Studio della regolazione trascrizionale della Stearoyl-CoA Desaturase 1 (SCD1) in Cellule Staminali Tumorali di adenocarcinoma del polmone

Le Cellule Staminali Tumorali (CSCs) rappresentano una sottopopolazione di cellule tumorali in grado di auto-rinnovarsi differenziandosi. Recenti evidenze suggeriscono che Stearoyl-CoA-desaturasi 1 (SCD1), enzima coinvolto nella sintesi degli acidi grassi monoinsaturi a partire dagli acidi grassi saturi, ha un ruolo in diverse neoplasie. Il gruppo di Ricerca studia da anni il ruolo di SCD1 nel tumore al polmone, il presente lavoro approfondisce gli aspetti legati alla sopravvivenza e alla propagazione delle CSCs confinando il sistema biologico all’adenocarcinoma polmonare. Nella prima parte della trattazione sono state utilizzate le linee primarie derivanti da versamento pleurico di adenocarcinoma del polmone, al fine di studiare gli effettori del pathway di Hippo, YAP e TAZ. In seguito all’inibizione di SCD1, le cellule 3D mostrano una riduzione di YAP/TAZ. La regolazione di YAP/TAZ da parte di SCD1 dipende anche dall'attività di β-catenina, poiché è stato dimostrato che la downregolazione di YAP/TAZ, indotta dal blocco di SCD1, può essere revertita con l'aggiunta del ligando esogeno. Affinché ci sia attivazione di SCD1 e di YAP/TAZ è necessario che il complesso di distruzione di β-catenina sia inattivo. In linea con i risultati in vitro, l'analisi immunoistochimica dei campioni di adenocarcinoma polmonare ha mostrato che i livelli di espressione di SCD1 co-variano con quelli di β-catenina e YAP/TAZ. I set di dati di espressione genica hanno permesso di osservare che gli elevati livelli di co-espressione di SCD1, β-catenina, YAP/TAZ e dei geni target hanno un forte valore prognostico negativo nell'adenocarcinoma polmonare. Infine, le analisi bioinformatiche, volte a identificare quali combinazioni geniche hanno avuto effetti sinergici sull'outcome clinico, hanno mostrato che una scarsa sopravvivenza è associata ad alta co-espressione di SCD1, β-catenina e YAP/TAZ e del gene target, birc5. Il recente lavoro pubblicato dimostra per la prima volta il coinvolgimento di SCD1 nella regolazione del pathway di Hippo nell’adenocarcinoma del polmone, e propone il metabolismo degli acidi grassi come regolatore chiave delle CSCs (Noto et al., 2017). Nella seconda parte del lavoro di Dottorato, lo studio approfondisce il meccanismo trascrizionale responsabile della regolazione di SCD1 nelle CSCs. Quindi, l’attenzione si è focalizzata sullo studio del promotore del gene SCD1. L’analisi bioinformatica è stata effettuata al fine di individuare i putativi siti di legame dei Fattori Trascrizionali implicati nella sua regolazione. I Fattori Trascrizionali identificati mediante analisi bioinformatica sono: Sterol-Regulated-Element-Binding-1 (SREPB-1), c-MYC, Fattore Nucleare Y (-YA e -YB) e il Fattore Nucleare kB (NF- kB). Sono state determinate le porzioni di arricchimento dei suddetti Fattori Trascrizionali mediante immunoprecipitazione della cromatina (ChIP-qPCR) nella regione di interesse del promotore. Dai risultati emerge che, il passaggio da cellule cresciute in condizioni di aderenza a sferoidi, presenta un importante arricchimento dei siti di legame dei Fattori Trascrizionali sul promotore di SCD1. Al fine di identificare i Fattori Trascrizionali che assumono importanza direttamente o indirettamente nelle CSCs, è stato effettuato il silenziamento genico per valutare se l’inibizione dell’attività possa esser riflessa sull’espressione di SCD1. Infatti, la regolazione di SCD1 in CSCs è influenzata dalla presenza di SREPB1 e NF-Y. Considerando i risultati ottenuti, l’obiettivo è stato quello di effettuare la mutagenesi sul promotore di SCD1, mediante il sistema CRISPR/Cas9 per ottenere dei cloni stabili che indicassero la porzione di promotore efficiente nella regolazione trascrizionale di SCD1 in CSCs. La generazione del clone PR_1Δ, in seguito a saggi funzionali, mostra una diminuzione dell’espressione di SCD1 e una ridotta capacità di formazione di cellule in 3D, poichè è stata deleta la porzione in cui sono presenti i siti di legame di SREBP1 e c-MYC e NF-kB. In futuro verranno approfondite le analisi di genomica funzionale del trascrittoma e di accessibilità della cromatina, sia circostanziate al promotore di SCD1 che ampliate nelle CSCs. Le informazioni che ne deriveranno ci permetteranno di rafforzare le informazioni e le terapie verso l’adenocarcinoma del polmone

