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1.
bioRxiv ; 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38895216

ABSTRACT

Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of Skp2 in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced tumor inflammation. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of Rb1 and Trp53. We showed that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. We further compared this model with OS models with functional disruption of Skp2: one with Skp2 knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We found reduction of T cell exhaustion and upregulation of interferon activation, along with evidence of replicative and endoplasmic reticulum-related stress in the Skp2 disruption models, and showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased activities of metastasis-related gene signatures in the Skp2-disrupted OS, which we validated by observation of a strong reduction in lung metastasis in the Skp2 knockout mice. Finally, we report several potential mechanisms of escape from targeting Skp2 in OS, including upregulation of Myc targets, DNA copy number amplification and overexpression of alternative E3 ligase genes, and potential alternative lineage activation. These mechanistic insights into OS tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.

2.
Oncogene ; 43(13): 962-975, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38355807

ABSTRACT

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.


Subject(s)
Bone Neoplasms , Osteosarcoma , Animals , Humans , Mice , Bone Neoplasms/drug therapy , Bone Neoplasms/genetics , Carcinogenesis , Cyclin-Dependent Kinase Inhibitor p27/genetics , Mice, Knockout , Osteosarcoma/drug therapy , Osteosarcoma/genetics , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Tumor Microenvironment
3.
Mol Cancer Ther ; 23(2): 223-234, 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-37871911

ABSTRACT

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.


Subject(s)
Bone Neoplasms , Osteosarcoma , Animals , Humans , Mice , Bone Neoplasms/genetics , Disease Models, Animal , Mice, Knockout , Mice, Transgenic , Osteosarcoma/genetics , Osteosarcoma/pathology , Prognosis , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Tumor Microenvironment/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
4.
bioRxiv ; 2023 May 12.
Article in English | MEDLINE | ID: mdl-37214958

ABSTRACT

Purpose: Osteosarcoma (OS) is an aggressive bone malignancy with a poor prognosis. One putative proto-oncogene in OS is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase. We previously demonstrated that SKP2 knockout in murine OS improved survival and delayed tumorigenesis. Here we aim to define the SKP2 drives transcriptional program and its clinical implication in OS. Experimental Design: We performed RNA-sequencing (RNA-seq) on tumors from a transgenic OS 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-/-). We validated our RNA-seq findings using qPCR and immunohistochemistry. To investigate the clinical implications of our results, we analyzed a human OS patient cohort ("NCI-TARGET OS") with RNA-seq and clinical data. Results: We found large differences in gene expression after SKP2 knockout. Strikingly, we observed increased expression of genes related to immune microenvironment infiltration in TKO tumors. We observed significant increases in 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 the changes. In OS 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 OS and the TCGA Sarcoma cohort. Conclusion: Our findings indicate that SKP2 may mediate immune exclusion from the OS tumor microenvironment, suggesting that SKP2 modulation in OS may induce anti-tumor immune activation.

5.
Br J Cancer ; 127(6): 969-975, 2022 10.
Article in English | MEDLINE | ID: mdl-35752713

ABSTRACT

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.


Subject(s)
CDC2-CDC28 Kinases , Neoplasms , CDC2-CDC28 Kinases/genetics , CDC2-CDC28 Kinases/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Humans , Neoplasms/genetics , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Protein , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
6.
Ann N Y Acad Sci ; 1490(1): 90-104, 2021 04.
Article in English | MEDLINE | ID: mdl-33594717

ABSTRACT

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.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Osteosarcoma/genetics , Osteosarcoma/pathology , S-Phase Kinase-Associated Proteins/metabolism , Animals , Carcinogenesis/genetics , Cells, Cultured , Gene Expression Regulation, Neoplastic/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Mas , Retinoblastoma Binding Proteins/genetics , Tumor Suppressor Protein p53/genetics
7.
Transl Oncol ; 13(10): 100809, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32623326

ABSTRACT

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.

8.
Cancer Res ; 80(11): 2355-2367, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32265224

ABSTRACT

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.


