Phosphorylation of paxillin at threonine 538 by PKCdelta regulates LFA1-mediated adhesion of lymphoid cells.

We investigated the PKCdelta-mediated phosphorylation of paxillin within its LIM4 domain and the involvement of this phosphorylation in activation of LFA-1 integrins of the Baf3 pro-B lymphocytic cell line. Using phosphorylated-threonine-specific antibodies, phosphorylated amino acid analysis and paxillin phosphorylation mutants, we demonstrated that TPA, the pharmacological analog of the endogenous second messenger diacyl glycerol, stimulates paxillin phosphorylation at threonine 538 (T538). The TPA-responsive PKC isoform PKCdelta directly binds paxillin in a yeast two-hybrid assay and phosphorylates paxillin at T538 in vitro and also co-immunoprecipitates with paxillin and mediates phosphorylation of this residue in vivo. Recombinant wild-type paxillin, its phospho-inhibitory T538A or phospho-mimetic T538E mutants were expressed in the cells simultaneously with siRNA silencing of the endogenous paxillin. These experiments suggest that phosphorylation of paxillin T538 contributes to dissolution of the actin cytoskeleton, redistribution of LFA-1 integrins and an increase in their affinity. We also show that phosphorylation of T538 is involved in the activation of LFA-1 integrins by TPA.

Mutational analysis of threonine 402 adjacent to the GXXXG dimerization motif in transmembrane segment 1 of ABCG2.

ABCG2 is an ATP-binding cassette half-transporter important in normal tissue protection, drug distribution, and excretion. ABCG2 requires homodimerization for function, though the mechanism for dimerization has not been elucidated. We conducted mutational analysis of threonine 402, three residues from the GXXXG motif in TM1, to study its potential role in ABCG2 dimerization (TXXXGXXXG). Single mutations to leucine (T402L) or arginine (T402R) did not have a significant impact on the ABCG2 protein. On the other hand, combining the T402 mutations with the GXXXG glycine to leucine mutations (T402L/G406L/G410L and T402R/G406L/G410L) resulted in a substantially reduced level of expression, altered glycosylation, degradation by a proteosome-independent pathway, and partial retention in the endoplasmic reticulum as suggested by immunostaining, Endo H sensitivity, and MG132 and bafilomycin failed effect. The T402L/G406L/G410L mutant when incubated with the ABCG2 substrate MX showed a shift on immunoblot analysis to the band representing the fully mature glycoprotein. The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. The same set of mutations also displayed impaired dimerization in the TOXCAT assay for TM1 compared to that of the wild type. Homology modeling of ABCG2 places the TXXXGXXXG motif at the dimer interface. These studies are consistent with a role for the extended TXXXGXXXG motif in ABCG2 folding, processing, and/or dimerization.

Arginine 383 is a crucial residue in ABCG2 biogenesis.

ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.

Clinical outcome in children with recurrent neuroblastoma treated with ABT-751 and effect of ABT-751 on proliferation of neuroblastoma cell lines and on tubulin polymerization in vitro.

BACKGROUND: ABT-751, an orally bioavailable sulfonamide, binds beta-tubulin to inhibit microtubule polymerization. We described response and event-free survival (EFS) in children with neuroblastoma and other solid tumors receiving ABT-751, assessed in vitro cytotoxicity of ABT-751 and evaluated the effect of ABT-751 on tubulin polymerization in peripheral blood mononuclear cells (PBMC) and pediatric tumor cell lines. PROCEDURE: Patients with neuroblastoma (n = 50) or other solid tumors (n = 26) enrolled on the ABT-751 pediatric phase I and pilot trials were reviewed. The sulforhodamine B (SRB) and ACEA Real-Time Cell Electronic Sensing (RT-CES) assays were used to determine the in vitro cytotoxicity. Pharmacodynamic effects on tubulin polymerization/depolymerization were assessed by Western blot and confocal microscopy using antibodies specific for post-translational modifications of polymerized tubulin. RESULTS: Forty-five patients with neuroblastoma were evaluated for anti-tumor response. No complete or partial responses were documented. The median EFS was 9.3 weeks for children with neuroblastoma and 3.3 weeks for children other solid tumors (P < 0.0001). The ABT-751 IC(50) was 0.6-2.6 mcM in neuroblastoma and 0.7-4.6 mcM in other solid tumor cell lines. Following drug exposure, polymerized tubulin decreased in a concentration- and time-dependent manner in cell lines. CONCLUSIONS: In children treated with ABT-751, the EFS is longer in children with neuroblastoma as compared to other diagnoses. In vitro, ABT-751 was cytotoxic at concentrations tolerable in children. Effects of ABT-751 on polymerization and microtubule structure were time- and dose-dependent but not dependent on tumor type.

