Proteomic Analysis of TNF-Alpha Resistant Human Breast Cancer Cells
Introduction: Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.
Methods: To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.
Results: Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.
Conclusions: This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.
Drug resistance represents a major obstacle to successful therapy of breast cancer, a leading cause of death among women in Western countries. It is well known that several ATP-binding cassette transporters, such as MDR (multidrug resistance), MRP (multidrug resistance associated protein), and BCRP (breast cancer resistance protein), are related to the development of drug resistance in breast cancers. However, many other proteins – including glutathione-S-transferase,, β2-microglobulin, heat shock protein (HSP)27, 14-3-3δ, and vimentin – have also been implicated in breast cancer drug resistance. These findings were based upon studies using various chemoresistant breast cancer cell lines such as adriamycin, verapamil, tamoxifen, vinblastine, and paclitaxel resistant MCF-7 cells. Although some aspects of the mechanisms of drug resistance have been characterized, the highly variable response to chemotherapy in the treatment of breast cancers remains poorly understood. Elucidating these drug resistance mechanisms is essential for improving tumor responses to clinical chemotherapies.
A growing area of interest that may reveal one such mechanism is the association of drug resistance with epithelial-mesenchymal transition (EMT) in cancer. EMT is the process by which adherent epithelial cells convert to motile mesenchymal cells and is essential in embryonic development. However, it appears that aberrant activation of EMT occurs in cancer progression, and is involved in highly aggressive, poorly differentiated breast cancers with increased potential for metastasis and recurrence. EMT has been linked to resistance to various drugs in cancer, including tamoxifen resistance in breast carcinoma cells, paclitaxel resistance in epithelial ovarian carcinoma cells, oxaliplatin resistance in colorectal cancer cells, gemcitibine resistance in pancreatic tumor cells, cetuximab resistance in hepatoma cells, and erlotinib resistance in non-small-cell lung carcinomas. The activities of several genes are known to contribute to EMT, including decreased expression of E-cadherin, and increased expression of snail, slug, and δ-EF1 (ZEB1). Increased expression of vimentin and N-cadherin are also seen in EMT. Evaluation of these markers in a drug-resistant cell line may shed light on the relationship between EMT and drug resistance.
TNF-α is a multifunctional cytokine that elicits a variety of biologic responses, such as inflammation and apoptosis. Additionally, TNF-α has been shown to induce EMT. Although TNF-α is not currently an anticancer agent for treatment of human cancers (because of side effects such as normal cell toxicity), low doses of TNF-α can markedly sensitize cancer cells to chemotherapy-induced apoptosis. We previously demonstrated that MCF-7 cell line variants exhibit differences in sensitivity to TNF-α and apoptosis induced by taxol and doxorubicin. Specifically, we demonstrated that apoptosis sensitive MCF-7-N cells (MCF-7 N variant) exhibited distinct differences in cell survival and apoptotic signaling when compared with inherently resistant MCF-7-M cells (MCF-7 M variant). We further demonstrated that apoptosis sensitive cells (MCF-7-N) could be driven to a resistant phenotype through prolonged exposure to increasing concentrations of TNF-α, leading to a stable, apoptosis-resistant phenotype (MCF-7-TNR) that was in part dependent upon mitogen-activated protein kinase (MAPK) and nuclear factor-κB signaling. Gene expression profiling revealed that MAPK kinase (MEK)5 was over-expressed in the TNF-α resistant MCF-7-M cells versus the TNF-α sensitive MCF-7-N cells. A similar upregulation of MEK5 in resistant cells was independently described in MCF-7-F cells, which developed resistance to the pure anti-estrogen fulvestrant through prolonged growth in fulvestrant-containing media. These studies demonstrate a potential role for the MEK5 pathway in the regulation of progression to drug resistance in breast cancer.
The MEK5/extracellular signal-regulated kinase (Erk)5 tandem is a component of MAPK cascades that mediate signals from various extracellular stimuli to the nucleus and regulate most cellular processes, including gene expression, proliferation, apoptosis, and motility. MAPK signaling may also play a role in EMT. Although MEK5/Erk5 signaling has not been extensively investigated, several studies suggest a role in cancer progression. For example, MEK5/Erk5 signaling has been demonstrated in prostate and breast cancer proliferation and tumorigenesis. Furthermore, inhibition of MEK5/Erk5 signaling in the MDA-MB-231 cell line, an aggressive breast cancer cell line with an EMT phenotype, induces apoptosis.
Based on these findings, which strongly indicate that MEK5/Erk5 signaling may mediate cancer progression to an aggressive phenotype, we further explored the involvement of MEK5/Erk5 signaling in resistance to apoptosis as well as EMT. To test the role played by MEK5/Erk5 activation in progression of breast carcinoma cells to a resistant phenotype, MCF-7 cells (N variant) were used to stably express a constitutive active MEK5 construct. These MCF-7-MEK5 cells exhibit resistance to TNF-α as compared with stable vector cells (MCF-7-VEC). Proteomic analysis based on two-dimensional electrophoresis (2-DE) and various mass spectrometric techniques has been employed in several studies of drug resistance of breast cancers. In this study we used a proteomics approach to define mechanisms of the MEK5 signaling cascade in the regulation of drug resistance. The differentially expressed proteins identified in proteomic analyses were confirmed at the gene expression level using reverse transcription RT-PCR assays. Results using immunofluorescence staining and gene expression analysis were consistent with an EMT phenotype in the MCF-7-MEK5 cells. These findings identify a potential role for the MEK5 pathway in coordinately promoting both an EMT phenotype and TNF-α resistance in breast cancer.
