Selective inhibition of integrin αvβ6 leads to rapid induction of urinary bladder tumors in cynomolgus macaques

Toxicological sciences : an official journal of the Society of Toxicology

2022 Dec 14

Guffroy, M;Trela, B;Kambara, T;Stawski, L;Chen, H;Luus, L;Montesinos, MS;Olson, L;He, Y;Maisonave, K;Carr, T;Lu, M;Ray, AS;Hazelwood, LA;
PMID: 36515490 | DOI: 10.1093/toxsci/kfac128

Administration of a novel and selective small molecule integrin αvβ6 inhibitor, MORF-627, to young cynomolgus monkeys for 28 days resulted in the rapid induction of epithelial proliferative changes in the urinary bladder of two animals, in the absence of test agent genotoxicity. Microscopic findings included suburothelial infiltration by irregular nests and/or trabeculae of epithelial cells, variable cytologic atypia, and high mitotic rate, without invasion into the tunica muscularis. Morphologic features and patterns of tumor growth were consistent with a diagnosis of early-stage invasive urothelial carcinoma. Ki67 immunohistochemistry demonstrated diffusely increased epithelial proliferation in the urinary bladder of several monkeys, including those with tumors, and αvβ6 was expressed in some epithelial tissues, including urinary bladder, in monkeys and humans. Spontaneous urothelial carcinomas are extremely unusual in young healthy monkeys, suggesting a direct link of the finding to the test agent. Inhibition of integrin αvβ6 is intended to locally and selectively block TGF-β signaling, which is implicated in epithelial proliferative disorders. Subsequent in vitro studies using a panel of integrin αvβ6 inhibitors in human bladder epithelial cells replicated the increased urothelial proliferation observed in monkeys, and was reversed through exogenous application of TGF-β. Moreover, analysis of in vivo models of liver and lung fibrosis revealed evidence of epithelial hyperplasia and cell cycle dysregulation in mice treated with integrin αvβ6 or TGF-β receptor I inhibitors. The cumulative evidence suggests a direct link between integrin αvβ6 inhibition and decreased TGF-β signaling in the local bladder environment, with implications for epithelial proliferation and carcinogenesis.

Crosstalk between transforming growth factor β-2 and Autotaxin in trabecular meshwork and different subtypes of glaucoma

Journal of biomedical science

2021 Jun 17

Igarashi, N;Honjo, M;Yamagishi, R;Kurano, M;Yatomi, Y;Igarashi, K;Kaburaki, T;Aihara, M;
PMID: 34140021 | DOI: 10.1186/s12929-021-00745-3

Elevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators.The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay. RNA scope was used to assess the expression of ATX and TGF-β2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-β2 regulates ATX expressions.TGF-β2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-β2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-β2 with concentration-dependent manner. In counter, ATX also induced TGF-β1, TGF-β2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-β and ATX inhibition.Trans-signaling of TGF-β2 regulates ATX expressions and thereby induced upregulations of TGF-βs or fibrosis of hTM. TGF-β2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Quantitation of TGF-β proteins in mouse tissues shows reciprocal changes in TGF-β1 and TGF-β3 in normal vs neoplastic mammary epithelium.

Oncotarget.

2016 Jun 21

Flanders KC, Yang YA, Herrmann M, Chen J, Mendoza N, Mirza AM, Wakefield LM.
PMID: 27203217 | DOI: 10.18632/oncotarget.9416

Transforming growth factor-βs (TGF-βs) regulate tissue homeostasis, and their expression is perturbed in many diseases. The three isoforms (TGF-β1, -β2, and -β3) have similar bioactivities in vitro but show distinct activities in vivo. Little quantitative information exists for expression of TGF-β isoform proteins in physiology or disease. We developed an optimized method to quantitate protein levels of the three isoforms, using a Luminex^® xMAP^®-based multianalyte assay following acid-ethanol extraction of tissues. Analysis of multiple tissues and plasma from four strains of adult mice showed that TGF-β1 is the predominant isoform with TGF-β2 being ~10-fold lower. There were no sex-specific differences in isoform expression, but some tissues showed inter-strain variation, particularly for TGF-β2. The only adult tissue expressing appreciable TGF-β3 was the mammary gland, where its levels were comparable to TGF-β1. In situ hybridization showed the luminal epithelium as the major source of all TGF-β isoforms in the normal mammary gland. TGF-β1 protein was 3-8-fold higher in three murine mammary tumor models than in normal mammary gland, while TGF-β3 protein was 2-3-fold lower in tumors than normal tissue, suggesting reciprocal regulation of these isoforms in mammary tumorigenesis.

Development of resistance to FAK inhibition in pancreatic cancer is linked to stromal depletion.

Gut

2019 May 10

Jiang H, Liu X, Knolhoff BL, Hegde S, Lee KB, Jiang H, Fields RC, Pachter JA, Lim KH, DeNardo DG.
PMID: 31076405 | DOI: 10.1136/gutjnl-2018-317424

Abstract

OBJECTIVE:

We investigated how pancreatic cancer developed resistance to focal adhesion kinase (FAK) inhibition over time.

