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RAS GTPase-activating protein-binding protein 1 (G3BP1) is a component of the RAS signaling pathway and a phosphorylation-dependent RNA/DNA endoribonuclease that links signal transduction and RNA metabolism. We identified G3BP1 as a nuclear interactor of platelet-derived growth factor receptor-β, forming a complex with BAF155, a component of the SWI/SNF chromatin remodeling complex, as well as with the transcription factor STAT3. Depletion of G3BP1 in human primary fibroblasts AG1523 reduced PDGF-BB-induced activation of STAT3 while increasing mRNA levels of FOS, MYC, and CCND1, which encode proteins involved in growth stimulation. Both STAT1 and cyclin D1 mRNA and protein levels were elevated upon G3BP1 knockdown, identifying G3BP1 as a negative regulator of STAT1 and cyclin D1 expression. G3BP1 depletion did not abolish PDGF-BB-induced proliferation of human primary fibroblasts. Thus, G3BP1 interacts with the SWI/SNF chromatin remodeling complex in the nucleus and regulates cell growth pathways by modulating STAT signaling and transcription of growth-associated genes.

Background

Platelet-derived growth factor receptor-beta (PDGFR beta) is a canonical marker of pericytes and stromal cells in most tissues. It is present on cancer associated fibroblasts (CAFs) in the tumor microenvironment and has thus been proposed as a potential therapeutic target both for anti-cancer drugs as well as for radioligand therapy (RLT). ATH001 is a novel Affibody molecule-based radiopharmaceutical in clinical development for targeting of PDGFR beta. We hypothesize that dimerization of ATH001 could improve affinity to PDGFR beta, leading to better characteristics for in vivo targeting. The dimeric construct, named ATH022, was generated by conjugation of two chemically synthesized ATH001 binders to a tri-functional linker comprising a DOTA chelator. Here, we present a comprehensive in vitro and in vivo evaluation of Gallium-68 and Indium-111 labeled ATH022, in direct comparison with ATH001.

Results

DOTA-ATH022 acted as a PDGFR beta antagonist and competed dose-dependently with the endogenous ligand PDGF-BB. Affinity of the dimer was improved approximately tenfold compared to DOTA-ATH001, mainly due to strongly decreased off-rate. Radiolabeled DOTA-ATH022 demonstrated higher specific binding and longer retention to U87 cells in vitro. Radiolabeled DOTA-ATH022 also exhibited elevated binding in PDGFR beta-positive tissues spleen and U87 tumors, that could be blocked by ATH001 in excess. Binding of DOTA-ATH022 in PDGFR beta avid and well-perfused spleen was 3 times higher than for DOTA-ATH001, but tumor binding was lower. In vivo retention in spleen was 5 times longer for Indium-111 labeled ATH022, in agreement with its in vitro binding characteristics.

Conclusions

DOTA-ATH022 is a novel dimerized version of ATH001, with significantly improved affinity towards PDGFR beta. Retention of radiolabeled DOTA-ATH022 in PDGFR beta-avid tissues and tumors was increased, however uptake in solid tumor tissue was not improved.

Transforming growth factor-β (TGF-β) exerts its cellular effects via binding to type I and type II kinase-associated receptors, whereby SMAD-dependent and SMAD-independent pathways are activated; amongst the latter is the Rho pathway with the downstream effectors Rho-associated coiled-coiled kinases 1 (ROCK1) and ROCK2. In the present study, we investigated whether ROCK1 and ROCK2 regulate TGF-β-SMAD signaling in breast cancer cells. We found that knockdown of ROCK2 or treatment with a highly selective ROCK2 kinase inhibitor (KD025) suppresses TGF-β-SMAD signaling, which was opposite to the effect of knockdown of ROCK1. Moreover, we demonstrate that overexpression of ROCK1 inhibits TGF-β-induced CAGA12-luc reporter expression, whereas a kinase-dead ROCK1 mutant or a ROCK inhibitor GSK42928A reversed this effect. Overexpression of ROCK2 enhanced TGF-β-induced CAGA12-luc reporter activity, while a kinase-dead ROCK2 mutant and KD025 reversed this effect. These observations suggest that the kinase activities of ROCK isoforms are needed for their inhibitory or stimulatory effects on TGF-β-SMAD signaling. In addition, we found that ROCK1 and ROCK2 have different subcellular localizations, and that SMAD3 interacts with ROCK1, but not with ROCK2. Furthermore, ROCK1 depletion in MDA-MB-231 cells promoted cell proliferation and reduced cell invasion, whereas, in contrast, ROCK2 depletion reduced cell proliferation and promoted cell invasion in vitro. Thus, our observations support the notion that the two ROCK isoforms have opposite effects on TGF-β-SMAD signaling.