Introduction: Despite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.
Methods: To compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.
Results: Proteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.
Conclusions: This study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.
Drug resistance represents a major obstacle to successful therapy of breast cancer, a leading cause of death among women in Western countries. It is well known that several ATP-binding cassette transporters, such as MDR (multidrug resistance), MRP (multidrug resistance associated protein), and BCRP (breast cancer resistance protein), are related to the development of drug resistance in breast cancers. However, many other proteins – including glutathione-S-transferase,, β2-microglobulin, heat shock protein (HSP)27, 14-3-3δ, and vimentin – have also been implicated in breast cancer drug resistance. These findings were based upon studies using various chemoresistant breast cancer cell lines such as adriamycin, verapamil, tamoxifen, vinblastine, and paclitaxel resistant MCF-7 cells. Although some aspects of the mechanisms of drug resistance have been characterized, the highly variable response to chemotherapy in the treatment of breast cancers remains poorly understood. Elucidating these drug resistance mechanisms is essential for improving tumor responses to clinical chemotherapies.
A growing area of interest that may reveal one such mechanism is the association of drug resistance with epithelial-mesenchymal transition (EMT) in cancer. EMT is the process by which adherent epithelial cells convert to motile mesenchymal cells and is essential in embryonic development. However, it appears that aberrant activation of EMT occurs in cancer progression, and is involved in highly aggressive, poorly differentiated breast cancers with increased potential for metastasis and recurrence. EMT has been linked to resistance to various drugs in cancer, including tamoxifen resistance in breast carcinoma cells, paclitaxel resistance in epithelial ovarian carcinoma cells, oxaliplatin resistance in colorectal cancer cells, gemcitibine resistance in pancreatic tumor cells, cetuximab resistance in hepatoma cells, and erlotinib resistance in non-small-cell lung carcinomas. The activities of several genes are known to contribute to EMT, including decreased expression of E-cadherin, and increased expression of snail, slug, and δ-EF1 (ZEB1). Increased expression of vimentin and N-cadherin are also seen in EMT. Evaluation of these markers in a drug-resistant cell line may shed light on the relationship between EMT and drug resistance.
TNF-α is a multifunctional cytokine that elicits a variety of biologic responses, such as inflammation and apoptosis. Additionally, TNF-α has been shown to induce EMT. Although TNF-α is not currently an anticancer agent for treatment of human cancers (because of side effects such as normal cell toxicity), low doses of TNF-α can markedly sensitize cancer cells to chemotherapy-induced apoptosis. We previously demonstrated that MCF-7 cell line variants exhibit differences in sensitivity to TNF-α and apoptosis induced by taxol and doxorubicin. Specifically, we demonstrated that apoptosis sensitive MCF-7-N cells (MCF-7 N variant) exhibited distinct differences in cell survival and apoptotic signaling when compared with inherently resistant MCF-7-M cells (MCF-7 M variant). We further demonstrated that apoptosis sensitive cells (MCF-7-N) could be driven to a resistant phenotype through prolonged exposure to increasing concentrations of TNF-α, leading to a stable, apoptosis-resistant phenotype (MCF-7-TNR) that was in part dependent upon mitogen-activated protein kinase (MAPK) and nuclear factor-κB signaling. Gene expression profiling revealed that MAPK kinase (MEK)5 was over-expressed in the TNF-α resistant MCF-7-M cells versus the TNF-α sensitive MCF-7-N cells. A similar upregulation of MEK5 in resistant cells was independently described in MCF-7-F cells, which developed resistance to the pure anti-estrogen fulvestrant through prolonged growth in fulvestrant-containing media. These studies demonstrate a potential role for the MEK5 pathway in the regulation of progression to drug resistance in breast cancer.
The MEK5/extracellular signal-regulated kinase (Erk)5 tandem is a component of MAPK cascades that mediate signals from various extracellular stimuli to the nucleus and regulate most cellular processes, including gene expression, proliferation, apoptosis, and motility. MAPK signaling may also play a role in EMT. Although MEK5/Erk5 signaling has not been extensively investigated, several studies suggest a role in cancer progression. For example, MEK5/Erk5 signaling has been demonstrated in prostate and breast cancer proliferation and tumorigenesis. Furthermore, inhibition of MEK5/Erk5 signaling in the MDA-MB-231 cell line, an aggressive breast cancer cell line with an EMT phenotype, induces apoptosis.
Based on these findings, which strongly indicate that MEK5/Erk5 signaling may mediate cancer progression to an aggressive phenotype, we further explored the involvement of MEK5/Erk5 signaling in resistance to apoptosis as well as EMT. To test the role played by MEK5/Erk5 activation in progression of breast carcinoma cells to a resistant phenotype, MCF-7 cells (N variant) were used to stably express a constitutive active MEK5 construct. These MCF-7-MEK5 cells exhibit resistance to TNF-α as compared with stable vector cells (MCF-7-VEC). Proteomic analysis based on two-dimensional electrophoresis (2-DE) and various mass spectrometric techniques has been employed in several studies of drug resistance of breast cancers. In this study we used a proteomics approach to define mechanisms of the MEK5 signaling cascade in the regulation of drug resistance. The differentially expressed proteins identified in proteomic analyses were confirmed at the gene expression level using reverse transcription RT-PCR assays. Results using immunofluorescence staining and gene expression analysis were consistent with an EMT phenotype in the MCF-7-MEK5 cells. These findings identify a potential role for the MEK5 pathway in coordinately promoting both an EMT phenotype and TNF-α resistance in breast cancer.
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