The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway.

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) alpha and beta. Gem binds ROKbeta independently of RhoA in the ROKbeta coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKbeta-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKbeta. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKbeta- and Rad opposed ROKalpha-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKbeta containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKbeta is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

Centrosomal amplification and aneuploidy induced by the antiretroviral drug AZT in hamster and human cells.

The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese hamster ovary (CHO) and normal human mammary epithelial cells (NHMECs) exposed to the antiretroviral drug zidovudine (3'-azido-3'-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC) strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (high incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound.

TDAG51 is an ERK signaling target that opposes ERK-mediated HME16C mammary epithelial cell transformation.

INTRODUCTION: Signaling downstream of Ras is mediated by three major pathways, Raf/ERK, phosphatidylinositol 3 kinase (PI3K), and Ral guanine nucleotide exchange factor (RalGEF). Ras signal transduction pathways play an important role in breast cancer progression, as evidenced by the frequent over-expression of the Ras-activating epidermal growth factor receptors EGFR and ErbB2. Here we investigated which signal transduction pathways downstream of Ras contribute to EGFR-dependent transformation of telomerase-immortalized mammary epithelial cells HME16C. Furthermore, we examined whether a highly transcriptionally regulated ERK pathway target, PHLDA1 (TDAG51), suggested to be a tumor suppressor in breast cancer and melanoma, might modulate the transformation process. METHODS: Cellular transformation of human mammary epithelial cells by downstream Ras signal transduction pathways was examined using anchorage-independent growth assays in the presence and absence of EGFR inhibition. TDAG51 protein expression was down-regulated by interfering small hairpin RNA (shRNA), and the effects on cell proliferation and death were examined in Ras pathway-transformed breast epithelial cells. RESULTS: Activation of both the ERK and PI3K signaling pathways was sufficient to induce cellular transformation, which was accompanied by up-regulation of EGFR ligands, suggesting autocrine EGFR stimulation during the transformation process. Only activation of the ERK pathway was sufficient to transform cells in the presence of EGFR inhibition and was sufficient for tumorigenesis in xenografts. Up-regulation of the PHLDA1 gene product, TDAG51, was found to correlate with persistent ERK activation and anchorage-independent growth in the absence or presence of EGFR inhibition. Knockdown of this putative breast cancer tumor-suppressor gene resulted in increased ERK pathway activation and enhanced matrix-detached cellular proliferation of Ras/Raf transformed cells. CONCLUSION: Our results suggest that multiple Ras signal transduction pathways contribute to mammary epithelial cell transformation, but that the ERK signaling pathway may be a crucial component downstream of EGFR activation during tumorigenesis. Furthermore, persistent activation of ERK signaling up-regulates TDAG51. This event serves as a negative regulator of both Erk activation as well as matrix-detached cellular proliferation and suggests that TDAG51 opposes ERK-mediated transformation in breast epithelial cells.

Platelets Promote Metastasis via Binding Tumor CD97 Leading to Bidirectional Signaling that Coordinates Transendothelial Migration.

Tumor cells initiate platelet activation leading to the secretion of bioactive molecules, which promote metastasis. Platelet receptors on tumors have not been well-characterized, resulting in a critical gap in knowledge concerning platelet-promoted metastasis. We identify a direct interaction between platelets and tumor CD97 that stimulates rapid bidirectional signaling. CD97, an adhesion G protein-coupled receptor (GPCR), is an overexpressed tumor antigen in several cancer types. Purified CD97 extracellular domain or tumor cell-associated CD97 stimulated platelet activation. CD97-initiated platelet activation led to granule secretion, including the release of ATP, a mediator of endothelial junction disruption. Lysophosphatidic acid (LPA) derived from platelets induced tumor invasiveness via proximal CD97-LPAR heterodimer signaling, coupling coincident tumor cell migration and vascular permeability to promote transendothelial migration. Consistent with this, CD97 was necessary for tumor cell-induced vascular permeability in vivo and metastasis formation in preclinical models. These findings support targeted blockade of tumor CD97 as an approach to ameliorate metastatic spread.