Cytokines from the transforming growth factor-beta (TGF-beta) superfamily are essential regulators of cell growth, survival, and differentiation, playing a pivotal role in mammalian embryonic development, adult tissue homeostasis, and progression of human diseases. Recently, an international symposium on TGF-beta Signaling in Development and Diseases was held in Nanchang, China, from October 21 to 23, 2025. This event showcased the latest advances in TGF-beta signaling and its pathophysiological functions. Over ten presentations at the symposium offered new insights on Smad-dependent and non-Smad TGF-beta signaling, its spatiotemporal regulation, and multifaceted roles of TGF-beta family cytokines in various pathophysiological contexts. The symposium also addressed potential strategies and opportunities for targeting the TGF-beta pathway in the treatment of human diseases.

Background

SRC homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), acting as a central node of many signaling pathways, has an emerging role in many diseases, including cancer. Transforming growth factor β (TGF-β) affects a wide spectrum of biological processes, including cell proliferation, apoptosis, differentiation and migration, during embryonic development and oncogenesis. TGF-β regulates cell proliferation in a cell-context-dependent manner; loss of TGF-β-mediated growth inhibition is a major characteristic of cancer cells. SHP2 regulates canonical and non-canonical TGF-β signaling in cancer cells and fibroblasts, however, the mechanism by which TGF-β activates SHP2 and the role of SHP2 in TGF-β-mediated growth inhibition has remained unclear.

Methods

The phosphorylation and activation of SHP2 was assessed after TGF-β stimulation of normal and breast cancer cells. The interaction between SHP2 and SRC was determined by co-immunoprecipitation (co-IP) assay. Pharmacological inhibition and gRNA-mediated knockout were applied to assess the role of SHP2 in TGF-β signaling, and RNA-sequencing to identify gene expression patterns in SHP2 knockout cells treated with TGF-β. Functional studies were performed using breast cancer cells to validate the role of SHP2 in TGF-β -mediated growth inhibition.

Results

We report that TGF-β activated SHP2 by promoting its tyrosine phosphorylation by SRC. SHP2 depletion in breast cancer cells reduced ubiquitination and degradation of TβRI by impeding the interaction between TβRI and SMAD7, which is a negative regulator of TβRI. Pharmacological or genetic inhibition of SHP2 facilitated TGF-β-induced SMAD2 phosphorylation and transcriptional responses. Consequently, inhibition of SHP2 profoundly enhanced TGF-β-induced cell growth arrest and senescence, including promotion of TGF-β-induced expression of the cell cycle inhibitor p15 at both gene and protein level.

Conclusions

Our findings uncover a mechanism by which SHP2 is activated by TGF-β in a SRC-dependent manner, and functions in a negative feedback mechanism to regulate TGF-β signaling.

