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1.
Environ Health Perspect ; 132(4): 47002, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38568856

RESUMEN

BACKGROUND: There is a suite of chemicals, including metals, pesticides, and personal care product compounds, which are commonly detected at high levels in US Center for Disease Control's National Health and Nutrition Examination Survey (NHANES) chemical biomarker screens. Whether these chemicals influence development of breast cancer is not well understood. OBJECTIVES: The objectives were to perform an unbiased concentration-dependent assessment of these chemicals, to quantify differences in cancer-specific genes and pathways, to describe if these differences occur at human population-relevant concentrations, and to specifically test for differences in markers of stemness and cellular plasticity. METHODS: We treated nontumorigenic mammary epithelial cells, MCF10A, with 21 chemicals at four concentrations (25 nM, 250 nM, 2.5µM, and 25µM) for 48 h. We conducted RNA-sequencing for these 408 samples, adapting the plexWell plate-based RNA-sequencing method to analyze differences in gene expression. We calculated gene and biological pathway-specific benchmark concentrations (BMCs) using BMDExpress3, identifying differentially expressed genes and generating the best fit benchmark concentration models for each chemical across all genes. We identified enriched biological processes and pathways for each chemical and tested whether chemical exposures change predicted cell type distributions. We contextualized benchmark concentrations relative to human population biomarker concentrations in NHANES. RESULTS: We detected chemical concentration-dependent differences in gene expression for thousands of genes. Enrichment and cell type distribution analyses showed benchmark concentration responses correlated with differences in breast cancer-related pathways, including induction of basal-like characteristics for some chemicals, including arsenic, lead, copper, and methyl paraben. Comparison of benchmark data to NHANES chemical biomarker (urine or blood) concentrations indicated an overlap between exposure levels and levels sufficient to cause a gene expression response. DISCUSSION: These analyses revealed that many of these 21 chemicals resulted in differences in genes and pathways involved in breast cancer in vitro at human exposure-relevant concentrations. https://doi.org/10.1289/EHP12886.


Asunto(s)
Neoplasias de la Mama , Perfilación de la Expresión Génica , Humanos , Femenino , Encuestas Nutricionales , Neoplasias de la Mama/inducido químicamente , Biomarcadores , ARN
2.
Database (Oxford) ; 20242024 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-38204359

RESUMEN

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that are highly expressed and extensively studied from the germline. piRNAs associate with PIWI proteins to maintain DNA methylation for transposon silencing and transcriptional gene regulation for genomic stability. Mature germline piRNAs have distinct characteristics including a 24- to 32-nucleotide length and a 2'-O-methylation signature at the 3' end. Although recent studies have identified piRNAs in somatic tissues, they remain poorly characterized. For example, we recently demonstrated notable expression of piRNA in the murine soma, and while overall expression was lower than that of the germline, unique characteristics suggested tissue-specific functions of this class. While currently available databases commonly use length and association with PIWI proteins to identify piRNA, few have included a chemical oxidation method that detects piRNA based on its 3' modification. This method leads to reproducible and rigorous data processing when coupled with next-generation sequencing and bioinformatics analysis. Here, we introduce piOxi DB, a user-friendly web resource that provides a comprehensive analysis of piRNA, generated exclusively through sodium periodate treatment of small RNA. The current version of piOxi DB includes 435 749 germline and 9828 somatic piRNA sequences robustly identified from M. musculus, M. fascicularis and H. sapiens. The database provides species- and tissue-specific data that are further analyzed according to chromosome location and correspondence to gene and repetitive elements. piOxi DB is an informative tool to assist broad research applications in the fields of RNA biology, cancer biology, environmental toxicology and beyond. Database URL:  https://pioxidb.dcmb.med.umich.edu/.


Asunto(s)
Biología Computacional , ARN de Interacción con Piwi , Animales , Ratones , Metilación de ADN , ARN , Células Germinativas
3.
Biol Chem ; 403(10): 969-982, 2022 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-35796294

RESUMEN

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Here we characterize a novel post-translational mechanism important for TMPRSS13 function: proteolytic cleavage within the extracellular TMPRSS13 stem region located between the transmembrane domain and the first site of N-linked glycosylation at asparagine (N)-250 in the scavenger receptor cysteine rich (SRCR) domain. Importantly, the catalytic competence of TMPRSS13 is essential for stem region cleavage, suggesting an autonomous mechanism of action. Site-directed mutagenesis of the 10 basic amino acids (four arginine and six lysine residues) in this region abrogated zymogen activation and catalytic activity of TMPRSS13, as well as phosphorylation, cell surface expression, and shedding. Mutation analysis of individual arginine residues identified R223, a residue located between the low-density lipoprotein receptor class A domain and the SRCR domain, as important for stem region cleavage. Mutation of R223 causes a reduction in the aforementioned functional processing steps of TMPRSS13. These data provide further insight into the roles of different post-translational modifications as regulators of the function and localization of TMPRSS13. Additionally, the data suggest the presence of complex interconnected regulatory mechanisms that may serve to ensure the proper levels of cell-surface and pericellular TMPRSS13-mediated proteolysis under homeostatic conditions.


