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Gli oncocitomi sono tumori epiteliali caratterizzati da un accumulo di mitocondri strutturalmente e funzionalmente compromessi, a prognosi generalmente benigna. Le cause genetiche della trasformazione oncocitaria sono tuttora sconosciute; pertanto, lo studio di oncocitomi in contesti familiari sindromici è utile nella ricerca dei determinanti genetici predisponenti il fenotipo. Diversi membri di una famiglia affetta da sindrome dell’iperparatiroidismo con tumore della mandibola (HPT-JT), dovuta ad un'ampia delezione in CDC73, hanno mostrato recidiva di tumori paratiroidei oncocitari. Il sequenziamento dell’esoma ha escluso mutazioni private della famiglia; all'interno della delezione ereditata, tuttavia, sono stati individuati elementi regolatori del gene glutaredossina 2 (GLRX2), codificante un'isoforma mitocondriale deputata alla deglutationilazione proteica reversibile -modificazione modulante l’attività di numerosi target- il cui ruolo nel cancro non è noto. La proteina è risultata assente in tutti i tumori e dimezzata nei tessuti sani dei soggetti. Per indagare se la sua assenza alteri la deglutationilazione proteica predisponendo al fenotipo oncocitario, sono stati generati modelli cellulari TPC1 e HCT116 GLRX2 KO in cui sono stati riscontrati un ridotto tasso proliferativo ed un'alterata glutationilazione proteica, particolarmente in seguito a stress ossidativo. Un esperimento pilota in vivo ha mostrato cellule KO oncocitoidi, con mitocondri morfologicamente alterati, suggerendo che l’alterazione redox innescata dall’assenza di GLRX2 possa indurre una disfunzione metabolica mitocondriale tale da mimare quelle osservate negli oncocitomi. L’analisi proteomica ha individuato diversi target di glutationilazione nei campioni KO identificando proteine del ciclo di Krebs e della catena respiratoria mitocondriale. In particolare, una marcata glutationilazione del complesso della piruvato deidrogenasi (PDHc) è stata correlata ad una ridotta sintesi di ATP dipendente d, Oncocytomas are epithelial tumors characterized by an accumulation of structurally and functionally compromised mitochondria, with a generally benign prognosis. The genetic causes behind oncocytic transformation are still unknown; therefore, the study of oncocytomas in syndromic familial contexts is particularly useful in the search for the genetic determinants of this phenotype. Several members of a family affected by hyperparathyroidism-jaw tumor syndrome (HPT-JT), due to a large deletion in CDC73, had recurrence of oncocytic parathyroid tumors. Exome sequencing ruled out private family mutations; however, within the inherited deletion, regulatory elements of the glutaredoxin 2 (GLRX2) gene have been identified. This gene encodes a mitochondrial isoform responsible for reversible protein deglutathionylation –a modification that modulates the activity of various targets- whose role in cancer is still unknown. The protein was absent in all tumors and halved in the healthy tissues of the subjects. To investigate whether its absence alters protein deglutathionylation predisposing to the oncocytic phenotype, TPC1 and HCT116 GLRX2 KO cell models were generated in which a reduced proliferative rate and altered glutathionylation of the proteome were found, particularly following oxidative stress. A pilot in vivo experiment showed oncocytic-like KO cells, with numerous altered mitochondria, consolidating the hypothesis that the redox alteration triggered by the absence of GLRX2 may induce a mitochondrial metabolic dysfunction mimicking those observed in oncocytomas. Proteomic analysis identified different glutathionylation targets in the KO samples such as proteins of the Krebs cycle and mitochondrial respiratory chain. Marked glutathionylation of the pyruvate dehydrogenase complex (PDHc) was correlated with a reduction in pyruvate-dependent ATP synthesis rate. Given the importance of oxidative stress in cancer pathophysiology and the role of glutathione in antioxidant res

