2 research outputs found
Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma
Abstract Backgrounds Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cellâfree DNA in human plasma. Aims First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsyâbased assays. Materials & Methods For the comparison we tested a solidâphase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primerâbased SNP detection system, for the detection of two wellâestablished cancerâcausing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5âmL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. Results In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%â26.7%) compared with the solidâphase purification approach (47.8%â65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10â3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primerâbased inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. Discussion A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. Conclusion Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primerâbased qPCR detection
The invariant chain CD74 protein is a cell surface binding partner of TIMPâ1 in breast cancer cells
Tissue inhibitor of metalloproteinasesâ1 (TIMPâ1) regulates the proteolytic activity of matrix metalloproteinases (MMPs), playing an important role in the homeostasis of the extracellular matrix. Beyond its wellâknown role in tissue maintenance, TIMPâ1 has been associated with multiple MMPâindependent cytokineâlike functions. The protein structure of TIMPâ1, with two distinct domains, one interacting with MMPs and another able to bind multiple partners, provides a rationale for this multifunctionality. The identification of CD63 as a cell surface receptor for TIMPâ1, able to mediate intracellular signaling through the Erk/MAPK axis, provided a molecular basis for the role of TIMPâ1 in cellular signaling. However, several lines of evidence suggest that TIMPâ1 may be able to associate with many interaction partners, thus attaining multiple functions. To enable the identification of previously unknown interaction partners that may underpin the core cellular functions of TIMPâ1, known as well as unknown, we performed a yeast twoâhybrid screening using a mammary gland complementary DNA (cDNA) library. We report here the identification of multiple interactors, including MHC class IIâassociated invariant chain Îł (CD74). We verified that CD74 interacts with TIMPâ1 in breast cancer cells and that this interaction contributes to cellular internalization of TIMPâ1 and mediates intracellular signaling through the Akt signaling axis in breast cancer cells. These data provide new insights into the complex nature of the functions of TIMPâ1 and their potential mechanistic basis