Impairment of cell adhesion and migration by inhibition of protein disulphide isomerases in three breast cancer cell lines.

Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified 'scratch wound' assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3-/- mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3-/- MEFs was less effective in promoting cell spreading and F-actin organisation or supporting 'scratch wound' closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression.

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE(+) and SopE(-) S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH-gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA-gfp reporter mirrored that of PprgH-gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.