L'inibizione del complesso respiratorio I (CI) è una strategia antitumorale emergente, sebbene la specificità e l’efficacia di nuovi farmaci restino poco investigate. La generazione di modelli cellulari tumorali nulli per il CI rivela la specificità di EVP 4593 e BAY 872243 nell’indurre gli effetti antiproliferativi non associati all’apoptosi, selettivamente via CI, riducendo eventuali effetti collaterali. Studi preliminari in vivo evidenziano un rallentamento della crescita tumorale negli animali trattati con EVP 4593, il quale emerge come l’inibitore più potente. Per il suo ruolo nella riprogrammazione metabolica, e la sua elevata frequenza di mutazioni nelle neoplasie umane, sono stati investigati i potenziali meccanismi di adattamento alla terapia anti-CI sulla base dello stato mutazionale di TP53. L’auxotrofia da aspartato, un hallmark metabolico delle cellule tumorali con un danno al CI, causa un blocco della sintesi proteica mTORC1-dipendente nelle linee cellulari con una p53 mutata o nulla, inducendo un collasso metabolico. Viceversa, l'attivazione del sensore energetico AMPK promuove un recupero parziale della sintesi di aspartato in linee cellulari con la forma wild type di P53, che è in grado di sostenere una migliore anaplerosi attraverso SCO2, fattore di assemblaggio del complesso respiratorio IV. Al fine di traslare questi risultati in un modello preclinico, si è ottimizzato l’ottenimento di colture di tumori umani espiantati tramite il bioreattore U-CUP. Il modello scelto è stato quello di carcinoma sieroso ad alto grado dell’ovaio (HGSOC), a partire da tessuto congelato, per l’elevata frequenza di mutazioni driver in TP53. I tessuti congelati preservano l'eterogeneità delle componenti cellulari del tessuto di origine e sono caratterizzati da cellule in attiva proliferazione senza attivazione di apoptosi. Dati preliminari mostrano un trend di riduzione dell’area tumorale nei tessuti trattati con EVP 4593 e supportano l’utilizzo del modello preclinico, Inhibition of respiratory complex I (CI) is an emerging anticancer strategy, whose specificity and efficacy of numerous inhibitors remain however elusive. Cancer cell models lacking CI reveal the specificity of EVP 4593 and BAY 872243 in inducing antiproliferative effects not associated with apoptosis, selectively via CI, reducing possible side effects. Preliminary in vivo studies show a slowing of tumor growth in animals treated with EVP 4593, which emerges as the most potent inhibitor. Because of its role in metabolic reprogramming, and its high mutation frequency in human malignancies, potential mechanisms of adaptation to anti-CI therapy based on TP53 mutational status have been investigated. Auxotrophy by aspartate, a metabolic hallmark of tumor cells with CI damage, causes a blockade of mTORC1-dependent protein synthesis in cell lines with mutated or null p53, inducing metabolic collapse. Conversely, activation of the energy sensor AMPK promotes partial recovery of aspartate synthesis in cell lines with the wild-type P53, which is able to sustain enhanced anaplerosis through SCO2, an assembly factor of respiratory complex IV. In order to translate these results into a preclinical model, cultures of explanted human tumors were optimized using the U-CUP bioreactor. The model chosen was high-grade serous ovarian cancer (HGSOC), from frozen tissue, because of the high frequency of driver mutations in TP53. Frozen tissues preserve the heterogeneity of cellular components of the tissue of origin and they are characterized by actively proliferating cells without activation of apoptosis. Preliminary data show a trend of tumor area reduction in EVP 4593-treated tissues and support the use of the preclinical model in the study of novel CI inhibitors by exploiting primary tissue from cancer patients.