Structure-function studies of the G-domain from human gem, a novel small G-protein.

Gem, a member of the Rad,Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 A resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.

Gem protein signaling and regulation.

Gem is a member of the RGK family of GTP-binding proteins within the Ras superfamily possessing a ras-like core and terminal extensions. We have used a variety of cell-based assays to investigate the physiological role of Gem and combined these assays with site-directed mutagenesis of Gem protein to identify the sites responsible for regulation of Gem activity. One function of Gem that has been explained is the inhibition of Rho kinase (ROK)-mediated cytoskeletal rearrangement. Transient expression of Gem in endothelial cells and stable transfection of fibroblasts resulted in decreased stress fiber formation and focal adhesion assembly. A neurite extension model using N1E-115 murine neuroblastoma showed that Gem inhibits actinomyosin-related contractility by specifically opposing ROKbeta activity. Phospho-specific antibodies were used in Western blot analysis to show that Gem prevents phosphorylation of the regulatory subunit of myosin light chain and myosin phosphatase by ROKbeta. On the contrary, LIMK, another substrate of ROKbeta, was unaffected by Gem expression as demonstrated by an in vitro kinase assay, suggesting that Gem exerts its effect by changing the substrate specificity of ROKbeta rather than by blocking its catalytic activity. Point mutations of Gem at serines 261 and 289 in the carboxyl-terminus inhibited Gem function, indicating that posttranslational phosphorylation of these serines regulates Gem's effect on cytoskeletal reorganization. Another biological role of Gem is inhibition of voltage-gated calcium channel activity. By use of a PC12 cell model combined with site-directed mutagenesis, we demonstrated that Gem inhibits growth hormone secretion stimulated by calcium influx through L-type calcium channels and that this function is dependent on GTP and calmodulin binding to Gem. The theory and method for the assays discussed previously are reviewed here.

Revelation of a cryptic major histocompatibility complex class II-restricted tumor epitope in a novel RNA-processing enzyme.

CD4+ T-cell responses against human tumor antigens are a potentially critical component of the antitumor immune response. Molecular methods have been devised for rapidly identifying MHC class II-restricted tumor antigens and elucidating the recognized epitopes. We describe here the identification of neo-poly(A) polymerase (neo-PAP), a novel RNA processing enzyme overexpressed in a variety of human cancers, by screening a melanoma-derived invariant chain fusion cDNA library with tumor-reactive CD4+ T lymphocytes. A cryptic nonmutated HLA-DRbeta1*0701-restricted neo-PAP epitope was processed through the endogenous MHC class II pathway. A unique point mutation effected a nonconservative substitution of a leucine for a proline residue at a structurally important site in neo-PAP that was remote from the recognized peptide, revealing a normally silent epitope for immune recognition. Genetic aberrations such as the described point mutation can have unexpected immunological consequences, in this case leading to immune recognition of a distant normal self epitope.

Tissue inhibitors of metalloproteinase 2 inhibits endothelial cell migration through increased expression of RECK.

The antiangiogenic function of the tissue inhibitors of metalloproteinases (TIMPs) has been attributed to their matrix metalloproteinase inhibitory activity. Here we demonstrate that TIMP-1 but not Ala+TIMP-1 inhibits both basal and vascular endothelial growth factor (VEGF)-stimulated migration of human microvascular endothelial cells (hMVECs), suggesting that this effect is dependent on direct inhibition of matrix metalloproteinase (MMP) activity. In contrast, TIMP-2 and mutant Ala+TIMP-2, which is devoid of MMP inhibitory activity, block hMVEC migration in response to VEGF-A stimulation. TIMP-2 and Ala+TIMP-2 also suppress basal hMVEC migration via a time-dependent mechanism mediated by enhanced expression of RECK, a membrane-anchored MMP inhibitor, which, in turn, inhibits cell migration. TIMP-2 treatment of hMVECs increases the association of Crk with C3G, resulting in enhanced Rap1 activation. hMVECs stably expressing Rap1 have increased RECK expression and display reduced cell migration compared with those expressing inactive Rap1(38N). RECK-null murine embryo fibroblasts fail to demonstrate TIMP-2-mediated decrease in cell migration despite activation of Rap1. TIMP-2-induced RECK decreases cell-associated MMP activity. Anti-RECK antibody increases MMP activity and reverses the TIMP-2-mediated reduction in cell migration. The effects of TIMP-2 on RECK expression and cell migration were confirmed in A2058 melanoma cells. These results suggest that TIMP-2 can inhibit cell migration via several distinct mechanisms. First, TIMP-2 can inhibit cell migration after VEGF stimulation by direct inhibition of MMP activity induced in response to VEGF stimulation. Secondly, TIMP-2 can disrupt VEGF signaling required for initiation of hMVEC migration. Third, TIMP-2 can enhance expression of RECK via Rap1 signaling resulting in an indirect, time-dependent inhibition of endothelial cell migration.