Inhibition of Stearoyl-CoA desaturase 1 reverts BRAF and MEK inhibition-induced selection of cancer stem cells in BRAF-mutated melanoma

Combination therapy with BRAF and MEK inhibitors significantly improves survival in BRAF mutated melanoma patients but is unable to prevent disease recurrence due to the emergence of drug resistance. Cancer stem cells (CSCs) have been involved in these long-term treatment failures. We previously reported in lung cancer that CSCs maintenance is due to altered lipid metabolism and dependent upon Stearoyl-CoA-desaturase (SCD1)-mediated upregulation of YAP and TAZ. On this ground, we investigated the role of SCD1 in melanoma CSCs

Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies

BACKGROUND: Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent. In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance. METHODS: Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies. The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting. The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays. RESULTS: In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth. We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells. CONCLUSIONS: On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response

Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma

miRNAs as candidate biomarker for the accurate detection of atypical endometrial hyperplasia/endometrial intraepithelial neoplasia

Endometrial cancer is the most common gynecologic malignancy in developed countries. Estrogen-dependent tumors (type I, endometrioid) account for 80% of cases and non-estrogen-dependent (type II, non-endometrioid) account for the rest. Endometrial cancer type I is generally thought to develop via precursor lesions along with the increasing accumulation of molecular genetic alterations. Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia is the least common type of hyperplasia but it is the type most likely to progress to type I cancer, whereas endometrial hyperplasia without atypia rarely progresses to carcinoma. MicroRNAs are a class of small, non-coding, single-stranded RNAs that negatively regulate gene expression mainly binding to 3'-untranslated region of target mRNAs. In the current study, we identified a microRNAs signature (miR-205, miR-146a, miR-1260b) able to discriminate between atypical and typical endometrial hyperplasia in two independent cohorts of patients. The identification of molecular markers that can distinguish between these two distinct pathological conditions is considered to be highly useful for the clinical management of patients because hyperplasia with an atypical change is associated with a higher risk of developing cancer. We show that the combination of miR-205, -146a, and -1260b has the best predictive power in discriminating these two conditions (>90%). With the aim to find a biological role for these three microRNAs, we focused our attention on a common putative target involved in endometrial carcinogenesis: the oncosuppressor gene SMAD4. We showed that miRs-146a, -205, and-1260b directly target SMAD4 and their enforced expression induced proliferation and migration of Endometrioid Cancer derived cell lines, Hec1 a cells. These data suggest that microRNAs-mediated impairment of the TGF-beta pathway, due to inhibition of its effector molecule SMAD4, is a relevant molecular alteration in endometrial carcinoma development. Our findings show a potential diagnostic role of this microRNAs signature for the accurate diagnosis of Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia and improve the understanding of their pivotal role in SMAD4 regulation

Human lung adenocarcinoma cell cultures derived from malignant pleural effusions as model system to predict patients chemosensitivity

BACKGROUND: Lung cancer is the leading cause of cancer related deaths and Malignant Pleural Effusion (MPE) is a frequent complication. Current therapies suffer from lack of efficacy in a great percentage of cases, especially when cancer is diagnosed at a late stage. Moreover patients' responses vary and the outcome is unpredictable. Therefore, the identification of patients who will benefit most of chemotherapy treatment is important for accurate prognostication and better outcome. In this study, using malignant pleural effusions (MPE) from non-small cell lung cancer (NSCLC) patients, we established a collection of patient-derived Adenocarcinoma cultures which were characterized for their sensitivity to chemotherapeutic drugs used in the clinical practice. METHODS: Tumor cells present in MPEs of patients with NSCLC were isolated by density gradient centrifugation, placed in culture and genotyped by next generation sequencing. In a subset of cases patient derived xenografts (PDX) were obtained upon tumor cell inoculation in rag2/IL2 knock-out mice. Isolated primary cultures were characterized and tested for drug sensitivity by in vitro proliferation assays. Additivity, antagonism or synergy for combinatorial treatments were determined by analysis with the Calcusyn software. RESULTS: We have optimized isolation procedures and culture conditions to expand in vitro primary cultures from Malignant Pleural Effusions (MPEs) of patients affected by lung adenocarcinomas, the most frequent form of non small cell lung cancer. Using this approach we have been able to establish 16 primary cultures from MPEs. Cells were banked at low passages and were characterized for their mutational pattern by next generation sequencing for most common driver mutations in lung cancer. Moreover, amplified cultures were shown to engraft with high efficiency when injected in immunocompromised mice. Cancer cell sensitivity to drugs used in standard chemotherapy regimens was assessed either individually or in combination. Differential chemosensitivity and different mutation profiles were observed which suggests that this isolation method could provide a platform for predicting the efficacy of chemotherapy in the clinical setting. Most importantly for six patients it was possible to establish a correlation between drug response in vitro and response to therapy in the clinic. CONCLUSIONS: Results obtained using primary cultured cells from MPEs underscore the heterogeneity of NSCLC in advanced stage as indicated by drug response and mutation profile. Comparison of data obtained from in vitro assays with patients' responses to therapy leads to the conclusion that this strategy may provide a potentially useful approach for evaluating individual chemosensitivity profile and tailor the therapy accordingly. Furthermore, combining MPE-derived primary cultures with their genomic testing allows to identify patients eligible to trials with novel targeted agents

