Transplant Biology

Adaptation to stressful conditions is key for the evolution and maintenance of organisms but also allows tumor cells to escape anti-cancer therapeutic interventions. The main research activity of my laboratory is aimed at understanding how normal- and cancer (stem) cells cope with unfavorable conditions by activating stress-protective programs. The identification of such adaptive signaling mechanisms is crucial for the design of therapeutic strategies aimed either at improving tissue regeneration and healthy aging or at developing more effective oncological therapies. As experimental models we use the epidermis and the corneal epithelium, as well as human cell lines derived from breast cancer, squamous cell carcinoma, and melanoma. At the molecular level, we mostly focus our research on the functions of Akt/mTOR signaling pathway, a master regulatory hub of cell metabolism, and its interplay with the transcriptional machinery governing epithelial “stemness”, such as p63/p73 transcription factors of the p53 family. Our research group has provided evidence that attenuation of both mTORC1 and mTORC2 signaling can be beneficial for the maintenance and expansion of normal (stem) cells that reside in stratified epithelia such as the epidermis and the corneal epithelium, at least in part, by enhancing their stress tolerance. 

In particular, we found that in the human cornea in vivo, Akt/mTOR signaling is prominent in the limbal stem cell niche located at the periphery of the corneal epithelium, where p63+ epithelial stem cells reside. Explants from this tissue region are currently used for generating ex vivo epithelial autografts for the treatment of pathological conditions leading to corneal opacity and blindness. In this context, we found that Akt signaling, through the Akt1 and Akt2 isoforms, plays a dual role in cultivated limbal stem cells, being Akt1 a positive regulator of stem cell self-renewal and maintenance, and Akt2 a driver of stem cell differentiation and exhaustion. Silencing of Akt2, the main nuclear Akt isoform in limbal-corneal cells, leads to a dramatic increase of functional stem cells lifespan. Mechanistically, this is mainly due to attenuation of mTORC1 signaling triggered by the FOXO family of stress-responsive transcription factors, which are negatively regulated by Akt2 in the limbal cell nucleus. The transcriptional activation of FOXOs subsequent to Akt2 depletion leads to enhanced expression of the TSC1 gene, a key suppressor of mTORC1 activity.  Our data are consistent with evidence obtained in other tissue types indicating that sustained activation of mTORC1 causes stem cell depletion, while attenuation of mTORC1 signaling protects stem cells under stressful conditions such as hypoxia, DNA damage and nutrients restriction. Since it is known that a major cause of failure of epithelial transplants in regenerative medicine is the paucity of stem cells in the epithelial grafts engineered ex vivo, our findings have potential translational implications, as culture of limbal cells in the presence of mTORC1 inhibitors (Rapalogs) may facilitate the expansion of functional stem cells in therapeutic settings. 

More recently, we have explored the roles of mTORC2 in epidermal homeostasis by generating a tissue-specific knockout mouse lacking the essential mTORC2-specific Rictor subunit in the epidermis. We found that Rictor/mTORC2 is dispensable for epidermal development, with mutant mice displaying subtle defects in epidermal cell proliferation and differentiation. However, we found that Rictor-KO epidermal cells are endowed with a remarkable resilience towards oxidative stress, are protected from DNA damage both in vitro and in vivo, and are prone to spontaneous immortalization in culture. This was somewhat surprising in light of the pro-survival role attributed to mTORC2 signaling in several physiopathological contexts. Nonetheless, Rictor-deficient keratinocytes exhibit changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). This, in turn, depends on hyperactivation of the Electron Transport Chain (ETC)-based Oxidative Phosphorylation (OXPHOS) fueled by glutaminolysis. Mechanistically, the resiliency of Rictor-deficient epidermal cells depends on these ROS increases through a biological response that fits the definition of mitohormesis, in which sub-lethal doses of mitochondrial ROS elicit adaptive mechanisms that protect cells from further oxidative insults. 