DESIGN:

Pancreatic ductal adenocarcinoma (PDAC) tumours from KPC mice (p48-CRE; LSL-KRasG12D/wt; p53flox/wt) treated with FAK inhibitor were analysed for the activation of a compensatory survival pathway in resistant tumours. We identified pathways involved in the regulation of signal transducer and activator of transcription 3 (STAT3) signalling on FAK inhibition by gene set enrichment analysis and verified these outcomes by RNA interference studies. We also tested combinatorial approaches targeting FAK and STAT3 in syngeneic transplantable mouse models of PDAC and KPC mice.

RESULTS:

In KPC mice, the expression levels of phosphorylated STAT3 (pSTAT3) were increased in PDAC cells as they progressed on FAK inhibitor therapy. This progression corresponded to decreased collagen density, lowered numbers of SMA+ fibroblasts and downregulation of the transforming growth factor beta (TGF-β)/SMAD signalling pathway in FAK inhibitor-treated PDAC tumours. Furthermore, TGF-β production by fibroblasts in vitro drives repression of STAT3 signalling and enhanced responsiveness to FAK inhibitor therapy. Knockdown of SMAD3 in pancreatic cancer cells abolished the inhibitory effects of TGF-β on pSTAT3. We further found that tumour-intrinsic STAT3 regulates the durability of the antiproliferative activity of FAK inhibitor, and combinatorial targeting of FAK and Janus kinase/STAT3 act synergistically to suppress pancreatic cancer progression in mouse models.

CONCLUSION:

Stromal depletion by FAK inhibitor therapy leads to eventual treatment resistance through the activation of STAT3 signalling. These data suggest that, similar to tumour-targeted therapies, resistance mechanisms to therapies targeting stromal desmoplasia may be critical to treatment durability.

Remodeling the tumor microenvironment via blockade of LAIR-1 and TGF-β signaling enables PD-L1-mediated tumor eradication

The Journal of clinical investigation

2022 Mar 01

Horn, LA;Chariou, PL;Gameiro, SR;Qin, H;Iida, M;Fousek, K;Meyer, TJ;Cam, M;Flies, D;Langermann, S;Schlom, J;Palena, C;
PMID: 35230974 | DOI: 10.1172/JCI155148

Collagens in the extracellular matrix (ECM) provide a physical barrier to tumor immune infiltration, while also acting as a ligand for immune inhibitory receptors. Transforming growth factor-β (TGF-β) is a key contributor to shaping the ECM by stimulating the production and remodeling of collagens. TGF-β-activation signatures and collagen-rich environments have both been associated with T-cell exclusion and lack of responses to immunotherapy. Here we describe the effect of targeting collagens that signal through the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) in combination with blockade of TGF-β and programmed cell death ligand 1 (PD-L1). This approach remodeled the tumor collagenous matrix, enhanced tumor infiltration and activation of CD8+ T cells, and repolarized suppressive macrophage populations resulting in high cure rates and long-term tumor-specific protection across murine models of colon and mammary carcinoma. The results highlight the advantage of direct targeting of ECM components in combination with immune checkpoint blockade therapy.

NCI-41356, a promising small inhibitor of HSPB5 in idiopathic pulmonary fibrosis?

Revue des Maladies Respiratoires

2023 Feb 01

Tanguy, J;Boutanquoi, P;Dondaine, L;Burgy, O;Bellaye, P;Beltramo, G;Garrido, C;Bonniaud, P;Goirand, F;
| DOI: 10.1016/j.rmr.2022.11.068

Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal disease of unknown aetiology. In France, it ranks among the most frequent interstitial pathologies and affects 6 out of 8 people per 100,000 each year. IPF is characterized by dysregulated healing mechanisms that leads to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. Nintedanib and Pirfenidone are the only currently available treatments even though they are only able to slow down the disease without being curative. In this context, inhibiting HSPB5, a low molecular weight heat shock protein known to be involved in the development of fibrosis, could constitute a potential therapeutic target. Our aim consist to explore how NCI-41356 (a chemical inhibitor of HSPB5) can limit the development of pulmonary fibrosis. Methods In vivo, fibrosis was assessed in mice injected intratracheally (i.t.) with Bleomycin (BLM) and treated with NaCl or NCI-41356 (3 times i.t. or 3 times a week i.v.). Fibrosis was evaluated by collagen quantification (Sircol, Sirius Red staining), Immunofluorescence, TGF-β gene expression (RNAscope). In vitro, TGF-β1 signaling was evaluated in epithelial cells treated by TGF-β1 with or without NCI-41356 (Western Blot, Immunofluorescence, Proximity ligation assay). Results In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and several pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 explaining NCI-41356 anti-fibrotic properties. Conclusion In this study, we determined that inhibition of HSPB5/SMAD4 could limit IPF in mice. NCI-41356 modulates SMAD4 nuclear translocation thus limiting TGF-β1 signaling and synthesis of collagen and pro-fibrotic markers. Further investigations with human fibrotic lung tissues are needed to determine if these results can be transposed in human.

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-β1 Signaling

Pharmaceuticals

2023 Jan 24

Tanguy, J;Boutanquoi, P;Burgy, O;Dondaine, L;Beltramo, G;Uyanik, B;Garrido, C;Bonniaud, P;Bellaye, P;Goirand, F;
| DOI: 10.3390/ph16020177

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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