Subject(s)
CDC2-CDC28 Kinases/antagonists & inhibitors , Lung Neoplasms/drug therapy , Retinoblastoma Protein/deficiency , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Small Cell Lung Carcinoma/drug therapy , Animals , CDC2-CDC28 Kinases/genetics , CDC2-CDC28 Kinases/metabolism , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Mice , Mice, Knockout , Molecular Targeted Therapy , Retinoblastoma Binding Proteins/deficiency , Retinoblastoma Binding Proteins/genetics , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/metabolism , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Xenograft Model Antitumor Assays
9.
Mol Pharmacol ; 95(1): 127-138, 2019 01.
Article in English | MEDLINE | ID: mdl-30409790

ABSTRACT

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.


Subject(s)
Cell Proliferation/drug effects , Chloroquinolinols/pharmacology , Lung Neoplasms/drug therapy , Lysosomes/drug effects , Membranes/drug effects , Permeability/drug effects , TOR Serine-Threonine Kinases/metabolism , Apoptosis/drug effects , Autophagy/drug effects , Cell Line, Tumor , Chloroquine/pharmacology , Humans , Lung Neoplasms/metabolism , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Phosphorylation/drug effects , Signal Transduction/drug effects
10.
Cancer Discov ; 5(12): 1262-70, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26370156

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/therapeutic use , Carrier Proteins/genetics , Gene Amplification , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Protein Kinase Inhibitors/therapeutic use , Adolescent , Age Factors , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Female , Gene Knockdown Techniques , Genetic Variation , Humans , In Situ Hybridization, Fluorescence , Inhibitory Concentration 50 , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Mice , Multiprotein Complexes/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Rapamycin-Insensitive Companion of mTOR Protein , TOR Serine-Threonine Kinases/antagonists & inhibitors , Xenograft Model Antitumor Assays
11.
ACS Med Chem Lett ; 6(2): 134-9, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25699157

ABSTRACT

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.

12.
J Foot Ankle Surg ; 54(2): 164-72, 2015.
Article in English | MEDLINE | ID: mdl-25619812

ABSTRACT

Intraoperatively, foot and ankle surgeons will encounter peroneal pathologic features in patients with asymptomatic lateral ankles. The purpose of the present study was to review the ankle magnetic resonance imaging (MRI) scans of patients without a history of ankle trauma or lateral ankle pain to determine which anatomic variants correlate with peroneal tendon pathologic features and noted pathophysiology. A total of 500 MRI scans were screened, 108 (41.90 ± 20.42) of which met the inclusion criteria. The peroneus brevis tendon was intact in 104 MRI scans (96.30%), and the peroneus longus tendon was intact in 108 (100.00%). The results of the present study have confirmed statistically significant correlations between the presence of an os perineum and tendinopathy of the peroneus longus [rs(106) = 0.27], undulating peroneal grooves and the severity of peroneal brevis tears [rs(106) = 0.32], a boomerang-shaped peroneus brevis tendon and increasing tendinopathy of the peroneal tendons [brevis (rs(106) = 0.37; longus rs(106) = 0.33], and low-lying muscle bellies and chronic injuries of the superior peroneal retinaculum (rϕ = 0.19). However, the present study did not find evidence to support the presumed correlations between peroneal tendon pathologic findings and hypertrophied peroneal tubercles, low-lying muscle bellies, or the peroneus quartus muscle. Adding to the published data, the present study found a statistically significant correlation between undulating peroneal grooves and an increasing prevalence of osteophytes within the peroneal groove [rs(106) = 0.32]. MRI findings of anatomic variants or peroneal pathologic features might be useful for injury prevention; however, we advise caution from using the findings alone to advocate surgical intervention. To definitively assess causation, prospective, long-term cohort studies are warranted.


