PTEN Lipid Phosphatase Activity Suppresses Melanoma Formation by Opposing an AKT/mTOR/FRA1 Signaling Axis.

Inactivating mutations in PTEN are prevalent in melanoma and are thought to support tumor development by hyperactivating the AKT/mTOR pathway. Conversely, activating mutations in AKT are relatively rare in melanoma, and therapies targeting AKT or mTOR have shown disappointing outcomes in preclinical models and clinical trials of melanoma. This has led to the speculation that PTEN suppresses melanoma by opposing AKT-independent pathways, potentially through noncanonical functions beyond its lipid phosphatase activity. In this study, we examined the mechanisms of PTEN-mediated suppression of melanoma formation through the restoration of various PTEN functions in PTEN-deficient cells or mouse models. PTEN lipid phosphatase activity predominantly inhibited melanoma cell proliferation, invasion, and tumor growth, with minimal contribution from its protein phosphatase and scaffold functions. A drug screen underscored the exquisite dependence of PTEN-deficient melanoma cells on the AKT/mTOR pathway. Furthermore, activation of AKT alone was sufficient to counteract several aspects of PTEN-mediated melanoma suppression, particularly invasion and the growth of allograft tumors. Phosphoproteomics analysis of the lipid phosphatase activity of PTEN validated its potent inhibition of AKT and many of its known targets, while also identifying the AP-1 transcription factor FRA1 as a downstream effector. The restoration of PTEN dampened FRA1 translation by inhibiting AKT/mTOR signaling, and FRA1 overexpression negated aspects of PTEN-mediated melanoma suppression akin to AKT. This study supports AKT as the key mediator of PTEN inactivation in melanoma and identifies an AKT/mTOR/FRA1 axis as a driver of melanomagenesis. SIGNIFICANCE: PTEN suppresses melanoma predominantly through its lipid phosphatase function, which when lost, elevates FRA1 levels through AKT/mTOR signaling to promote several aspects of melanomagenesis.

Genetic tools for the stable overexpression of circular RNAs.

Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.

Squaring the circle: circRNAs in melanoma.

Non-coding RNAs are emerging as critical molecules in the genesis, progression, and therapy resistance of cutaneous melanoma. This includes circular RNAs (circRNAs), a class of non-coding RNAs with distinct characteristics that forms through non-canonical back-splicing. In this review, we summarize the features and functions of circRNAs and introduce the current knowledge of the roles of circRNAs in melanoma. We also highlight the various mechanisms of action of the well-studied circRNA CDR1as and describe how it acts as a melanoma tumor suppressor. We further discuss the utility of circRNAs as biomarkers, therapeutic targets, and therapeutic agents in melanoma and outline challenges that must be overcome to comprehensively characterize circRNA functions.

A MAPK/miR-29 Axis Suppresses Melanoma by Targeting MAFG and MYBL2.

The miR-29 family of microRNAs is encoded by two clusters, miR-29b1~a and miR-29b2~c, and is regulated by several oncogenic and tumor suppressive stimuli. While in vitro evidence suggests a tumor suppressor role for miR-29 in melanoma, the mechanisms underlying its deregulation and contribution to melanomagenesis have remained elusive. Using various in vitro systems, we show that oncogenic MAPK signaling paradoxically stimulates transcription of pri-miR-29b1~a and pri-miR-29b2~c, the latter in a p53-dependent manner. Expression analyses in melanocytes, melanoma cells, nevi, and primary melanoma revealed that pri-miR-29b2~c levels decrease during melanoma progression. Inactivation of miR-29 in vivo with a miRNA sponge in a rapid melanoma mouse model resulted in accelerated tumor development and decreased overall survival, verifying tumor suppressive potential of miR-29 in melanoma. Through integrated RNA sequencing, target prediction, and functional assays, we identified the transcription factors MAFG and MYBL2 as bona fide miR-29 targets in melanoma. Our findings suggest that attenuation of miR-29b2~c expression promotes melanoma development, at least in part, by derepressing MAFG and MYBL2.