Activation Of TGF-β Pathway Research Articles

BRISC-Mediated PPM1B-K63 Deubiquitination and Subsequent TGF-β Pathway Activation Promote High-Fat/High-Sucrose Diet-Induced Arterial Stiffness.

Metabolic syndrome heightens cardiovascular disease risk primarily through increased arterial stiffness. We previously demonstrated the involvement of YAP (Yes-associated protein) in high-fat/high-sucrose diet (HFHSD)-induced arterial stiffness via modulation of PPM1B (protein phosphatase Mg2+/Mn2+-dependent 1B)-lysine63 (K63) deubiquitination. In this study, we aimed to elucidate the role and mechanisms underlying PPM1B deubiquitination in HFHSD-induced arterial stiffness. Enzymes governing PPM1B deubiquitination were identified through siRNA screening and mass spectrometry. Glutathione S-transferase pull-down, coimmunoprecipitation, protein purification, and immunofluorescence were used to explore the mechanism underlying PPM1B deubiquitination. Doppler ultrasound was used to evaluate HFHSD-induced arterial stiffness in mice, and telemetry was used to record pulsatile (systolic and diastolic) blood pressure. Smooth muscle cell-specific PPM1B overexpression attenuated HFHSD-induced arterial stiffness in mice in a PPM1B-K326-K63-linked polyubiquitination-dependent manner. Mechanistically, ABRO1 (Abraxas brother 1; a core BRCC36 [BRCA1/BRCA2-containing complex subunit 36] isopeptidase complex component) directly bound YAP and underwent liquid-liquid phase separation with YAP and PPM1B in a YAP-dependent manner, which in turn promoted PPM1B deubiquitination. Furthermore, smooth muscle cell-specific Abro1-knockout mice and Brcc3-knockout mice showed attenuated HFHSD-induced arterial stiffness and activation of transforming growth factor-β-Smad signaling. We elucidated the PPM1B deubiquitination mechanisms and highlighted a potential therapeutic target for metabolic syndrome-related arterial stiffness.

MiR-184, a downregulated ovary-elevated miRNA transcriptionally activated by SREBF2, exerts anti-apoptotic properties in ovarian granulosa cells through inducing SMAD3 expression

Follicular atresia is the primary threat to female fertility. miRNAs are dysregulated in granulosa cells (GCs) during follicular atresia, and have emerged as crucial regulators of the initiation and progression of follicular atresia. However, the downregulated ovary-elevated (OE) miRNAs and their biological functions in ovary remain elusive. Here, 13 downregulated OE miRNAs were systematically identified by integrating tissue expression high-throughput data and comparative transcriptome analyses, among which miR-184 was specifically highly expressed in ovary but dramatically downregulated during follicular atresia. Low miR-184 levels were also positively correlated with follicular atresia. Based on the in vitro GC and follicle culture system, we found that miR-184 suppressed GC apoptosis and follicular atresia. Mechanistically, miR-184 induced SMAD3 transcription by acting as a saRNA, and also stabilized SMAD3 mRNA by directly binding to its 5′-UTR, which promoted TGF-β pathway activity and its anti-apoptotic effect. In addition, miR-184 was transcribed independently of host gene, which was activated by SREBF2 in an H3K4me3-dependent manner. Comparative analysis revealed that SREBF2 expression and H3K4me3 enrichment on miR-184 promoter in GCs from atretic follicles were dramatically reduced, which leads to the downregulation of miR-184 during follicular atresia. Moreover, the expression pattern, function, target, and regulatory mechanism of miR-184 among mammals are highly conserved and universal. Taken together, our findings demonstrate that miR-184, transcriptionally activated by SREBF2 in an H3K4me3-dependent manner, exerts anti-atretic effects by inducing SMAD3 expression, highlighting that it is a promising regulator for improving follicular development, ovarian health and female fertility.

Exploring the relation between TGF-β pathway activity and response to checkpoint inhibition in patients with metastatic melanoma.

