Table of ContentsMethodsResultsDiscussionThe cancer stem cell (CSC) hypothesis is considered a reasonable explanation for the rapid multiplication of cells in the disease. These are self-sufficient cells that appear to have increased tumorigenicity and self-renewal, allowing them to grow more quickly than normal cells. Although the subject of CSC is debated and not yet completely understood, it is clear that cancer cells are supported by a number of signaling mechanisms which may be paracrine, such as Wnt/beta-catenin signaling, or by a signal of cell to cell, like Notch.Say no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get the original essay The Wnt (wingless/integrated) signaling pathway is an evolutionarily conserved signaling pathway. It plays a role in vital processes for survival such as embryonic development, tissue regeneration in organs with high turnover such as the skin, the intestine and in the development of pluripotent stem cells such as hematopoietic stem cells, but also in the tumorigenesis. In the canonical pathway, the absence of Wnt ligands leads to the phosphorylation of beta-catenin by GSK3, which leads to the degradation of beta-catenin by proteasomes. When Wnt ligands are present, they bind to Frizzled (Fzd) receptors, recruit kinases and Disheveled (Dvl) protein, which inhibits beta-catenin degradation. This results in translocation of beta-catenin to the nucleus, thereby initiating replication. Nearly 50% of breast cancers, including triple negative breast cancer, have been shown to have increased beta-catenin levels due to active Wnt signaling caused by overexpressed Wnt receptors and signaling molecules. . Thus, regulation of the canonical Wnt signaling pathway to control cancer proliferation has become a major goal. The Notch signaling pathway consists of four Notch receptors, named Notch, which are activated by the binding of one of their five major ligands, Delta-like ligand (DLL1), Delta-like ligand (DLL3), Delta-like ligand (DLL3), type Delta 4 (DLL4), Jagged-1 (JAG1) and Jagged-2 (JAG2) from an adjacent cell. This leads to γ-secretase-induced cleavage of the Notch intracellular domain (NICD), which is translocated into the nucleus, interacts with the CSL using the co-activator Mastermind (MAM), and initiates transcription. Notch signaling was first discovered to play a role in acute lymphoblastic leukemia cancer, after which it played a role in many other cancers, including breast cancer. Many studies have been carried out to investigate the correlation of Notch signaling pathway in breast cancer prognosis. Notch also interacts with other signaling pathways like the Wnt pathway, further increasing its usefulness for cancer cells. Thus, inhibition of Notch signaling has been tested to block the pathway and abrogate cancer cell multiplication by various methods, including antibodies 18 and γ-secretase inhibitors. This study used three proteins that inhibit Wnt signaling, namely Wnt inhibitory factor-1. (WIF1), Secreted Frizzled Related Protein 2 (SFRP2) and Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1), as well as a Notch signaling protein, Jagged-1 (Jag1), to be expressed by the adeno-associated viral vectors of serotype 6 (AAV -6) as decoys to block in vitro signaling in triple negative breast cancer cells.A decoy involves the use of the binding domain of a receptor/protein, without the signaling apparatus. This acts as an inhibitor by binding the ligand and preventing it from binding to the receptor itself. However, there are some drawbacks to using decoys, such as expressing decoy proteins as a single domain. To address this issue, collagen domains of C1qTNF3 were used, which trimerize and help increase decoy expression levels. MethodsMolecular cloning: Human DKK1, WIF1, JAG1, and SFRP2 cDNA were ordered from DNASU (Arizona). Plasmids were digested using restriction enzymes. PCR products of the above genes and the AAV vector CTR1 (containing the inverted terminal repeat/ITR sequence) were digested with the restriction enzymes BamH1 and EcoR1 and purified by gel extraction. The ligation products were then transformed into DH5α competent cells. Stocks of glycerol were constituted and frozen for each of the constructions. Minipreps using the QIAGEN miniprep kit were performed and verified by digestion and sequencing. Maxipreps were performed using QIAGEN maxiprep kits and glycerol stocks from miniprep samples. Transfection: HEK293T cells were used for transfection. They were cultured in Dulbeco's modified Eagle's medium (DMEM) (ThermoFisher, Waltham, MA) containing 10% fetal bovine serum (FBS) and 1% PenStrep one day before, to achieve approximately 80% confluency of the cells at the time of transfection. Polyethyleneimine (PEI) was used to transfect the cells and the plate was incubated at 37°C overnight. Media was changed after 4 p.m. Cells and conditioned media were collected after 24 hours. Cells were resuspended using 500 μl of phosphate-buffered saline (PBS). Cells were lysed by sonication, with an amplitude of 70 for 12 seconds in 1 second pulses. Western blot: Cell lysate samples were prepared with a reducing agent. Conditioned media were prepared with and without reducing agent. SDS-PAGE was performed with 10% Bis-Tris gel and MOPS buffer. The proteins were transferred to a PVDF membrane at 100 V for 50 minutes. The membrane was then blocked using 0.5% casein-PBS buffer solution to block nonspecific sites. 2 μl of mouse anti-FLAG M2 antibody diluted 1/2000 were incubated with the membrane for 1 hour at room temperature. The membrane was then washed once using 100 ml of TBS-TWEEN 20 buffer and 0.01% sodium dodecyl sulfate (SDS), followed by two washes using only TBS-TWEEN20 buffer. This was followed by incubation with AlexaFluor 680 goat anti-mouse antibody diluted 1:5000 in 50 ml PBS-casein buffer with 0.01% SDS. The membrane was developed using Li-Cor Odyssey. AAV/minivirus production: DKK1, WIF1, JAG1, and SFRP2 DNA were co-transfected with AAV6 capsid helper plasmid into HEK293T cells at 70% confluence in a six-well plate. Polyethyleneimine was used to transfect a total of 2.7 μg of DNA, 1.8 μg of the helper plasmid (containing Rep and Cap sequences for AAV6), and 0.9 μg of protein DNA into HEK293T cells. The supernatant, which contains the minivirus, was collected after 72 hours. Transduction: For the in vitro triple negative breast cancer (TNBC) experiment, MDA-MB231 cancer cells were used. Cells were cultured using DMEM with 10% FBS. Each well contained HEK293T cells with 2 ml of DMEM. Virus transduction was carried out in six-well plates. 100 μl of minivirus was added to the cells. They.