Targeted inhibition of SCFSKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma.

Osteosarcoma(OS) is a highly aggressive bone cancer for which treatment has remained essentially unchanged for decades. Although OS is characterized by extensive genomic heterogeneity and instability, RB1 and TP53 have been shown to be the most commonly inactivated tumor suppressors in OS. We previously generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which largely recapitulates human OS with nearly complete penetrance. SKP2 is a repression target of pRb and serves as a substrate recruiting subunit of the SCFSKP2 complex. In addition, SKP2 plays a central role in regulating the cell cycle by ubiquitinating and promoting the degradation of p27. We previously reported the DKOAA transgenic model, which harbored a knock-in mutation in p27 that impaired its binding to SKP2. Here, we generated a novel p53-Rb1-SKP2 triple-knockout model (TKO) to examine SKP2 function and its potential as a therapeutic target in OS. First, we observed that OS tumorigenesis was significantly delayed in TKO mice and their overall survival was markedly improved. In addition, the loss of SKP2 also promoted an apoptotic microenvironment and reduced the stemness of DKO tumors. Furthermore, we found that small-molecule inhibitors of SKP2 exhibited anti-tumor activities in vivo and in OS organoids as well as synergistic effects when combined with a standard chemotherapeutic agent. Taken together, our results suggest that SKP2 inhibitors may reduce the stemness plasticity of OS and should be leveraged as next-generation adjuvants in this cancer.

SKP2 Knockout in Rb1/p53-Deficient Mouse Models of Osteosarcoma Induces Immune Infiltration and Drives a Transcriptional Program with a Favorable Prognosis.

Osteosarcoma is an aggressive bone malignancy with a poor prognosis. One putative proto-oncogene in osteosarcoma is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase. We previously demonstrated that Skp2 knockout in murine osteosarcoma improved survival and delayed tumorigenesis. Here, we performed RNA sequencing (RNA-seq) on tumors from a transgenic osteosarcoma mouse model with conditional Trp53 and Rb1 knockouts in the osteoblast lineage ("DKO": Osx1-Cre;Rb1lox/lox;p53lox/lox) and a triple-knockout model with additional Skp2 germline knockout ("TKO": Osx1-Cre;Rb1lox/lox;p53lox/lox;Skp2-/-), followed by qPCR and immunohistochemistry validation. To investigate the clinical implications of our results, we analyzed a human osteosarcoma patient cohort ("NCI-TARGET OS") with RNA-seq and clinical data. We found large differences in gene expression after SKP2 knockout. Surprisingly, we observed increased expression of genes related to immune microenvironment infiltration in TKO tumors, especially the signature genes for macrophages and to a lesser extent, T cells, B cells, and vascular cells. We also uncovered a set of relevant transcription factors that may mediate these changes. In osteosarcoma patient cohorts, high expression of genes upregulated in TKO was correlated with favorable overall survival, which was largely explained by the macrophage gene signatures. This relationship was further supported by our finding that SKP2 expression was negatively correlated with macrophage infiltration in the NCI-TARGET osteosarcoma and the TCGA Sarcoma cohorts. Overall, our findings indicate that SKP2 may mediate immune exclusion from the osteosarcoma tumor microenvironment, suggesting that SKP2 modulation in osteosarcoma may induce antitumor immune activation.

Targeting the untargetable: RB1-deficient tumours are vulnerable to Skp2 ubiquitin ligase inhibition.

Proteins that regulate the cell cycle are accumulated and degraded in a coordinated manner during the transition from one cell cycle phase to the next. The rapid loss of a critical protein, for example, to allow the cell to move from G1/G0 to S phase, is often regulated by its ubiquitination and subsequent proteasomal degradation. Protein ubiquitination is mediated by a series of three ligases, of which the E3 ligases provide the specificity for a particular protein substrate. One such E3 ligase is SCFSkp1/Cks1, which has a substrate recruiting subunit called S-phase kinase-associated protein 2 (Skp2). Skp2 regulates cell proliferation, apoptosis, and differentiation, can act as an oncogene, and is overexpressed in human cancer. A primary target of Skp2 is the cyclin-dependent kinase inhibitor p27 (CDKN1b) that regulates the cell cycle at several points. The RB1 tumour suppressor gene regulates Skp2 activity by two mechanisms: by controlling its mRNA expression, and by an effect on Skp2's enzymatic activity. For the latter, the RB1 protein (pRb) directly binds to the substrate-binding site on Skp2, preventing protein substrates from being ubiquitinated and degraded. Inactivating mutations in RB1 are common in human cancer, becoming more frequent in aggressive, metastatic, and drug-resistant tumours. Hence, RB1 mutation leads to the loss of pRb, an unrestrained increase in Skp2 activity, the unregulated decrease in p27, and the loss of cell cycle control. Because RB1 mutations lead to the loss of a functional protein, its direct targeting is not possible. This perspective will discuss evidence validating Skp2 as a therapeutic target in RB1-deficient cancer.