Immune checkpoint inhibition (ICI) is highly effective for the treatment of melanoma, but intrinsic resistance is present in a subgroup of patients. TGF-β pathway activity may play a role in this resistance by preventing T-cells from entering the tumor microenvironment, causing immune escape. We investigated the association of TGF-β signal transduction pathway activity with resistance to ICI treatment in advanced melanoma. Furthermore, other pathway activities were analyzed to better understand their potential role in ICI resistance. The activity of 8 signaling pathways (TGF-β, Hedgehog, MAPK, AR, NOTCH, PI3K, JAK/STAT1-2, and NFkB) was analyzed from tumor tissue from patients with advanced melanoma. Pathway activity scores (PAS) were explored for associations with survival and response to ICI in 34 patients (19 non-responders and 15 responders). A second, independent method to investigate the predictive value of TGF-β pathway activation was conducted by determining levels of phosphorylated SMAD2. The mean TGF-β PAS of responders vs non-responders was 53.9 vs 56.8 (P = 0.265). No significant relation with progression-free survival was detected for TGF-β activity (P = 0.078). No association between pSMAD2 staining and treatment response or survival was identified. In contrast, Hedgehog scores of responders versus non-responders were 35.7 vs 41.6 (P = 0.038). High Hedgehog PAS was the sole significant predictor of resistance to ICI (OR 0.88, P = 0.033) and worse progression-free survival (HR 1-1.1, P = 0.012). TGF-β pathway activation showed no significant relation with treatment response to ICI or survival in patients with advanced melanoma. Hedgehog PAS was identified as a possible biomarker associated with both treatment response and survival.

PIEZO1 attenuates Marfan syndrome aneurysm development through TGF-β signaling pathway inhibition via TGFBR2.

Marfan syndrome (MFS) is a hereditary disorder primarily caused by mutations in the FBN1 gene. Its critical cardiovascular manifestation is thoracic aortic aneurysm (TAA), which poses life-threatening risks. Owing to the lack of effective pharmacological therapies, surgical intervention continues to be the current definitive treatment. In this study, the role of Piezo-type mechanosensitive ion channel component 1 (Piezo1) in MFS was investigated and the activation of PIEZO1 was identified as a potential treatment for MFS. PIEZO1 expression was detected in MFS mice (Fbn1C1041G/+) and patients. Piezo1 conditional knockout mice in vascular smooth muscle cells of MFS mice (MFS × CKO) was generated, and bioinformatics analysis and experiments in vitro and in vivo were performed to investigate the role of Piezo1 in MFS. PIEZO1 expression decreased in the aortas of MFS mice; MFS × CKO mice showed aggravated TAA, inflammation, extracellular matrix remodelling, and TGF-β pathway activation compared to MFS mice. Mechanistically, PIEZO1 knockout exacerbated the activation of the TGF-β signalling pathway by inhibiting the endocytosis and autophagy of TGF-β receptor 2 mediated by Rab GTPase 3C. Additionally, the pharmacological activation PIEZO1 through Yoda1 prevented TGF-β signalling pathway activation and reversed TAA in MFS mice. Piezo1 deficiency aggravates MFS aneurysms by promoting TGF-β signalling pathway activation via TGF-β receptor 2 endocytosis and a decrease in autophagy. These data suggest that PIEZO1 may be a potential therapeutic target for MFS treatment.

A patient stratification signature mirrors the immunogenic potential of high grade serous ovarian cancers

BackgroundWhile high-grade serous ovarian cancer (HGSC) has proven largely resistant to immunotherapy, sporadic incidents of partial and complete response have been observed in clinical trials and case reports. These observations suggest that a molecular basis for effective immunity may exist within a subpopulation of HGSC. Herein, we developed an algorithm, CONSTRU (Computing Prognostic Marker Dependencies by Successive Testing of Gene-Stratified Subgroups), to facilitate the discovery and characterization of molecular backgrounds of HGSC that confer resistance or susceptibility to protective anti-tumor immunity.MethodsWe used CONSTRU to identify genes from tumor expression profiles that influence the prognostic power of an established immune cytolytic activity signature (CYTscore). From the identified genes, we developed a stratification signature (STRATsig) that partitioned patient populations into tertiles that varied markedly by CYTscore prognostic power. The tertile groups were then analyzed for distinguishing biological, clinical and immunological properties using integrative bioinformatics approaches.ResultsPatient survival and molecular measures of immune suppression, evasion and dysfunction varied significantly across STRATsig tertiles in validation cohorts. Tumors comprising STRATsig tertile 1 (S-T1) showed no immune-survival benefit and displayed a hyper-immune suppressed state marked by activation of TGF-β, Wnt/β-catenin and adenosine-mediated immunosuppressive pathways, with concurrent T cell dysfunction, reduced potential for antigen presentation, and enrichment of cancer-associated fibroblasts. By contrast, S-T3 tumors exhibited diminished immunosuppressive signaling, heightened antigen presentation machinery, lowered T cell dysfunction, and a significant CYTscore-survival benefit that correlated with mutational burden in a manner consistent with anti-tumor immunoediting. These tumors also showed elevated activity of DNA damage/repair, cell cycle/proliferation and oxidative phosphorylation, and displayed greater proportions of Th1 CD4 + T cells. In these patients, but not those of S-T1 or S-T2, validated predictors of immunotherapy response were prognostic of longer patient survival. Further analyses showed that STRATsig tertile properties were not explained by known HGSC molecular or clinical subtypes or singular immune mechanisms.ConclusionsSTRATsig is a composite of parallel immunoregulatory pathways that mirrors tumor immunogenic potential. Approximately one-third of HGSC cases classify as S-T3 and display a hypo-immunosuppressed and antigenic molecular composition that favors immunologic tumor control. These patients may show heightened responsiveness to current immunotherapies.

