ABSTRACT
Histone deacetylase inhibitors (HDACIs) represent a class of promising agents that can improve radiotherapy in cancer treatment. However, the full therapeutic potential of HDACIs as radiosensitizers has been restricted by limited efficacy in solid malignancies. In this study, we report the development of nanoparticle (NP) formulations of HDACIs that overcome these limitations, illustrating their utility to improve the therapeutic ratio of the clinically established first generation HDACI vorinostat and a novel second generation HDACI quisinostat. We demonstrate that NP HDACIs are potent radiosensitizers in vitro and are more effective as radiosensitizers than small molecule HDACIs in vivo using mouse xenograft models of colorectal and prostate carcinomas. We found that NP HDACIs enhance the response of tumor cells to radiation through the prolongation of γ-H2AX foci. Our work illustrates an effective method for improving cancer radiotherapy treatment.
Subject(s)
Chemoradiotherapy , Histone Deacetylase Inhibitors/therapeutic use , Nanoparticles/chemistry , Neoplasms/therapy , Animals , Cell Line, Tumor , Chemistry, Pharmaceutical , Histone Deacetylase Inhibitors/pharmacology , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Hydroxamic Acids/therapeutic use , Male , Mice, Nude , Nanoparticles/ultrastructure , Neoplasms/pathology , Radiation-Sensitizing Agents/pharmacology , Radiation-Sensitizing Agents/therapeutic use , VorinostatABSTRACT
The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti-K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants.
Subject(s)
Genes, ras , Neoplasms/genetics , RNA Splicing , Amino Acid Sequence , Cell Line, Tumor , Humans , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
The metastasis-associated tyrosine phosphatase PRL-3/PTP4A is upregulated in numerous cancers, but the mechanisms modulating PRL-3 activity other than its expression levels have not been investigated. Here we report evidence for both Src-dependent tyrosine phosphorylation of PRL-3 and Src-mediated regulation of PRL-3 biological activities. We used structural mutants, pharmacological inhibitors and siRNA to demonstrate Src-dependent phosphorylation of endogenous PRL-3 in SW480 colon cancer cells. We also demonstrated that PRL-3 was not tyrosine phosphorylated in SYF mouse embryo fibroblasts deficient in Src, Yes and Fyn unless Src was re-expressed. Further, we show that platelet-derived growth factor (PDGF) can stimulate PRL-3 phosphorylation in a Src-dependent manner. Finally, we show that PRL-3-induced cell motility, Matrigel invasion and activation of the cytoskeleton-regulating small GTPase RhoC were abrogated in the presence of the phosphodeficient PRL-3 mutant Y53F, or by use of a Src inhibitor. Thus, PRL-3 requires the activity of a Src kinase, likely Src itself, to promote these cancer-associated phenotypes. Our data establish a model for the regulation of PRL-3 by Src that supports the possibility of their coordinate roles in signaling pathways promoting invasion and metastasis, and supports simultaneous use of novel molecularly targeted therapeutics directed at these proteins.
Subject(s)
Immediate-Early Proteins/metabolism , Models, Biological , Platelet-Derived Growth Factor/metabolism , Protein Tyrosine Phosphatases/metabolism , src-Family Kinases/metabolism , Animals , Cell Line, Tumor , Collagen , Drug Combinations , Humans , Immunoprecipitation , Laminin , Mice , Neoplasm Invasiveness/physiopathology , Phosphorylation , Proteoglycans , RNA, Small Interfering , ras Proteins/metabolism , rhoC GTP-Binding Protein , src-Family Kinases/geneticsABSTRACT
Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27Kip1. Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.
Subject(s)
Adenocarcinoma/pathology , Carcinoma, Pancreatic Ductal/pathology , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins c-pim-1/physiology , Proto-Oncogene Proteins/physiology , Radiation Tolerance , Signal Transduction/physiology , ras Proteins/physiology , Adenocarcinoma/radiotherapy , Carcinoma, Pancreatic Ductal/radiotherapy , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p27/analysis , Humans , Neoplasm Invasiveness , Pancreatic Neoplasms/radiotherapy , Phosphorylation , Proto-Oncogene Proteins c-pim-1/analysis , Proto-Oncogene Proteins p21(ras) , bcl-Associated Death Protein/metabolismABSTRACT
BACKGROUND: TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Thallion's proprietary DECIPHER® technology, a genomics and bioinformatics platform that predicts the chemical structures of secondary metabolites based on gene sequences obtained by scanning bacterial genomes. Our recent studies suggest that TLN-4601 inhibits the Ras-ERK MAPK pathway post Ras prenylation and prior to MEK activation. The Ras-ERK MAPK signaling pathway is a well-validated oncogenic cascade based on its central role in regulating the growth and survival of cells from a broad spectrum of human tumors. Furthermore, RAS isoforms are the most frequently mutated oncogenes, occurring in approximately 30% of all human cancers, and KRAS is the most commonly mutated RAS gene, with a greater than 90% incidence of mutation in pancreatic cancer. RESULTS: To evaluate whether TLN-4601 interferes with K-Ras signaling, we utilized human pancreatic epithelial cells and demonstrate that TLN-4601 treatment resulted in a dose- and time-dependent inhibition of Ras-ERK MAPK signaling. The compound also reduced Ras-GTP levels and induced apoptosis. Finally, treatment of MIA PaCa-2 tumor-bearing mice with TLN-4601 resulted in antitumor activity and decreased tumor Raf-1 protein levels. CONCLUSION: These data, together with phase I/II clinical data showing tolerability of TLN-4601, support conducting a clinical trial in advanced pancreatic cancer patients.
