James J. Fiordalisi

PRL Tyrosine Phosphatases Regulate Rho Family GTPases to Promote Invasion and Motility

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.

K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif

Significance The KRAS oncogene is mutated more frequently in human cancer than any other. The KRAS transcript is alternatively spliced to give rise to two products, K-Ras4A and K-Ras4B, both of which are oncogenic when KRAS is mutated. We detected significant amounts of each transcript in human tumor cells and colorectal carcinomas. We found that K-Ras4A is targeted to the plasma membrane by dual targeting motifs distinct from those of K-Ras4B. Because interfering with membrane association of Ras proteins remains one of the most attractive approaches to anti-Ras therapy, efforts in this direction will have to disrupt both the K-Ras4A and the K-Ras4B membrane-targeting pathways. 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.

Mammalian expression vectors for Ras family proteins: Generation and use of expression constructs to analyze Ras family function

Publisher Summary This chapter discusses the mammalian expression vectors for Ras family proteins. The chapter presents some observations concerning the strengths and weaknesses of several mammalian protein expression vectors, both commercially available and “homemade.” In addition to discussing the advantages and disadvantages of particular features of mammalian protein expression vectors, the chapter also compares and contrast them functionally with respect to biological readouts commonly used in the study of Ras protein function, including protein expression, signaling activity in enzyme-linked transcriptional trans -activation reporter assays, and transforming ability in fibroblast focus-forming assays. The chapter also discusses several techniques for generating and manipulating protein expression constructs. Several methods for introducing plasmid DNA into mammalian cells, including transfection with a variety of reagents and infection using retroviral packaging vectors is also discussed. A major conclusion from these analyses is that no one vector is ideally suited for all applications or cell lines. Those useful for stable expression may be inadequate for transient expression. Species and cell type differences can greatly influence the usefulness of a particular vector. The vector-specific nature of some assays also prompts a caution regarding observations made from exogenous overexpression of Ras proteins from heterologous promoters. The general tendency is to utilize a particular expression vector because it yields a positive response for a specific assay. If vast overexpression is required to achieve a response, consideration should be given to whether such a response represents a physiologically relevant function of Ras.

The oncogenic kinase Pim-1 is modulated by K-Ras signaling and mediates transformed growth and radioresistance in human pancreatic ductal adenocarcinoma cells

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.

Binding of native κ-neurotoxins and site-directed mutants to nicotinic acetylcholine receptors

The κ-neurotoxins are useful ligands for the pharmacological characterization of nicotinic acetylcholine receptors because they are potent antagonists at only a subgroup of these receptors containing either α3- or α4-subunits (IC50 ≤ 100 nM). Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus [κ-bungarotoxin (κ-Bgt), κ2-Bgt, κ3-Bgt] and Bungarus flaviceps [κ-flavitoxin (κ-Fvt)]. Two approaches were taken to examine the binding of these toxins to nicotinic receptors. First, venom-derived κ-Fvt and κ-Bgt were radioiodinated and the specific binding was measured of these toxins to overlapping synthetic peptides (16–20 amino acids in length) prepared based on the known sequence of the nicotinic receptor α3-subunit. At least two main regions of interaction between the toxins and the receptor subunit were identified, both lying in the N-terminal region of the subunit that is exposed to the extracellular space. The second approach examined the importance of several sequence positions in κ-Bgt for binding to α3-containing receptors in autonomic ganglia and α1-containing muscle receptors. This was done using site-directed mutants of κ-Bgt produced by an Escherichia coli expression system. Arg-34 and position 36 were important for binding to both receptor subtypes, while replacing Gln-26 with Trp-26 (an invariant in α-neurotoxins) increased affinity for the muscle receptor by 8-fold. The results confirm that κ-neurotoxins bind potently to the α3-subunit and bind with considerably reduced affinity (Kd ≈ 10 μM) to muscle receptors. Site-directed mutagenesis of recombinant κ-Bgt is thus an important approach for the study of structure-function relationships between κ-Bgt and nicotinic receptors.

Using inhibitors of prenylation to block localization and transforming activity

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.

REK, A GENE EXPRESSED IN RETINA AND BRAIN, ENCODES A RECEPTOR TYROSINE KINASE OF THE AXL/TYRO3 FAMILY

Rek (retina-expressed kinase) has been identified as a putative novel receptor-type tyrosine kinase of the Axl/Tyro3 family with a potential role in neural cell development. rek clones were isolated from a chick embryonic brain cDNA library with a DNA probe obtained by reverse transcriptase-polymerase chain reaction of mRNA from Müller glia-like cells cultured from chick embryonic retina. Sequence analysis indicated that Rek is a protein of 873 amino acids with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains with eight predicted N-glycosylation sites. Two consensus src homology 2 domain binding sites are present in the cytoplasmic domain, suggesting that Rek activates several signal transduction pathways. Northern analysis of rek mRNA revealed a 5.5-kilobase transcript in chick brain, retina, and kidney and in primary cultures of retinal Müller glia-like cells. Rek protein was identified by immunoprecipitation and immunoblotting as a 140-kDa protein expressed in the chick retina at embryonic days 6-13, which corresponded to the major period of neuronal and glial differentiation. Transfection of rek cDNA into COS cells resulted in transient expression of a putative precursor of 106 kDa that autophosphorylated in immune complex protein kinase assays. Overexpression of rek cDNA in mouse NIH3T3 fibroblasts resulted in activation of the 140-kDa rek kinase and induction of morphologically transformed foci. These properties indicated that Rek has oncogenic potential when overexpressed, but its normal function is likely to be related to cell-cell recognition events governing the differentiation or proliferation of neural cells.

Nanoparticle formulations of histone deacetylase inhibitors for effective chemoradiotherapy in solid tumors

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.

Src-Mediated Phosphorylation of the Tyrosine Phosphatase PRL-3 Is Required for PRL-3 Promotion of Rho Activation, Motility and Invasion

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.

TLN-4601 suppresses growth and induces apoptosis of pancreatic carcinoma cells through inhibition of Ras-ERK MAPK signaling

Background TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Thallions 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.