RESUMO
It is unknown how cues from the tumor microenvironment can regulate post-transcriptional mechanisms, such as alternative splicing, that control genes that drive malignant growth. The induction of cyclooxygenase 2 (Cox-2) by integrin α3ß1 in breast cancer cells can promote tumor progression. We have used RNAi to suppress α3ß1 in human MDA-MB-231 breast cancer cells and then investigated changes in global gene expression. Numerous mRNAs, including Cox-2, show altered expression and/or alternative exon usage (AEU) in α3ß1-deficient cells. AEU included patterns predicted to render an mRNA susceptible to degradation, such as 3'-UTR variations or retention of elements that target an mRNA for nonsense-mediated decay (NMD). PCR-based analysis of α3ß1-deficient cells confirmed changes in Cox-2 mRNA that might target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 3'-UTR. Moreover, Cox-2 mRNA has reduced stability in α3ß1-deficient cells, which is partially reversed by knockdown of the essential NMD factor UPF1. Our study identifies α3ß1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that has potential for exploitation as a therapeutic target.
Assuntos
Ciclo-Oxigenase 2/genética , Integrina alfa3beta1/fisiologia , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/genética , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Íntrons , Laminina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , RNA Helicases , RNA Mensageiro/metabolismo , Transativadores/genética , Transativadores/metabolismo , TranscriptomaRESUMO
Integrin receptors for cell adhesion to extracellular matrix have important roles in promoting tumor growth and progression. Integrin alpha3beta1 is highly expressed in breast cancer cells in which it is thought to promote invasion and metastasis; however, its roles in regulating malignant tumor cell behavior remain unclear. In the current study, we used short-hairpin RNA (shRNA) to show that suppression of alpha3beta1 in a human breast cancer cell line, MDA-MB-231, leads to decreased tumorigenicity, reduced invasiveness, and decreased production of factors that stimulate endothelial cell migration. Real-time PCR revealed that suppression of alpha3beta1 caused a dramatic reduction in expression of the cyclooxygenase-2 (COX-2) gene, which is frequently overexpressed in breast cancers and has been exploited as a therapeutic target. Decreased COX-2 was accompanied by reduced prostaglandin E2 (PGE(2)), a major prostanoid produced downstream of COX-2 and an important effector of COX-2 signaling. shRNA-mediated suppression of COX-2 showed that it has a role in tumor cell invasion and cross-talk to endothelial cells. Furthermore, treatment with PGE(2) restored these functions in alpha3beta1-deficient MDA-MB-231 cells. These findings identify a role for alpha3beta1 in regulating two properties of tumor cells that facilitate cancer progression: invasiveness and ability to stimulate endothelial cells. They also reveal a novel role for COX-2 as a downstream effector of alpha3beta1 in tumor cells, thereby identifying alpha3beta1 as a potential therapeutic target to inhibit breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo-Oxigenase 2/genética , Células Endoteliais/patologia , Integrina alfa3beta1/antagonistas & inibidores , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/biossíntese , Células Endoteliais/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During cutaneous wound healing, epidermal keratinocytes play essential roles in the secretion of factors that promote angiogenesis. However, specific cues in the wound microenvironment that trigger the production of pro-angiogenic factors by keratinocytes, and the cellular receptors that mediate this response, remain unclear. In this study, we exploited a model of conditional integrin knockout to demonstrate impaired wound angiogenesis in mice that lack alpha3beta1 integrin in epidermis. In addition, we used genetic and shRNA approaches to determine that alpha3beta1-integrin deficiency in keratinocytes leads to reduced mRNA and protein expression of the pro-angiogenic factor mitogen-regulated protein 3 (MRP3; also known as PRL2C4), and to demonstrate that this regulation provides a mechanism of keratinocyte-to-endothelial-cell crosstalk that promotes endothelial-cell migration. Finally, we showed that the impaired wound angiogenesis in epidermis-specific alpha3-integrin-knockout mice is correlated with reduced expression of MRP3 in wounded epidermis. These findings identify a novel role for alpha3beta1 integrin in promoting wound angiogenesis through a mechanism of crosstalk from epidermal to endothelial cells, and they implicate MRP3 in this integrin-dependent crosstalk. Such a mechanism represents a novel paradigm for integrin-mediated regulation of wound angiogenesis that extends beyond traditional roles for integrins in cell adhesion and migration.
