Targeting systems vulnerabilities in uveal melanoma by CRISPR Cas/9 focal adhesion kinase (FAK) genome editing and therapeutic inhibition

Dottorato di Ricerca in Medicina Traslazionale. Ciclo XXXIl Melanoma Uveale rappresenta la neoplasia intraoculare più frequente nell’età adulta. Colpisce circa 2,500 individui ogni anno negli USA ed il 50% dei pazienti affetti da tale neoplasia sviluppa metastasi entro 5 anni dalla diagnosi. Non essendo state ancora identificate terapie efficaci, la sopravvivenza in presenza di metastasi è di circa 6 mesi. Il Melanoma Uveale è geneticamente caratterizzato dalla presenza di mutazioni somatiche attivanti a carico degli oncogeni GNAQ e GNA11, che codificano per due diverse subunità α delle proteine G. Tali mutazioni sono state identificate rispettivamente in circa il 94% dei casi di Melanoma Cutaneo ed il 4% dei casi di Melanoma Uveale. Sulla base di tali osservazioni, nel presente lavoro di tesi è stato valutato il ruolo esercitato da una proteina citoplasmatica ad attività tirosin-chinasica associata ai recettori per le integrine denominata FAK (focal adhesion kinase), nella progressione del Melanoma Uveale, sia in vitro che in vivo. In particolare, mediante analisi bioinformatica (www.cbioportal.com) delle alterazioni genomiche di campioni estratti da pazienti affetti da melanoma uveale (n=80), è stato inizialmente determinato che il gene codificante per FAK (PTK2) risulta over-espresso nel 56% dei casi. Inoltre, il presente studio condotto in cellule di Melanoma Uveale OMM1.3 (GNAQ/11 mutate) e in cellule ingegnerizzate per l’espressione di un recettore di membrana accoppiato a proteine-G (Gαq) attivato esclusivamente da ligandi sintetici denominate HEK293 DREADD/Gq, ha dimostrato il coinvolgimento di segnali mediati da GNAQ nell’attivazione di FAK attraverso il reclutamento del fattore coinvolto nello scambio di nucleotidi guaninici denominato TRIO e la proteina appartenente alla super-famiglia di Ras denominata Rho-A. A riprova, saggi biologici hanno dimostrato l’efficacia di specifici inibitori di FAK nei processi di proliferazione cellulare sia in cellule di Melanoma Uveale derivanti da lesioni primarie che da metastasi epatiche. Attraverso l’innovativo approccio genetico denominato CRISPR/Cas 9 genome editing (Clustered Regularly Interspaced Short Palindromic Repeats), il silenziamento dell’espressione di FAK ha ridotto significativamente la crescita del melanoma uveale in modelli sperimentali utilizzati in vivo. Collettivamente, i risultati ottenuti indicano che FAK può essere considerato un potenziale target terapeutico per il trattamento del Melanoma Uveale e di altre neoplasie caratterizzate da mutazioni oncogeniche a carico delle subunità αq/α11 dei recettori di membrana accoppiati a proteine G.Università della Calabri

Focal Adhesion Kinase Fine Tunes Multifaced Signals toward Breast Cancer Progression

Breast cancer represents the most common diagnosed malignancy and the main leading cause of tumor-related death among women worldwide. Therefore, several efforts have been made in order to identify valuable molecular biomarkers for the prognosis and prediction of therapeutic responses in breast tumor patients. In this context, emerging discoveries have indicated that focal adhesion kinase (FAK), a non-receptor tyrosine kinase, might represent a promising target involved in breast tumorigenesis. Of note, high FAK expression and activity have been tightly correlated with a poor clinical outcome and metastatic features in several tumors, including breast cancer. Recently, a role for the integrin-FAK signaling in mechanotransduction has been suggested and the function of FAK within the breast tumor microenvironment has been ascertained toward tumor angiogenesis and vascular permeability. FAK has been also involved in cancer stem cells (CSCs)-mediated initiation, maintenance and therapeutic responses of breast tumors. In addition, the potential of FAK to elicit breast tumor-promoting effects has been even associated with the capability to modulate immune responses. On the basis of these findings, several agents targeting FAK have been exploited in diverse preclinical tumor models. Here, we recapitulate the multifaceted action exerted by FAK and its prognostic significance in breast cancer. Moreover, we highlight the recent clinical evidence regarding the usefulness of FAK inhibitors in the treatment of breast tumors

Correction: Rigiracciolo, D.C., et al., IGF-1/IGF-1R/FAK/YAP Transduction Signaling Prompts Growth Effects in Triple-Negative Breast Cancer (TNBC) Cells. Cells 2020, 9, 1010

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IGF-1/IGF-1R/FAK/YAP Transduction Signaling Prompts Growth Effects in Triple-Negative Breast Cancer (TNBC) Cells

Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype that currently lacks targeted treatment options. The role played by the insulin-like growth factor-1 (IGF-1) and its cognate receptor IGF-1R in TNBC has been reported. Nevertheless, the molecular mechanisms by which the IGF-1/IGF-1R system may contribute to TNBC progression still remains to be fully understood. By computational analysis of the vast cancer genomics information in public databases (TCGA and METABRIC), we obtained evidence that high IGF-1 or IGF-1R levels correlate with a worse clinical outcome in TNBC patients. Further bioinformatics analysis revealed that both the focal adhesion and the Hippo pathways are enriched in TNBC harboring an elevated expression of IGF-1 or IGF-1R. Mechanistically, we found that in TNBC cells, the IGF-1/IGF-1R system promotes the activation of the FAK signal transduction pathway, which in turn regulates the nuclear accumulation of YAP (yes-associated protein/yes-related protein) and the expression of its target genes. At the biological level, we found that the IGF-1/IGF-1R-FAK-YAP network cascade triggers the growth potential of TNBC cells, as evaluated in different experimental systems. Overall, our results suggest that the IGF-1/IGF-1R/FAK/YAP axis may contribute to the progression of the aggressive TNBC subtype

Whole-genome CRISPR screening identifies PI3K/AKT as a downstream component of the oncogenic GNAQ–focal adhesion kinase signaling circuitry

G proteins and G protein-coupled receptors activate a diverse array of signal transduction pathways that promote cell growth and survival. Indeed, hot spot-activating mutations in GNAQ/GNA11, encoding Gαq proteins, are known to be driver oncogenes in uveal melanoma (UM), for which there are limited effective therapies currently available. Focal adhesion kinase (FAK) has been recently shown to be a central mediator of Gαq-driven signaling in UM, and as a result, is being explored clinically as a therapeutic target for UM, both alone and in combination therapies. Despite this, the repertoire of Gαq/FAK-regulated signaling mechanisms have not been fully elucidated. Here, we used a whole-genome CRISPR screen in GNAQ-mutant UM cells to identify mechanisms that, when overactivated, lead to reduced sensitivity to FAK inhibition. In this way, we found that the PI3K/AKT signaling pathway represented a major resistance driver. Our dissection of the underlying mechanisms revealed that Gαq promotes PI3K/AKT activation via a conserved signaling circuitry mediated by FAK. Further analysis demonstrated that FAK activates PI3K through the association and tyrosine phosphorylation of the p85 regulatory subunit of PI3K and that UM cells require PI3K/AKT signaling for survival. These findings establish a novel link between Gαq-driven signaling and the stimulation of PI3K as well as demonstrate aberrant activation of signaling networks underlying the growth and survival of UM and other Gαq-driven malignancies

Assessment of androgen receptor, IGF-IR and insulin receptor expression in male patients with severe peripheral artery disease

Background Peripheral artery disease (PAD) of the lower limbs is a common condition that can affect quality of life. Androgen receptor (AR) can exert sex-specific effects on metabolic system, endothelial function and vascular tone. IGF-I receptor (IGF-IR) and insulin receptor (IR) may also be involved in the aforementioned functions. The aim of this study was to evaluate AR, IGF-IR and IR expression in the arterial vessel walls of PAD patients. Results: This is a cross-sectional study examining 30 males with PAD undergoing open surgery procedures. Mean age was 75.9±8.8y. All patients belonged to Rutherford stage 4-6. Median expression levels of IR, IGF-IR and AR significantly decreased from stage 4 to 6 (p < 0.05). Significance: the study evidenced a progressive decrease of IR, IGF-IR and AR expression as the severity of disease increased. Altered levels of IR, IGF-IR and AR following PAD may be useful for the clinical evaluation of these patients

GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells Toward Breast Tumor Progression

The FGF2/FGFR1 paracrine loop is involved in the cross-talk between breast cancer cells and components of the tumor stroma as cancer-associated fibroblasts (CAFs). By quantitative PCR (qPCR), western blot, immunofluorescence analysis, ELISA and ChIP assays, we demonstrated that 17&beta;-estradiol (E2) and the G protein estrogen receptor (GPER) agonist G-1 induce the up-regulation and secretion of FGF2 via GPER together with the EGFR/ERK/c-fos/AP-1 signaling cascade in (ER)-negative primary CAFs. Evaluating the genetic alterations from METABRIC and TCGA datasets, we then assessed that FGFR1 is the most frequently amplified FGFRs family member and its amplification/expression associates with shorter survival rates in breast cancer patients. Therefore, in order to assess the functional FGF2/FGFR1 interplay between CAFs and breast cancer cells, we generated the FGFR1-knockout MDA-MB-231 cells using CRISPR/Cas9 genome editing strategy. Using conditioned medium from estrogen-stimulated CAFs, we established that the activation of FGF2/FGFR1 paracrine signaling triggers the expression of the connective tissue growth factor (CTGF), leading to the migration and invasion of MDA-MB-231 cells. Our findings shed new light on the role elicited by estrogens through GPER in the activation of the FGF2/FGFR1 signaling. Moreover, our findings may identify further biological targets that could be considered in innovative combination strategies halting breast cancer progression