ABSTRACT
The proper subcellular localization and biological activity of most Ras and Rho family small GTPases are dependent on their posttranslational modification by isoprenylation. Farnesyltransferase (FTase) and geranylgeranyl transferase I (GGTase I) are the prenyltransferases that catalyze the irreversible attachment of C15 farnesyl (Ras, Rnd) or C20 (R-Ras, Ral, Rap, Rho, Rac, Cdc42) isoprenoid lipid moieties to these small GTPases and other proteins. Therefore, pharmacological inhibitors of FTase (FTIs) and GGTase I (GGTIs) have been developed to prevent these modifications and thereby to block the lipid-mediated association of Ras and Rho proteins with cellular membranes and the consequent signaling and transforming activities. In addition, other small molecule inhibitors such as farnesyl thiosalicylic acid (FTS) can compete with the isoprenoid moiety of small GTPases for membrane binding sites. Finally, endogenous regulatory proteins such as RhoGDIs can bind to and mask the prenyl groups of small GTPases, leading to their sequestration from membranes. We describe here methods to use each of these categories of prenylation inhibitors to manipulate and investigate the subcellular localization patterns and transforming potential of these Ras and Rho family GTPases.
Subject(s)
Enzyme Inhibitors/pharmacology , Prenylation/drug effects , Alkyl and Aryl Transferases/antagonists & inhibitors , Animals , Dimethylallyltranstransferase/antagonists & inhibitors , Farnesol/analogs & derivatives , Farnesol/pharmacology , Farnesyltranstransferase/antagonists & inhibitors , GTP Phosphohydrolases/antagonists & inhibitors , Guanine Nucleotide Dissociation Inhibitors/pharmacology , Mice , NIH 3T3 Cells , Protein Transport/drug effects , Salicylates/pharmacology , ras Proteins/antagonists & inhibitors , rho GTP-Binding Proteins/antagonists & inhibitors , rho-Specific Guanine Nucleotide Dissociation InhibitorsABSTRACT
Phosphatase found in regenerating liver (PRL)-1, PRL-2, and PRL-3 [also known as PTP4A1, PTP4A2, and PTP4A3, respectively] constitute a unique family of putative protein tyrosine phosphatases (PTPs) modified by farnesylation. PRL-3 is amplified and its message is up-regulated in colorectal carcinoma metastases. Its ectopic expression promotes invasive and metastatic properties, supporting a causal link between PRL-3 and late-stage cancer development. However, neither PRL phosphatase substrates nor their signaling pathways have been defined. To address possible mechanisms for the biological activity of PRL-3, we sought to identify its downstream targets, reasoning that regulators of motility and invasion, such as the Rho family of small GTPases, might be logical candidates. We found that levels of active RhoA and RhoC were increased 4- to 7-fold in SW480 colorectal carcinoma cells expressing exogenous PRL-1 and PRL-3, and that PRL-mediated motility and Matrigel invasion were blocked by pharmacologic inhibition of Rho kinase (ROCK), a key Rho effector. In contrast, the activity of Rac was reduced by PRL PTPs, whereas Cdc42 activity was unaffected. PRL-3 stimulated transcription driven by the serum response element in a Rho-dependent manner. We also confirmed that the ability of PRL PTPs to induce invasion and motility is dependent on farnesylation. Catalytic PRL-3 mutants (C104A or D72A) were impaired in PRL-3-induced invasion and Rho activation, indicating that these properties require phosphatase activity. We conclude that PRL PTPs stimulate Rho signaling pathways to promote motility and invasion. Characterization of PRL activity and regulatory pathways should enhance efforts to understand and interfere with PRL-mediated events in invasion and metastasis.