Oxidative stress drives endometrial fibrosis via TGF-β1/MAPK signaling pathway in breast cancer.

Breast cancer patients have high serum reactive oxygen species (ROS) levels, which exert toxicity on the ovary. However, it is still unclear whether tumor-derived ROS play a role in endometrial development and function in breast cancer. Breast cancer patients and healthy controls were recruited and endometrial thickness was measured by transvaginal ultrasound (TVUS). Xenograft tumors of the breast cancer cell line MDA-MB-231 in a female BALB/c nude mice model were established, and the therapeutic mechanism of vitamin C (VC) was investigated on uterine pathology invivo and the contribution of co-culture of breast cancer cell and endometrial epithelial cell on this process was examined invitro. Median thickness in endometria was lower in breast cancer patients and tumor-bearing mice compared to controls. A gene signature of uteri in tumor-bearing mice demonstrated differential expression of genes (DEGs) regulating extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT), and activation of TGF-β and MAPK signaling pathways. In addition, ROS, EMT- and ECM-related protein levels were enhanced in uteri in tumor-bearing mice, as well as in Ishikawa cells which were co-cultured with MDA-MB-231 cells compared to controls. Supplementation with VC reduced endometrial damage, inhibited the EMT process and collagen deposition, and maintained better histologic architecture of uteri in tumor-bearing mice via inactivation of the TGF-β1/p38MAPK pathway. In women with breast cancer oxidative stress in the endometrium results in a fibrotic response as a consequence of EMT. VC could alleviate endometrial fibrosis via TGF-β1/p38MAPK pathway and provide new predictive and therapeutic targets for fertility preservation in younger breast cancer patients.

The Neurodevelopmental Protein POGZ Suppresses Metastasis in Triple-Negative Breast Cancer by Attenuating TGFβ Signaling.

The pogo transposable element-derived zinc finger protein, POGZ, is notably associated with neurodevelopmental disorders through its role in gene transcription. Many proteins involved in neurological development are often dysregulated in cancer, suggesting a potential role for POGZ in tumor biology. Here, we provided experimental evidence that POGZ influences the growth and metastatic spread of triple-negative breast cancers (TNBC). In well-characterized models of TNBC, POGZ exerted a dual role, both as a tumor promoter and metastasis suppressor. Mechanistically, loss of POGZ potentiated TGFβ pathway activation to exert cytostatic effects while simultaneously increasing the mesenchymal and migratory properties of breast tumors. Although POGZ levels are elevated in human breast cancers, the most aggressive forms of TNBC tumors, including those with increased mesenchymal and metastatic properties, exhibit dampened POGZ levels, and low POGZ expression was associated with inferior clinical outcomes in these tumor types. Taken together, these data suggest that POGZ is a critical suppressor of the early stages of the metastatic cascade. Significance: The POGZ neurodevelopmental protein plays dual functions in triple-negative breast cancers as a tumor promoter and metastasis suppressor, inhibiting TGFβ-regulated EMT to limit breast cancer metastatic progression.

Abstract Tu061: Pericyte-mediated perivascular fibrosis in the pressure-overloaded heart is dependent on TGF-β signaling and is restrained by ITGB1.

Background: Pericytes exhibit remarkable phenotypic plasticity and have been implicated in the pathogenesis of fibrosis. Cardiac pericytes are involved in repair and fibrotic remodeling of the infarcted heart; however, their role in the cardiomyopathy induced by pressure overload is not known. We hypothesized that cardiac pericytes may contribute to remodeling of the pressure-overloaded heart by converting into fibroblasts and we examined the molecular signals mediating pericyte activation. Methods and Results: Transverse aortic constriction (TAC) protocols were performed to study the role of pericytes in the pressure-overloaded heart. Using fibroblast:pericyte dual reporter mice and lineage tracing experiments with the inducible NG2 CreER mice (to track pericytes) combined with PDGFRa EGFP reporter mice (to reliably label fibroblasts), we found no pericyte to fibroblast conversion in the pressure-overloaded heart. scRNA-seq demonstrated that pericyte lineage cells do not express fibroblast identity genes after TAC but acquire a fibrogenic phenotype expressing high levels of matricellular genes, growth factors, adhesion molecules and integrins. Bioinformatic analysis identified transforming growth factor (TGF)-b and integrin beta-1 (ITGB1) as important upstream regulators of the transcriptional profile of pericytes after TAC. Pericyte-specific loss of transforming growth factor beta receptor 2 (TGFBR2) attenuated dysfunction and perivascular fibrosis after TAC. In contrast, pericyte-specific ITGB1 loss had deleterious effects increasing mortality, promoting dysfunction, inducing microvascular rarefaction and stimulating a pro-inflammatory and fibrogenic pericyte phenotype. Conclusions: In the pressure-overloaded heart, pericytes acquire a fibrogenic phenotype without converting to fibroblasts. Pericyte-specific activation of TGF-β pathways mediates perivascular myocardial fibrosis after TAC. In contrast, pericyte-specific ITGB1 signaling exerts protective actions, preserving microvascular structure, attenuating inflammation and inhibiting fibrosis.