Histologic analysis of idiopathic pulmonary fibrosis by morphometric and fractal analysis

: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disorder, ultimately leading to respiratory failure and death. Despite great research advances in understanding the mechanisms underlying the disease, its diagnosis, and its treatment, IPF still remains idiopathic without known biological or histological markers able to predict disease progression or response to treatment. The histologic hallmark of IPF is usual interstitial pneumonia (UIP), with its intricate architectural distortion and temporal inhomogeneity. We hypothesize that normal lung alveolar architecture can be compared to fractals, such as the Pythagoras tree with its fractal dimension (Df), and every pathological insult, distorting the normal lung structure, could result in Df variations. In this study, we aimed to assess the UIP histologic fractal dimension in relationship to other morphometric parameters in newly diagnosed IPF patients and its possible role in the prognostic stratification of the disease. Clinical data and lung tissue specimens were obtained from twelve patients with IPF, twelve patients with non-specific interstitial pneumonia (NSIP), and age-matched "healthy" control lung tissue from patients undergoing lung surgery for other causes. Histology and histomorphometry were performed to evaluate Df and lacunarity measures, using the box counting method on the FracLac ImageJ plugin. The results showed that Df was significantly higher in IPF patients compared to controls and fibrotic NSIP patients, indicating greater architectural distortion in IPF. Additionally, high Df values were associated with higher fibroblastic foci density and worse prognostic outcomes in IPF, suggesting that Df may serve as a potential novel prognostic marker for IPF. The scalability of Df measurements was demonstrated through repeated measurements on smaller portions from the same surgical biopsies, which were selected to mimic a cryobiopsy. Our study provides further evidence to support the use of fractal morphometry as a tool for quantifying and determining lung tissue remodeling in IPF, and we demonstrated a significant correlation between histological and radiological Df in UIP pattern, as well as a significant association between Df and FF density. Furthermore, our study demonstrates the scalability and self-similarity of Df measurements across different biopsy types, including surgical and smaller specimens

Circulating Vitamin D levels status and clinical prognostic indices in COVID-19 patients

BACKGROUND: Several immune mechanisms activate in COVID-19 pathogenesis. Usually, coronavirus infection is characterized by dysregulated host immune responses, interleukine-6 increase, hyper-activation of cytotoxic CD8 T lymphocytes. Interestingly, Vitamin D deficiency has been often associated with altered immune responses and infections. In the present study, we evaluated Vitamin D plasma levels in patients affected with different lung involvement during COVID-19 infection.METHODS: Lymphocyte phenotypes were assessed by flow cytometry. Thoracic CT scan involvement was obtained by an image analysis program.RESULTS: Vitamin D levels were deficient in (80%) of patients, insufficient in (6.5%) and normal in (13.5%). Patients with very low Vitamin D plasma levels had more elevated D-Dimer values, a more elevated B lymphocyte cell count, a reduction of CD8+T lymphocytes with a low CD4/CD8 ratio, more compromised clinical findings (measured by LIPI and SOFA scores) and thoracic CT scan involvement.CONCLUSIONS: Vitamin D deficiency is associated with compromised inflammatory responses and higher pulmonary involvement in COVID-19 affected patients. Vitamin D assessment, during COVID-19 infection, could be a useful analysis for possible therapeutic interventions.TRIAL REGISTRATION: 'retrospectively registered'

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells