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Figure 5 from Tumor-Secreted ADAMTSL4 Activates Latent TGFβ1 to Drive Cancer Cachexia Juliano Machado (25154222) Vignesh Karthikaisamy (25154225) Hermine Mohr (25154228) Doris Kaltenecker (25154231) Pia Benedikt (25154234) Pauline Morigny (25154237) Amit Mhamane (25154240) Julia Geppert (25154243) Amy Rose Fumo (25154246) Kerstin Haase (14123058) Estefania Simoes (25154249) Joanna D.C.C. Lima (18725115) Anastasia Georgiadi (25154252) Achim Krüger (25154255) José Pinhata Otoch (25154258) Marc E. Martignoni (25154261) Vickie E. Baracos (25154264) Mariam Jamal-Hanjani (15386766) Marilia C.L. Seelaender (25154267) Olga Prokopchuk (25154270) Julia Szendrödi (25154273) Maria Rohm (25154276) Stephan Herzig (25154279) Mauricio Berriel Diaz (15037094) Cancer Molecular and Cellular Biology Gastrointestinal Cancers Colorectal cancer Lung Cancer Metabolism Metabolic pathways ADAMTSL4 targets LAP of proTGFβ1 to promote myotube atrophy via latency activation and TGFBR1-dependent signaling. A and B, LRC-TriCEPS experiments were performed using C2C12 myotubes to identify surface interactors of recombinant human ADAMTSL4-CT (rhADAMTSL4-CT). Volcano plots of LC-MS/MS data comparing rhADAMTSL4-CT against glycine (negative control; A) or TRFE (positive control; B) show enrichment of membrane-associated proteins, including proTGFβ1 (UniProt P04202), as significant targets of rhADAMTSL4-CT (see also Supplementary Table S3 and Supplementary Data S9). C, Co-IP assays were performed in HEK293T cells transiently transfected with tagged constructs, followed by IP from whole-cell lysates (WCL). Using HA-tagged LAP (HA-LAP), ADAMTSL4-CT (His- or V5-tagged) was specifically detected in the precipitated complex, demonstrating a direct interaction between ADAMTSL4 and the LAP of proTGFβ1. D, Co-IP using FLAG-tagged mature TGFβ1 (FLAG–TGFβ1) did not show interaction with ADAMTSL4-CT, despite robust detection in input controls, indicating that ADAMTSL4 selectively binds LAP but not mature TGFβ1. E, Schematic representation of constructs used in NanoBiT luciferase complementation assays. ADAMTSL4 was fused to the small fragment (SmBiT), whereas LAP was fused to the large fragment (LgBiT). Upon protein–protein interaction, proximity of SmBiT and LgBiT enables luciferase reconstitution in the presence of substrate, resulting in luminescence proportional to binding. F, NanoBiT assay demonstrating interaction between ADAMTSL4-CT (ATL4-CT) and LAP in live cells. G, Schematic representation of the NanoBiT-based dissociation assay. A full-length proTGFβ1 construct was engineered to fuse LgBiT to LAP and SmBiT to the TGFβ1 domain. This design preserves physiologic endoplasmic reticulum (ER)–Golgi processing and proteolytic cleavage while maintaining the noncovalent association of LAP and mature TGFβ1 within the SLC. Disruption of the LAP–TGFβ1 interaction results in separation of LgBiT and SmBiT, leading to decreased luminescence and enabling quantitative assessment of protein–protein dissociation. H and I, ADAMTSL4 promotes dissociation of mature TGFβ1 from LAP, as measured by reduced NanoBiT luminescence signal. J, Total free TGFβ1 protein levels measured by ELISA in supernatants of C2C12 myotubes treated with vehicle or rhADAMTSL4-CT (250 ng/mL). K and L,Tgfb1 and Fbxo32 mRNA expression in C2C12 myotubes treated with vehicle or rhADAMTSL4-CT, with or without lentiviral knockdown of proTgfb1. M, Representative immunofluorescence images of C2C12 myotubes treated with vehicle or rhADAMTSL4-CT, with or without lentiviral knockdown of proTgfb1. N, Western blot analysis of phosphorylated SMAD2 (p-SMAD2) as a readout of TGFβ pathway activation in C2C12 myotubes treated with rhADAMTSL4-CT in the presence or absence of the TGFβ1–neutralizing antibody 1D11 (1.5 μg/mL). O–R, qPCR analysis of Tgfb1, Ctgf, Fbxo32, and Map1lc3 in myotubes treated with rhADAMTSL4-CT ± TGFβ1–neutralizing antibody 1D11 (1.5 μg/mL). S–X, Functional inhibition of TGFBR1 using galunisertib (10 μmol/L), LY-364947 (10 μmol/L), or SB-431542 (10 μmol/L) in the context of rhADAMTSL4-CT treatment: (S) Western blot analysis of p-SMAD2; (T and U) Ctgf and Fbxo32 mRNA expression; (V) quantification of myotube diameter; (W) representative immunofluorescence images. X, Schematic summary of the mechanistic framework uncovered in this study. ADAMTSL4 binds the LAP of proTGFβ1, as demonstrated by co-IP and NanoBiT interaction assays. This interaction promotes dissociation of the latent TGFβ1 complex, leading to the release of bioactive TGFβ1. Functionally, ADAMTSL4-induced TGFβ1 activation is inhibited by rhLAP, demonstrating the requirement of LAP engagement. Downstream signaling is blocked by the TGFβ1–neutralizing antibody 1D11, confirming ligand-dependent activation. Pharmacologic inhibition of TGFBR1 (galunisertib, LY-364947, SB-431542) and genetic suppression of proTgfb1 further establish that ADAMTSL4-driven effects on SMAD2 activation and myotube atrophy are mediated through canonical TGFβ signaling. Together, these data support a model in which ADAMTSL4 activates latent TGFβ1 via LAP binding, triggering TGFBR1-dependent signaling and atrophy-related gene programs. Data are presented as the mean ± SEM. Each dot represents an individual biological replicate. Statistical analysis: one-way ANOVA with Tukey multiple-comparisons test (F, H, and I), two-way mixed-effects model with Tukey multiple-comparisons test (J–L and N–R), and two-way mixed-effects model with Holm–Šídák multiple-comparisons test (S–V). Significance is denoted as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. [E, Created in BioRender. Machado, J. (2026) <a target="_blank" href="https://biorender.com/xis2eju">https://BioRender.com/xis2eju</a>; G, Created in BioRender. Machado, J. (2026) <a target="_blank" href="https://biorender.com/wqwc3bb">https://BioRender.com/wqwc3bb</a>; X, Created in BioRender. Machado, J. (2026) <a target="_blank" href="https://biorender.com/l2gs5ov">https://BioRender.com/l2gs5ov</a>.] 2026-10-01T00:00:00Z Image Figure 10.1158/2159-8290.34040801 https://figshare.com/articles/figure/Figure_5_from_Tumor-Secreted_ADAMTSL4_Activates_Latent_TGF_1_to_Drive_Cancer_Cachexia/34040801 CC BY