22 results on '"Larios JM"'
Search Results
2. Abstract P2-02-19: Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients
- Author
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Paoletti, C, primary, Cani, AK, additional, Aung, K, additional, Darga, EP, additional, Cannell, EM, additional, Hovelson, DH, additional, Yazdani, M, additional, Blevins, AR, additional, Tokudome, N, additional, Larios, JM, additional, Thomas, DG, additional, Brown, ME, additional, Gersch, C, additional, Schott, AF, additional, Robinson, DR, additional, Chinnaiyan, AM, additional, Bischoff, F, additional, Hayes, DF, additional, Rae, JM, additional, and Tomlins, SA, additional
- Published
- 2016
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3. Targeting ESR1 mutation-induced transcriptional addiction in breast cancer with BET inhibition.
- Author
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Udden SN, Wang Q, Kumar S, Malladi VS, Wu SY, Wei S, Posner BA, Geboers S, Williams NS, Liu Y, Sharma JK, Mani RS, Malladi S, Parra K, Hofstad M, Raj GV, Larios JM, Jagsi R, Wicha MS, Park BH, Gupta GP, Chinnaiyan AM, Chiang CM, and Alluri PG
- Subjects
- Cell Proliferation, Female, Fulvestrant pharmacology, Fulvestrant therapeutic use, Humans, Mutation, Protein Domains, Transcription, Genetic, Breast Neoplasms drug therapy, Breast Neoplasms genetics, Breast Neoplasms pathology, Estrogen Receptor alpha genetics
- Abstract
Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration-approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant-induced endocrine therapy resistance in breast cancer.
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- 2022
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4. Successful treatment of dermato-neuro syndrome with plasmapheresis.
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Larios JM, Ciuro J, Sam Varghese T, and Lyons SE
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- Aged, Brain Diseases diagnosis, Brain Diseases etiology, Clinical Deterioration, Diagnosis, Differential, Humans, Male, Neurologic Examination methods, Patient Care Management methods, Respiration, Artificial methods, Syndrome, Treatment Outcome, Coma diagnosis, Coma etiology, Coma therapy, Plasmapheresis methods, Scleromyxedema complications, Scleromyxedema physiopathology, Seizures diagnosis, Seizures etiology, Seizures therapy
- Abstract
Altered mental status can have many causes ranging from emergent intracranial pathologies to more insidious, systemic toxic aetiologies. We report a rare case of dermato-neuro syndrome in a 71-year-old man with a known history of scleromyxoedema. The patient initially presented with encephalopathy which quickly progressed to generalised tonic-clonic seizures and coma. While his presentation fits with other, although rare, cases of dermato-neuro syndrome, it is imperative to rule out lethal, more common causes of altered mentation. Due to the rarity and difficulty in diagnosis of dermato-neuro syndrome, there is a significant debate regarding the optimal management as there are no standardised treatment protocols. In our case, the patient was successfully treated with plasmapheresis resulting in improved neurologic function., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2020. No commercial re-use. See rights and permissions. Published by BMJ.)
- Published
- 2020
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5. Short-term CDK4/6 Inhibition Radiosensitizes Estrogen Receptor-Positive Breast Cancers.
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Pesch AM, Hirsh NH, Chandler BC, Michmerhuizen AR, Ritter CL, Androsiglio MP, Wilder-Romans K, Liu M, Gersch CL, Larios JM, Pierce LJ, Rae JM, and Speers CW
- Subjects
- Animals, Apoptosis, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Proliferation, Chemoradiotherapy, Female, Humans, Mice, Mice, SCID, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Breast Neoplasms radiotherapy, Cyclin-Dependent Kinase 4 antagonists & inhibitors, Cyclin-Dependent Kinase 6 antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Radiation Tolerance drug effects, Radiation-Sensitizing Agents pharmacology, Receptors, Estrogen metabolism
- Abstract
Purpose: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor-positive (ER
+ ) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models., Experimental Design: Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy., Results: Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21-1.52) and led to a decrease in the surviving fraction of cells at 2 Gy ( P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08-1.68) and abemaciclib-treated (rER: 1.19-2.05) cells. Combination treatment decreased RAD51 foci formation ( P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency ( P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94-1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth., Conclusions: These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence., (©2020 American Association for Cancer Research.)- Published
- 2020
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6. Targeted degradation of activating estrogen receptor α ligand-binding domain mutations in human breast cancer.
