Focal adhesion kinase (FAK) phosphorylation is a key regulator of embryonal rhabdomyosarcoma (ERMS) cell viability and migration

Abstract

Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children. Pathogenesis of RMS is associated with aggressive growth pattern and increased risk of morbidity and mortality. There are two main subtypes or RMS: embryonal and alveolar. The embryonal type is characterized by distinct molecular aberrations, including alterations in the activity of certain protein kinases. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays a vital role in focal adhesion (FA) assembly to promote cytoskeleton dynamics and regulation of cell motility. It is regulated by multiple phosphorylation sites: tyrosine 397, Tyr 576/577, and Tyr 925. Tyrosine 397 is the autophosphorylation site that regulates FAK localization at the cell periphery to facilitate the assembly and formation of the FA complex. The kinase activity of FAK is mediated by the phosphorylation of Tyr 576/577 within the kinase domain activation loop. Aberrations of FAK phosphorylation have been linked to the pathogenesis of different types of cancers. In this regard, pY397 upregulation is linked to increase ERMS cell motility, invasion, and tumorigenesis.

Methods: In this study, we have used an established human embryonal muscle rhabdomyosarcoma cell line RD as a model to examine FAK phosphorylation profiles to characterize its role in the pathogenies of RMS.

Results: Our findings revealed a significant increase of FAK phosphorylation at pY397 in RD cells compared to control cells (hTERT). On the other hand, Tyr 576/577 phosphorylation levels in RD cells displayed a pronounced reduction. Our data showed that Y925 residue exhibited no detectable change. The in vitro analysis showed that the FAK inhibitor, PF-562271 led to G1 cell-cycle arrest induced cell death (IC₅₀, ~ 12 µM) compared to controls. Importantly, immunostaining analyses displayed a noticeable reduction of Y397 phosphorylation following PF-562271 treatment. Our data also showed that PF-562271 suppressed RD cell migration in a dose-dependent manner associated with a reduction in Y397 phosphorylation.

Conclusions: The data presented herein indicate that targeting FAK phosphorylation at distinct sites is a promising strategy in future treatment approaches for defined subgroups of rhabdomyosarcoma.

Keywords: Cell migration; Focal adhesion kinase (FAK); Phosphorylation; Rhabdomyosarcoma.

Fig. 1

Focal adhesion kinase (FAK) phosphorylation status in RD and hTERT cells. RD or hTERT cells were collected and analyzed by Western blot. Protein phosphorylation was detected using anti-phospho-specific antibodies (pY397, pY576/577, and pY925). Bands were quantified by scanning densitometry and normalized to total FAK protein and to the loading control. Values represent means ± SEM for n = 4 independent experiments. *Significantly different from the vehicle control (Student’s t test, p < 0.05)

Fig. 2

Immunocytochemistry of RD cells for pY397 FAK following treatment with PF-562271. RD cells fixed and stained with anti-pY397-FAK (red channel) to examine phosphorylation dynamics following vehicle control (DMSO) and PF-562271 (10 μM) (a). Cytoskeletal localization of pY397-FAK was detected by staining F-actin cytoskeleton with AlexaFluor488-phalloidin (green channel). b Higher resolution images were taken for pY397 following vehicle control (left panel) and PF-562271 (10 μM) (right panel). Arrows indicated signal localization in the nucleus and F-actin cytoskeleton. For each independent plate of cells, 10 random visual fields were acquired from each whole-well scan, and cells in 8 images were quantified from each field. Scale bars 30 μm

Fig. 3

PF-562271-induced cell cytotoxicity in RD cells. a Human rhabdomyosarcoma (RD) cell lines or hTERT cells were treated with increasing concentration of PF-562271 [1 × 10⁻⁴ to 100, μM] for 96 h. Cellular viability was calculated as (%) by comparing the absorbance ratio (percentage) of the treated cells normalized to the control (DMSO) treated cells. b Western-blot analysis of FAK-pY397 in treated RD cells with a concentration gradient of (1 nM–100 µM) of PF-562271. FAK phosphorylation signal was normalized to total β-actin loading control protein

Fig. 4

PF-562271 effects on RD cell cycle. DNA histogram of the RD cell cycle was analyzed using flow cytometry with PI staining following PF562271 [10 µM] or vehicle control (DMSO) treatments (a). Phases represent G1%, S%, and G2% phases were analyzed for both conditions (b)

Fig. 5

RD cell migration and FAK phosphorylation (pY397) were suppressed by FAK inhibition. Cell-migration assay was completed by incubating RD cells (7 × 10³) with increasing concentration of PF-562271 [0–25 µM] for 24 h. Cell migration was calculated and expressed as the percentage of number of migrated cells into the total number of cells. % Migration was normalized to vehicle control treatment (upper panel). FAK phosphorylation was analyzed by SDS-PAGE with anti-pY397-FAK and normalized to total β-actin loading control protein (lower panel). Values represent means ± SEM for n = 4 independent experiments. *Significantly different from the vehicle control (Student’s t test, p < 0.05)