Background Birt-Hogg-Dubé (BHD) syndrome is a rare genetic syndrome caused by pathogenic or likely pathogenic germline variants in the FLCN gene. Patients with BHD syndrome have an increased risk of fibrofolliculomas, pulmonary cysts, pneumothorax and renal cell carcinoma. There is debate regarding whether colonic polyps should be added to the criteria. Previous risk estimates have mostly been based on small clinical case series. Methods A comprehensive review was conducted to identify studies that had recruited families carrying pathogenic or likely pathogenic variants in FLCN. Pedigree data were requested from these studies and pooled. Segregation analysis was used to estimate the cumulative risk of each manifestation for carriers of FLCN pathogenic variants. Results Our final dataset contained 204 families that were informative for at least one manifestation of BHD (67 families informative for skin manifestations, 63 for lung, 88 for renal carcinoma and 29 for polyps). By age 70 years, male carriers of the FLCN variant have an estimated 19% (95% CI 12% to 31%) risk of renal tumours, 87% (95% CI 80% to 92%) of lung involvement and 87% (95% CI 78% to 93%) of skin lesions, while female carriers had an estimated 21% (95% CI 13% to 32%) risk of renal tumours, 82% (95% CI 73% to 88%) of lung involvement and 78% (95% CI 67% to 85%) of skin lesions. The cumulative risk of colonic polyps by age 70 years old was 21% (95% CI 8% to 45%) for male carriers and 32% (95% CI 16% to 53%) for female carriers. Conclusions These updated penetrance estimates, based on a large number of families, are important for the genetic counselling and clinical management of BHD syndrome.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer characterized by increased mortality. Here, we show for the first time that anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase of the insulin receptor superfamily, plays a pivotal role in the pathogenesis of cSCC. Our data demonstrate that the overexpression of the constitutively active, mutated ALK, ALK$^{ F1174L }$ , is sufficient to initiate the development of cSCC and is 100% penetrant. Moreover, we show that cSCC development upon ALK$^{ F1174L }$ overexpression is independent of the cell-of-origin. Molecularly, our data demonstrate that ALK$^{ F1174L }$ cooperates with oncogenic Kras$^{ G12D }$ and loss of p53, well-established events in the biology of cSCC. This cooperation results in a more aggressive cSCC type associated with a higher grade histological morphology. Finally, we demonstrate that Stat3 is a key downstream effector of ALK$^{ F1174L }$ and likely plays a role in ALK$^{ F1174L }$ -driven cSCC tumorigenesis. In sum, these findings reveal that ALK can exert its tumorigenic potential via cooperation with multiple pathways crucial in the pathogenesis of cSCC. Finally, we show that human cSCCs contain mutations in the ALK gene. Taken together, our data identify ALK as a new key player in the pathogenesis of cSCC, and this knowledge suggests that oncogenic ALK signaling can be a target for future clinical trials.

Il Complesso I (CI) mitocondriale è uno dei target metabolici più promettenti nelle terapie anti- cancro. In particolare, la metformina è un inibitore noto del CI, capace di inibire la crescita delle cellule tumorali, ma non di eradicare la patologia. Recentemente, l’associazione metformina ed ipoglicemia si è rivelata letale per i tumori, sebbene l’efficacia terapeutica del trattamento sinergico possa essere influenzata dall’accumulo di alterazioni genetiche nei più noti drivers della tumorigenesi. Abbiamo così investigato l’effetto dello stress metabolico indotto dalla restrizione di glucosio in un pannello di linee cellulari tumorali con un severo deficit sul CI e con un diverso stato genetico di TP53. Il deficit del CI associato alla carenza di glucosio inducono un abbattimento dei livelli di espressione della proteina p53 mutata, ma non della controparte wild-type. Il fenomeno biologico osservato non dipende né da un blocco trascrizionale, né dall’innesco di vie di degradazione intracellulare, come proteasoma ed autofagia. La scomparsa di p53 mutata, invece, sembra dipendere da un blocco generale della sintesi proteica, verosimilmente indotto dallo stress energetico e nutrizionale. Nella controparte p53 wild-type, invece, si osserva solo una parziale riduzione della sintesi proteica, suggerendo l’innesco di possibili vie di adattamento per compensare il danno sul CI. La carenza di amminoacidi è una