The interaction of SKP2 with p27 enhances the progression and stemness of osteosarcoma.

Osteosarcoma is a highly aggressive malignancy for which treatment has remained essentially unchanged for years. Our previous studies found that the F-box protein SKP2 is overexpressed in osteosarcoma, acting as a proto-oncogene; p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases and a downstream substrate of SKP2-mediated ubiquitination. Overexpression of SKP2 and underexpression of p27 are common characteristics of cancer cells. The SCFSKP2 E3 ligase ubiquitinates Thr187-phosphorylated p27 for proteasome degradation, which can be abolished by a Thr187Ala knock-in (p27T187A KI) mutation. RB1 and TP53 are two major tumor suppressors commonly coinactivated in osteosarcoma. We generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which developed osteosarcoma with full penetrance. When p27T187A KI mice were crossed on to the DKO background, p27T187A protein was found to accumulate in osteosarcoma tumor tissues. Furthermore, p27T187A promoted apoptosis in DKO tumors, slowed disease progression, and significantly prolonged overall survival. RNA sequencing analysis also linked the SCFSKP2 -p27T187A axis to potentially reduced cancer stemness. Given that RB1 and TP53 loss or coinactivation is common in human osteosarcoma, our study suggests that inhibiting the SKP2-p27 axis may represent a desirable therapeutic strategy for this cancer.

Skp2 depletion reduces tumor-initiating properties and promotes apoptosis in synovial sarcoma.

Synovial sarcoma (SS) is an aggressive soft-tissue cancer with a poor prognosis and a propensity for local recurrence and distant metastasis. In this study, we investigated whether S phase kinase-associated protein (Skp2) plays an oncogenic role in tumor initiation, progression, and metastasis of SS. Our study revealed that Skp2 is frequently overexpressed in SS specimens and SS18-SSX transgenic mouse tumors, as well as correlated with clinical stages. Next, we identified that genetic depletion of Skp2 reduced mesenchymal and stemness markers, and inhibited the invasive and proliferative capacities of SS cell lines. Furthermore, Skp2 depletion markedly suppressed the growth of SS xenografts tumors. Treatment of SS cell lines with the skp2 inhibitor flavokawain A (FKA) reduced Skp2 expression in a dose-dependent manner and resulted in cell cycle arrest and apoptosis. FKA also suppressed the invasion and tumor-initiating properties in SS, similar to the effects of Skp2 knockdown. In addition, a combination of FKA and conventional chemotherapy showed a synergistic therapeutic efficacy. Taken together, our results suggest that Skp2 plays an essential role in the biology of SS by promoting the mesenchymal state and cancer stemness. Given that chemotherapy resistance is often associated with cancer stemness, strategies of combining Skp2 inhibitors with conventional chemotherapy in SS may be desirable.

Targeted Inhibition of the E3 Ligase SCFSkp2/Cks1 Has Antitumor Activity in RB1-Deficient Human and Mouse Small-Cell Lung Cancer.

The RB1 tumor suppressor gene is mutated in highly aggressive tumors including small-cell lung cancer (SCLC), where its loss, along with TP53, is required and sufficient for tumorigenesis. While RB1-mutant cells fail to arrest at G1-S in response to cell-cycle restriction point signals, this information has not led to effective strategies to treat RB1-deficient tumors, as it is challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, which was confirmed as the mechanism of protection by using knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind/inhibit Skp2 have in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using genetic and pharmacologic approaches, antitumor activity was seen with Skp2 loss or inhibition in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. Our data highlight a downstream actionable target in RB1-deficient cancers, for which there are currently no targeted therapies available. SIGNIFICANCE: There are no effective therapies for SCLC. The identification of an actionable target downstream of RB1, inactivated in SCLC and other advanced tumors, could have a broad impact on its treatment.