Use of Ciliogenesis to Detect Aneugens: The Role of Primary Cilia.

Primary cilia arise from the centrosomes of quiescent or post-mitotic cells, and serve as sensory organelles that communicate mechanical and chemical stimuli from the environment to the interior of the cell. Cilium formation may, therefore, become a useful end point signaling exposure to genotoxins or aneugens. Here we have used the aneugen, zidovudine (AZT), an antiretroviral drug that induces DNA replication arrest and centrosomal amplification (>2 centrosomes per quiescent cell), to evaluate cilia formation in retinal epithelial (pigmented) cells. Since cilia are derived from centrosomes, and aneugens can induce centrosomal amplification, the production of multiple cilia arising from multiple centrosomes may reveal the aneugenic nature of the agents. Cells were exposed to AZT to induce centrosomal amplification, cultured without serum to allow the centrioles to develop cilia, and immunostained to visualize cilia and centrosomes. Nuclear DNA was stained with DAPI. Preliminary observations suggest that cells with multiple centrosomes are able to generate extra cilia.

Tumor expression of 4-1BB ligand sustains tumor lytic T cells.

Inadequate costimulation by solid tumors is generally believed to induce immune tolerance during primary tumor growth. We looked for tumor-specific immunity vs. tolerance in patients with Ewing's sarcoma. Circulating T cells from patients with progressively growing Ewing's tumors displayed MHC restricted tumor-induced proliferation and robust tumor lysis. Tumor-reactive T cells reside within the memory CD3+CD8+ subset and are CD28-/4-1BB+. Autologous Ewing's tumors expressed 4-1BBL, and tumor-induced T cell proliferation and activation required costimulation by 4-1BBL. Stimulation of PBL with anti-CD3/4-1BBL, but not anti-CD3/anti-CD28 induced tumor lytic effectors. Similarly, in a xenograft model, anti-CD3/4-1BBL expanded T cells controlled primary growth and prevented metastasis of autologous tumors while nonactivated and anti-CD3/anti-CD28 activated CD8+ cells did not. These results question prevailing models of tumor induced tolerance accompanying progressive tumor growth; rather, we show coexistence of progressive tumor growth and anti-tumor immunity, with costimulation provided by the tumor itself. They further demonstrate a potential new therapeutic role for 4-1BBL mediated costimulation in expanding tumor reactive CTLs for use in the adoptive immunotherapy of cancer.

Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.

The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca(2+) influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca(2+) influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.

Role of nucleotide excision repair and p53 in zidovudine (AZT)-induced centrosomal deregulation.

The nucleoside reverse transcriptase inhibitor zidovudine (AZT) induces genotoxic damage that includes centrosomal amplification (CA > 2 centrosomes/cell) and micronucleus (MN) formation. Here we explored these end points in mice deficient in DNA repair and tumor suppressor function to evaluate their effect on AZT-induced DNA damage. We used mesenchymal-derived fibroblasts cultured from C57BL/6J mice that were null and wild type (WT) for Xpa, and WT, haploinsufficient and null for p53 (6 different genotypes). Dose-responses for CA formation, in cells exposed to 0, 10, and 100 µM AZT for 24 hr, were observed in all genotypes except the Xpa((+/+)) p53((+/-)) cells, which had very low levels of CA, and the Xpa((-/-)) p53((-/-)) cells, which had very high levels of CA. For CA there was a significant three-way interaction between Xpa, p53, and AZT concentration, and Xpa((-/-)) cells had significantly higher levels of CA than Xpa((+/+)) cells, only for p53((+/-)) cells. In contrast, the MN and MN + chromosomes (MN + C) data showed a lack of AZT dose response. The Xpa((-/-)) cells, with p53((+/+)) or ((+/-)) genotypes, had levels of MN and MN + C higher than the corresponding Xpa((+/+)) cells. The data show that CA is a major event induced by exposure to AZT in these cells, and that there is a complicated relationship between AZT and CA formation with respect to gene dosage of Xpa and p53. The loss of both genes resulted in high levels of damage, and p53 haploinsufficicency strongly protected Xpa((+/+)) cells from AZT-induced CA damage.

