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A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation.

ABSTRACT: Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRAS^G12V exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRAS^G12V-expressing cells. This represents an applicable approach to explore protein-protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells.

SUBMITTER: Wang LT

PROVIDER: S-EPMC8610992 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation.

Wang Li-Ting LT Proulx Marie-Ève MÈ Kim Anne D AD Lelarge Virginie V McCaffrey Luke L

Scientific reports 20211123 1

Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally assoc ...[more]

PMID: 34815476

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Project description:Chondrosarcomas represent a heterogeneous group of primary bone cancers that are characterized by hyaline cartilaginous neoplastic tissue and are predominantly resistant to radiation and chemotherapy. However, adjuvant radiotherapy is often recommended in inoperable cases or after incomplete resections. To improve the efficiency of treatment, the present study tested a combination therapy with ionizing radiation (IR) and the proteasome inhibitor bortezomib. Using a three-dimensional (3D) spheroid model, 0-20 Gy of IR was applied to chondrosarcoma cells and healthy human chondrocytes. Following combined treatment with IR and bortezomib, the cell cycle distribution, apoptotic induction, the survivin pathway, autophagy and DNA damage were evaluated. Both cell types exhibited a slight decrease in viability following increasing doses of IR; the chondrosarcoma cells demonstrated a significant dose-dependent increase in the expression levels of the DNA damage marker histone H2AX phosphorylation at serine 139 (γH2AX). The combination treatment with bortezomib significantly decreased the cell viability after 48 h compared with that in irradiated cells. High-dose IR induced a G2/M phase arrest, which was accompanied by a decrease in the number of cells at the G1 and S phase. Co-treatment with bortezomib changed the distribution of the cell cycle phases. The mRNA expression levels of the proapoptotic genes Bcl-2-associated X protein (Bax) and Bak were significantly increased by bortezomib treatment and combination therapy with IR. In addition, the combination therapy resulted in a synergistic decrease of the expression levels of survivin and its corresponding downstream pathway molecules, including heat shock protein 90, X-linked inhibitor of apoptosis protein, smad 2 and smad 3. Comparative analyses of γH2AX at 1 and 24 h post-IR revealed efficient DNA repair in human chondrosarcoma cells. Therefore, additional bortezomib treatment may only temporarily improve the radiation sensitivity of chondrosarcoma cells. However, the inhibition of the survivin pathway by the combined treatment with IR and bortezomib, observed in the present study, revealed a novel aspect in the tumor biology of chondrosarcoma 3D spheroid cultures and may represent a potential target for therapy.

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A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation.

Publications

A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation.

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