Our laboratory is presently focused on the identification of adaptive mechanisms at the basis of cancer therapeutic resistance. For instance, nearly 50% of cutaneous melanomas carry activating mutations of the BRAF oncogene, and the combination of BRAF- and MEK-inhibitors (BRAF/MEKi) is frequently used in their clinical management. This therapeutic approach often induces a rapid regression of tumors at advanced stages of disease, but has the major drawback of the nearly inevitable acquisition of therapeutic resistance. This can be driven by multiple adaptive and/or genetic mechanisms of tumor cells that often converge on the upregulation of mitochondrial bioenergetics and NAD+ biosynthetic pathways.  Building on the knowledge gained in Rictor-depleted epidermal cells, we hypothesized that mTORC2 depletion may protect BRAFV600E metastatic melanoma (MM) cells from therapeutic stress induced by targeted therapy by promoting similar changes in mitochondrial bioenergetics. This is seemingly counter-intuitive, as mTORC2 is regarded as an oncogenic driver in several tumor types including MM, because of its capacity to stimulate Akt pro-oncogenic functions and to promote glucose metabolisms and lipid biogenesis. However, bioinformatics analysis of the TCGA database revealed that in the context of cutaneous MM, tumors expressing low RICTOR levels are associated with a poorer patients’ prognosis as compared to high-RICTOR expressing counterparts. Gene-set enrichment analysis (GSEA) of Low-RICTOR MM specimens also evidenced a prominent molecular signature suggestive of activation of ETC-based mitochondrial OXPHOS.  We found that mTORC2-deficient BRAFV600E MM cells obtained through RICTOR knockdown are indeed intrinsically tolerant to BRAF/MEKi, and anticipate the acquisition of BRAFi resistance after prolonged drug exposure both in vitro and in vivo, indicating that mTORC2 counteracts the acquisition of targeted therapy resistance in MM. Unbiased proteomic analysis of RICTOR-deficient cells has revealed increased expression and/or differential post-translational modification of proteins involved in mitochondrial OXPHOS and NAD metabolism. Consistently, RICTOR-deficient cells show enhanced mitochondrial respiratory potential and increased expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, the rate-limiting enzyme of the NAD+ salvage pathway.  Notably, pharmacological inhibition of either NAMPT or the ETC in RICTOR-deficient cells is sufficient to restore sensitivity to BRAFi. We also found that in drug naïve BRAFV600E MM cells, the endogenous RICTOR protein is downregulated in the few proliferating cells that survive a prolonged BRAFi exposure.  Because these cells represent the seeds of BRAFi-resistant cell populations, this suggest that downregulation of endogenous RICTOR provides an early adaptation mechanism that allows MM cells to escape BRAF inhibition. This adaptive resistance mechanism may set the basis for the occurrence of further genetic and/or epigenetic alterations that permanently stabilize BRAF/MEKi-resistance in tumors. Overall, our findings unveil a novel tumor suppressive function for RICTOR/mTORC2, whose inactivation promotes the acquisition of MM therapeutic resistance.  We propose that measurement of intra-tumor RICTOR levels may have a prognostic value, and help to predict the responsiveness of BRAFV600E MM to targeted therapy. Moreover, our findings suggest that the NAMPT-ETC axis may represent a specific therapeutic vulnerability of low-RICTOR MM, with potential therapeutic implications.

Like in melanoma, also in epithelial cancers the lethality of tumors largely depends on the ability of cancer cells to survive therapeutic intervention thanks to adaptive stress protective programs that foster the maintenance of “persister” cell populations reigniting tumor growth and metastasis. The dynamic reprogramming of the transcriptional landscape plays a key role in the stress adaptation capacity and therapeutic resistance of epithelial tumors, often involving changes in the expression of genes that protect cells from oxidative damage. Increasing evidence indicates that persister cancer cells can be effectively sensitized to ferroptosis, a non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation, being “addicted” to anti-ferroptotic mechanisms for their survival to therapeutic stress. Importantly, being ferroptosis an immunogenic form of cell death, tumor cells engaging anti-ferroptotic mechanisms also acquire immune evasive properties via mechanisms that are only partially defined. As a consequence, the induction of ferroptosis may enhance the responses of tumors to immunotherapies.

Evidence from the literature and very recent data from our laboratory point to the whole p53 family of transcription factors (p53, p63 and p73) as major transcriptional regulators of redox genes whose expression can dictate the sensitivity or resistance of cells to ferroptotic stimuli. One prominent gene in this network is SLC7A11, encoding for the functional subunit of the cystine/glutamate antiporter system xc- involved in glutathione biosynthesis. SLC7A11 is highly expressed in tumors and persister cancer cells, and its activity plays a key role in the protection of cells from ferroptosis, thus representing a therapeutic target in ferroptosis-based anti-cancer strategies. Whereas p53 inhibits SLC7A11 expression, our recent data indicate that in squamous cell carcinoma models, ΔNp63 (and to a lesser extent p73), may exert an even broader control of the glutathione metabolic pathway, including a direct positive regulation of SLC7A11 expression. Thus, epithelial cancers overexpressing p63 and/or p73 may be protected from cancer therapy, at least in part, by engaging anti-ferroptotic transcriptional programs. We are presently trying to define the transcriptome changes of epithelial cells derived from breast cancers and squamous cell carcinomas, in response to oxidative stress as a function of p53 family members expression.