Effects of HER2 Transmembrane and Juxtamembrane Domain Mutations on HER2 Activation of MCF7 cells
Open AccessEffects of HER2 Transmembrane and Juxtamembrane Domain Mutations on HER2 Activation of MCF7 cells ErbB2, also commonly known as HER2, is an aggressive subtype of invasive breast carcinoma (IBC). It belongs to the EGFR (epidermal growth factor receptor) family, which functions for cell differentiation, proliferation, and growth of normal cells. The ERBB2 gene encodes HER2, a transmembrane protein, which has the potential to promote cancer cell growth. Gene amplification, protein overexpression, and mutations are the principle mechanisms of HER2 alternation making HER2 a major target for clinical therapies. Correlation of HER2 amplification and overexpression is analogous and is actively seen in approximately 20-30% of breast carcinoma cases. Literature establishes that auto-phosphorylation of the tyrosine residues in the carboxyl tail of HER2 cascades various signals that promote cell survival and proliferation. The HER2 extracellular domain and kinase domain have major impacts on its downstream signaling mechanisms. However, smaller sequences of HER2, the Transmembrane domain (TMD), and the Juxtamembrane domain (JMD) can comprise certain mutations that disrupt crucial roles leading to tumorigenesis along and subjection of the cell surface to perturbation and deformation. This paper is an effort towards studying the effects of specific TMD mutations (V659E and G660D) and JMD mutations (R678Q and Q709L) on the activation of HER2. R678Q is the third most common mutation in HER2 while other chosen mutations are not as prevalent as other kinase domain and extracellular domain mutations. This study is an attempt to analyze the activating mechanisms of TMD and JMD, hence, the most potent mutations from previous research data in these regions were selected for the experiment. These mutations are precisely considered due to its specificity in alteration of the sequence that leads to the gain of function of HER2 enhancing its expression and signaling. Due to the potent nature established in clinical data, these mutations are thought to be better candidates in specific cancers for therapy. This study demonstrates HER2 expression levels in two cell lines: MCF7, a carcinogenic cell line, and MCF10A, a non-cancerogenic cell line as a vector for analysis. For HER2 protein level analysis, BCA assay and western blots were used after cloning the desired mutation in Wild Type (Wt) HER2 plasmid. From the experiments performed above, a certain pattern of increase in expression and activation of HER2 is observed in most of the mutations compared to the parental HER2 in MCF7 cells. The positive control used in the experiment is a high HER2 expressor, SKBR3 cell line. From established research and theoretical studies performed on HER2, the mutations are expected to surpass the SKBR3 HER2 expression at a significant rate. While the results from the experiments post-analysis show increased expression in this study, the findings have lower confidence in comparison to the theoretical and supporting documents acquired from research and studies. Nonetheless, JMD and TMD mutations did show an overall increase and change in the expression level of HER2 activation as compared to the WtHER2, demonstrating these domains as crucial regions for the activation of this protein.
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Shrestha_gwu_0075M_15133.pdf | 2020-08-04 | Open Access |
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