Pro-fibrotic effect of the susceptible gene PCSK5 in vascular fibrosis of Takayasu arteritis via TGF-β and SMAD3 signaling pathway activation

BackgroundVascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-β) activation is critical. Understanding TGF-β activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-β activation and vascular fibrosis development. MethodsIn TAK patients, PCSK5 and TGF-β expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-β in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients. ResultsPatients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-β and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-β (pro-TGF-β) to the mature form by binding the pro-TGF-β cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-β binding, decreasing TGF-β activation and ECM expression, which was also partially validated in leflunomide-treated patients. ConclusionThe findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-β and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-β binding, presenting a new TAK treatment approach.

Titin mechanical unloading disrupts sarcomere tensional homeostasis triggering fast, non-canonical myocardial fibrosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): We acknowledge funding from Ministerio de Ciencia e Innovación (Madrid, Spain) through grant FJC2021 047055-I and European Research Council (ERC) through grant H2020-ERC-2020-COG (ProtMechanics-Live). Background/Introduction Stresses exerted during myocardial contraction and relaxation cycles are mainly braced by mechanical proteins located in the extracellular matrix (ECM) and cardiomyocytes (CMs). Titin (TTN) is a gigantic protein that scaffolds the sarcomere (i.e. the basic unit of contraction). Since most of the CM cytoskeleton is occupied by sarcomeres, titin is an essential structural hub for the maintenance of a homeostatic level of mechanical tension. Purpose It has been shown that mechanical disruption of the ECM affects the equilibrium forces between CMs and the ECM, leading to morphological changes in sarcomeres. However, potential alteration of tensional homeostasis resulting from defective mechanics of CM proteins remains unknown due to the lack of tools for in vivo studies. Therefore, the aim of this study is to explore the in vivo response of the myocardium to the abrupt cessation of the mechanical properties of titin. Methods For this purpose, we have experimentally challenged CMs to a tensional unloading by means of a titin cleavage model (TEVs-TTN) [1], both in vitro, using neonatal CM (NMCM), and in vivo. In this model, a cassette with a tobacco etch virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (myoAAV), we were able to cleave titin in living CMs, ceasing only its mechanical properties. Samples were analyzed using an array of techniques such as histology, immunofluorescence and bulk and single-nucleus RNAseq. Results Our results show that while in NMCM we obtain a complete titin cleavage, in vivo we observe mosaic expression of TEVp and no more than 30% cleavage of the protein. In both cases, TEVp expression in TEVs-TTN does not affect cell viability. However, mild myocardial titin cleavage leads to a fibrotic response in less than six days, characterized by an increase in interstitial collagen and an expansion of cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this fibrotic response occurs without activation of the canonical TGFβ pathway. Conclusion Taken together, our results suggest that the loss of titin mechanical-structural function in cardiomyocytes derived from the cleavage of a single peptide bond elicits a fast fibrotic compensatory response in the myocardium through intercellular communication with fibroblasts.

Serum albumin exposure enhances cell invasiveness and paclitaxel resistance in human neuroblastoma cells, with attenuation by valeriana-type iridoid glycosides.

Neuroblastoma, a prevalent extracranial solid tumor in children, arises from undifferentiated nerve cells. While tumor vasculature, often characterized by increased permeability, influences metastasis and recurrence, the direct impact of blood-borne molecules on tumor progression remains unclear. In the present study, we focused on the effect of exposure to albumin, one of the most abundant proteins in the serum, on human neuroblastoma cells. Albumin exposure elevated oxidative stress and led to mitochondria dysfunction via the activation of TGFβ and PI3K pathways, accompanied by an increase in the metastatic and invasive properties of neuroblastoma cells. Proteins relevant to the induction of autophagy were upregulated in response to prolonged albumin exposure. Additionally, pre-exposure to albumin before treatment resulted in increased resistance to paclitaxel. Two valeriana-type iridoid glycosides, patrisophoroside and patrinalloside, recently isolated from Nardostachys jatamansi significantly mitigated the effect of albumin on oxidative stress, cell invasiveness, and chemoresistance. These findings illuminate the potential role of blood-borne molecules, such as albumin, in the progression and metastasis of neuroblastoma, as well as the possible therapeutic implications of valeriana-type iridoid glycosides in anti-cancer treatment.