- Author
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Gonzalez TL, Hancock M, Sun S, Gersch CL, Larios JM, David W, Hu J, Hayes DF, Wang S, and Rae JM
- Subjects
- Breast Neoplasms genetics, Breast Neoplasms pathology, CRISPR-Cas Systems, Cell Proliferation drug effects, Estrogen Receptor alpha antagonists & inhibitors, Estrogen Receptor alpha metabolism, Female, Humans, Proteolysis, Tumor Cells, Cultured, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, Estrogen Antagonists pharmacology, Estrogen Receptor alpha genetics, Estrogens pharmacology, Mutation
- Abstract
Purpose: Studies have identified several estrogen receptor α (ERα) ligand-binding domain (LBD) somatic mutations in endocrine therapy resistant, metastatic ER-positive breast cancers. The most common mutations, Tyr537Ser (Y537S) and Asp538Gly (D538G), are detected in ~ 30% of endocrine resistant metastatic breast cancer patients. These ESR1 mutations induce the agonist conformation of ERα, confer an estrogen-independent phenotype, and promote drug resistance to antiestrogens., Methods: ER-positive, estrogen-dependent MCF-7 cells were engineered to express either the Y537S or D538G mutants using CRISPR knock-in (cY537S and cD538G). These cells were used to screen several estrogen receptor degrader (ERD) compounds synthesized using the Proteolysis Targeting Chimeras (PROTAC) method to induce degradation of ERα via the ubiquitin-proteasome pathway., Results: Wild-type MCF-7 and ERα LBD mutant cells were treated with ERD-148 (10 pM-1 µM) and assayed for cellular proliferation using the PrestoBlue cell viability assay. ERD-148 attenuated ER-dependent growth with IC
50 values of 0.8, 10.5, and 6.1 nM in MCF-7, cY537S, and cD538G cells, respectively. Western blot analysis showed that MCF-7 cells treated with 1 nM ERD-148 for 24 h exhibited reduced ERα protein expression as compared to the mutants. The ER-regulated gene, GREB1, demonstrated significant downregulation in parental and mutant cells after 24 h of ERD-148 treatment at 10 nM. Growth of the ER-negative, estrogen-independent MDA-MB-231 breast cancer cells was not inhibited by ERD-148 at the ~ IC90 observed in the ER-positive cells., Conclusion: ERD-148 inhibits the growth of ER-positive breast cancer cells via downregulating ERα with comparable potency to Fulvestrant with marginal non-specific toxicity.- Published
- 2020
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7. Correction to: Targeted degradation of activating estrogen receptor α ligand-binding domain mutations in human breast cancer.
- Author
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Gonzalez TL, Hancock M, Sun S, Gersch CL, Larios JM, David W, Hu J, Hayes DF, Wang S, and Rae JM
- Abstract
In the original publication of the article, the spelling of the sixth author's given name was incorrect. The corrected author name should read as "Wadie David". The original article has been corrected.
- Published
- 2020
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8. Functional characterization of the G162R and D216H genetic variants of human CYP17A1.
- Author
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Capper CP, Liu J, McIntosh LR, Larios JM, Johnson MD, Hollenberg PF, Osawa Y, Auchus RJ, and Rae JM
- Subjects
- Catalytic Domain, HEK293 Cells, Half-Life, Humans, Protein Conformation, Steroid 17-alpha-Hydroxylase chemistry, Ubiquitination, Mixed Function Oxygenases metabolism, Mutation, Steroid 17-alpha-Hydroxylase genetics, Steroid 17-alpha-Hydroxylase metabolism
- Abstract
Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5 h, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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9. Comprehensive Mutation and Copy Number Profiling in Archived Circulating Breast Cancer Tumor Cells Documents Heterogeneous Resistance Mechanisms.
