Journal: bioRxiv
Article Title: In Vivo Multiplexed Modeling Reveals Diverse Roles of the TBX2 Subfamily and Egr1 in Ras -Driven Lung Adenocarcinoma
doi: 10.1101/2025.03.15.642187
Figure Lengend Snippet: A) Schematic representation of the molecular basis of TBX2-associated signaling pathways in cancer. TBX2 , TBX3 , and TBX5 contribute to the dysregulation of multiple cancer hallmarks, including cell proliferation, senescence, apoptosis, invasion, metastasis, and the pro-inflammatory response. EGR1 , TNFaip3 , ATF3 , Tbx4, and Chd2 are all modulated by TBX2 signaling in lung cancer cell lines and recurrently dysregulated in human lung cancers. While EGR1 , TNFAIP3, and ATF3 all directly modulate key carcinogenic pathways, the mechanisms by which TBX4 and CHD2 influence cancer remain largely unexplored( , , – ). AKT (AKT serine/threonine kinase), BAX (BCL2 associated X, apoptosis regulator), HDAC (Histone deacetylase), MDM2 (MDM2 proto-oncogene, E3 ubiquitin protein ligase), MMP9 (Matrix metallopeptidase 9), NDRG1 (N-myc downstream-regulated gene 1), p14ARF (Cyclin-dependent kinase inhibitor 2A, isoform p14ARF), p 21CIP1 (Cyclin-dependent kinase inhibitor 1A), p53 (Tumor protein p53), PAsrp (Phosphatidylserine receptor protein), PRC2 (Polycomb repressive complex 2), SNCG (Synuclein gamma), and TNFa (Tumor necrosis factor alpha). B) Experimental schematic of the Tuba-seq approach used to investigate combinatorial inactivation of potential LUAD regulators in vivo. Tumor initiation was achieved through intratracheal intubation with Lenti-sgTS-Pool/Cre in Kras LSL-G12D ,Rosa26 CAG-LSL-Cas9-GFP mice. The Lenti-sgTS-Pool contained two inert sgRNA vectors along with a positive control (sg Rb1 ) and a negative control (sg Pcna ). Each of the eight genes studied was targeted using two distinct sgRNAs. Each sgRNA vector included a unique sgID and a random barcode, allowing quantification of individual tumor sizes via deep sequencing. The genotype, time points and lentiviral titers are as indicated. C) Representative images of lung lobes from mice at 6- and 20-weeks post-tumor initiation. Images include fluorescence dissecting scope views, H&E-stained sections, and TTF1-stained (lung epithelial marker) sections of lung lobes. Scale bars are indicated on each image. D) Quantification of the percent tumor area in representative mice revealed a significant increase at 20 weeks compared to 6 weeks (Wilcoxon test, p<0.05). Each dot represents an individual mouse, with horizontal bars indicating the mean tumor area.
Article Snippet: The table lists the sgRNA sequences used to target each of the genes analyzed in this study and their corresponding sgIDs. sgRNAs targeting Rb1 (Addgene #89647), Neo1 (Addgene #67594), Neo3 (Addgene #89653), and Pcna , has been adopted from previous studies( ).
Techniques: Protein-Protein interactions, Histone Deacetylase Assay, Ubiquitin Proteomics, In Vivo, Positive Control, Negative Control, Plasmid Preparation, Sequencing, Fluorescence, Staining, Marker
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