Targeting CXADR-mediated AKT signaling suppresses tumorigenesis and enhances chemotherapy efficacy in Ewing sarcoma

Background: 

Ewing sarcoma (EwS) is a malignant cancer that mainly affects children and young adults. Though it primarily targets bone and soft-tissue, distant metastasis is the main cause of death. The main drivers for this metastasis remain poorly understood, limiting the efficacy of current treatments. There is therefore a need to identify the genes that influence EwS progression and metastasis in order to develop new precise therapies.  

Metastasis in carcinomas is mediated by epithelial-to-mesenchymal transition (EMT). During this process, cells proliferate less and acquire a more migratory and invasive phenotype. In sarcomas, current literature suggests that tumor cells can switch to a more mesenchymal or epithelial phenotype depending on internal or external signals. One of the critical pathways that these activation signals rely on is AKT.  

In the preprint highlighted here, the authors propose that Coxsackievirus and Adenovirus receptor (CXADR) is an activator of AKT signaling in EwS. This viral receptor has several roles as a component of the tight junction complex. With extracellular, transmembrane and intracellular domains, CXADR is responsible for cell-cell adhesion and interactions with other signaling proteins. In this study, the authors report that CXADR inhibition in EwS reduces AKT signaling, leading to reduced tumorigenic features and metastatic phenotypes both in vivo and in vitro. Considering that high CXADR levels has also been linked to increased metastasis and poor outcomes in EwS patients, coupling of current treatment regimes with AKT inhibition may provide as a promising new combination therapy based on patient-specific CXADR expression. 

Key Findings: 

Three genes, including CXADR, were associated with worst patient survival and metastasis 

To determine clinically relevant genes in EwS that promote metastasis, the authors first performed differential expression analysis on RNAseq data of EwS tumors from patients displaying best and worst outcomes. From an initial 369 differentially expressed genes, the authors filtered against a second, independent EwS cohort before GO enrichment analysis; three genes, namely ANKLE1, BRSK2 and CXADR, emerged as being associated with worst patient survival and metastasis. Due to significantly higher expression found in both EwS cell lines and in metastatic patients, CXADR was prioritized. 

CXADR knockdown shows promising results both in vitro and in vivo 

To examine the role CXADR might play in EwS, the authors performed various in vitro and in vivo experiments. Through siPOOL and doxycycline (DOX)-inducible shRNA gene silencing, CXADR knockdown (KD) in MHH-ES-1 and SK-N-MC cell lines resulted in significant inhibition of cell proliferation. Excitingly, when MHH-ES-1 and SK-N-MC cells carrying 2 different DOX-inducible shRNAs against CXADR were injected into NSG mice, a significant reduction in local tumor weight and growth was seen, as well as increased necrotic area and overall number of apoptotic cells. Orthotopic injection of KD cells in mice also showed a remarkable decrease in tumor growth at the primary leg location, inhibiting metastatic spread to distant organs. 

CXADR may promote EMT through PI3K-AKT signaling with PTEN 

The authors next sought to understand how CXADR levels mediate the observed phenotype. For this, MHH-ES-1 and SK-N-MC cells carrying shRNA-mediated DOX-inducible CXADR KD were subjected to RNAseq and subsequent fGSEA analyses, revealing an association between ‘epithelial-mesenchymal-transition’ (EMT) and high CXADR expression. Furthermore, 5 significantly regulated PI3K-AKT signaling gene sets were shared between the cell lines upon CXADR KD.  This suggests that CXADR may promote EMT via AKT pathway activation. Concordantly, western blot analysis of CXADR silencing over time corroborated decreased pAKT protein levels and inactivation of other downstream targets like GSK3β. Although little is known about how CXADR regulates AKT, co-IP of CXADR in MHH-ES-1 and SK-N-MC cells confirmed PTEN as a co-complex partner. Importantly, the authors also found CXADR expression to be significantly correlated to a more active AKT signature in their second EwS patient cohort.   

Role of CXADR in EMT-like processes holds clinical relevance 

Finally, the authors probed if CXADR regulation of the AKT pathway is clinically valid using Capivasertib, a pan-AKT inhibitor approved for metastatic breast cancer. Interestingly, both drug-response and clonogenic growth assays in various EwS cell lines, A-673, SK-ES-1, MHH-ES-1 and SK-N-MC, all revealed CXADR levels to be a determinant factor of treatment efficacy, with higher CXADR expression associated with better response to Capivasertib. This effect can be enhanced 2-3-fold via combination therapy with Vincristine, as tested in long-term clonogenic growth assays in both MHH-ES-1 and SK-N-MC cell lines. All in all, these results support the role of CXADR in EMT-like processes through PI3K-AKT signaling with PTEN.  

What we like about this preprint: 

Current treatment strategies for Ewing sarcoma patients with metastatic disease are highly toxic and lead to poor outcomes. Therefore, precision oncology approaches provide an opportunity for patients to receive personalized treatments and improve said outcomes. This preprinted study identified a clinically relevant gene candidate through the analysis of EwS patients displaying extreme outcomes (best outcome versus worst outcome). The authors used a variety of available resources such as: i) target identification using data from the Ewing Sarcoma Cell Line Atlas (ESCLA) project, ii) hypothesis testing using CXADR knockdowns and iii) translational exploration using a clinically approved therapy already on the market. By using a combination of in vivo and in vitro experiments, the authors present an encouraging biomarker for patient stratification and a therapeutic target that can be incorporated in current EwS standard of care. This preprint presents a powerful example of bench-to-bedside research strategies.   

Questions for the authors: 

1. While it was unexpectedly discovered through in vitro drug response assays, do the authors have a hypothesis on why Capivasertib showed a response relative to the level of CXADR expression? How would this finding further affect patient stratification strategies for different EwS outcomes and cases?
2. It is exciting that combination therapy of Vincristine with Capivasertib can lead to increased inhibitory effects in tested cells that is possibly explained by simultaneous disruption on PI3K-AKT signaling and mitosis. However, do the authors have a hypothesis on why the combination of Doxorubicin and Capivasertib showed little to no synergistic impact on all the cell lines tested? Additionally, did they consider cyclophosphamide as another combination therapy agent?
3. Overall, the authors indeed provide a useful blueprint showcasing the impact data integration can have on personalized medicine research. To that same effect, what are the authors’ thoughts on integrating proteomics approaches to complement their transcriptomics data reported from tumors and gain more insights from protein-level expression of CXADR in EwS patients?

Tags: ewing sarcoma, pediatric cancer

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