Hydrophobic Phenoxazines Shut Down PI3K/Akt/mTOR/P70S6/S6 Kinase Signalling Pathway and Induces Massive Apoptosis in Rhabdomyosarcoma Cells

Akt plays an important role in many types of cancers and has been identified as a therapeutic target. Several types of cancers have posed a major threat to human health. Conventional treatments suffer from limitations of side effects, poor responses and drugresistance. Phenoxazines have shown diverse biological activities and promising agents in anti-cancer, anti-viral and antibacterial therapy. In this study, we evaluated the effect of phenoxazine derivatives on rhabdomyosarcoma cells. Hydrophobic phenoxazines shut down Akt/mTOR/p70S6/S6 kinase pathway and induce apoptosis in rhabdomyosarcoma cells. There is activation of Akt pathway in rhabdomyosarcoma cell lines which have tumorigenic potential. These cell lines are sensitive to phenoxazines. The phenoxazine derivatives are compared for their ability to inhibit Akt phosphorylation in these cells. The lipophilicity of these compounds increased significantly by increasing the chain length to (-CH2)5 or (-CH2)6 from the corresponding (-CH2)3 or (-CH2)4 at N10 -position of the phenoxazine ring. The ability of various phenoxazine derivatives to inhibit Akt phosphorylation in rhabdomyosarcoma cells follows the order: N10-hexyl > N10-pentyl > N10-butyl > N10-propyl. Within the series, -Cl in C-2 position on the phenoxazine ring demonstrated a higher potency compared to phenoxazines with –H in C-2 position, suggesting that chlorine is playing a critical role on the growth inhibition.

CircVAMP3: A circRNA with a Role in Alveolar Rhabdomyosarcoma Cell Cycle Progression

Circular RNAs (circRNAs), a class of covalently closed RNAs formed by a back-splicing reaction, have been involved in the regulation of diverse oncogenic processes. In this article we describe circVAMP3, a novel circular RNA overexpressed in RH4, a representative cell line of alveolar rhabdomyosarcoma. We demonstrated that circVAMP3 has a differential m6A pattern opposed to its linear counterpart, suggesting that the two isoforms can be differently regulated by such RNA modification. Moreover, we show how circVAMP3 depletion in alveolar rhabdomyosarcoma cells can impair cell cycle progression, through the alteration of the AKT-related pathways, pointing to this non-coding RNA as a novel regulator of the alveolar rhabdomyosarcoma progression and as a putative future therapeutic target.

Role for the Histone Demethylase KDM4B in Rhabdomyosarcoma via CDK6 and CCNA2: Compensation by KDM4A and Apoptotic Response of Targeting Both KDM4B and KDM4A

Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach.

The c-Myc/AKT1/TBX3 Axis Is Important to Target in the Treatment of Embryonal Rhabdomyosarcoma

Rhabdomyosarcoma is a highly aggressive malignant cancer that arises from skeletal muscle progenitor cells and is the third most common solid tumour in children. Despite significant advances, rhabdomyosarcoma still presents a therapeutic challenge, and while targeted therapy has shown promise, there are limited options because the molecular drivers of rhabdomyosarcoma are poorly understood. We previously reported that the T-box transcription factor 3 (TBX3), which has been identified as a druggable target in many cancers, is overexpressed in rhabdomyosarcoma patient samples and cell lines. To identify new molecular therapeutic targets to treat rhabdomyosarcoma, this study investigates the potential oncogenic role(s) for TBX3 and the factors responsible for upregulating it in this cancer. To this end, rhabdomyosarcoma cell culture models in which TBX3 was either stably knocked down or overexpressed were established and the impact on key hallmarks of cancer were examined using growth curves, soft agar and scratch motility assays, as well as tumour-forming ability in nude mice. Our data show that TBX3 promotes substrate-dependent and -independent proliferation, migration and tumour formation. We further reveal that TBX3 is upregulated by c-Myc transcriptionally and AKT1 post-translationally. This study identifies c-Myc/AKT1/TBX3 as an important axis that could be targeted for the treatment of rhabdomyosarcoma.