Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

Abstract

Background: Increased cell mobility is a signature when tumor cells undergo epithelial-to-mesenchymal transition. TGF-β is a key stimulating factor to promote the transcription of a variety of downstream genes to accelerate cancer progression and metastasis, including osteopontin (OPN) which exists in several functional forms as different splicing variants. In non-small cell lung cancer cells, although increased total OPN expression was observed under various EMT conditions, the exact constitution and the underlining mechanism towards the generation of such OPN splicing isoforms was poorly understood.

Methods: We investigated the possible mechanisms of osteopontin splicing variant and its role in EMT and cancer metastasis using NSCLC cell line and cell and molecular biology techniques.

Results: In this study, we determined that OPNc, an exon 4 excluded shorter form of Opn gene products, appeared to be more potent to promote cell invasion. The expression of OPNc was selectively increased to higher abundance during EMT following TGF-β induction. The switching from OPNa to OPNc could be enhanced by RUNX2 (a transcription factor that recognizes the Opn gene promoter) overexpression, but appeared to be strictly in a HDAC dependent manner in A549 cells. The results suggested the increase of minor splicing variant of OPNc required both (1) the enhanced transcription from its coding gene driven by specific transcription factors; and (2) the simultaneous modulation or fluctuation of the coupled splicing process that depends to selective classed of epigenetic regulators, predominately HDAC family members.

Conclusion: Our study not only emphasized the importance of splicing variant for its role in EMT and cancer metastasis, but also helped to understand the possible mechanisms of the epigenetic controls for defining the levels and kinetic of gene splicing isoforms and their generations.

Keywords: EMT; HDAC; OPN; RUNX2; Splicing.

Fig. 1

Upregulation of RUNX2 associated with increased OPN expression during the EMT progression in TGF-β treated A549 cells. a TGF-β treatments for 48 h increased cell migration. b TGF-β treatments induced significant changes in the expression of major EMT marker genes at both mRNA (left) and protein (right) levels. c The mRNA (upper) and protein (lower) expression of OPN and RUNX2 were synchronically increased following TGF-β treatments

Fig. 2

RUNX2 overexpression enhanced the expression of OPN, especially of splicing isoform c. a Schematic illustration of isoform-specific primers used for the quantitative analyses of OPN-SIs. b TGF-β induction increased the opn transcript levels unproportionally among different splicing isoforms. c TGF-β preferentially promoted OPNc splicing in A549 cells. d Western blot of RUNX2 in A549 cells transfected with an overexpression plasmid. e Overexpression of RUNX2 altered the OPN-SIs and OPNt mRNA levels. f RUNX2 overexpression selectively increased OPNc transcript levels. g Knockdown of RUNX2 expression attenuated TGF-β induced opn expression in A549 cells

Fig. 3

Knock down of SRSF1 reduced mRNA levels of OPN-SIs. a RNAi of SRSF1 decreased OPNc levels in A549 cells. b Inhibition of OPNb and OPNc splicing from reporter assays following SRSF1 siRNA transfection. c Western blot of SRSF1 in A549 cells transfected with targeted siRNAs

Fig. 4

RUNX2 dependent OPNc splicing required normal activities of HDAC1 or HDAC2. a Treatment of A549 cells with HDACs inhibitor TSA suppressed OPNc splicing induced by RUNX2 overexpression. b Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells following TSA treatment. c Inhibition of HDAC1 activity by NaB deprived RUNX2 induced OPNc splicing. d Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells treated with NaB. e Knockdown of HDAC1 or HDAC2, but not HDAC3, decreased RUNX2-induced OPNc splicing. f Western blots of HDAC1, HDAC2 and HDAC3 from A549 cells transfected with the targeted siRNAs

Fig. 5

OPNc overexpression was most potent to induce an invasive phenotype in A549 cells among OPN-SIs. a Overexpression of OPNb and OPNc significantly increased the invasiveness of transfected cells. b Overexpression of OPN-SIs promoted cell mobility and migration. c Immunoblots of EMT marker proteins in cells with overexpression of OPN-SIs

Fig. 6

TGF-β induced OPNc expression enhanced the mobility of SK-MES-1 cells with a dependence to HDACs. a TGF-β treatments for 48 h increased the invasiveness of SK-MES-1 cells. b TGF-β induction increased OPNt and OPN-SIs levels with a preference to OPNc. c TGF-β promoted OPNc splicing with most significance in SK-MES-1 cells. d Knockdown of HDAC1 or HDAC2 significantly decreased the RUNX2-induced OPNc splicing. e The HDAC1 and HDAC2 expression were markedly reduced in the SK-MES-1 cells transfected with the targeted siRNAs. f Overexpression of OPN-SIs significantly promoted the migration of SK-MES-1 cells

Fig. 7

OPNc expression in association with clinical features of NSCLC patients. a Demographic information of collected NSCLC tissues and statistics from quantitative PCR analyses of OPNc expression. b Overall survival of patients with NSCLC cancer in OPNc high and OPNc low groups