Transcriptome analysis revealed SMURF2 as a prognostic biomarker for oral cancer.

The activation of TGF-β pathway can facilitate tumorigenesis. Understanding the TGF-related genes (TRGs) in oral cancer and determining their prognostic value is of utmost importance. The TRGs were selected to develop a prognostic model based on lasso regression. Oral cancer patients were classified into high-risk and low-risk groups based on the risk model. Subsequently, multivariate COX regression was employed to identify the prognostic marker. Additionally, the expression of SMURF2 was validated using quantitative real-time polymerase chain reaction (qRT-PCR) and the Human Protein Atlas (HPA) database. To investigate the relationship between SMURF2 expression and immune cell infiltrations, we conducted single-sample Gene Set Enrichment Analysis (ssGSEA) analyses. We identified 16 differentially expressed TRGs in oral cancer, all of which showed upregulation. From these, we selected eight TRGs as prognostic signatures. Furthermore, the high-risk group demonstrated lower infiltration levels of immune cells, immune score, and higher tumor purity. Interestingly, we also found that SMURF2 serves as an independent prognostic biomarker. SMURF2 was upregulated in oral cancer, as confirmed by public databases and qRT-PCR analysis. Importantly, our results indicate a close association between SMURF2 expression and the immune microenvironment. The 8-TRG signature prognosis model that we constructed has the ability to predict the survival rate and immune activity of oral cancer patients. SMURF2 could be effective in recognizing prognosis and evaluating immune efficacy for oral cancer.

First-in-human study of GFH018, a small molecule inhibitor of transforming growth factor-β receptor I inhibitor, in patients with advanced solid tumors

BackgroundTransforming growth factor-β (TGF-β) is a cytokine with multiple functions, including cell growth regulation, extracellular matrix production, angiogenesis homeostasis adjustment and et al. TGF-β pathway activation promotes tumor metastasis/progression and mediates epithelial-mesenchymal transmission suppressing immunosurveillance in advanced tumors. GFH018, a small molecule inhibitor blocking TGF-β signal transduction, inhibits the progression and/or metastasis of advanced cancers. This first-in-human study evaluated the safety, tolerability, pharmacokinetics (PK), and efficacy of GFH018 monotherapy in patients with advanced solid tumors.MethodsThis phase I, open-label, multicenter study used a modified 3+3 dose escalation and expansion design. Adult patients with advanced solid tumors failing the standard of care were enrolled. Starting at 5 mg, eight dose levels up to 85 mg were evaluated. Patients received GFH018 BID (14d-on/14d-off) starting on the 4th day after a single dose on cycle 1, day 1. Subsequent cycles were defined as 28 days. The study also explored the safety of 85 mg BID 7d-on/7d-off. Adverse events were graded using NCI criteria for adverse events (NCI-CTCAE v5.0). PK was analyzed using a noncompartmental method. Efficacy was evaluated using RECIST 1.1. Blood samples were collected for biomarker analysis.ResultsFifty patients were enrolled and received at least one dose of GFH018. No dose-limiting toxicity occurred, and the maximum tolerated dose was not reached. Forty-three patients (86.0%) had at least one treatment-related adverse event (TRAE), and three patients (6.0%) had ≥ G3 TRAEs. The most common TRAEs (any grade/grade ≥3) were AST increased (18%/0%), proteinuria (14%/2%), anemia (14%/2%), and ALT increased (12%/0%). No significant cardiotoxicity or bleeding was observed. GFH018 PK was linear and dose-independent, with a mean half-life of 2.25–8.60 h from 5 – 85 mg. Nine patients (18.0%) achieved stable disease, and one patient with thymic carcinoma achieved tumor shrinkage, with the maximum target lesion decreased by 18.4%. Serum TGF-β1 levels were not associated with clinical responses. The comprehensive recommended dose for Phase II was defined as 85 mg BID 14d-on/14d-off.ConclusionsGFH018 monotherapy presented a favorable safety profile without cardiac toxicity or bleeding. Modest efficacy warrants further studies, including combination strategies.Trial registrationClinicalTrial. gov (https://www.clinicaltrials.gov/), NCT05051241. Registered on 2021-09-02.

