James P. Madigan

Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification.

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.

Global and specific transcriptional repression by the histone H3 amino terminus in yeast

The yeast CHA1 promoter is activated in the presence of serine or threonine. Activation requires the Cha4p activator, and it results in perturbation of a nucleosome that incorporates the TATA element under noninducing conditions. We show that in yeast lacking the amino terminus of histone H3, the promoter is constitutively active and the chromatin is concomitantly perturbed. This derepression occurs in the absence of elevated intracellular levels of serine or threonine and is not observed in cells lacking Rpd3p, Tup1p, or the amino terminus of histone H4. Furthermore, derepression in the absence of the H3 amino terminus requires the primary activator of this promoter, Cha4p, which we show by chromatin immunoprecipitation to be constitutively bound to the CHA1 promoter in WT yeast. Thus, the H3 amino terminus is required to prevent Cha4p from activating CHA1 in the absence of inducer. We also present results of a microarray experiment showing that the H3 amino terminus has a substantial repressive effect on a genome-wide scale.

Regulation of Rnd3 localization and function by protein kinase Cα-mediated phosphorylation

The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by post-translational modifications such as prenylation and phosphorylation. We show in the present study that, upon PKC (protein kinase C) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCalpha-null cells or to a non-phosphorylatable mutant of Rnd3. We further show that PKCalpha directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signalling from the Rho-ROCK (Rho-kinase) pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization.

Genome-Wide Analysis of the Relationship between Transcriptional Regulation by Rpd3p and the Histone H3 and H4 Amino Termini in Budding Yeast

ABSTRACT The histone amino termini have emerged as key targets for a variety of modifying enzymes that function as transcriptional coactivators and corepressors. However, an important question that has remained largely unexplored is the extent to which specific histone amino termini are required for the activating and repressive functions of these enzymes, Here we address this issue by focusing on the prototypical histone deacetylase, Rpd3p, in the budding yeast Saccharomyces cerevisiae. We show that targeting Rpd3p to a reporter gene in this yeast can partially repress transcription when either the histone H3 or the histone H4 amino terminus is deleted, indicating that the “tails” are individually dispensable for repression by Rpd3p. In contrast, we find that the effect of rpd3 gene disruption on global gene expression is considerably reduced in either a histone H3Δ1-28 (H3 lacking the amino-terminal 28 amino acids) or a histone H4(K5,8,12,16Q) (H4 with lysine residues 5, 8, 12, and 16 changed to glutamine residues) background compared to the wild-type background, indicating a requirement for one or both of these histone tails in Rpd3p-mediated regulation for many genes. These results suggest that acetylation of either the H3 or the H4 amino terminus could suffice to allow the activation of such genes. We also examine the relationship between H3 tails and H4 tails in global gene expression and find substantial overlap among the gene sets regulated by these histone tails. We also show that the effects on genome-wide expression of deleting the H3 or H4 amino terminus are similar but not identical to the effects of mutating the lysine residues in these same regions. These results indicate that the gene regulatory potential of the H3 and H4 amino termini is substantially but not entirely contained in these modifiable lysine residues.

The Transforming Rho Family GTPase Wrch-1 Disrupts Epithelial Cell Tight Junctions and Epithelial Morphogenesis

ABSTRACT Wrch-1, an atypical and transforming Rho GTPase, regulates cellular activities including proliferation and actin organization, but its functions and effectors remain poorly characterized. We show here that Wrch-1 distributes along the apical and basolateral membranes in MDCK cells and binds the cell polarity protein Par6 in a GTP-dependent manner. Activated Wrch-1 negatively regulates the kinetics of tight junction (TJ) assembly during epithelial cell polarization but has no detectable effect on overall cell polarity in confluent monolayers. It also causes a dramatic cytoskeletal reorganization and multilayering in cells grown in two-dimensional culture and disrupts cystogenesis of cells grown in three-dimensional (3D) culture. Similarly, short hairpin RNA-mediated knockdown of Wrch-1 perturbs cystogenesis in 3D culture, suggesting that tight regulation of Wrch-1 activity is necessary for normal epithelial morphogenesis. A weakly transforming effector domain mutant of activated Wrch-1 that inhibits Par6 binding abrogates the ability of Wrch-1 to disrupt TJ formation, actin organization, and epithelial morphogenesis. We hypothesize that Wrch-1-induced morphological and growth transformation may occur in part through Par6-mediated disruption of TJs and actin organization.