Lysosome Membrane Permeabilization and Disruption of the Molecular Target of Rapamycin (mTOR)-Lysosome Interaction Are Associated with the Inhibition of Lung Cancer Cell Proliferation by a Chloroquinoline Analog.

Lysosomes degrade cellular proteins and organelles and regulate cell signaling by providing a surface for the formation of critical protein complexes, notably molecular target of rapamycin (mTOR) complex 1 (mTORC1). Striking differences in the lysosomes of cancer versus normal cells suggest that they could be targets for drug development. Although the lysomotropic drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been widely investigated, studies have focused on their ability to inhibit autophagy. We synthesized a novel compound, called EAD1, which is structurally related to CQ but is a 14-fold more potent inhibitor of cell proliferation. Here we find that EAD1 causes rapid relocation, membrane permeabilization (LMP), and deacidification of lysosomes, and it induces apoptosis and irreversibly blocks proliferation of human lung cancer H460, H520, H1299, HCC827, and H1703 cells. EAD1 causes dissociation of mTOR from lysosomes and increases mTOR's perinuclear versus cytoplasmic localization, changes previously shown to inactivate mTORC1. The effect on mTOR was not seen with HCQ, even at >10-fold greater concentrations. Phosphorylation of a downstream target of mTORC1, ribosomal protein S6, was inhibited by EAD1. Although EAD1 also inhibited autophagy, it retained full antiproliferative activity in autophagy-deficient H1650 lung cancer cells, which have a biallelic deletion of Atg7, and in H460 Atg7-knockout cells. As Atg7 is critical for the canonical autophagy pathway, it is likely that inhibition of autophagy is not how EAD1 inhibits cell proliferation. Further studies are needed to determine the relationship of LMP to mTORC1 disruption and their relative contributions to drug-induced cell death. These studies support the lysosome as an underexplored target for new drug development.

RICTOR Amplification Defines a Novel Subset of Patients with Lung Cancer Who May Benefit from Treatment with mTORC1/2 Inhibitors.

UNLABELLED: We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months. SIGNIFICANCE: RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Discovery of autophagy inhibitors with antiproliferative activity in lung and pancreatic cancer cells.

The autophagy inhibitors chloroquine (CQ) and hydroxychloroquine (HCQ) have single agent antiproliferative activity against human cancer cell lines; however, low potency may limit their antitumor efficacy clinically. We synthesized a series of chloroquine analogs that retained the 4-aminoquinoline subunit and incorporated different substituted triazoles into the target structure. These compounds were tested for growth inhibition against H460 and HCC827 human lung cancer and BxPC3 pancreatic cancer cells. The most potent compound, EAD1, had an IC50 of 5.8 µM in the BxPC3 cells and was approximately 8-fold more potent than CQ and HCQ. EAD1 inhibited autophagy, as judged by the cellular accumulation of the autophagy-related autophagosome proteins LC3-II and p62 and induced apoptosis. The increases in LC3-II levels by the analogues were highly correlated with their growth inhibitory IC50s, suggesting that autophagy blockade is closely linked to inhibition of cell proliferation. EAD1 is a viable lead compound for evaluation of the antitumor activity of autophagy inhibitors in vivo.

The autophagy inhibitor chloroquine overcomes the innate resistance of wild-type EGFR non-small-cell lung cancer cells to erlotinib.

INTRODUCTION: The epidermal growth factor receptor (EGFR) inhibitor erlotinib is much less effective in non-small-cell lung cancer (NSCLC) tumors with wild-type EGFR, than in tumors with activating EGFR mutations. Autophagy is a tightly regulated lysosomal self-digestion process, which may alternatively promote cell survival or type II cell death. This study assessed the role of autophagy in erlotinib-mediated cytotoxicity. METHODS: We used wild-type EGFR erlotinib-sensitive and erlotinib-resistant NSCLC cell lines to determine whether inhibiting autophagy by a therapeutic agent potentiated the antitumor activity of erlotinib in vitro and in vivo. RESULTS: Erlotinib at a clinically relevant concentration (2 µM) induced autophagy in NSCLC cells with wild-type EGFR, and the degree of induction was greater in cells that were resistant than sensitive, suggesting that autophagy is cytoprotective. This was confirmed by knockdown of the autophagy-related gene Atg-5, and by using the autophagy inhibitor chloroquine (CQ), both of which increased the cytotoxicity of erlotinib. The synergistic activity of CQ was not because of the potentiation of erlotinib's effects on autophagy, cell-cycle arrest, and inhibition of both EGFR or downstream signaling of EGFR. Rather, CQ markedly activated apoptosis in the cells. The ability of CQ to potentiate the antitumor activity of erlotinib was also seen in mice bearing NSCLC tumor xenografts. CONCLUSIONS: The ability to adapt to anti-EGFR therapy by triggering autophagy may be a key determinant for resistance to erlotinib in wild-type EGFR NSCLC. Inhibition of autophagy by CQ represents a novel strategy to broaden the spectrum of erlotinib efficacy in wild-type EGFR NSCLC tumors.