Transcription factor SNAI1 guides plasticity and invasiveness in cancer. Using a complete SNAI1 knockout in mesenchymal, triple-negative breast cancer cells, unbiased genome-wide transcriptomic analysis revealed a marked under-expression of integrin-based adhesion and endocytic components. Utilizing this knockout cell model, complementary breast cancer cell models and functional screening of multiple differentially expressed genes, we found that the pioneering transcription factor FOXA1, whose expression is repressed by SNAI1, associates with several key mediators of the cellular phenotype. FOXA1 represses the small GTPase ARF6 and its exchange factor PSD4. In addition, some of the integrin and matrix metalloproteinase genes are regulated by the transcriptional FOXA1 signal. Accordingly, SNAI1 knockout cells presented poor adhesion to collagen type I or fibronectin, formed defective invadopodia and focal adhesions with weakened FAK/SRC signaling. SNAI1 knockout cells performed ineffective receptor-mediated internalization, including nanoparticle and extracellular vesicle (EV) uptake, exhibited reduced lysosomal content, lacked multivesicular bodies enriched in intraluminal vesicles and showed decreased EV secretion. Gain-of-function experiments demonstrated that SNAI1 has an impact on the PSD4/ARF6 signaling module, using FOXA1 as an intermediate factor to regulate EV release by tumor cells. We propose that the SNAI1-FOXA1 transcriptional mechanism operates at the level of membrane and vesicular trafficking control, which interlinks cell plasticity, adhesion and invasiveness through the extracellular environment, with the associated process of EV secretion.

Metastatic castration-resistant prostate cancer (mCRPC) remains the primary cause of prostate cancer-related mortality. Despite the availability of treatments, the molecular mechanisms underlying tumor invasion and metastasis are not fully understood, highlighting the need for novel therapeutic strategies. In this study, we developed fully human monoclonal antibodies (mAbs) that prevent the proteolytic cleavage of the transforming growth factor-beta (TGFβ) type I receptor (TβRI) by steric hindrance. This cleavage, mediated by the metalloprotease ADAM17 (a disintegrin and metalloprotease domain 17; also known as TACE), results in the generation of a soluble intracellular domain (TβRI-ICD) that is translocated to the nucleus of castration-resistant prostate cancer (CRPC) cells and promotes epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. High levels of TGFBR1 correlated with poor survival in two independent clinical cohorts of patients with mCRPC, and a strong positive correlation between TGFBR1 and ADAM17 expression was observed. In a preclinical human orthotopic mCRPC mouse model, treatment with therapeutic mAbs effectively prevented the nuclear accumulation of TβRI-ICD, inhibited EMT, and suppressed tumor growth, invasion, and metastasis. Notably, the therapeutic effect was comparable to that of docetaxel, a current standard-of-care chemotherapy, without noticeable side effects on body weight, proximal aorta or heart function detected in immune-deficient mice. These findings suggest that targeting TβRI cleavage using specific mAbs is a novel precision medicine approach for the treatment of mCRPC. By selectively blocking the prometastatic activity of TβRI-ICD without disrupting physiological TGF beta signaling, this strategy may provide a safer and more effective alternative to existing therapies for advanced prostate cancer.

Cancer signaling encompasses a wide array of entangled molecular cascades that promote oncogenic progression and counteract the effect of tumor suppressors. Transforming growth factor β (TGFβ) induces complex and stage-dependent effects throughout tumor progression. During pre-malignant hyperplastic growth, TGFβ restricts cell proliferation and inflammation, while on the other hand, TGFβ promotes migration and distal metastasis of cancer cells. To dissect the temporal chromatin-based transcriptional response to TGFβ, we employed 3D culture models of isogenic human breast epithelial cells, exemplified by non-oncogenic MCF-10A (MI) and their HRAS-transformed counterpart (MII). Genome-wide chromatin accessibility profiling revealed an extensive chromatin opening induced by TGFβ at transcription start sites and enhancer elements in both models, with a marked enrichment of SOX4 binding motifs in oncogenic cells. Transcriptomic analyses unexpectedly revealed the upregulation of DNA replication and DNA damage response pathways, following TGFβ stimulation of oncogenic MII 3D cultures. Canonical TGFβ-driven programs, including epithelial-mesenchymal transition and metabolic reprogramming, were activated in both models. Notably, single-cell RNA-seq of primary breast tumors confirmed co-expression of SOX4 and cell cycle regulators. Mechanistically, we show that TGFβ induces the interaction between the MH2 domain of SMAD3 and the intrinsically disordered regions of SOX4, co-activating downstream gene targets. Validating the genome-wide analyses, we found that resistance of breast cancer cells to the CDK4/6 inhibitor palbociclib conferred by TGFβ stimulation was functionally dependent on SOX4. Collectively, our findings reveal an apparent oncogenic function of TGFβ in promoting cell cycle progression and drug resistance through SOX4, highlighting the pro-tumorigenic role of TGFβ signaling in breast cancer progression.

