Alveolar rhabdomyosarcoma–associated PAX3-FOXO1 promotes tumorigenesis via Hippo pathway suppression

Abstract

Alveolar rhabdomyosarcoma (aRMS) is an aggressive sarcoma of skeletal muscle characterized by expression of the paired box 3-forkhead box protein O1 (PAX3-FOXO1) fusion oncogene. Despite its discovery nearly two decades ago, the mechanisms by which PAX3-FOXO1 drives tumor development are not well characterized. Previously, we reported that PAX3-FOXO1 supports aRMS initiation by enabling bypass of cellular senescence checkpoints. We have now found that this bypass occurs in part through PAX3-FOXO1-mediated upregulation of RASSF4, a Ras-association domain family (RASSF) member. RASSF4 expression was upregulated in PAX3-FOXO1-positive aRMS cell lines and tumors. Enhanced RASSF4 expression promoted cell cycle progression, senescence evasion, and tumorigenesis through inhibition of the Hippo pathway tumor suppressor MST1. We also found that the downstream Hippo pathway target Yes-associated protein 1 (YAP), which is ordinarily restrained by Hippo signaling, was upregulated in RMS tumors. These data suggest that Hippo pathway dysfunction promotes RMS. This work provides evidence for Hippo pathway suppression in aRMS and demonstrates a progrowth role for RASSF4. Additionally, we identify a mechanism used by PAX3-FOXO1 to inhibit MST1 signaling and promote tumorigenesis in aRMS. Introduction Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Over the past 30 years, clinical trials in North America, Europe, and Australia have identified superior treatment strategies leading to the improved survival of discrete groups of RMS patients. A distinctly worse outcome is encountered for patients with the alveolar histologic variant of RMS (aRMS), who have a 5-year survival rate of less than 50% (1). Even more dismal is the survival for those whose tumors express the signature paired box 3-forkhead box protein O1 (PAX3-FOXO1) fusion gene; in the metastatic setting, their survival rate at 4 years is less than 10% (2). Although PAX3-FOXO1 (and the related fusion protein PAX7-FOXO1) was identified in the 1990s (3-5) and intensely studied in terms of its regulation, downstream targets, and cellular phenotypic effects, a unified understanding of how the fusion gene and its resulting oncoprotein contribute so profoundly to aRMS tumorigenesis remains obscure.

Deregulation of the Hippo pathway in soft-tissue sarcoma promotes FOXM1 expression and tumorigenesis

“…Both genetic and pharmacologic inhibition of YAP/TEAD and FOXM1 result in sarcoma cell proliferation defects, suggesting that these targets represent promising therapeutic interventions for mesenchymal tumors. Very recent studies have shown that YAP overexpression and function plays a role in the pediatric malignancies alveolar embryonal rhabdomyosarcoma (aRMS) and embryonal rhabdomyosarcoma (eRMS) (23,24). Together with these findings, we conclude that the Hippo pathway is potentially important in multiple sarcoma subtypes.…”

Netrin-1 exerts oncogenic activities through enhancing Yes-associated protein stability

“…Imaginably, the cancer cells can secrete the intracellular netrin-1 and activate the receptors on the plasma membrane via autocrine, leading to YAP signaling regulation. It is also reported that YAP activity in modulation of proliferation and senescence is regulated by Ras association domain family 1A (RASSF1A) (28) and RASSF4 (29), respectively. The RASSF family of proteins consists of 10 members, most of which are considered as tumor suppressor proteins that undergo loss of expression through promoter methylation in various types of cancers.…”

RASSF tumor suppressor gene family: Biological functions and regulation

“…Required for apoptosis and growth inhibition [8] Inhibits MST2 activity [93] Modulates the MAP kinase signal downstream of the Ras signal [77] K-Ras [8] ST1, MST2 [177] MST1 [75] Not available 5A (Nore1A) 1q32 47.1 kDa Suppresses tumour growth via apoptosis induction or cell cycle delay [7,82] Regulates microtubule formation [182] Induces degradation of HIPK1 oncoprotein [183] Required for the TNFa mediated apoptosis and full activation of MST1 [80] Ras, Carma1 [182,184] MST1 [43,177] MST2 [177] tubulin, Aurora A [182] Mdm2 [183] Itch [185] Yes [80] resistant to TNFa-induced apoptosis, fail to activate Mst1 in vivo 5C (Nore1B, RAPL) 1q32 30.4 kDa…”

“…Current studies demonstrate that RASSF4 is broadly expressed in normal tissues [8,73,74] and the reduction of RASSF4 expression due to promoter specific hypermethylation was detected in tumor cell lines and primary tumors [8,73,74] suggesting that RASSF4 might act as a tumor suppressor. However, a recent paper revealed a pro-growth role for RASSF4 in alveolar rhabdomyosarcoma (aRMS) [75]. One of the subtypes of aRMS is characterized by paired box-3-forkhead box protein O1 (PAX3-FOXO1) -a chimeric protein, arising as a result of chromosomal translocation.…”

“…A further study revealed suppressed Hippo signaling in aRMS, that was reflected in high expression level of YAP (Yes-associated protein 1). Although attempts to find a clear cause and effect relationship between YAP and RASSF4 biology failed, YAP was shown clearly to be involved in senescence regulation [75]. Interestingly, earlier studies have shown that RASSF4 associated directly with activated K-Ras and induced cell death in 293T embryonic kidney and MCF-7 breast cancer cells [8], indicating a tumor suppressor role of RASSF4.…”