Paracrine stimulation of endothelial cell motility and angiogenesis by platelet-derived deoxyribose-1-phosphate.

OBJECTIVE: Micromolar concentrations of the proangiogenic metabolite deoxyribose-1-phosphate (dRP) were detected in platelet supernatants by mass spectrometry. In this study, we assessed whether the release of dRP by platelets stimulates endothelial cell migration and angiogenesis. METHODS AND RESULTS: Protein-free supernatants from thrombin-stimulated platelets increased human umbilical vein endothelial cell migratory activity in transmigration and monolayer repair assays. This phenomenon was ablated by genetic silencing of dRP-generating uridine phosphorylase (UP) and thymidine phosphorylase (TP) or pharmacological inhibition of UP and restored by exogenous dRP. The stimulation of endothelial cell migration by platelet-derived dRP correlated with upregulation of integrin ß(3), which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer α(v)ß(3). The physiological relevance of dRP release by platelets was confirmed in a chick chorioallantoic membrane assay, where the presence of this metabolite in platelet supernatants strongly induced capillary formation. CONCLUSIONS: Platelet-derived dRP stimulates endothelial cell migration by upregulating integrin ß(3) in a reactive oxygen species-dependent manner. As demonstrated by our in vivo experiments, this novel paracrine regulatory pathway is likely to play an important role in the stimulation of angiogenesis by platelets.

Antiangiogenic and antitumor activity of 6-(2-aminoethyl)amino-5-chlorouracil, a novel small-molecule inhibitor of thymidine phosphorylase, in combination with the vascular endothelial growth factor-trap.

PURPOSE: Tumors produce multiple proangiogenic factors, making it unlikely that agents targeting a single angiogenic pathway will be sufficient to treat the spectrum of tumors that occur clinically. Platelet-derived endothelial cell growth factor has angiogenic activity in vitro and in vivo and is overexpressed in most human cancers, where its expression has been correlated with increased microvessel density, more aggressive tumors, and poorer patient prognosis. Platelet-derived endothelial cell growth factor is identical to the enzyme thymidine phosphorylase (TP), and unlike other angiogenic factors, the proangiogenic actions of TP are dependent on its enzyme activity. EXPERIMENTAL DESIGN: A potent and specific small-molecule inhibitor of the catalytic activity of TP, 6-(2-aminoethyl)amino-5-chlorouracil (AEAC), was tested for antiangiogenic and antitumor activity in human cancer xenografts in vivo. RESULTS: Oral administration of AEAC caused 40% to 50% reductions in the growth of A549 non-small cell lung cancer and PANC-1 pancreatic cancer xenografts, but it was not active against a second pancreatic tumor, BxPC-3. AEAC reduced the microvessel density in the tumors, providing evidence for an antiangiogenic action. Equal or better activity was seen when the mice were treated with the vascular endothelial growth factor (VEGF)-Trap, a soluble VEGF decoy receptor, and the combination of AEAC and VEGF-Trap produced additive antitumor activity that was significantly greater than the VEGF-Trap alone. In the A549 tumors, the combination produced tumor regressions. CONCLUSION: These studies show antitumor activity for a drug targeting TP and suggest that inhibitors of TP could be used to augment the clinical efficacy of drugs targeting the VEGF pathway.

Interference with endothelial cell function by JG-03-14, an agent that binds to the colchicine site on microtubules.