Subject(s)
Ankle , Tendon Injuries/epidemiology , Tendon Injuries/pathology , Adult , Age Factors , Female , Humans , Incidence , Magnetic Resonance Imaging , Male , Middle Aged , Osteophyte/epidemiology , Osteophyte/pathology , Osteophyte/physiopathology , Retrospective Studies , Risk Factors , Sex Factors , Tendon Injuries/physiopathology , Young Adult
13.
J Proteome Res ; 13(10): 4325-38, 2014 Oct 03.
Article in English | MEDLINE | ID: mdl-25198380

ABSTRACT

Ralstonia eutropha H16 is a denitrifying microorganism able to use nitrate and nitrite as terminal electron acceptors under oxygen deprivation. To identify proteins showing an altered expression pattern in response to oxygen supply, R. eutropha cells grown aerobically and anaerobically were compared in a comprehensive proteome and transcriptome approach. Nearly 700 proteins involved in several processes including respiration, formation of cell appendages, and DNA and cofactor biosynthesis were found to be differentially expressed. A combination of 1D gel-LC and conventional 2D gel analysis of six consecutive sample points covering the entire denitrification sequence revealed a detailed view on the shifting abundance of the key proteins of denitrification. Denitrification- or anaerobiosis-induced alterations of the respiratory chain included a distinct expression pattern for multiple terminal oxidases. Alterations in the central carbon metabolism were restricted to a few key functions including the isoenzymes for aconitase and isocitrate dehydrogenase. Although R. eutropha is a strictly respiratory bacterium, the abundance of certain fermentation enzymes was increased. This work represents a comprehensive survey of denitrification on the proteomic and transcriptomic levels and provides unique insight into how R. eutropha adapts its metabolism to low oxygen conditions.


Subject(s)
Bacterial Proteins/metabolism , Cupriavidus necator/physiology , Denitrification , Oxygen/metabolism , Proteomics , Transcriptome , Bacterial Proteins/genetics , Cupriavidus necator/genetics , Cupriavidus necator/metabolism , Gene Expression Profiling , Transcription, Genetic
14.
Foot Ankle Spec ; 7(4): 277-285, 2014 08 01.
Article in English | MEDLINE | ID: mdl-25005701

ABSTRACT

The hindfoot and ankle are dynamic structures to which the interplay of tendinous pathologies is scarcely understood. Five hundred consecutive ankle magnetic resonance imaging examinations, obtained between December 27, 2011 and April 9, 2013, were reviewed. Patients without a history of hindfoot or ankle trauma or lateral ankle pain were included. The 108 MRIs that met the inclusion and exclusion criteria were then re-evaluated by 2 musculoskeletal radiologists. Of these, 55.56% demonstrated pathology of the Achilles tendon (AT), 44.44% demonstrated pathology of the posterior tibial tendon (PTT), 35.19% demonstrated pathology of the peroneus brevis (PB), and 37.96% demonstrated pathology of the peroneus longus (PL). In our asymptomatic patient population, 16 (14.81%) patients demonstrated concomitant pathology of the AT, PTT, and peroneal tendons. There were positive, moderate correlations between graded pathology of the AT and the PTT, rs(106) = 0.32, P = .001; the AT and PB, rs(106) = 0.38, P = 0.001; and the AT and PL, rs(106) = 0.46, P = .001. However, there were no statistically significant correlations between pathology of the PTT and PB, rs(106) = 0.17, P = .08, or the PTT and PL, rs(106) = 0.14, P = .15. These findings suggest an intimate relationship between the AT, PTT, and the peroneal tendons. These individual anatomic structures may have underappreciated functional relationships that could lead to future investigations. LEVEL OF CLINICAL EVIDENCE: Level IV.