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Paoletti C, Cani AK, Larios JM, Hovelson DH, Aung K, Darga EP, Cannell EM, Baratta PJ, Liu CJ, Chu D, Yazdani M, Blevins AR, Sero V, Tokudome N, Thomas DG, Gersch C, Schott AF, Wu YM, Lonigro R, Robinson DR, Chinnaiyan AM, Bischoff FZ, Johnson MD, Park BH, Hayes DF, Rae JM, and Tomlins SA
- Subjects
- Breast Neoplasms pathology, Female, Humans, Mutation, Neoplastic Cells, Circulating pathology, Breast Neoplasms genetics, DNA Copy Number Variations genetics, Neoplastic Cells, Circulating metabolism
- Abstract
Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 ( n = 5/32), harbored the known activating ESR1 p.Y537S mutation ( n = 26/32), or harbored a novel ESR1 p.A569S ( n = 1/32). ESR1 p.A569S was modestly activating in vitro , consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients. Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110-22. ©2017 AACR ., (©2017 American Association for Cancer Research.)
- Published
- 2018
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10. Heterogeneous estrogen receptor expression in circulating tumor cells suggests diverse mechanisms of fulvestrant resistance.
- Author
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Paoletti C, Larios JM, Muñiz MC, Aung K, Cannell EM, Darga EP, Kidwell KM, Thomas DG, Tokudome N, Brown ME, Connelly MC, Chianese DA, Schott AF, Henry NL, Rae JM, and Hayes DF
- Subjects
- Aromatase Inhibitors pharmacology, Aromatase Inhibitors therapeutic use, Biomarkers, Tumor metabolism, Cell Line, Tumor, Estradiol pharmacology, Fulvestrant, Humans, Neoplastic Cells, Circulating drug effects, Neoplastic Cells, Circulating pathology, Treatment Outcome, Drug Resistance, Neoplasm drug effects, Estradiol analogs & derivatives, Neoplastic Cells, Circulating metabolism, Receptors, Estrogen metabolism
- Abstract
Fulvestrant is a dose dependent selective estrogen receptor (ER) down-regulator (SERD) used in ER-positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch(®) system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch(®) CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
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11. The CYP17A1 inhibitor abiraterone exhibits estrogen receptor agonist activity in breast cancer.
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Capper CP, Larios JM, Sikora MJ, Johnson MD, and Rae JM
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- Antineoplastic Combined Chemotherapy Protocols pharmacokinetics, Breast Neoplasms drug therapy, Breast Neoplasms genetics, Cell Line, Tumor, Cell Proliferation drug effects, Estradiol pharmacology, Female, Fulvestrant, Gene Expression Regulation, Neoplastic drug effects, Humans, MCF-7 Cells, Neoplasm Proteins genetics, Androstenes pharmacology, Breast Neoplasms metabolism, Estradiol analogs & derivatives, Receptors, Estrogen metabolism
- Abstract
Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone's ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.
- Published
- 2016
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12. Surgical skills assessment of applicants to general surgery residency.