JG-03-14, a novel tetrasubstituted pyrrole with microtubule-depolymerizing and anti-proliferative activities, was tested for its effect on endothelial cell (EC) functions in vitro. JG-03-14 was a potent inhibitor of EC vessel-like tube formation on extracellular matrix (IC(50) of 40nM) and caused the involution of established vessels, potential anti-angiogenic and vascular-disrupting activities, respectively. These actions were not due to the inhibition of EC proliferation or to the induction of apoptosis by JG-03-14. While similar effects were observed with the microtubule-depolymerizing and vascular-disrupting drug combretastatin-A4 (CoA4), JG-03-14 had a more selective effect on tube formation, relative to its cytotoxic actions, than did CoA4. Potential molecular mechanisms for JG-03-14's anti-vascular actions were explored. In contrast to the taxanes, which also have anti-vascular actions, JG-03-14 did not disrupt focal adhesion formation or block VEGF-induced phosphorylation of focal adhesion kinase. It did, however, inhibit VEGF-induced phosphorylation of VE-cadherin and reduce the association of beta-catenin with VE-cadherin. It caused cell retraction, intercellular gaps, and abnormally elongated adherens junctions at low concentrations, and prominent, but reversible, plasma membrane blebbing at higher concentrations. These results suggest that JG-03-14 may affect vascular morphogenesis by disrupting the interaction of adjacent endothelial cells, possibly as a consequence of effects on VE-cadherin, beta-catenin, and/or actin. They also provide the first report of anti-vascular activity for this class of compounds.

Antivascular actions of microtubule-binding drugs.

Microtubule-binding drugs (MBD) are widely used in cancer chemotherapy and also have clinically relevant antiangiogenic and vascular-disrupting properties. These antivascular actions are due in part to direct effects on endothelial cells, and all MBDs (both microtubule-stabilizing and microtubule-destabilizing) inhibit endothelial cell proliferation, migration, and tube formation in vitro, actions that are thought to correspond to therapeutic antiangiogenic actions. In addition, the microtubule-destabilizing agents cause prominent changes in endothelial cell morphology, an action associated with rapid vascular collapse in vivo. The effects on endothelial cells occur in vitro at low drug concentrations, which do not affect microtubule gross morphology, do not cause microtubule bundling or microtubule loss and do not induce cell cycle arrest, apoptosis, or cell death. Rather, it has been hypothesized that, at low concentrations, MBDs produce more subtle effects on microtubule dynamics, block critical cell signaling pathways, and prevent the microtubules from properly interacting with transient subcellular assemblies (focal adhesions and adherens junctions) whose subsequent stabilization and/or maturation are required for cell motility and cell-cell interactions. This review will focus on recent studies to define the molecular mechanisms for the antivascular actions of the MBDs, information that could be useful in the identification or design of agents whose actions more selectively target the tumor vasculature.

Taxotere-induced inhibition of human endothelial cell migration is a result of heat shock protein 90 degradation.

In addition to effects on tumor cell proliferation and apoptosis, microtubule-binding agents are potent inhibitors of angiogenesis. The cancer chemotherapeutic drug Taxotere (docetaxel) inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) migration in vitro at concentrations substantially lower than required to cause cell cycle arrest or apoptosis. Here, we show that Taxotere caused the ubiquitination and subsequent proteasomal degradation of heat shock protein 90 (Hsp90) in HUVEC. This prevented signaling from the focal adhesions and VEGF receptors and inhibited integrin activation. Taxotere prevented the VEGF-induced phosphorylation of focal adhesion kinase, Akt, and endothelial nitric oxide synthase (eNOS), all of which are Hsp90 client proteins. Taxotere completely blocked the VEGF-induced increase in eNOS activity, and the addition of a NO donor reversed the inhibitory effect of Taxotere on VEGF-induced migration. A similar reversal occurred with a proteasomal inhibitor of Hsp90 degradation. Furthermore, overexpression of Hsp90 rescued HUVEC from the inhibition of VEGF-induced migration by Taxotere. Previous studies have suggested that tubulin is also a client protein of Hsp90, and immunocytochemical analysis showed that Taxotere caused the dissociation of Hsp90 from tubulin. We suggest that uncomplexed Hsp90 is more susceptible to ubiquitination and subsequent proteasomal degradation than the bound form. Although inhibitors of Hsp90 are currently under clinical investigation as antitumor agents, this seems to be the first account of a drug that reduces Hsp90 function by enhancing its proteasomal degradation. Further, the loss of Hsp90 and the inactivation of Hsp90 client proteins are previously undescribed actions of Taxotere that may contribute to its antiangiogenic activity.