Histone deacetylase inhibitors influence chemotherapy transport by modulating expression and trafficking of a common polymorphic variant of the ABCG2 efflux transporter.

Histone deacetylase inhibitors (HDI) have exhibited some efficacy in clinical trials, but it is clear that their most effective applications have yet to be fully determined. In this study, we show that HDIs influence the expression of a common polymorphic variant of the chemotherapy drug efflux transporter ABCG2, which contributes to normal tissue protection. As one of the most frequent variants in human ABCG2, the polymorphism Q141K impairs expression, localization, and function, thereby reducing drug clearance and increasing chemotherapy toxicity. Mechanistic investigations revealed that the ABCG2 Q141K variant was fully processed but retained in the aggresome, a perinuclear structure, where misfolded proteins aggregate. In screening for compounds that could correct its expression, localization, and function, we found that the microtubule-disrupting agent colchicine could induce relocalization of the variant from the aggresome to the cell surface. More strikingly, we found that HDIs could produce a similar effect but also restore protein expression to wild-type levels, yielding a restoration of ABCG2-mediated specific drug efflux activity. Notably, HDIs did not modify aggresome structures but instead rescued newly synthesized protein and prevented aggresome targeting, suggesting that HDIs disturbed trafficking along microtubules by eliciting changes in motor protein expression. Together, these results showed how HDIs are able to restore wild-type functions of the common Q141K polymorphic isoform of ABCG2. More broadly, our findings expand the potential uses of HDIs in the clinic.

TMPRSS2- driven ERG expression in vivo increases self-renewal and maintains expression in a castration resistant subpopulation.

Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1(hi)/EpCAM(+) basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium.

LPA receptor heterodimerizes with CD97 to amplify LPA-initiated RHO-dependent signaling and invasion in prostate cancer cells.

CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Gα12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target.

Centrosome amplification induced by the antiretroviral nucleoside reverse transcriptase inhibitors lamivudine, stavudine, and didanosine.

In cultured cells, exposure to the nucleoside reverse transcriptase inhibitor (NRTI) zidovudine (AZT) induces genomic instability, cell cycle arrest, micronuclei, sister chromatid exchanges, and shortened telomeres. In previous studies, we demonstrated AZT-induced centrosome amplification (>2 centrosomes/cell). Here, we investigate centrosome amplification in cells exposed to other commonly used NRTIs. Experiments were performed using Chinese Hamster ovary (CHO) cells, and two normal human mammary epithelial cell (NHMEC) strains: M99005 and M98040, which are high and low incorporators of AZT into DNA, respectively. Cells were exposed for 24 hr to lamivudine (3TC), stavudine (d4T), didanosine (ddI), and thymidine, and stained with anti-pericentrin antibody. Dose response curves were performed to determine cytotoxicity and a lower concentration at near plasma levels and a 10 fold higher concentration were chosen for the experiments. In CHO cells, there was a concentration-dependent, significant (P < 0.05) increase in centrosome amplification for each of the NRTIs. In NHMEC strain M99005, an NRTI-induced increase (P < 0.05) in centrosome amplification was observed for the high concentrations of each NRTI and the low doses of 3TC and ddI. In NHMEC strain M98040, the high doses of ddI and d4T showed significant increases in centrosome amplification. Functional viability of amplified centrosomes was assessed by arresting microtubule nucleation with nocodazole. In cells with more than two centrosomes, the ability to recover microtubule nucleation was similar to that of unexposed cells. We conclude that centrosome amplification is a consequence of exposure to NRTIs and that cells with centrosome amplification are able to accomplish cell division.