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Panait L, Larios JM, Brenes RA, Fancher TT, Ajemian MS, Dudrick SJ, and Sanchez JA
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- Aptitude, Computer Simulation, Female, Humans, Laparoscopy education, Male, Surveys and Questionnaires, User-Computer Interface, Aptitude Tests, Educational Measurement methods, General Surgery education, Internship and Residency standards, Motor Skills
- Abstract
Background: Manual skill proficiency is not currently employed in selecting residents for general surgery training programs. The study objective was to assess whether the technical skill levels of applicants to a general surgery residency program are higher than those of internal medicine residents., Material and Methods: Forty-two applicants to a community general surgery program underwent manual skill testing on interview day. Four laparoscopic tasks on a virtual reality (VR) simulator (LapSim, Goteborg, Sweden) were tested. Performance scores were computer-generated. Participants' previous experience with other manual dexterity activities was assessed via a questionnaire. Applicants' self-perception of their surgical skills was correlated with their skill dexterity scores on the simulator. Candidates' simulator scores were also compared with those of a group of internal medicine interns (n = 9) and a group of mid-level surgical residents, PGY 2-3 (n = 7)., Results: Simulator scores of the applicants were significantly lower than those of mid-level surgical residents in all VR tasks (P < 0.05). The internal medicine interns scored higher that the surgery candidates in three of four simulator tasks. Participation in other manual dexterity activities was not associated with increased dexterity scores., Conclusion: This study suggests that surgical dexterity levels do not correlate with the self-assessed skill levels or with previous experience with other manual dexterity activities. Moreover, there appears to be no self-selection of applicants for surgery residency based on actual surgical skills. Selection criteria for surgical training, which incorporate technical proficiency skills, may potentially better discriminate those applicants with an aptitude for a surgical specialty., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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13. The androgen metabolite 5alpha-androstane-3beta,17beta-diol (3betaAdiol) induces breast cancer growth via estrogen receptor: implications for aromatase inhibitor resistance.
- Author
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Sikora MJ, Cordero KE, Larios JM, Johnson MD, Lippman ME, and Rae JM
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- Androgens metabolism, Androgens pharmacology, Blotting, Western, Breast Neoplasms drug therapy, Cell Line, Tumor, Estrogen Receptor Modulators pharmacology, Estrogen Receptor alpha drug effects, Female, Humans, Reverse Transcriptase Polymerase Chain Reaction, Androstane-3,17-diol metabolism, Aromatase Inhibitors therapeutic use, Breast Neoplasms metabolism, Cell Proliferation drug effects, Drug Resistance, Neoplasm physiology, Estrogen Receptor alpha metabolism
- Abstract
The aromatase inhibitors (AIs) are used to treat estrogen receptor-positive (ER+) breast tumors in post-menopausal women, and function by blocking the conversion of adrenal androgens to estrogens by the enzyme CYP19 aromatase. Breast cancer patients receiving AI therapy have circulating estrogen levels below the level of detection; however, androgen concentrations remain unchanged. We were interested in studying the effects of androgens on breast cancer cell proliferation under profound estrogen-deprived conditions. Using in vitro models of estrogen-dependent breast cancer cell growth we show that the androgens testosterone and 5alpha-dihydrotestosterone induce the growth of MCF-7, T47D and BT-474 cells in the absence of estrogen. Furthermore, we demonstrate that under profound estrogen-deprived conditions these breast cancer cells up-regulate steroidogenic enzymes that can metabolize androgens to estrogens. Lastly, we found that the downstream metabolite of 5alpha-dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), is estrogenic in breast cancer cells, and induces growth and ER-signaling via activation of ERalpha. In conclusion, our results show that breast cancer cells deprived of estrogen up-regulate steroidogenic enzymes and metabolize androgens to estrogen-like steroids. The generation of estrogen-like steroids represents a potential mechanism of resistance to aromatase inhibitors.
- Published
- 2009
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14. Expression levels and activation of a PXR variant are directly related to drug resistance in osteosarcoma cell lines.