The microtubule binding drug laulimalide inhibits vascular endothelial growth factor-induced human endothelial cell migration and is synergistic when combined with docetaxel (taxotere).

Laulimalide, a natural product from marine sponges, is a microtubule-stabilizing agent that binds to tubulin at a site distinct from that of the taxoids. In the present study, we found that laulimalide inhibited human umbilical vein endothelial cell (HUVEC) tubule formation and vascular endothelial growth factor (VEGF)-induced HUVEC migration, key components of the angiogenic process. These occurred at concentrations substantially lower than that which inhibited HUVEC proliferation. When combined, laulimalide and docetaxel (Taxotere) synergistically inhibited migration and tubule formation, but their combined effect on proliferation was antagonistic. Possible mechanism(s) by which laulimalide inhibited VEGF-induced HUVEC migration were explored. Similar to docetaxel, laulimalide had no effect on the VEGF-induced tyrosine phosphorylation of the VEGF receptor Flk-1/KDR (VEGFR-2). Low concentrations of laulimalide substantially blocked subsequent VEGFR-2 downstream events, as did docetaxel, including the phosphorylation of the Tyr397 and Tyr407 residues of focal adhesion kinase (FAK), the association of VEGFR-2 with FAK and Hsp90, and the Tyr31 phosphorylation of paxillin. Laulimalide inhibited integrin activation; however, compared with docetaxel, it had a weaker inhibitory effect on the VEGF-induced association of VEGFR-2 with the alpha5beta1 integrin. Compared with docetaxel, laulimalide more potently caused a reduction in the constitutive levels (i.e., in the absence of VEGF) of phosphorylated paxillin and more potently inhibited the association of RhoA with the alpha5beta1 integrin. In conclusion, although both docetaxel and laulimalide inhibited integrin-associated signaling pathways that mediated VEGF-induced cell migration, their actions on the signaling cascade seemed not to be identical. These complementary actions could account for their synergistic effects on HUVEC.

Gene expression profiling-based prediction of response of colon carcinoma cells to 5-fluorouracil and camptothecin.

5-Fluorouracil (5-FU) is the most common chemotherapeutic agent used in the treatment of colorectal cancer, yet objective response rates are low. Recently, camptothecin (CPT) has emerged as an effective alternative therapy. Decisive means to determine treatment, based on the likelihood of response to each of these agents, could greatly enhance the management of this disease. Here, the ability of cDNA microarray-generated basal gene expression profiles to predict apoptotic response to 5-FU and CPT was determined in a panel of 30 colon carcinoma cell lines. Genes whose basal level of expression correlated significantly with 5-FU- and CPT-induced apoptosis were selected, and their predictive power was assessed using a "leave one out" jackknife cross-validation strategy. Selection of the 50 genes best correlated with 5-FU-induced apoptosis, but not 50 randomly selected genes, significantly predicted response to this agent. Importantly, this gene expression profiling approach predicted response more effectively than four previously established determinants of 5-FU response: thymidylate synthase and thymidine phosphorylase activity; and p53 and mismatch repair status. Furthermore, reanalysis of the database demonstrated that selection of the 149 genes best correlated with CPT-induced apoptosis maximally and significantly predicted response to this agent. These studies demonstrate that the basal gene expression profile of colon cancer cells can be used to predict and distinguish response to multiple chemotherapeutic agents and establish the potential of this methodology as a means by which rational decisions regarding choice of therapy can be approached.

Expression of the angiogenic factor thymidine phosphorylase in THP-1 monocytes: induction by autocrine tumor necrosis factor-alpha and inhibition by aspirin.

