FAK Signaling in Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children and adolescents. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 is at high risk. The fusion negative subgroup, FN-RMS, has a good prognosis when non-metastatic. Despite a multimodal therapeutic approach, FP-RMS and metastatic FN-RMS often show a dismal prognosis with 5-year survival of less than 30%. Therefore, novel targets need to be discovered to develop therapies that halt tumor progression, reducing long-term side effects in young patients. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates focal contacts at the cellular edges. It plays a role in cell motility, survival, and proliferation in response to integrin and growth factor receptors' activation. FAK is often dysregulated in cancer, being upregulated and/or overactivated in several adult and pediatric tumor types. In RMS, both in vitro and preclinical studies point to a role of FAK in tumor cell motility/invasion and proliferation, which is inhibited by FAK inhibitors. In this review, we summarize the data on FAK expression and modulation in RMS. Moreover, we give an overview of the approaches to inhibit FAK in both preclinical and clinical cancer settings.

Keywords: FAK; FAK inhibitors; cell invasion; cell migration; focal adhesion complex; kinase; myogenesis; rhabdomyosarcoma; sarcoma.

Figure 1

Schematic representation of focal adhesion kinase (FAK). The three main domains of FAK are depicted: The N-terminal 4.1, ezrin, radixin, moesin homology domain (FERM) (in blue), central kinase domain (in green), and focal-adhesion targeting (FAT) domain (in yellow). Domain boundaries are shown. F1, F2, and F3 represent the three lobes of the FERM domain and the regions for binding with transcription factors (such as GATA4 and NANOG) and E3 ubiquitin ligase factors (such as CHIP). Other binding sites are those for MDM2 (E3 ubiquitin ligase), MBD2 (methyl-CpG binding domain protein 2, an epigenetic factor) and MEF2 (transcription factor). Outside the FERM domain the p53-binding site is shown. A nuclear localization sequence (NLS) is located at the F2 FERM lobe, while two nuclear export sequences (NES) are at the F1 FERM lobe and in the kinase domain. PRR1, 2, and 3 represent proline-rich regions (i.e., two polyproline (PxxP) motifs) that interact with the Src homology SH3 domains of several proteins, including Src. Several phosphorylation sites are depicted, among which include Y397, the autophosphorylation site, and Y925, both binding sites for Src. N: N-terminus. C: C-terminus.

Figure 2

Schematic representation of FAK involvement in tumor growth and metastasis. (a) FAK is autophosphorylated in response to growth factor receptors and integrins activation and activated by Src. (b) Active FAK promotes tumor cell invasion and metastasis, activating PI3K-AKT-mTOR signaling cascade, which results in increased NFkB transcriptional activity. (c) Active FAK also stimulates cytoskeletal remodeling and focal adhesion formation/turnover, inducing SRC-dependent phosphorylation of paxillin and p130cas, leading to the formation of a focal adhesion complex which includes phosphorylated/active FAK, paxillin, and p130cas. SRC also stimulates ERK signaling cascade which results in the ETS transcription factor-dependent induction of cyclin D1 (CycD1) expression which in turn promotes tumor cell survival and growth. (d) Nuclear FAK acts as a scaffold protein for the p53–MDM2 interaction, inducing p53 ubiquitination and its proteasomal degradation which results in apoptosis inhibition. Figure realized with BioRender.com.

Figure 3

FAK regulation during myoblasts differentiation. The expression of FAK and its phosphorylation are related to specific points in the differentiation of myoblasts into myotubes. FAK is activated by growth factor receptors and regulates the development of myoblasts and the formation of muscle fibers. During the proliferation phase, the activation of growth factor receptors leads to FAK phosphorylation/activation, needed for MYOD expression, while the expression of MYOG is blocked by the binding of MBD2 to its promoter. In the early differentiation phase, phosphorylated-FAK fraction decreases and FAK cytoplasmic interaction with MBD2 promotes the translocation of the FAK/MBD2 complex into the nucleus, where MBD2 interaction with the MYOG promoter is prevented, leading to MYOG expression. During myotubes formation, FAK levels increase, even if not at the same level as in the proliferative phase. In this terminal phase, characterized by MyHC expression, integrins activation induces multiple pathways such as PI3K/AKT/mTOR which crosstalk with FAK to control protein synthesis and the size of the muscle fibers. Figure realized with BioRender.com.

Figure 4

FAK involvement in rhabdomyosarcoma. (A) FAK is phosphorylated and activated upon binding of growth factors to the RTK receptors and integrins activation. Phosphorylated FAK promotes cell growth and protein synthesis and blocks apoptosis through activation of PI3K-AKT-mTOR signaling. The FAK–Src complex (i) induces the ERK1/2 pathway, leading to the reduction of phosphorylated TSC2 which results in increased mTOR activity; and (ii) blocks TSC2 directly. (B) In rhabdomyosarcoma, MET, which is a transcriptional target of PAX3-FOXO1 (P3F), is overexpressed and induces signal cascades, promoting motility and invasion also through FAK activation. In these tumor cells, the “MyomiRs” miR-1/miR-206 are downregulated and when forcedly expressed, they target MET mRNA and block its translation, resulting in a block of invasion and motility. Figure realized with BioRender.com.