RESUMEN
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 cell-free 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 biopsy-based assays. MATERIALS & METHODS: For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing 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 0.5 mL 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%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based 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 primer-based qPCR detection.
RESUMEN
Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the proteolytic activity of matrix metalloproteinases (MMPs), playing an important role in the homeostasis of the extracellular matrix. Beyond its well-known role in tissue maintenance, TIMP-1 has been associated with multiple MMP-independent cytokine-like functions. The protein structure of TIMP-1, 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 TIMP-1, able to mediate intracellular signaling through the Erk/MAPK axis, provided a molecular basis for the role of TIMP-1 in cellular signaling. However, several lines of evidence suggest that TIMP-1 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 TIMP-1, known as well as unknown, we performed a yeast two-hybrid screening using a mammary gland complementary DNA (cDNA) library. We report here the identification of multiple interactors, including MHC class II-associated invariant chain γ (CD74). We verified that CD74 interacts with TIMP-1 in breast cancer cells and that this interaction contributes to cellular internalization of TIMP-1 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 TIMP-1 and their potential mechanistic basis.