Subject(s)
Adenocarcinoma/enzymology , Cell Cycle Proteins/metabolism , Cell Movement/physiology , Colonic Neoplasms/enzymology , Immediate-Early Proteins/metabolism , Neoplasm Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Cell Cycle Proteins/genetics , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA-Binding Proteins/metabolism , Enzyme Activation , Humans , Immediate-Early Proteins/genetics , Membrane Proteins , Mice , NIH 3T3 Cells , Neoplasm Invasiveness , Neoplasm Proteins/genetics , Protein Prenylation , Protein Tyrosine Phosphatases/genetics , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Transfection , cdc42 GTP-Binding Protein/metabolism , ras Proteins , rhoC GTP-Binding ProteinABSTRACT
Modification of small GTPases by lipids is required for their proper subcellular localization and biological activity. Lipids added post-translationally include both farnesyl and geranylgeranyl isoprenoids and the fatty acid palmitate. Thus, specific small molecule inhibitors of these processes cause mislocalization of small GTPases and impair their biological activity. Common biochemical methods of determining the lipid modification status or inhibitor sensitivity of small GTPases, such as in vitro prenylation assays, SDS-PAGE mobility shifts or metabolic labeling, although highly useful in their own right, cannot distinguish differences among specific subpopulations of cells, link lipid modification status with other properties of interest, or provide spatio-temporal information. An alternative method takes advantage of the tight link between small GTPase lipid modification and subcellular localization. The innate localization pattern of the enhanced green fluorescent protein, a common epitope tag frequently used in live cell imaging, is altered by fusion to modified but not unmodified small GTPases. We describe here a technique that takes advantage of these properties to monitor post-translational modifications of these proteins in a rapid, visual manner in live cells.
Subject(s)
GTP Phosphohydrolases/genetics , Green Fluorescent Proteins/genetics , Lipid Metabolism , Molecular Probes , Protein Processing, Post-Translational/physiology , Animals , GTP Phosphohydrolases/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , Mice , NIH 3T3 Cells , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is characterized by dramatic premature aging and accelerated cardiovascular disease. HGPS is almost always caused by a de novo point mutation in the lamin A gene (LMNA) that activates a cryptic splice donor site, producing a truncated mutant protein termed "progerin." WT prelamin A is anchored to the nuclear envelope by a farnesyl isoprenoid lipid. Cleavage of the terminal 15 aa and the farnesyl group releases mature lamin A from this tether. In contrast, this cleavage site is deleted in progerin. We hypothesized that retention of the farnesyl group causes progerin to become permanently anchored in the nuclear membrane, disrupting proper nuclear scaffolding and causing the characteristic nuclear blebbing seen in HGPS cells. Also, we hypothesized that blocking farnesylation would decrease progerin toxicity. To test this hypothesis, the terminal CSIM sequence in progerin was mutated to SSIM, a sequence that cannot be farnesylated. SSIM progerin relocalized from the nuclear periphery into nucleoplasmic aggregates and produced no nuclear blebbing. Also, blocking farnesylation of authentic progerin in transiently transfected HeLa, HEK 293, and NIH 3T3 cells with farnesyltransferase inhibitors (FTIs) restored normal nuclear architecture. Last, treatment of both early- and late-passage human HGPS fibroblasts with FTIs resulted in significant reductions in nuclear blebbing. Our results suggest that treatment with FTIs represents a potential therapy for patients with HGPS.
Subject(s)
Cell Nucleus/metabolism , Cell Nucleus/pathology , Lamin Type A/chemistry , Lamin Type A/genetics , Lamin Type A/metabolism , Progeria/metabolism , Progeria/pathology , Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/metabolism , Animals , Cell Line , Cell Nucleus/drug effects , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Humans , Mice , Mutation/genetics , Progeria/drug therapy , Progeria/genetics , Protein Prenylation/drug effectsABSTRACT
RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.
Subject(s)
Cell Transformation, Neoplastic/pathology , Proto-Oncogene Proteins p21(ras)/physiology , ral GTP-Binding Proteins/physiology , ATP-Binding Cassette Transporters/metabolism , Animals , Carrier Proteins/metabolism , Cell Line , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , GTPase-Activating Proteins/metabolism , Gene Expression/genetics , Guanosine Triphosphate/metabolism , Humans , Mice , Mice, SCID , Neoplasm Transplantation/pathology , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Protein Binding/physiology , Protein Transport/physiology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RNA, Small Interfering/genetics , Transfection , Vesicular Transport Proteins , ral GTP-Binding Proteins/genetics , ral GTP-Binding Proteins/metabolism , ral Guanine Nucleotide Exchange Factor/genetics , ral Guanine Nucleotide Exchange Factor/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , DNA-Binding Proteins , Drug Resistance/genetics , Protein Prenylation/physiology , Proto-Oncogene Proteins p21(ras)/drug effects , Proto-Oncogene Proteins p21(ras)/metabolism , ras Proteins/drug effects , ras Proteins/metabolism , 3T3 Cells , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Division , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase , Gene Expression Profiling , Humans , Mice , Mutation , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Recombinant Fusion Proteins , Transcription Factors/metabolism , Transfection , ets-Domain Protein Elk-1 , ras Proteins/geneticsABSTRACT
Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.