15.
Foot Ankle Spec ; 7(6): 449-56, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25005703

ABSTRACT

UNLABELLED: With the increased prevalence of obesity, there has been a parallel rise in musculoskeletal disorders. However, the effect of body mass index (BMI) on pathology of the hindfoot and ankle is scarcely understood. The purpose of the present report was to compare the number of tendinous and ligamentous pathologies within the hindfoot and ankle between overweight (BMI ≥ 25.00 kg/m(2)) and nonoverweight (BMI < 25.00 kg/m(2)) atraumatic patients. We hypothesized that overweight patients would demonstrate more tendinous and ligamentous pathologies compared with their nonoverweight counterparts. Five hundred consecutive magnetic resonance images were reviewed. One hundred eight patients met the inclusion and exclusion criteria. Sixty-six patients were overweight and 42 patients were nonoverweight. Ninety-eight percent of overweight patients demonstrated pathology of a tendinous or ligamentous nature, whereas 62% of nonoverweight patients demonstrated pathology of a tendinous or ligamentous nature. Thus, the prevalence of pathology was 1.59 times higher among overweight patients compared with nonoverweight patients. Moreover, controlling for age, overweight patients demonstrated approximately twice as many tendinous and ligamentous pathologies compared with nonoverweight patients (adjusted mean ± SD = 4.44 ± 2.14 vs 1.98 ± 2.07, respectively), which was statistically significantly different (P < .001). To definitively assess causation and the clinical evolution of hindfoot and ankle pathology, prospective, longitudinal cohort studies are warranted. LEVEL OF EVIDENCE: Diagnostic, Level III: Case series.


Subject(s)
Ankle Joint/pathology , Ligaments, Articular/pathology , Magnetic Resonance Imaging , Overweight/pathology , Tendons/pathology , Adult , Body Mass Index , Female , Humans , Male , Retrospective Studies , Weight-Bearing , Young Adult
16.
J Foot Ankle Surg ; 53(4): 429-33, 2014.
Article in English | MEDLINE | ID: mdl-24796886

ABSTRACT

Given the frequency and burden of ankle sprains, the pathologic features identified on magnetic resonance imaging (MRI) scans are widely known in the symptomatic population. Ankle MRI pathologic features in the asymptomatic population, however, are poorly understood. Such examinations are rarely undertaken unless an ankle has been injured or is painful. We report the systematic MRI findings from the reports of 108 consecutive asymptomatic lateral ankles (104 patients). Our purpose was to (1) report the prevalence of osteochondral lesions of the talus (OLTs) and pathologic features of the medial and lateral ligaments, peroneal tendons, and superior peroneal retinaculum (SPR); (2) correlate the presence of OLTs with the pathologic features of the medial and lateral ligaments, peroneal tendons, and SPR; and (3) correlate ligamentous discontinuity with the peroneal pathologic features, OLTs, and SPR pathologic features. A total of 16 OLTs (14.81%) were present (13 medial and 3 lateral). Of the 16 patients with OLTs, 8 (50.00%) had concomitant peroneal pathologic findings. Healthy medial and lateral ligaments were noted in 41 patients (37.96%), and ligamentous discontinuity was grade I in 25 (23.15%), II in 32 (29.63%), III in 5 (4.63%), and grade IV in 5 patients (4.63%). A weak positive correlation was found between attenuation or tears of the superficial deltoid and medial OLTs (phi coefficient = 0.23, p = .0191) and a moderate positive correlation between tears of the posterior talofibular ligament and lateral OLTs (phi coefficient = 0.30, p = .0017). Additionally, a moderate positive correlation between ligamentous discontinuity and tendinopathy of the peroneus brevis was noted [Spearman's coefficient(106) = 0.29, p = .0024]. These findings add to the evidence of concomitant pathologic features in the asymptomatic population. To definitively assess causation and evaluate the clinical evolution of radiologic findings, future, prospective, longitudinal cohort studies are necessary.


Subject(s)
Ankle Joint/pathology , Cartilage Diseases/diagnosis , Ligaments, Articular/pathology , Soft Tissue Injuries/diagnosis , Talus/pathology , Tendons/pathology , Adult , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Retrospective Studies , Young Adult
17.
J Thorac Oncol ; 8(6): 693-702, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23575415

ABSTRACT

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.


Subject(s)
Autophagy/drug effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Chloroquine/pharmacology , Drug Resistance, Neoplasm/drug effects , Lung Neoplasms/drug therapy , Quinazolines/pharmacology , Animals , Antimalarials/pharmacology , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Blotting, Western , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle/drug effects , Cell Proliferation/drug effects , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride , Flow Cytometry , Humans , Lung Neoplasms/pathology , Mice , Mice, Nude , Protein Kinase Inhibitors/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
18.
Appl Environ Microbiol ; 78(22): 7884-90, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22941075

ABSTRACT

With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong P(cbbL) promoter of the R. eutropha H16 cbb(c) operon (coding for autotrophic CO(2) fixation). In batch cultures growing on H(2) and CO(2) as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with (13)CO(2) and (15)NH(4)Cl, and the (13)C/(15)N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [(13)C/(15)N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.