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Mensah-Osman EJ, Thomas DG, Tabb MM, Larios JM, Hughes DP, Giordano TJ, Lizyness ML, Rae JM, Blumberg B, Hollenberg PF, and Baker LH
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- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Antineoplastic Agents pharmacology, Blotting, Western, Cell Line, Tumor, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System metabolism, Doxorubicin pharmacology, Etoposide pharmacology, Humans, Osteosarcoma genetics, Pregnane X Receptor, Protein Isoforms drug effects, RNA, Messenger analysis, RNA, Small Interfering, Receptors, Steroid drug effects, Reverse Transcriptase Polymerase Chain Reaction, Rifampin pharmacology, Drug Resistance, Neoplasm physiology, Osteosarcoma metabolism, Protein Isoforms metabolism, Receptors, Steroid metabolism
- Abstract
Background: Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors., Methods: Polymerase chain reaction (PCR) and Western blot analysis were used to determine PXR mRNA and protein expression, respectively. Real-time PCR and CYP3A catalytic activity using 7-benzyl-trifluoromethyl coumarin (BFC) as the probe substrate were used to measure the induction of P450 3A4 or MDR1. siRNA transfections were performed for PXR and cytotoxicity determined by a colorimetric based assay or Annexin v-Fitc staining., Results: Differences were observed in the molecular size of the PXR protein expressed in sarcoma cell lines when compared with the wildtype PXR expressed in normal liver, kidney, or small intestine. A polyclonal PXR antibody raised against the N-terminus of the wildtype PXR did not detect PXR expressed in these sarcoma cell lines. In the osteosarcoma cell lines, etoposide and doxorubicin were better inducers of P450 3A4 and MDR1 than rifampin. siRNA against PXR down-regulated P450 3A4 expression only in the osteosarcoma cell line. Cytotoxicity assays showed that the resistance of the osteosarcoma cell lines to etoposide correlated with PXR protein expression levels and activation of P450 3A4 and could be prevented by ketoconazole., Conclusion: The results suggest that PXR plays a critical role in the regulation of P450 3A4 expression in osteosarcoma and that its expression and activation in these tumors may influence the effect of chemotherapeutic agents on the induction of target genes implicated in drug resistance.
- Published
- 2007
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15. GREB1 is a novel androgen-regulated gene required for prostate cancer growth.
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Rae JM, Johnson MD, Cordero KE, Scheys JO, Larios JM, Gottardis MM, Pienta KJ, and Lippman ME
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- Cell Line, Tumor, Chromatin Immunoprecipitation, DNA, Neoplasm drug effects, DNA, Neoplasm genetics, Dose-Response Relationship, Drug, Estrogens pharmacology, Gene Expression Regulation, Neoplastic drug effects, Humans, Male, Neoplasm Proteins analysis, Neoplasms, Hormone-Dependent chemistry, Neoplasms, Hormone-Dependent genetics, Neoplasms, Hormone-Dependent pathology, Neoplasms, Hormone-Dependent physiopathology, Oligonucleotide Array Sequence Analysis, Prostatic Neoplasms chemistry, Prostatic Neoplasms pathology, RNA, Messenger analysis, RNA, Messenger genetics, RNA, Small Interfering pharmacology, Reverse Transcriptase Polymerase Chain Reaction, Androgens pharmacology, Cell Proliferation drug effects, Neoplasm Proteins genetics, Neoplasm Proteins physiology, Prostatic Neoplasms genetics, Prostatic Neoplasms physiopathology
- Abstract
Background: Gene regulated in breast cancer 1 (GREB1) is a novel estrogen-regulated gene shown to play a pivotal role in hormone-stimulated breast cancer growth. GREB1 is expressed in the prostate and its putative promoter contains potential androgen receptor (AR) response elements., Methods: We investigated the effects of androgens on GREB1 expression and its role in androgen-dependent prostate cancer growth., Results: Real-time PCR demonstrated high level GREB1 expression in benign prostatic hypertrophy (BPH), localized prostate cancer (L-PCa), and hormone refractory prostate cancer (HR-PCa). Androgen treatment of AR-positive prostate cancer cells induced dose-dependent GREB1 expression, which was blocked by anti-androgens. AR binding to the GREB1 promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Suppression of GREB1 by RNA interference blocked androgen-stimulated LNCaP cell proliferation., Conclusions: GREB1 is expressed in proliferating prostatic tissue and prostate cancer, is regulated by androgens, and suppression of GREB1 blocks androgen-induced growth suggesting GREB1 may be critically involved in prostate cancer proliferation.