caratteristica dei tumori solidi che potrebbe essere esacerbata in condizioni di deficit generali della catena respiratoria mitocondriale. In particolare, l’inibizione del CI causa auxotrofia da aspartato, metabolita limitante per la proliferazione, condizione che potrebbe generare il blocco della sintesi proteica osservato. L’incremento di espressione dei livelli del trasportatore aspartato/glutatammato mediata da p53 mutata compensa l’auxotrofia da aspartato, identificando un meccanismo di adattamento al deficit del CI. Dunque, i risultati ottenuti sottolineano l’i, Mitochondrial Complex I (CI) is one of the most promising metabolic targets in anti-cancer therapies. Metformin is a known inhibitor of CI inhibiting cancer cell growth, but not of eradicating the disease. Recently, the combination of metformin and hypoglycaemia has been shown to be lethal in tumours, although the therapeutic efficacy of synergistic treatment may be affected by the accumulation of genetic alterations in the known drivers of tumorigenesis. We thus investigated the effect of metabolic stress induced by glucose restriction in a panel of tumour cell lines with a severe CI deficiency and with a different genetic status of TP53. The CI deficiency associated with glucose restriction induced a decrease in the expression levels of the mutated p53 protein, but not of its wild-type counterpart. The biological phenomenon observed depends neither on a transcriptional blockade nor on the triggering of intracellular degradation pathways such as proteasome and autophagy. The decrease of mutated p53 seems to depend on a general blockade of protein synthesis, probably induced by energy and nutritional stress. In the wild type p53 counterpart, on the other hand, only a partial reduction in protein synthesis is observed, suggesting the triggering of possible adaptive pathways to compensate for the damage on the CI. Amino acid deprivation is a feature of solid tumours that could be exacerbated under conditions of mitochondrial respiratory chain deficiency. The CI inhibition causes aspartate auxotrophy, a condition that could generate the observed blockade of protein synthesis. Increased expression of aspartate/glutamate transporter levels mediated by mutated p53 could compensate for aspartate auxotrophy, identifying a possible mechanism of adaptation to CI deficiency. Thus, the results obtained underline the importance of implementing anti-complex I therapy in cancer, as different p53 status may alter treatment efficacy.

Targeting metabolism has become a valid anticancer strategy to pursue. Among others, respiratory complex-I (CI) has been identified as a valid target. In this context, a genetic ablation of mitochondrial CI enzyme in colorectals (HCT) and osteosarcomas (143B) was associated with the lack of HIF-1α stabilization and subsequent inability to adapt to hypoxic environment, suggesting this may contribute to the anti-tumorigenic effect of CI deficiency. However, most recent data imply that, despite the lack of HIF-1α, the progression of CI-deficient tumors seems to be supported by components of tumor-microenvironment (TME), in particular tumor associated macrophages (TAMs). Thus, the aim of this thesis was (i) to prove that the lack of HIF-1α stabilization and subsequent inability to adapt to hypoxic environment is a generalized phenomenon in murine, human cancers and in an orthotopic system and may contribute to the anti-tumorigenic effect of CI deficiency (ii) to prove that the CI-deficient tumors activate macrophages to progress despite HIF-1α destabilization (iii) to identify common targetable factors responsible for macrophage recruitment among the CI deficient 143B and HCT models. By introducing a non-degradable form of HIF-1α (HIF-TM) in CI deficient models, we demonstrated that HIF-TM rescued their tumorigenic potential, mature vasculature and vessel size. Moreover, in 143B CI deficient model (143B-/-), lack of HIF-1 activity correlated with macrophage migration inhibitory factor (MIF) downregulation and macrophage abundance. This indicated that the disruption of HIF-1-MIF axis leading to vasculature remodeling and TAM recruitment is one of the adaptive mechanisms activated by the 143B-/- tumors. However, this mechanism may not be generalized to the epithelial HCT model. Large-scale omics approach on CI deficient and CI competent 143B and HCT xenografts derived supernatants identified nine common metabolites secreted specifically from both the CI deficient models