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.

The Protein Phosphatase 2A Inhibitor LB100 Sensitizes Ovarian Carcinoma Cells to Cisplatin-Mediated Cytotoxicity

Despite early positive response to platinum-based chemotherapy, the majority of ovarian carcinomas develop resistance and progress to fatal disease. Protein phosphatase 2A (PP2A) is a ubiquitous phosphatase involved in the regulation of DNA-damage response (DDR) and cell-cycle checkpoint pathways. Recent studies have shown that LB100, a small-molecule inhibitor of PP2A, sensitizes cancer cells to radiation-mediated DNA damage. We hypothesized that LB100 could sensitize ovarian cancer cells to cisplatin treatment. We performed in vitro studies in SKOV-3, OVCAR-8, and PEO1, -4, and -6 ovarian cancer lines to assess cytotoxicity potentiation, cell-death mechanism(s), cell-cycle regulation, and DDR signaling. In vivo studies were conducted in an intraperitoneal metastatic mouse model using SKOV-3/f-Luc cells. LB100 sensitized ovarian carcinoma lines to cisplatin-mediated cell death. Sensitization via LB100 was mediated by abrogation of cell-cycle arrest induced by cisplatin. Loss of the cisplatin-induced checkpoint correlated with decreased Wee1 expression, increased cdc2 activation, and increased mitotic entry (p-histone H3). LB100 also induced constitutive hyperphosphorylation of DDR proteins (BRCA1, Chk2, and γH2AX), altered the chronology and persistence of JNK activation, and modulated the expression of 14-3-3 binding sites. In vivo, cisplatin sensitization via LB100 significantly enhanced tumor growth inhibition and prevented disease progression after treatment cessation. Our results suggest that LB100 sensitizes ovarian cancer cells to cisplatin in vitro and in vivo by modulation of the DDR pathway and cell-cycle checkpoint abrogation. Mol Cancer Ther; 14(1); 90–100. ©2014 AACR.

Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier

Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the BPB. In the present study, we show that in vitro models of the placenta possess ABCG2 activity and can specifically transport D-luciferin, the endogenous substrate of firefly luciferase. To test ABCG2 transport activity at the BPB, we devised a breeding strategy to generate a bioluminescent pregnant mouse model to demonstrate transporter function in vivo. We found that coadministering the ABCG2 inhibitors Ko143 and gefitinib with D-luciferin increased bioluminescent signal from fetuses and placentae, whereas the control P-gp inhibitor DCPQ had no effect. We believe that our bioluminescent pregnant mouse model will facilitate greater understanding of the BPB and ABCG2 activity in health and disease.

Targeting ras for anticancer drug discovery

Publisher Summary Several recent research observations have rekindled interest in targeting Ras for cancer treatment. Genome-wide cancer genome studies have revealed that the genes most commonly mutated in cancers were already identified in more systematic studies. Another observation involves the association of KRAS mutations with resistance to treatment with inhibitors of the epidermal growth factor receptor (EGFR). The structural and biochemical distinctions between normal and mutant Ras are well delineated. Approaches for blocking Ras have focused on either inhibition of Ras membrane association or downstream effector signaling. This chapter summarizes the specific targets under evaluation for each of these directions. Cell permeable inhibitors are identified and shown to exhibit target based and anti-tumor activity in cell culture and/or mouse models of cancer. These analyses demonstrate tumor prevention rather than inhibition of an already developed tumor. The study provides an overview of the status of the development of inhibitors of Ras effector signaling. With renewed appreciation and interest in Ras as a therapeutic target for cancer treatment, and with promising leads for indirect approaches for blocking Ras function, there remains strong optimism that anti-Ras therapies will finally reach the clinic.

Abstract A25: Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition

PAX-fusion negative rhabdomyosarcoma (RMS) arises from skeletal muscle precursors that have failed to differentiate normally despite the expression of the myogenic master transcription factor, MYOD1. The cure rate for relapsed or refractory fusion negative RMS is poor despite aggressive multi-modality treatment. Novel treatment approaches such as the use of targeted therapies including those that induce skeletal muscle differentiation might improve overall survival for patients with fusion negative RMS. Genetic studies have shown that the most common single nucleotide variant in fusion negative RMS is an oncogenic change in one of the RAS isoforms, namely NRAS, HRAS or KRAS. In this study, we hypothesized that targeting aberrant RAS activity releases the differentiation block in fusion negative RMS and sought to unravel the underlying epigenetic mechanisms through which RAS signaling drives oncogenic transcription in RMS. To achieve this goal, we combined high-throughput drug screening with biochemical, RNAseq and ChIPseq assays across a panel of RMS cell lines driven by oncogenic RAS mutations. Critically, we demonstrated that expression of oncogenic RAS was necessary for survival of these RAS-mutated RMS cells. In addition, overexpression of mutant RAS isoforms in C2C12 myoblasts inhibited myogenic differentiation induced by low-serum conditions. This differentiation block was mediated primarily by engagement of the RAF-MEK-ERK MAP kinase pathway. In corroboration with these observations, an unbiased screen of the ability of small molecules to impact cell viability demonstrated that inhibitors of the MAP kinase pathway were the most potently selective class of molecules for RAS-mutated RMS. In particular, trametinib, an allosteric, non-ATP competitive inhibitor of MEK1/2, was the most consistently potent MEK inhibitor in RAS-mutated RMS cell lines. Trametinib treatment induced G1 arrest and skeletal muscle differentiation in RAS-mutated RMS cell lines. Trametinib also slowed tumor growth and prolonged survival in xenograft models of RAS-mutated RMS. To determine the mechanism by which MEK inhibition induced skeletal muscle differentiation in RAS-mutated RMS, we analyzed changes in gene expression, transcription factor deposition and histone modification in RMS cells treated with trametinib. Trametinib treatment increased expression of myogenic transcription factors, such as MYOG and MEF2C, and decreased expression of transcription factors important for proliferation, such as MYC and ID3, in RAS-mutated RMS cells. ChIPseq experiments demonstrated that this transcriptional reprogramming was driven in part by changes in the active enhancer landscape, since H3K27ac deposition at MYH3, TTNT2 and other muscle-specific loci increased with trametinib treatment. Both MYC and MYOD1 bound the active enhancers induced by trametinib treatment in RAS-mutated RMS, despite an overall decrease in MYC expression. Finally, we found significant ERK2 deposition on the MYOG promoter in the untreated cells. ERK2 is known to recruit the Polycomb repressive machinery at developmental loci in embryonic stem cells and therefore aberrant ERK2 activity may facilitate repression of MYOG expression in RAS-mutated RMS. In summary, our data support a model of RAS-driven RMS in which aberrant ERK activity drives tumor cell proliferation, in part through increased expression and stability of MYC, and prevents myogenic differentiation, in this case through alterations in the enhancer landscape and interactions with the Polycomb repressive machinery. Future work is aimed at identifying rational combinations of trametinib and direct epigenetic modulators that synergistically drive RAS-mutated RMS differentiation with the goal of providing measurable clinical benefit in relapsed or refractory RAS-mutated RMS. Citation Format: Marielle E. Yohe, Berkley E. Gryder, Jack F. Shern, Young K. Song, Hongling Liao, Hsein-Chao Chou, Sivasish Sindiri, Arnulfo Mendoza, Xiaohu Zhang, Rajarashi Guha, Diana C. Haines, James P. Madigan, Jun S. Wei, Marc Ferrer, Craig J. Thomas, Javed Khan. Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A25.