Integrins are heterodimeric transmembrane proteins composed of alpha and beta subunits that mediate cell-cell and cell-extracellular matrix (ECM) interactions. Several integrins are overexpressed in human cancers and their ECM recognition motif, arginine-glycine-aspartate (RGD), is being utilized for tumour imaging and targeting.
To explore the expression and function of RGD-binding integrins in corticotroph tumours.
We determined the expression of RGD-binding integrins by qPCR in 18 corticotroph tumours and compared transcript levels with gonadotroph tumours (n = 16) and normal pituitaries (n = 2). To study the role of integrins, we established their expression profile in murine corticotroph tumour AtT-20 cells by RT-PCR and investigated the effect of their inhibition with RNA interference on human POMC promoter activity and cell viability (WST1 colorimetric assay). We used fluorescence microscopy to assess RGD peptide binding in these cells.
Corticotroph tumours express αv (ITGAV), β1 (ITGB1), β5 (ITGB5), β8 (ITGB8), and α8 (ITGA8). Integrins αv, β1, β5 are overexpressed in corticotroph compared to gonadotroph tumours, where expression was almost undetectable (P < 0.0001) and human normal pituitary (P < 0.001). The expression of β8 was higher in corticotroph only compared to gonadotroph tumours (P = 0.04), but not to the normal pituitary. We found that AtT-20 cells express all these four integrins. Knocking down each αv, β1, and β5, decreased human POMC promoter activity compared to scramble control (% suppression 63 ± 22, 54 ± 23, and 69 ± 28 respectively; P < 0.05), while β8 had little effect. Knocking down αv and β1 had a small but significant effect on AtT-20 cell viability (% suppression 15.92 ± 1.6 and 27.4 ± 1.4 respectively; P < 0.05). Using immunofluorescence, we observed that an RGD peptide conjugated with the near-infrared fluorophore cyanine-5.5 could bind to and label AtT20 cells, with no deleterious effects on AtT-20 cell viability (WST1 assay) and function (determined by POMC promoter activity).
This study shows that corticotroph tumours express the genes encoding the alpha and beta subunits of the RGD-binding integrins αvβ1, αvβ5, and αvβ8. We have preliminary evidence that these integrins may regulate POMC promoter activity. RGD peptide conjugates potential as corticotroph tumour imaging agents.
22 May 2021 - 26 May 2021