The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates 1 – 3 . However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy 4 , 5 , is phosphorylated by mTORC1 via a substrate-specific mechanism mediated by RagGTPases. Thus, TFEB phosphorylation is strictly dependent on amino acid-mediated activation of RagC/D GTPase but, unlike other mTORC1 substrates such as S6K and 4E-BP1, insensitive to growth factor-induced Rheb activity. This mechanism plays a crucial role in Birt-Hogg-Dubé (BHD) syndrome, a disorder caused by mutations of the RagC/D activator folliculin (FLCN) and characterized by benign skin tumors, lung and kidney cysts and renal cell carcinoma 6 , 7 . We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and paradoxical mTORC1 hyperactivity observed in BHD syndrome. Remarkably, depletion of TFEB in a kidney-specific mouse model of BHD syndrome fully rescued the disease phenotype and associated lethality and normalized mTORC1 activity. Together, these findings identify a substrate-specific control mechanism of mTORC1, whose dysregulation leads to kidney cysts and cancer.

Nanodelivery systems usually improve the biodistribution of drugs, leading to reduced side effects and enhanced therapeutic efficacy. However, only a small portion of the injected nanoparticle dose accumulates in pathological tissue. Challenges in drug delivery arise due to a multitude of transport obstacles in the body, including the endothelium, the extracellular matrix, and the cell membrane. In general, nanoparticles are designed to overcome only a few biological barriers, making them inadequate for localized drug delivery. Accordingly, a multifunctional and multicomponent systems are required to effectively address a wide variety of transport obstacles. A suitable approach to obtain high levels of multifunctionality is to bring together the nanoscale with the microscale, resulting in post-nano strategies for drug delivery. This review discusses several such post-nano approaches, with an emphasis on the multistage vector (MSV) platform. The MSV consists of three components on different spatial scales, each intended to address biological barriers that exist in a specific compartment in the body. The first stage vector is a microparticle that is designed to navigate in the vascular compartment. The second stage vector consists of nanoparticles that are released from the microparticle into the tissue interstitium, where they address biological barriers in extracellular and intracellular compartments. The final component of the system is a small molecule therapeutic agent. A new generation of microparticle-based strategies with expanded applications has recently been developed, including injectable nanoparticle generators and silicon particles for immunotherapy. Notably, the advantage of incorporating microstructures in drug delivery vehicles is apparent from the observation that superior functionality only appears on the microscale, highlighting the inherent functional limitations of nanostructures.

Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.