ABSTRACT
Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.
Subject(s)
Phosphatidylinositol 3-Kinases , Uterine Cervical Neoplasms , Animals , Cell Line, Tumor , Cell Movement , ErbB Receptors , Female , Humans , Mice , Mice, Nude , Prognosis , Uterine Cervical Neoplasms/drug therapyABSTRACT
Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.
Subject(s)
Humans , Animals , Female , Rats , Uterine Cervical Neoplasms/drug therapy , Phosphatidylinositol 3-Kinases , Prognosis , Cell Movement , Cell Line, Tumor , ErbB Receptors , Mice, NudeABSTRACT
Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.
Subject(s)
Alternative Splicing/genetics , Drug Resistance, Neoplasm/genetics , Osteopontin/genetics , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , RNA Splicing/genetics , Taxoids/pharmacology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell Survival/genetics , Docetaxel , Down-Regulation/genetics , Epithelial-Mesenchymal Transition/genetics , Humans , Male , Prostate/drug effects , Signal Transduction/genetics , Up-Regulation/geneticsABSTRACT
Human osteopontin is subject to alternative splicing, which generates three isoforms, termed OPNa, OPNb and OPNc. These variants show specific expression and roles in different cell contexts. We present an overview of current knowledge of the expression profile of human OPN splicing isoforms (OPN-SIs), their tissue-specific roles, and the pathways mediating their functional properties in different pathophysiological conditions. We also describe their putative application as biomarkers, and their potential use as therapeutic targets by using antibodies, oligonucleotides or siRNA molecules. This synthesis provides new clues for a better understanding of human OPN splice variants, their roles in normal and pathological conditions, and their possible clinical applications.
Subject(s)
Alternative Splicing , Neoplasms/genetics , Osteopontin/metabolism , Signal Transduction , Humans , Neoplasms/diagnosis , Neoplasms/therapy , Protein IsoformsABSTRACT
Osteopontin splicing isoforms (OPN-SI) present differential expression patterns and specific tumor roles. Our aims were to characterize OPN-SI expression in prostate cancer (PCa) and benign prostate hyperplasia (BPH) tissues, besides evaluating their potential as biomarkers for PCa diagnosis and prognostic implications. Prostatic tissue specimens were obtained from 40 PCa and 30 benign prostate hyperplasia (BPH) patients. Quantitative real time PCR (qRT-PCR) was used to measure OPN-SI mRNA expression. Immunohistochemical analysis was performed using an anti-OPNc polyclonal antibody. Biostatistical analyses evaluated the association of OPN-SI and total Prostate Specific Antigen (PSA) serum levels with clinical and pathological data. PCa tissue samples presented significantly higher levels of OPNa, OPNb and OPNc transcripts (p<0.01) than in BPH specimens. OPN-SI mRNA expression were positively correlated with Gleason Score (p<0.01). ROC curves and logistic regression analyses demonstrated that OPN-SI and PSA were able to distinguish PCa from BPH patients (p<0.01). The OPNc isoform was the most upregulated variant and the best marker to distinguish patients' groups, presenting sensitivity and specificity of 90% and 100%, respectively. Immunohistochemistry analysis also demonstrated OPNc upregulation in PCa samples as compared to BPH tissues. OPNcprotein was also strongly stained PCa tissues presenting High Gleason Score. Multivariate analysis indicated that OPNc expression levels above the cut-off value presented a chance 4-fold higher for PCa occurrence. We conclude that OPN-SI were overexpressed in PCa tissues, strongly associated with PCa occurrence and with tumor cell differentiation. Our results suggest OPNc splicing isoform as an important biomarker contributing to improve PCa diagnosis and prognosis, besides providing insights into early steps of PCa carcinogenesis.
Subject(s)
Osteopontin/genetics , Prostatic Hyperplasia/genetics , Prostatic Neoplasms/genetics , RNA Splicing/genetics , RNA, Messenger/genetics , Adult , Aged , Aged, 80 and over , Antibodies , Biomarkers, Tumor/blood , Cell Differentiation , Diagnosis, Differential , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Staging , Predictive Value of Tests , Prognosis , Prostatic Hyperplasia/diagnosis , Prostatic Neoplasms/diagnosis , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Real-Time Polymerase Chain Reaction , Sensitivity and SpecificitySubject(s)
Humans , Genetic Heterogeneity , Osteopontin , Ovarian Neoplasms , Prostate , Prostatic Neoplasms , Protein IsoformsABSTRACT
Mutations in different exons of ret proto-oncogene are responsible for the development of medullary thyroid carcinoma (MTC). The mutations can occur as sporadic or as part of multiple endocrine neoplasia (MEN) type 2 hereditary syndromes. Here we report the first focused study of sporadic MTC in Brazilian patients regarding clinical and molecular analysis of ret proto-oncogene. Our study seeks to estimate the risk of hereditary MTC cases among apparently sporadic cases in a Brazilian population and describe ret genetic variants in their germinative lineage. Germinative sequence variants were screened by DNA sequencing and denaturing gradient gel electrophoresis (DGGE) analysis of exons 10, 11, 13, 14, 15, and 16 of 24 Brazilian patients with apparently sporadic MTC. We identified 1 inherited case of 24 (4%) patients with apparently sporadic MTC. Polymorphisms for the ret proto-oncogene coding region were identified in codon 769 of exon 13 (LeuCTT--> LeuCTG) at a frequency of 13% (3/24) and in codon 904 of exon 15 (SerTCC--> SerTCG) at a frequency of 16.6% (4/24). The observed frequency (4%) of inherited disease among apparent sporadic MTC strengthens routine application of ret proto-oncogene germinative DNA screening in all cases of apparently sporadic MTC ascertained at Brazilian cancer hospitals.