Abstract 2495: CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages

Abstract The TGFβ receptor inhibitor galunisertib (GAL) exhibited promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the randomized phase 2 H9H-MC-JBAJ study. However, the identification of predictive biomarkers remains of unique importance. We used a 279 multi-analyte panel and confirmed CCL3 as the most significant plasma protein for chemoresistance in patients receiving gemcitabine (GEM) [overall survival (mOS) (95%CI), high vs low, 3.6 (2.5-4.0) vs 10.1 (7.2-17.0) months, HR (95%CI)= 3.92 (2.14-7.20); p<0.001]. GAL dramatically reverted this resistance and CCL3 was the most significant predictive marker for the combination of this agent plus GEM [mOS (95%CI), GAL + GEM vs. placebo + GEM, 9.0 (5.5-12.1) vs 3.6 (2.5-4.0) months, HR (95%CI)= 0.39 (0.23-0.68); p<0.001]. We delved into the mechanisms behind this exceptional response in PDAC with this prognostically negative ecotype sustained by CCL3 in 6 different murine PDAC models. Mice bearing high-Ccl3 tumors had a significantly shorter mOS, a higher infiltration of M2-macrophages (TAMs), and a more mesenchymal/basal-like phenotype than did models with undetectable Ccl3.Ccl3 was stably expressed in DT4313 and PAN610, and silenced in RC416 cells. In co-culture models, DT4313Ccl3 and PAN610Ccl3 had a more mesenchymal phenotype and induced a M2 polarization of RAW264.7 murine TAMs than did controls. Mice bearing DT4313Ccl3 or PAN610Ccl3 tumors had a significantly shorter mOS, an higher infiltration of M2-polarized TAMs and a more mesenchymal/basal-like phenotype than controls. The expression of Tgfβ1 ligand and the phosphorylation of Smad2 were triggered only in TAMs if co-cultured with Ccl3-high RC416scr, DT4313Ccl3 or PAN610Ccl3 cells. This activation of Tgfβ pathway was blocked by maraviroc, a selective inhibitor of Ccl3-receptor Ccr5. In co-culture, GAL reverted the mesenchymal phenotype of PDAC cells and the M2 polarization of TAMs. By using an 111 different cytokine immunoassay, Leukemia Inhibitory Factor (Lif) was the most significantly factor overexpressed by TAMs if stimulated by Ccl3. GAL suppressed this Lif expression. In mice bearing DT4313Ccl3 tumors GEM was ineffective, whereas it prolonged mOS in those with DT4313NTC tumors. Conversely, GAL plus GEM doubled mOS duration vs. GEM, modulated M2- TAMs infiltration, the mesenchymal/basal-like phenotype and plasma Lif only in mice bearing DT4313Ccl3 tumors, but not in those with DT4313NTC controls. In summary, we demonstrated that tumor-derived Ccl3 activates an autocrine Tgfβ signaling in TAMs, triggering their M2-phenotype and secretion of Lif. This Tgfβ-Lif axis sustains paracrinally a mesenchymal/basal-like PDAC ecotype resistant to GEM. Inhibition of Tgfβ signaling modulates this poor prognosis ecotype, and explains the exceptional response to GAL in patients with high-CCL3 PDAC. Citation Format: Silvia Pietrobono, Monica Bertolini, Veronica De Vita, Enza Scarlato, Federica Fazzini, Fabio Sabbadini, Domenico Mangiameli, Alberto Quinzii, Simona Casalino, Davide Melisi. CCL3 predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining a basal-like ecotype enriched in LIF-producing macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2495.

FAT1 upregulation is correlated with an immunosuppressive tumor microenvironment and predicts unfavorable outcome of immune checkpoint therapy in non-small cell lung cancer

BackgroundPrevious studies found that FAT1 was recurrently mutated and aberrantly expressed in multiple cancers, and the loss function of FAT1 promoted the formation of cancer-initiating cells in several cancers. However, in some types of cancer, FAT1 upregulation could lead to epithelial-mesenchymal transition (EMT). The role of FAT1 in cancer progression, which appears to be cancer-type-specific, is largely unknown. MethodsQRT-PCR and immunochemistry were used to verify the expression of FAT1 in non-small cell lung cancer (NSCLC). QRT-PCR and Western blot were used to detect the influence of siFAT1 knockdown on the expression of potential targets of FAT1 in NSCLC cell lines. GEPIA, KM-plotter, CAMOIP, and ROC-Plotter were used to evaluate the association between FAT1 and clinical outcomes based on expression and clinical data from TCGA and immune checkpoint inhibitors (ICI) treated cohorts. ResultsWe found that FAT1 upregulation was associated with the activation of TGF-β and EMT signaling pathways in NSCLC. Patients with a high FAT1 expression level tend to have a poor prognosis and hard to benefit from ICI therapy. Genes involved in TGF-β/EMT signaling pathways (SERPINE1, TGFB1/2, and POSTN) were downregulated upon knockdown of FAT1. Genomic and immunologic analysis showed that high cancer-associated fibroblast (CAF) abundance, decreased CD8+ T cells infiltration, and low TMB/TNB were correlated with the upregulation of FAT1, thus promoting an immunosuppressive tumor microenvironment (TME) which influence the effect of ICI-therapy. ConclusionOur findings revealed the pattern of FAT1 upregulation in the TME of patients with NSCLC, and demonstrated its utility as a biomarker for unfavorable clinical outcomes, thereby providing a potential therapeutic target for NSCLC treatment.

