RESUMO
The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fibronectinas/biossíntese , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células A549 , Animais , Anoikis/fisiologia , Neoplasias da Mama/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Agregação Celular/fisiologia , Linhagem Celular Tumoral , Fibronectinas/genética , Fibronectinas/metabolismo , Xenoenxertos , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , NADPH Oxidase 4/biossíntese , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise Serial de Tecidos , Regulação para CimaRESUMO
BACKGROUND: Normal cells are sensitive to anoikis, which is a cell detachment-induced apoptosis. However, cancer cells acquire anoikis resistance that is essential for successful metastasis. This study aimed to demonstrate the function and potential mechanism of NADPH oxidase 4 (NOX4) and EGFR activation in regulating anoikis resistance in lung cancer. METHODS: Cells were cultured either in the attached or suspended condition. Cell viability was measured by cell counting and live and dead cell staining. Expression levels of NOX4 and EGFR were measured by PCR and immunoblotting. Reactive oxygen species (ROS) levels were measured by flow cytometry. Effects of NOX4 overexpression or NOX4 knockdown by si-NOX4 on anoikis sensitivity were explored. Levels of NOX4 and EGFR in lung cancer tissues were evaluated by IHC staining. RESULTS: NOX4 was upregulated but EGFR decreased in suspended cells compared with attached cells. Accordingly, ROS levels were increased in suspended cells, resulting in the activation of Src and EGFR. NOX4 knockdown decreased activation of Src and EGFR, and thus sensitised cells to anoikis. NOX4 overexpression increased EGFR levels and attenuated anoikis. NOX4 expression is upregulated and is positively correlated with EGFR levels in the lung cancer patient tissues. CONCLUSIONS: NOX4 upregulation confers anoikis resistance by ROS-mediated activation of EGFR and Src, and by maintaining EGFR levels, which is critical for cell survival.
Assuntos
Anoikis/genética , Receptores ErbB/fisiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , NADPH Oxidases/fisiologia , Células A549 , Anoikis/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Receptores ErbB/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , NADPH Oxidase 4 , NADPH Oxidases/antagonistas & inibidores , Metástase Neoplásica , RNA Interferente Pequeno/farmacologia , Células Tumorais CultivadasRESUMO
Metastasis is a multistep process including dissociation of cancer cells from primary sites, survival in the vascular system, and proliferation in distant target organs. As a barrier to metastasis, cells normally undergo an apoptotic process known as "anoikis," a form of cell death due to loss of contact with the extracellular matrix or neighboring cells. Cancer cells acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory and lymphatic systems to disseminate throughout the body. Because recent technological advances enable us to detect rare circulating tumor cells, which are anoikis resistant, currently, anoikis resistance becomes a hot topic in cancer research. Detailed molecular and functional analyses of anoikis resistant cells may provide insight into the biology of cancer metastasis and identify novel therapeutic targets for prevention of cancer dissemination. This paper comprehensively describes recent investigations of the molecular and cellular mechanisms underlying anoikis and anoikis resistance in relation to intrinsic and extrinsic death signaling, epithelial-mesenchymal transition, growth factor receptors, energy metabolism, reactive oxygen species, membrane microdomains, and lipid rafts.