aggressive behavior in wound healing and invasion assays and, on the contrary, expression of phosphodeficient CRK-II serine 41 mutant in A549 cells resulted in reduced cell motility and invasiveness. We also provide evidence that PAK1 mediates CRK-II serine 41 phosphorylation. RNAi mediated silencing of PAK1 increased p120-catenin level in A549 and H157 cells. Furthermore, PAK1 silencing decreased cell motility and invasiveness in A549 cells. These effects were abrogated in A549 cells expressing phosphomimetic CRK-II serine 41. In summary, these data provide evidence for the role of PAK1 in the promotion of cell motility, cell invasiveness and the down regulation of p120-catenin through CRK serine 41 phosphorylation in NSCLC cells.