Abstract 6395: Metabolic dysregulation as a determinant of prognosis and response to sorafenib in liver cancer

Abstract Background & Aims: Elevated metabolic activity is a defining characteristic of liver cancer. Nevertheless, our understanding of the molecular underpinnings of high metabolic activity in liver cancer and its impact on clinical outcomes and treatment responses remains limited. We aimed to elucidate the metabolic features of liver cancer associated with clinical outcomes, particularly overall survival and responsiveness to sorafenib treatment. Methods: Utilizing cross-species comparisons of genomic data, we integrated gene expression profiles from liver tissues in metabolically challenged mice and from patients with cirrhosis and liver cancer. To categorize patients, we developed a genomic predictor and applied it to gene expression data from a cirrhosis cohort (n=216) and large liver cancer cohorts (n=1390). Comprehensive statistical and informatics analyses were conducted to evaluate clinical significance, including overall survival and responses to standard treatments. Moreover, we integrated genomic data derived from patient-derived xenograft (PDX) models into our analytical framework. Results: Our integrated data analysis unveiled three distinct metabolic subtypes of tissues: high, moderate, and low metabolic subtypes. In the cirrhosis cohort, the high metabolic subtype was significantly associated with early liver cancer development. Among liver cancer cohorts, the high metabolic subtype exhibited characteristics such as poor survival, heightened hepatic stem cell features, increased genomic instability, and elevated expression of AFP, KRT19, EPCAM, and SALL4. Notably, when liver cancer patients undergoing systemic sorafenib treatment were stratified using our genomic predictor, those with low and moderate metabolic activity derived significant benefits from sorafenib therapy. Conversely, the high metabolic subtype displayed substantial resistance to sorafenib, suggesting that metabolic activity may dictate the response of liver cancer cells to sorafenib treatment. Gene network analysis of the metabolic expression signature identified multiple signaling pathways that potentially contribute to sorafenib resistance in liver cancer cells. Our analysis predicted the activation of pathways such as the TGF-β pathway (TGFB1), consistent with previous findings of TGF-β pathway activation in highly metabolic cancer cells. Additionally, our analysis identified YAP1, HIF1A, and MAPK pathways as activated pathways in sorafenib-resistant liver cancer. Conclusions: This study identifies clinically and metabolically distinct subtypes of liver cancer, biomarkers associated with these subtypes, and mechanisms underlying metabolic-mediated resistance of liver cancer cells to sorafenib. Furthermore, the metabolic subtypes observed in PDX models provide valuable tools for selecting appropriate preclinical models for future research endeavors. Citation Format: Hyewon Park, Sowon Park, Sung-Hwan Lee, Yun Seong Jeong, Bohwa Sohn, Sun Young Yim, Ju-Seog Lee. Metabolic dysregulation as a determinant of prognosis and response to sorafenib in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6395.

Lipopolysaccharide-induced chronic inflammation increases female serum gonadotropins and shifts the pituitary transcriptomic landscape.

Female reproductive function depends on a choreographed sequence of hormonal secretion and action, where specific stresses such as inflammation exert profound disruptions. Specifically, acute LPS-induced inflammation inhibits gonadotropin production and secretion from the pituitary, thereby impacting the downstream production of sex hormones. These outcomes have only been observed in acute inflammatory stress and little is known about the mechanisms by which chronic inflammation affects reproduction. In this study we seek to understand the chronic effects of LPS on pituitary function and consequent luteinizing and follicle stimulating hormone secretion. A chronic inflammatory state was induced in female mice by twice weekly injections with LPS over 6 weeks. Serum gonadotropins were measured and bulk RNAseq was performed on the pituitaries from these mice, along with basic measurements of reproductive biology. Surprisingly, serum luteinizing and follicle stimulating hormone was not inhibited and instead we found it was increased with repeated LPS treatments. Analysis of bulk RNA-sequencing of murine pituitary revealed paracrine activation of TGFβ pathways as a potential mechanism regulating FSH secretion in response to chronic LPS. These results provide a framework with which to begin dissecting the impacts of chronic inflammation on reproductive physiology.

