The Role of Nestin in Basal-like Breast Cancer Pathogenesis and Progression
1 Purpose
Nestin is a member of the class VI family of intermediate filament proteins, originally identified as a marker of neural stem cells and subsequently demonstrated to be expressed in breast cancer (BC) and other cancer types. Loss of Nestin expression would result in a reduction and/or loss of a basal-like phenotype and consequently, a loss of aggressive features.
To validate this hypothesis in vitro and to gain insights into the mechanisms by which Nestin influences the basal-like phenotype of BC, we generated a Nestin knockout triple-negative breast cancer (TNBC) cell line model (MDA-MB-231) by CRISPR/Cas9 gene editing. Proteomic profiling of Nestin knockout (NesKO) and wild-type (NesWT) cell lysates revealed a total of 113 proteins that were differentially expressed (arbitrary cut-off of 2-fold change) in NesKO as compared to NesWT. The downregulated proteins in NesKO cells were significantly enriched for pathways involved in epithelial-mesenchymal transition (EMT), ECM organization, cell-ECM interactions as well as mitochondrial metabolism and protein synthesis. On the other hand, upregulated proteins in NesKO cells were enriched for pathways involved in keratinization, hemidesmosome assembly, glycogen metabolism and Type I IFN-stimulated signaling.
The proteomic profile of NesKO cells suggests that Nestin deficiency results in ‘decreased stemness’ or a more ‘differentiated’ phenotype with altered cell-ECM interactions that could negatively regulate cell migration and/or invasion. Further, NesKO cells also exhibit increased metabolism, cellular stress and decreased levels of proteins essential for maintaining mitochondrial health and function that could impact cell growth and proliferation. To further validate these findings and to analyze the effect(s) of Nestin deficiency on tumor growth and metastases in vivo, we propose a xenograft mouse model with NesWT and NesKO clones to track tumor development and metastatic spread in them.
2 Distress
Mice will develop breast cancer and metastases, but they will be euthanised before the general condition would be impaired. For this reason we classify the severity of the experiment as moderate.
3 Expected benefit
The experiment will give us an insight in the role of Nestin in breast cancer pathogenesis and progression
4 Number of animals, and what kind
16 female immuno-defficient mice.
5 How to adhere to 3R
The main objective of the experiment is to identify if Nestin deficiency has an impact on tumor growth and metastases in vivo. Tumor development and metastatic spread cannot be tracked in vitro.
The smallest possible groups to provide a statistically significant power will be used.
The implantation of cancer cells will be achieved under anaesthesia. The animals will receive analgesia post implantation and will be monitored closely by project members. Experienced technicians will monitor the animals and they will very early discover any sign of illness and take the decision of euthanasia, if necessary. All cages will have environmental enrichment available at Vivarium.
Nestin is a member of the class VI family of intermediate filament proteins, originally identified as a marker of neural stem cells and subsequently demonstrated to be expressed in breast cancer (BC) and other cancer types. Loss of Nestin expression would result in a reduction and/or loss of a basal-like phenotype and consequently, a loss of aggressive features.
To validate this hypothesis in vitro and to gain insights into the mechanisms by which Nestin influences the basal-like phenotype of BC, we generated a Nestin knockout triple-negative breast cancer (TNBC) cell line model (MDA-MB-231) by CRISPR/Cas9 gene editing. Proteomic profiling of Nestin knockout (NesKO) and wild-type (NesWT) cell lysates revealed a total of 113 proteins that were differentially expressed (arbitrary cut-off of 2-fold change) in NesKO as compared to NesWT. The downregulated proteins in NesKO cells were significantly enriched for pathways involved in epithelial-mesenchymal transition (EMT), ECM organization, cell-ECM interactions as well as mitochondrial metabolism and protein synthesis. On the other hand, upregulated proteins in NesKO cells were enriched for pathways involved in keratinization, hemidesmosome assembly, glycogen metabolism and Type I IFN-stimulated signaling.
The proteomic profile of NesKO cells suggests that Nestin deficiency results in ‘decreased stemness’ or a more ‘differentiated’ phenotype with altered cell-ECM interactions that could negatively regulate cell migration and/or invasion. Further, NesKO cells also exhibit increased metabolism, cellular stress and decreased levels of proteins essential for maintaining mitochondrial health and function that could impact cell growth and proliferation. To further validate these findings and to analyze the effect(s) of Nestin deficiency on tumor growth and metastases in vivo, we propose a xenograft mouse model with NesWT and NesKO clones to track tumor development and metastatic spread in them.
2 Distress
Mice will develop breast cancer and metastases, but they will be euthanised before the general condition would be impaired. For this reason we classify the severity of the experiment as moderate.
3 Expected benefit
The experiment will give us an insight in the role of Nestin in breast cancer pathogenesis and progression
4 Number of animals, and what kind
16 female immuno-defficient mice.
5 How to adhere to 3R
The main objective of the experiment is to identify if Nestin deficiency has an impact on tumor growth and metastases in vivo. Tumor development and metastatic spread cannot be tracked in vitro.
The smallest possible groups to provide a statistically significant power will be used.
The implantation of cancer cells will be achieved under anaesthesia. The animals will receive analgesia post implantation and will be monitored closely by project members. Experienced technicians will monitor the animals and they will very early discover any sign of illness and take the decision of euthanasia, if necessary. All cages will have environmental enrichment available at Vivarium.