- Published
- 2006
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16. Genes regulated by estrogen in breast tumor cells in vitro are similarly regulated in vivo in tumor xenografts and human breast tumors.
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Creighton CJ, Cordero KE, Larios JM, Miller RS, Johnson MD, Chinnaiyan AM, Lippman ME, and Rae JM
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- Age Factors, Animals, Breast Neoplasms metabolism, Cell Line, Tumor, Estrogen Receptor alpha analysis, Estrogen Receptor alpha biosynthesis, Estrogen Receptor alpha genetics, Gene Expression Profiling, Humans, Mice, Mice, Nude, Proto-Oncogene Proteins c-myc metabolism, RNA, Messenger metabolism, Receptors, Progesterone biosynthesis, Receptors, Progesterone genetics, Transcriptional Activation, Xenograft Model Antitumor Assays, Breast Neoplasms genetics, Estradiol pharmacology, Gene Expression Regulation, Neoplastic
- Abstract
Background: Estrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors., Results: We generated DNA microarray-based gene expression profiles from three estrogen receptor alpha (ERalpha)-positive breast cancer cell lines stimulated by 17beta-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from our preclinical models were co-expressed with ERalpha in a panel of ERalpha+ breast tumor mRNA profiles, when corrections were made for patient age, as well as with progesterone receptor. Furthermore, the E2-regulated genes were significantly enriched for transcriptional targets of the myc oncogene and were found to be coordinately expressed with Myc in human tumors., Conclusion: Our results provide significant validation of a widely used in vitro model of estrogen signaling as being pathologically relevant to breast cancers in vivo.
- Published
- 2006
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17. GREB 1 is a critical regulator of hormone dependent breast cancer growth.
- Author
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Rae JM, Johnson MD, Scheys JO, Cordero KE, Larios JM, and Lippman ME
- Subjects
- Animals, Breast Neoplasms metabolism, Cell Line, Tumor, Estradiol, Estrogen Receptor alpha metabolism, Female, Humans, Mice, Mice, Nude, Oligonucleotide Array Sequence Analysis, RNA Interference, Random Allocation, Breast Neoplasms genetics, Estrogen Receptor alpha genetics, Gene Expression Regulation, Neoplastic genetics, Neoplasm Proteins genetics, Neoplasm Proteins metabolism
- Abstract
Background: Estrogen plays a central role in breast cancer pathogenesis and many potent risk factors for the development of the disease can be explained in terms of increased lifetime exposure to estrogen. Although estrogen regulated genes have been identified, those critically involved in growth regulation remain elusive.METHODS. To identify candidate genes involved in estrogen stimulated breast cancer growth, DNA microarray based gene expression profiles were generated from three estrogen receptor alpha (ER alpha) positive breast cancer cell lines grown under multiple stimulatory and inhibitory conditions., Results: Only three genes were significantly induced by 17beta-estradiol (E2) relative to control in all three cell lines: GREB 1, stromal cell-derived factor 1 (SDF-1) and trefoil factor 1 (pS2). Quantitative real-time PCR assays confirmed that in all three cell lines, GREB 1 was induced by E2, but not by the antiestrogens tamoxifen (TAM) or ICI 182,780. GREB 1 expression level was strongly correlated with ER alpha positivity in 39 breast cancer cell lines of known ER alpha expression status. GREB 1 induction by E2 was rapid (7.3 fold by 2 h for MCF-7) and mirrored the fraction of cells entering S-phase when released from an estrogen deprivation induced cell arrest. Suppression of GREB 1 using siRNA blocked estrogen induced growth in MCF-7 cells and caused a paradoxical E2 induced growth inhibition., Conclusion: These data suggest that GREB 1 is critically involved in the estrogen induced growth of breast cancer cells and has the potential of being a clinical marker for response to endocrine therapy as well as a potential therapeutic target.
- Published
- 2005
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18. Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase.