Studies on the effect and mechanism of CD147 on melanoma stem cells.

Melanoma is the most aggressive form of skin cancer. Melanoma stem cells (MSCs) are one of the driving forces of melanoma invasion and metastasis. Therefore, it is of great significance to explore the mechanisms that maintain the stemness of MSCs. In this study, CD147-positive (CD147+) MSCs derived from A375 cell line were characterized. Side population (SP) and non-SP cells were sorted from A375 cells. Quantitative real-time polymerase chain reaction and Western blot analysis were conducted to determine the expression of CD147 in SP and non-SP cells. Subsequently, CD147+ and CD147-negative (CD147-) cells were isolated from SP cells. Stem cell characteristics and metastatic potential of CD147+/- antigen-presenting cells were identified by sphere-forming, wound-healing, and transwell assays. Western blot analysis was performed to evaluate the protein levels of transforming growth factor-beta1 (TGFβ1) and neurogenic locus notch homolog protein 1 (Notch1) signaling pathway. Xenograft tumor experiments were conducted to investigate the tumorigenic capacity of CD147+ cells in vivo. CD147 was highly expressed in SP cells of A375 cell line. CD147+ cells have stronger abilities for sphere forming, migration, and invasion in vitro. The protein levels of TGFβ1, notch1, jagged1, and Hes1 were higher in CD147+ cells than in CD147- cells. Moreover, the CD147+ cells showed stronger tumorigenic and metastatic potential in vivo. SP cells of A375 cell line expressed high levels of CD147, and CD147+ SP cells possessed much stronger stem-like characteristics and motility, which is linked to the activation of TGFβ and notch pathways.

Thrombospondin 2 is a key determinant of fibrogenesis in non-alcoholic fatty liver disease.

Hepatic overexpression of the thrombospondin 2 gene (THBS2) and elevated levels of circulating thrombospondin 2 (TSP2) have been observed in patients with chronic liver disease. This study aimed to identify the specific cells expressing THBS2/TSP2 in non-alcoholic fatty liver disease (NAFLD) and investigate the underlying mechanism behind THBS2/TSP2 upregulation. Comprehensive NAFLD liver gene datasets, including single-cell RNA sequencing (scRNA-seq), in-house NAFLD liver tissue, and LX-2 cells derived from human hepatic stellate cells (HSCs), were analysed using a combination of computational biology, genetic, immunological, and pharmacological approaches. Analysis of the genetic dataset revealed the presence of 1433 variable genes in patients with advanced fibrosis NAFLD, with THBS2 ranked among the top 2 genes. Quantitative polymerase chain reaction (qPCR) examination of NAFLD livers showed a significant correlation between THBS2 expression and fibrosis stage (r = .349, p < .001). In support of this, scRNA-seq data and insitu hybridization demonstrated that the THBS2 gene was highly expressed in HSCs of NAFLD patients with advanced fibrosis. Pathway analysis of the gene dataset revealed THBS2 expression to be associated with the transforming growth factor beta (TGFβ) pathway and collagen gene activation. Moreover, the activation of LX-2 cells with TGFβ increased THBS2/TSP2 and collagen expression independently of the TGFβ-SMAD2/3 pathway. THBS2 gene knockdown significantly decreased collagen expression in LX-2 cells. THBS2/TSP2 is highly expressed in HSCs and plays a role in regulating fibrogenesis in NAFLD patients. THBS2/TSP2 may therefore represent a potential target for anti-fibrotic therapy in NAFLD.

TGF-β induces matrisome pathological alterations and EMT in patient-derived prostate cancer tumoroids

Extracellular matrix (ECM) tumorigenic alterations resulting in high matrix deposition and stiffening are hallmarks of adenocarcinomas and are collectively defined as desmoplasia. Here, we thoroughly analysed primary prostate cancer tissues obtained from numerous patients undergoing radical prostatectomy to highlight reproducible structural changes in the ECM leading to the loss of the glandular architecture. Starting from patient cells, we established prostate cancer tumoroids (PCTs) and demonstrated they require TGF-β signalling pathway activity to preserve phenotypical and structural similarities with the tissue of origin. By modulating TGF-β signalling pathway in PCTs, we unveiled its role in ECM accumulation and remodelling in prostate cancer. We also found that TGF-β-induced ECM remodelling is responsible for the initiation of prostate cell epithelial-to-mesenchymal transition (EMT) and the acquisition of a migratory, invasive phenotype. Our findings highlight the cooperative role of TGF-β signalling and ECM desmoplasia in prompting prostate cell EMT and promoting tumour progression and dissemination.