Dataset Information

Phosphoproteomics identifies potential downstream targets of the integrin α2β1 inhibitor BTT-3033 in prostate stromal cells.

ABSTRACT:

Background

Integrin α2β1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide) was recently reported to inhibit neurogenic and thromboxane A2-induced human prostate smooth muscle contraction, and thus represents a target with a different inhibition spectrum than that of α1-blockers in benign prostate hyperplasia (BPH) treatments. Clarifying the underlying mechanisms of the inhibition effects will provide insights into the role of integrin α2β1 in prostate contraction and enable new intracellular targets for smooth muscle contraction to be explored.

Methods

ProteomeHD was used to predict and enrich the top co-regulated proteins of integrin α2 (ITGA2). A phosphoproteomic analysis was conducted on human prostate stromal cells (WPMY-1) treated with 1 or 10 µM of BTT-3033 or solvent for controls. A clustering analysis was conducted to identify the intracellular targets that were inhibited in a dose-dependent manner. Gene ontology (GO) and annotation enrichments were conducted to examine any functional alterations and identify possible downstream targets. A Kinase-substrate enrichment analysis (KSEA) was conducted to identify kinases-substrate relationships.

Results

Enrichments of the actin cytoskeleton and guanosine triphosphatases (GTPases) signaling were predicted from the co-regulated proteins with ITGA2. LIM domain kinases, including LIM domain and actin-binding 1 (LIMA1), zyxin (ZYX), and thyroid receptor-interacting protein 6 (TRIP6), which are functionally associated with focal adhesions and the cytoskeleton, were present in the clusters with dose-dependent phosphorylation inhibition pattern. 15 substrates were dose-dependently inhibited according to the KSEA, including polo-like kinase 1 (PLK1), and GTPases signaling proteins, such as disheveled segment polarity protein 2 (DVL2).

Conclusions

In this study, we proposed that the mechanisms underlying the contractile and proliferative effects of integrin α2β1 are the LIM domain kinases, including the ZYX family, and substrates, including PLK1 and DVL2.

SUBMITTER: Li B

PROVIDER: S-EPMC8506561 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Publications

Phosphoproteomics identifies potential downstream targets of the integrin α2β1 inhibitor BTT-3033 in prostate stromal cells.

Li Bingsheng B Li Pan P Xia Weiping W You Baiyang B Yu Qingfeng Q Zhang Bo B Huang Ru R Wang Ruixiao R Liu Yuhan Y Chen Zhi Z Gan Yu Y He Yao Y Hennenberg Martin M Stief Christian G CG Chen Xiang X

Annals of translational medicine 20210901 17

<h4>Background</h4>Integrin α2β1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide) was recently reported to inhibit neurogenic and thromboxane A2-induced human prostate smooth muscle contraction, and thus represents a target with a different inhibition spectrum than that of α1-blockers in benign prostate hyperplasia (BPH) treatments. Clarifying the underlying mechanisms of the inhibition effects will provide insights into the rol ...[more]

PMID: 34733932

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Project description:BackgroundThe P21 activated kinases (PAK) are frequently dysregulated in cancer and have central roles in oncogenic signalling, prompting the development of PAK inhibitors (PAKi) as anticancer agents. However, such compounds have not reached clinical use because, at least partially, there is a limited mechanistic understanding of their mode of action. Here, we aimed to characterize functional and molecular responses to PAKi (PF-3758309, FRAX-486 and IPA-3) in multiple acute myeloid leukaemia (AML) models to gain insights on the biochemical pathways affected by these inhibitors in this disease and identify determinants of response in patient samples.MethodsWe mined phosphoproteomic datasets of primary AML, and used proteomics and phosphoproteomics to profile PAKi impact in immortalized (P31/Fuj and MV4-11), and primary AML cells from 8 AML patients. These omics datasets were integrated with gene dependency data to identify which proteins targeted by PAKi are necessary for the proliferation of AML. We studied the effect PAKi on cell cycle progression, proliferation, differentiation and apoptosis. Finally, we used phosphoproteomics data as input for machine learning models that predicted ex vivo response in two independent datasets of primary AML cells (with 36 and 50 cases, respectively) to PF-3758309 and identify markers of response.ResultsWe found that PAK1 activation- measured from phosphoproteomics data- was predictive of poor prognosis in primary AML cases. PF-3758309 was the most effective PAKi in reducing proliferation and inducing apoptosis in AML cell lines. In cell lines and primary cells, PF-3758309 inhibited PAK, AMPK and PKCA activities, reduced c-MYC transcriptional activity and the expression of ribosomal proteins, and targeted the FLT3 pathway in FLT3-ITD mutated cells. In primary cells, PF-3758309 reduced STAT5 phosphorylation at Tyr699. Functionally, PF-3758309 reduced cell-growth, induced apoptosis, blocked cell cycle progression and promoted differentiation in a model-dependent manner. ML modelling accurately classified primary AML samples as sensitive or resistant to PF-3758309 ex vivo treatment, and highlighted PHF2 phosphorylation at Ser705 as a robust response biomarker.ConclusionsIn summary, our data define the proteomic, molecular and functional responses of primary and immortalised AML cells to PF-3758309 and suggest a route to personalise AML treatments based on PAK inhibitors.

Dataset Information

Phosphoproteomics identifies potential downstream targets of the integrin α2β1 inhibitor BTT-3033 in prostate stromal cells.

Background

Methods

Results

Conclusions

Publications

Phosphoproteomics identifies potential downstream targets of the integrin α2β1 inhibitor BTT-3033 in prostate stromal cells.

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