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Thannickal VJ, Lee DY, White ES, Cui Z, Larios JM, Chacon R, Horowitz JC, Day RM, and Thomas PE
- Subjects
- Cell Adhesion physiology, Cell Differentiation physiology, Cells, Cultured, Extracellular Matrix Proteins pharmacology, Fetus cytology, Fibroblasts cytology, Fibroblasts enzymology, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, Humans, Phosphorylation drug effects, Signal Transduction drug effects, Transforming Growth Factor beta1, src-Family Kinases metabolism, Integrins metabolism, Lung cytology, Protein-Tyrosine Kinases metabolism, Signal Transduction physiology, Transforming Growth Factor beta pharmacology
- Abstract
Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.
- Published
- 2003
- Full Text
- View/download PDF
19. Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts.
- Author
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Larios JM, Budhiraja R, Fanburg BL, and Thannickal VJ
- Subjects
- Biphenyl Compounds pharmacology, Catalase metabolism, Catalase pharmacology, Cell Line, Dimerization, Fibroblasts drug effects, Fluorescent Dyes, Horseradish Peroxidase metabolism, Horseradish Peroxidase pharmacology, Humans, Hydrogen Peroxide pharmacology, Lung, Onium Compounds pharmacology, Oxidation-Reduction, Extracellular Matrix Proteins metabolism, Fibroblasts metabolism, Hydrogen Peroxide metabolism, Transforming Growth Factor beta pharmacology, Tyrosine analogs & derivatives, Tyrosine metabolism
- Abstract
The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.
- Published
- 2001
- Full Text
- View/download PDF
20. Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.
- Author
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Thannickal VJ, Day RM, Klinz SG, Bastien MC, Larios JM, and Fanburg BL
- Subjects
- Cell Division drug effects, Cells, Cultured, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Humans, Hydrogen Peroxide metabolism, Lung cytology, Lung metabolism, Multienzyme Complexes metabolism, NADH, NADPH Oxidoreductases metabolism, Protein-Lysine 6-Oxidase metabolism, Signal Transduction, Tachykinins, Fibroblast Growth Factor 2 pharmacology, Lung drug effects, Reactive Oxygen Species metabolism, Transforming Growth Factor beta pharmacology, ras Proteins metabolism
- Abstract
Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production.
- Published
- 2000
- Full Text
- View/download PDF
21. [Decile distribution of risk of mortality from ischemic cardiopathy in the male population registered at a health care center].
- Author
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Sánchez de Toro Larios JM, Navarro Martínez A, Hernández Ruipérez T, Martínez Ros MT, López Piñera M, and Ferrer Mora A
- Subjects
- Adult, Age Distribution, Health Facilities, Humans, Male, Middle Aged, Risk Factors, Myocardial Ischemia mortality
- Abstract
Objective: To distribute the male population registered at our health centre into deciles of risk of death from ischaemic heart disease, using only the data in the clinical history., Design: A crossover, retrospective and observational study, without random distribution., Setting: Urban health centre., Patients and Other Participants: The work material was 2848 clinical histories of men aged between 25 and 55 1420 of these were histories with up-to-date data because the men had had a consultation during the preceding year., Measurements and Main Results: The method proposed by Shaper to evaluate cardiovascular risk was used. The most prevalent risk factor was tobacco dependency, at 50.1% (CI 95%, 47.5-52.7), whereas hypertension and diabetes did not exceed 14.1% (CI, 12.3-15.9) and 2.5% (CI, 0.016-3.31), respectively, 90% of those under 35 were in the 10-30 deciles, while about half the over-45s were in the 40-90 deciles., Conclusions: Use of the Shaper method enabled us to distribute our male patients according to risk and identify 4% (2.56-5.37) as being at high risk of ischaemic heart disease.
- Published
- 1998
22. [Alcohol and ischemic cardiopathy].
- Author
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Sánchez de Toro Larios JM
- Subjects
- Female, Humans, Male, Alcohol Drinking adverse effects, Myocardial Ischemia prevention & control
- Published
- 1995
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