The angiogenic factor thymidine phosphorylase (TP) is highly expressed in human monocytes and macrophages, and its expression has been linked to the pathology and progression of solid tumors, rheumatoid arthritis, and gastric ulcers. In this study, TP mRNA and enzyme activity were found to be up-regulated upon the induction of differentiation of the human monocyte cell line THP-1 by phorbol 12-myristate 13-acetate (PMA). TP expression in THP-1 cells was similarly increased by tumor necrosis factor-alpha (TNFalpha). Because monocytes and macrophages are a predominant source of TNFalpha, the up-regulation of TP upon THP-1 differentiation could have been caused by the autocrine production of TNFalpha. In support of this hypothesis, PMA increased TNFalpha mRNA levels; furthermore, the increase in TP expression with PMA treatment was partially blocked by a neutralizing antibody to TNFalpha, particularly at the earlier time points. This data also suggested there may be additional mechanisms regulating TP expression upon PMA treatment of the cells. The induction of TP by TNFalpha was mimicked by an antibody to the TNFalpha receptor R2 (TNF-R2; p75), but not by an antibody to TNF-R1 (p55), suggesting that the TNF-R2 plays a role in the regulation of TP expression. The PMA-induced increase in TP expression was blocked by aspirin but not by the related agent indomethacin, suggesting that aspirin's effect was not caused by the inhibition of cellular cyclooxygenases. An alternative mechanism by which aspirin inhibits gene expression is the modulation of the transcription factor NFkappaB, and the TNFalpha-induced increase in TP mRNA was blocked by a cell-permeable NFkappaB inhibitory peptide. Furthermore, TNFalpha increased and aspirin (but not indomethacin) decreased NFkappaB DNA-binding activity in THP-1 cells. In conclusion, the modulation of TP expression in monocytes by pro- and anti-inflammatory agents suggests that its angiogenic-related actions could contribute to the inflammatory response associated with a number of pathophysiological conditions.

Thymidine phosphorylase and 2-deoxyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3.

Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-deoxyribose. The mechanisms by which 2-deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin alpha 5 beta 1 or alpha v beta 3, whereas VEGF-induced endothelial cell migration was only blocked by the alpha v beta 3 antibody. Further, thymidine phosphorylase and 2-deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin alpha 5 beta 1 and, in intact cells, increased the co-localization of focal adhesion kinase with alpha 5 beta 1. Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to alpha 5 beta 1 and alpha v beta 3, directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of alpha 5 beta 1 was also increased by thymidine phosphorylase and 2-deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-deoxyribose on signaling pathways associated with endothelial cell migration.

Mechanisms by which tumor cells and monocytes expressing the angiogenic factor thymidine phosphorylase mediate human endothelial cell migration.

The angiogenic factor thymidine phosphorylase (TP) is highly expressed in many human solid tumors, and the level of its expression is associated with tumor neovascularization, invasiveness, and metastasis and with shorter patient survival time. TP promotes endothelial cell (EC) migration in vitro and angiogenesis in vivo, and these have been linked to its enzymatic activity. The mechanism by which TP stimulates EC migration was investigated using human umbilical vein ECs (HUVECs). TP induced concentration-dependent HUVEC migration, which required a TP gradient and thymidine and which was abrogated by the TP inhibitor CIMU (5-chloro-6(1-imidazolylmethyl)uracil). The chemotactic actions of TP plus thymidine were duplicated by the TP metabolite, 2-deoxyribose-1-phosphate (dR-1-P), and 10-fold more potently by its subsequent metabolite, 2-deoxyribose (2dR). Migration induced by dR-1-P, but not 2dR, was blocked by an alkaline phosphatase inhibitor, suggesting that the actions of dR-1-P first required its conversion to 2dR. In the migration assay, [5'-3H]dThd was metabolized to dR-1-P (96%) and 2dR (3.8%), and a gradient of both metabolites was maintained between the lower and upper chambers over the entire 5-h assay. TP expression in human solid tumors occurs in both tumor epithelial cells and in tumor-associated macrophages. The migration assay was adapted to use TP-transfected carcinoma cells to stimulate HUVEC migration, and they were found to induce more migration than did control vector-transfected cells. Human monocyte cells U937 and THP1, which constitutively expressed high levels of TP, also strongly induced HUVEC migration in the coculture assay. CIMU inhibited tumor-cell and monocyte-induced migration. In contrast, a neutralizing antibody to TP had no effect on cell-stimulated HUVEC migration, even though it completely blocked the migration mediated by purified TP. Thus, the intracellular actions of TP were sufficient to stimulate HUVEC chemotaxis. In contrast to purified TP, when incubated with [5'-3H]-thymidine, cells expressing TP released up to 20-fold more 2dR into the medium than dR-1-P. These studies demonstrate that TP-expressing cells mediate EC migration via the intracellular metabolism of thymidine and subsequent extracellular release of 2dR, which forms a chemotactic gradient.