PMC

Examples of human MM in tissue microarrays co-stained using antibodies for SV40 T-antigen (blue/nuclear) and CD44 (red). Of the 34 MMs in the array, approximately 50% were SV40+, and of these SV40+ tumors (~100%) were CD44-. Of the 17 SV40- tumors, approximately 53% were CD44+ (all CD44+ tumors in the tissue array). A. Sample panel of SV40-, CD44+ tumors. B. Sample panel of SV40+, CD44- tumors. The arrow indicates nuclear SV40 T-antigen staining.

Immunofluorescence image shows greater uptake of BODIPY FL hyaluronic acid by MMs with higher Fra-1 expression levels (SV40-) (A). Internalization of the BODIPY FL HA probe (green) was visualized by fluorescence confocal microscopy. This internalization is reduced in the SV40+ line with low basal Fra-1 (red) expression (B) or those with high basal Fra-1 expression after Fra-1 knock-down (right panel).

Fra-1 and CD44 are critical to MM cell migration. Wound assays showed that in the SV40- cell lines MM1 (panel A) or MM6 cell line (panel B) shFra-1 and shCD44 transfected cells showed less migration than empty vector (EV) controls. * = P ≤ 0.05 in comparison to 24 h controls.

MMs with high basal Fra-1 expression have also higher CD44 expression. Western blots show constitutive levels of Fra-1 (three bands show different levels of phosphorylation), CD44, and levels of α-Tubulin (control for protein loading) in confluent cells maintained for 24 h in serumless medium or after 4 h with the addition of 10% FBS (columns +).

Tissue microarray slide showing that Fra-1 staining (intensity score of 1–3 compared to 0 as observed in 3 normal lungs) is expressed in the majority of human mesotheliomas (MM), (42/43). Images in A and B show immunostaining of Fra-1 (red) in the upper panel. Bottom panels show merged images of Fra-1, p-ERK1/2 and nuclear staining (SYTOX green).

Use of shRNA constructs to knock-down Fra-1 confirm Fra-1-dependent CD44 expression in MMs. A. Western blots of SV40- (MM1) and SV40+ (MM3) lines showing empty vector (EV) controls or RNAi shFra-1 constructs. B. Immunofluorescence image showing CD44 (red) levels MM cell lines. C. Agarose gels on an SV40- cell line showing the efficiency of the RNAi constructs (shFra-1 and shCD44) on knockdown of CD44 and Fra-1 measured by a semi-quantitative RT-PCR reaction and using GAPDH as a control.

CD44 expression is dependent on Fra-1 and the pathways that control Fra-1 expression. (A) Western blot showing the effect of the PI3K and Src inhibitors in the CD44 expression of an SV40+ and SV40- cell line. (B) Western blots showing that CD44 expression in the SV40- MM line follows the pattern of its Fra-1 expression. The use of a dominant negative construct for Fra-1 (dnFra-1) decreases CD44 expression, but not further decreases are observed after PD98059 or LY294002 is added to the dnFra-1 stable cell line.

ERK1/2, Src and PI3K regulation of Fra-1 expression in MM cell lines is tumor line-specific. A. RT-QPCR showing Src, ERK1/2, and PI3K-dependent decreases in Fra-1 in MM1, Src and ERK1/2-dependent decreases of Fra-1 in MM2, and PI3K-dependent decreases in Fra-1 in lines MM1, MM3 and MM4. B. Luciferase assays show PI3K-dependent Fra-1 promoter activation in MM3 cells. C. Western blot analysis of Fra-1 shows inhibition by the PI3K inhibitor, LY294002 at 20 μM. D. EMSA shows a dose dependent decrease in Fra-1 in the AP-1 complex after treatment of the MM3 line with the PI3K inhibitor, LY294002 at 10 and 20 μM * = P ≤ 0.05 in comparison to untreated control (0).