Liprin-α1 modulates cancer cell signaling by transmembrane protein CD82 in adhesive membrane domains linked to cytoskeleton

Abstract

Background: PPFIA1 is located at the 11q13 region commonly amplified in cancer. The protein liprin-α1 encoded by PPF1A1 contributes to the adhesive and invasive structures of cytoskeletal elements and is located at the invadosomes in cancer cells. However, the precise mechanism of liprin-α1 function in cancer progression has remained elusive.

Methods: Invasion regulating activity of liprin-α1 was examined by analyzing the functions of squamous cell carcinoma of head and neck (HNSCC) cell lines in three-dimensional collagen I after RNAi mediated gene knockdown. Transcriptome profiling and Gene Set Enrichment Analysis from HNSCC and breast cancer cells were used to identify expression changes relevant to specific cellular localizations, biological processes and signaling pathways after PPFIA1 knockdown. The significance of the results was assessed by relevant statistical methods (Wald and Benjamini-Hochberg). Localization of proteins associated to liprin-α1 was studied by immunofluorescence in 2D and 3D conditions. The association of PPFIA1 amplification to HNSCC patient survival was explored using The Cancer Genome Atlas data.

Results: In this study, we show that liprin-α1 regulates biological processes related to membrane microdomains in breast carcinoma, as well as protein trafficking, cell-cell and cell-substrate contacts in HNSCC cell lines cultured in three-dimensional matrix. Importantly, we show that in all these cancer cells liprin-α1 knockdown leads to the upregulation of transmembrane protein CD82, which is a suppressor of metastasis in several solid tumors.

Conclusions: Our results provide novel information regarding the function of liprin-α1 in biological processes essential in cancer progression. The results reveal liprin-α1 as a novel regulator of CD82, linking liprin-α1 to the cancer cell invasion and metastasis pathways.

Keywords: Breast cancer; CD82; Head and neck cancer; Invasion; Liprin-α1; PPFIA1; RNA sequencing; Three-dimensional cell culture.

Fig. 1

UT-SCC-42B and UT-SCC-19B carcinoma cells were embedded in 3D collagen after shPPFIA1_69 or shScr (control) transduction and cultured for seven days. a Representative phase contrast light micrographs showed less cellular outgrowths and invasive phenotype in colonies transduced with shPPFIA1_69 compared to shScr control cells. b Quantification of cell invasive growth measured by relative area of the colonies; mean ± SEM; three collagen preparations/stable control (shScr) or knockdown (shPPFIA1_69) cell line. *P < 0.001, unpaired Student’s t-test. c Western blot confirmed the efficacy of liprin-α1 knockdown in UT-SCC-19B and UT-SCC-42B cell lines. d Quantification of relative colony and lumen area of UT-SCC-42B cells in 3D collagen with shScr and shPPFIA1_97; mean ± SEM; three collagen preparations/stable control (shScr) or knockdown (shPPFIA1_97) cell line. *P < 0.01, unpaired Student’s t-test. Scale bar 10 μm. e Lumen formation was diminished in liprin-α1 knockdown cell colonies compared to shScr cells as visualized by Z-stacks. f Representative confocal micrographs illustrated liprin-α1 (green) localization in the cytosol and at the cell-extracellular matrix contacts including focal adhesion-like cellular outgrowths. Phalloidin staining (filamentous actin, red) visualizes reduced growth and changes in cytoskeletal elements after liprin-α1 knockdown. g Confocal micrographs showed vinculin localization at the cell colonies. Scale bar 50 μm

Fig. 2

a Venn diagram showed a number of shared genes in UT-SCC-42A/B and MDA-MB-231 cell lines grown in 3D collagen I after liprin-α1 silencing. b Venn diagram illustrated the number of shared genes in UT-SCC-42A/B and MDA-MB-231 cell lines in 3D collagen I and in UT-SCC-24B cell line grown in 2D cell culture after liprin-α1 silencing. c Selected clusters from pathway analysis comparing liprin-α1 knockdown (shPPFIA1) cells to control cells (shScr). Ranking of the clusters was carried out by normalized enrichment scores (NES)

Fig. 3

a Heat map of top ranked differentially expressed genes in shPPFIA1 vs shScr MDA-MB-231 cells using RNA sequencing. Genes were ranked based on the p-value and fold change. Color coding from blue to red depicts gene expression differencies between shPPFIA1 and shScr cells from low to high expression. b Gene expression analysis showed upregulation of SORL1 and CD82 at mRNA level in MDA-MB-231 cell lines, and western blot validation showed upregulation of CD82 protein expression in breast and HNSCC cancer cell lines. SORL1 protein expression was increased in HNSCC cells after liprin-α1 knockdown from different biological replicates. Tubulin and vinculin were used as loading controls. Error bar was calculated as standard deviation from different constructs, and value was considered statistically significant when *P < 0,05 calculated as unpaired student’s t-test. c Western blot showed the upregulation of CD82 in breast and HNSCC cell line and SORL1 in HNSCC cell lines. d Immunofluorescence staining showed upregulation and localization of CD82 to intracellular vesicle-like structures or cell edge in MDA-MB-231 cells after liprin-α1 knockdown. CD82 was at the cell-cell contact sites and near the cell edge in shPPFIA1 UT-SCC-42B cell colonies grown in three-dimensional collagen I. Scale bar is 10 μm and magnification 63×

Fig. 4

a Immunofluorescence staining of CD82 and liprin-α1 in UT-SCC-42A cells cultured in 2D showed localization of CD82 in intracellular vesicle-like structures and at the cell membrane after liprin-α1 knockdown (shPPFIA1). Liprin-α1 was localized in invadosome structures or after the leading edge in shScr control cells. b Immunofluorescence staining of CD82 and liprin-α1 in UT-SCC-42A control (shScr) and liprin-α1 knockdown (shPPFIA1) cells in 3D culture. In shScr cell colonies, liprin-α1 was located either as cytosolic or on the edges of the cellular outgrowths in adhesion- or invadosome-like structures. ShPPFIA1 cells showed localization of CD82 mainly at the cell membrane, in cell-cell and in cell-extracellular matrix contacts. Scale bar is 10 μm, and magnification 63×