Asunto(s)
Proteínas de la Membrana , Procesamiento Proteico-Postraduccional , Arginina/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de la Membrana/metabolismo , Proteolisis
4.
J Biol Chem ; 297(4): 101227, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34562451

RESUMEN

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.


Asunto(s)
Membrana Celular/enzimología , Precursores Enzimáticos/metabolismo , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis , Serina Endopeptidasas/metabolismo , Animales , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Precursores Enzimáticos/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Dominios Proteicos , Transporte de Proteínas/genética , Serina Endopeptidasas/genética
5.
Oncogene ; 39(41): 6421-6436, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32868877

RESUMEN

Breast cancer progression is accompanied by increased expression of extracellular and cell-surface proteases capable of degrading the extracellular matrix as well as cleaving and activating downstream targets. The type II transmembrane serine proteases (TTSPs) are a family of cell-surface proteases that play critical roles in numerous types of cancers. Therefore, the aim of this study was to identify novel and uncharacterized TTSPs with differential expression in breast cancer and to determine their potential roles in progression. Systematic in silico data analysis followed by immunohistochemical validation identified increased expression of the TTSP family member, TMPRSS13 (transmembrane protease, serine 13), in invasive ductal carcinoma patient tissue samples compared to normal breast tissue. To test whether loss of TMPRSS13 impacts tumor progression, TMPRSS13 was genetically ablated in the oncogene-induced transgenic MMTV-PymT tumor model. TMPRSS13 deficiency resulted in a significant decrease in overall tumor burden and growth rate, as well as a delayed formation of detectable mammary tumors, thus suggesting a causal relationship between TMPRSS13 expression and the progression of breast cancer. Complementary studies using human breast cancer cell culture models revealed that siRNA-mediated silencing of TMPRSS13 expression decreases proliferation, induces apoptosis, and attenuates invasion. Importantly, targeting TMPRSS13 expression renders aggressive triple-negative breast cancer cell lines highly responsive to chemotherapy. At the molecular level, knockdown of TMPRSS13 in breast cancer cells led to increased protein levels of the tumor-suppressive protease prostasin. TMPRSS13/prostasin co-immunoprecipitation and prostasin zymogen activation experiments identified prostasin as a potential novel target for TMPRSS13. Regulation of prostasin levels may be a mechanism that contributes to the pro-oncogenic properties of TMPRSS13 in breast cancer. TMPRSS13 represents a novel candidate for targeted therapy in combination with standard of care chemotherapy agents in patients with hormone receptor-negative breast cancer or in patients with tumors refractory to endocrine therapy.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Ductal de Mama/patología , Neoplasias Mamarias Experimentales/patología , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Mama/patología , Carcinoma Ductal de Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Conjuntos de Datos como Asunto , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Serina Endopeptidasas/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética
6.
Sci Rep ; 10(1): 13896, 2020 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-32807808

RESUMEN

Cancer progression is often accompanied by increased levels of extracellular proteases capable of remodeling the extracellular matrix and promoting pro-cancerous signaling pathways by activating growth factors and receptors. The type II transmembrane serine protease (TTSP) family encompasses several proteases that play critical roles in cancer progression; however, the expression or function of the TTSP TMPRSS13 in carcinogenesis has not been examined. In the present study, we found TMPRSS13 to be differentially expressed at both the transcript and protein levels in human colorectal cancer (CRC). Immunohistochemical analyses revealed consistent high expression of TMPRSS13 protein on the cancer cell surface in CRC patient samples; in contrast, the majority of normal colon samples displayed no detectable expression. On a functional level, TMPRSS13 silencing in CRC cell lines increased apoptosis and impaired invasive potential. Importantly, transgenic overexpression of TMPRSS13 in CRC cell lines increased tolerance to apoptosis-inducing agents, including paclitaxel and HA14-1. Conversely, TMPRSS13 silencing rendered CRC cells more sensitive to these agents. Together, our findings suggest that TMPRSS13 plays an important role in CRC cell survival and in promoting resistance to drug-induced apoptosis; we also identify TMPRSS13 as a potential new target for monotherapy or combination therapy with established chemotherapeutics to improve treatment outcomes in CRC patients.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Invasividad Neoplásica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética
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