Primers for PCR amplification and sequencing are listed (Supplemental Table 2)
Primers for PCR amplification and sequencing are listed (Supplemental Table 2). during the current study are available from the corresponding author on reasonable request. Abstract Background pathway can occur through PI3-kinase, and mutations of N6022 the encoding gene are known to be oncogenic. Mutations in co-occur with hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. Results Treatment with HER2-targeted therapy reveals that mutations in the kinase domain name (H1047R) but not the helical domain name (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain name mutation, as exhibited by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, the cell harbors. Only mutations in the kinase domain name fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the mutational status and levels of neuregulin-1 expression in patients Cdc14A2 with (E542K and E545K) that binds the p85 regulatory subunit of PI3K . Helical and kinase domain name mutations account for about 85% of all mutations seen in in breast cancer . These mutations are sometimes combined in clinical outcome association studies [18, 19] based in part on laboratory studies showing that overexpression of the two mutation types in biological models produces comparable response phenotypes [20, 21]. However, they are functionally distinct with crystallographic studies of p110 showing conformational differences between the two hotspot mutations [16, 22]. We sought to better understand the impact of helical (E545K) and kinase (1047R) domain name mutations around the therapeutic response of HER2+ cells to targeted inhibitors. We have previously reported generation of cell lines in which mutations are overexpressed by conventional transgene expression and that mutant cell lines show increased resistance to lapatinib . These cells required combination therapy using lapatinib plus an AKT inhibitor to restore sensitivity to the same levels as control cells. However, this methodology is limited because transgenic cells retain two endogenous wildtype copies of as well as an unknown number of transgenic mutant alleles. We report here the generation of isogenic knockin mutants of each hotspot mutation, which has the major advantage of maintaining gene expression under control of the endogenous promoter, resulting in physiological levels of expression of the mutants. This is distinct from the majority of studies that have introduced mutations via transfection or transduction, which result in overexpression of the mutants. We report below our findings of the substantial differences of the impact of kinase versus helical domain name mutations of PIK3CA on signaling and therapeutic responses to HER2-targeted receptor tyrosine kinase inhibitors in the HER2-amplified cell line, SK-BR-3. Methods N6022 Plasmids and AAV production Plasmids for adeno-associated virus (AAV) production made up of either E545K or H1047R mutation of PIK3CA were a generous gift of Dr. Ben Ho Park. AAV production was done in HEK293T cells and contamination of SK-BR-3 cells with AAV-PIK3CA was performed as previously described . pSpCas9(BB)-2A-Puro (PX459) was a gift from Feng Zhang (Addgene plasmid # 48139) and the protocol for generating knockout cells has been described . CRISPR guideRNA sequences to target PTEN have been N6022 designed previously . Cell lines and compounds All cell lines were purchased from ATCC (Manassas, VA), cultured in the prescribed medium, and incubated at 37?C in 5% CO2. Cell line identity was confirmed by genotyping, and all cultures were tested to ensure absence of mycoplasma contamination. SK-BR-3 selection conditions were 0.5?mg/mL for G418 and 0.25g/mL for puromycin. Lapatinib, neratinib, the AKT inhibitor GSK690693, and the MEK inhibitors GSK1120212 and PD0325901 were purchased from Selleck Chemicals (Houston, TX). DNA isolation and sequencing Genomic DNA was isolated from cells using the Qiagen (Germantown, MD) DNeasy Blood & Tissue Kit. Primers for PCR amplification and sequencing are listed (Supplemental Table 2). Prior to sequencing, all amplified PCR products were gel isolated on Invitrogen 2% agarose (Thermo Fisher Scientific (Waltham, MA)) gels to ensure purity. Cell growth assays Cells were seeded in Corning (Corning, NY) 96-well white-walled clear-bottom plates. Drug was added 24?h after seeding. Within each plate, each drug concentration was randomly arrayed in triplicate using a liquid-handling robot as described previously . Cell Titer-Glo (Promega (Fitchburg, WI)) was used according to the manufacturers.