The transforming growth factor β (TGF-β) pathway is a developmental signaling network that regulates tissue homeostasis and malfunctions in human diseases, including cancer. TGF-β signals via two receptors, which activate SMAD and alternative signaling pathways. We show that TGF-β induces the expression of the mammalian long noncoding RNA (lncRNA) VIM-AS1 (Vimentin antisense RNA1) variant-2 (v.2) via a transcriptional SMAD-GATA6-SPI1 complex. VIM-AS1 v.1 and v.2 localize in different cell compartments, including the nuclear border. Unbiased whole transcriptomic analysis and functional gain and loss of function assays establish that VIM-AS1 v.2 enhances TGF-β signaling. Mechanistically, VIM-AS1 v.2 interacts with the nucleoporin Nup358/RanBP2, contributing to the binding of Nup358/RanBP2 to SMAD2/3 and enhancing SMAD nuclear accumulation. In the context of cancer biology, VIM-AS1 did not affect the antiproliferative actions of TGF-β, yet had an impact on the epithelial-mesenchymal transition gene program, and increased the invasion and motility of tumor cells, whereas its silencing sensitized cancer cells to chemotherapeutic agents. The molecular mechanism highlights how a lncRNA can modulate the nuclear pore's capacity to import SMAD complexes, by facilitating their capture by Nup358/RanBP2 and thereby enhancing nuclear accumulation of SMADs with distinct isoform composition, thus promoting selectively TGF-β signaling responses.

Background: Activated hepatic stellate cells (aHSCs) are the key cell population in the injured liver driving fibrogenesis. aHSCs express platelet-derived growth factor receptor beta (PDGFRβ), which is absent from quiescent HSCs. PDGFRβ is therefore an attractive target of PET tracers for imaging of fibrogenesis. Here, we present the pharmacological characterization of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 in preparation for clinical translation and further confirm PDGFRβ as a biomarker of activated HSCs in liver disease by single cell sequencing.

Methods: The expression of PDGFRβ in subpopulations of HSCs was evaluated in scRNAseq datasets from both a mouse and human liver samples. DOTA-Cys-ATH001 was evaluated for affinity and mechanism of binding to PDGFRβ. [⁶⁸Ga]Ga-DOTA-Cys-ATH001 was evaluated for binding in vitro in mouse and human liver biopsies.The in vivo stability, biodistribution, pharmacokinetics, dosimetry and microdosing toxicology were evaluated in rats and pigs.

Results: PDGFRβ expression was specifically upregulated in activated HSCs. [⁶⁸Ga] Ga-DOTA-Cys-ATH001 could differentiate fibrotic liver from healthy liver.The binding co-localized with tissue areas positive for collagen deposition and PDGFRβ immunostaining. Based on the microdosing toxicology study the no observed adverse effect level was at least 1000 μg/kg, suggesting that the intended clinical PET scan dose is safe for use. Dosimetry calculations of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 predicted an effective dose in human amenable to repeated examinations.

Conclusions: The data presented here suggests that PDGFRβ PET imaging with [⁶⁸Ga] Ga-DOTA-Cys-ATH001 has potential for non-invasive detection of activated HSCs. Clinical translation of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 is ongoing.