Subject(s)
Arginine/metabolism , Cupriavidus necator/metabolism , Isotope Labeling/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bioreactors/microbiology , Carbon Dioxide/metabolism , Fermentation , Gene Expression , Hydrogen/metabolism , Metabolic Engineering , Peptide Synthases/genetics , Peptide Synthases/metabolism , Plasmids , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Synechocystis/enzymology , Synechocystis/genetics
19.
J Proteome Res ; 10(6): 2767-76, 2011 Jun 03.
Article in English | MEDLINE | ID: mdl-21561103

ABSTRACT

The soil-dwelling lithoautotrophic bacterium Ralstonia eutropha H16 utilizes hydrogen as the key source of energy during aerobic growth on hydrogen and carbon dioxide. We examined the soluble and membrane protein complements of lithoautotrophically grown cells and compared them to the protein complements of cells grown organoheterotrophically on succinate. (14)N/(15)N-based inverse metabolic labeling in combination with GeLC-MS led to the identification of 1452 proteins, 1174 of which could be quantitated. Far more proteins were found to be more abundant in the lithoautotrophically than in the organoheterotrophically grown cells. In addition to the induction of the key enzymes of hydrogen oxidation and carbon dioxide fixation, we observed several characteristic alterations in the proteome correlated with lithoautotrophic growth. (I) Genes for three terminal oxidases were upregulated. (II) NAD(P) transhydrogenase and enzymes for the accumulation of poly(3-hydroxybutyrate) (PHB) showed increased protein abundance. (III) Lithoautotrophically grown cells were equipped with an enhanced inventory of transport systems. (IV) The expression of cell surface appendages involved in cell movement was markedly increased, while proteins involved in cell adhesion were decreased. Our data show that the hydrogen-based lifestyle of R. eutropha H16 relies on an extensive protein repertoire adapting the organism to the alternative energy and carbon sources.


Subject(s)
Adaptation, Physiological , Bacterial Proteins/metabolism , Cupriavidus necator/physiology , Membrane Proteins/metabolism , Proteome/metabolism , Carbon Dioxide/metabolism , Carrier Proteins/metabolism , Culture Media , Cupriavidus necator/growth & development , Cupriavidus necator/metabolism , Electron Transport/physiology , Gene Expression Profiling , Hydrogen/metabolism , Molecular Motor Proteins/metabolism , NADP/metabolism , Sigma Factor/metabolism , Succinic Acid , Tandem Mass Spectrometry
20.
Arterioscler Thromb Vasc Biol ; 30(12): 2631-8, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20884872

ABSTRACT

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.


Subject(s)
Blood Platelets/metabolism , Cell Movement , Chorioallantoic Membrane/blood supply , Endothelial Cells/metabolism , Neovascularization, Physiologic , Paracrine Communication , Ribosemonophosphates/metabolism , Animals , Blood Platelets/drug effects , Cell Movement/drug effects , Cells, Cultured , Chick Embryo , Endothelial Cells/drug effects , Enzyme Inhibitors/pharmacology , Gas Chromatography-Mass Spectrometry , Gene Silencing , Humans , Integrin alphaV/metabolism , Integrin alphaVbeta3/metabolism , Integrin beta3/metabolism , Neovascularization, Physiologic/drug effects , Paracrine Communication/drug effects , Reactive Oxygen Species/metabolism , Thrombin/metabolism , Thymidine Phosphorylase/antagonists & inhibitors , Thymidine Phosphorylase/genetics , Thymidine Phosphorylase/metabolism , Time Factors , Uridine Phosphorylase/antagonists & inhibitors , Uridine Phosphorylase/genetics , Uridine Phosphorylase/metabolism
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