MiR-182-3p targets TRF2 and impairs tumor growth of triple-negative breast cancer.

The telomeric repeat-binding factor 2 (TRF2) is a telomere-capping protein that plays a key role in the maintenance of telomere structure and function. It is highly expressed in different cancer types, and it contributes to cancer progression. To date, anti-cancer strategies to target TRF2 remain a challenge. Here, we developed a miRNA-based approach to reduce TRF2 expression. By performing a high-throughput luciferase screening of 54 candidate miRNAs, we identified miR-182-3p as a specific and efficient post-transcriptional regulator of TRF2. Ectopic expression of miR-182-3p drastically reduced TRF2 protein levels in a panel of telomerase- or alternative lengthening of telomeres (ALT)-positive cancer cell lines. Moreover, miR-182-3p induced DNA damage at telomeric and pericentromeric sites, eventually leading to strong apoptosis activation. We also observed that treatment with lipid nanoparticles (LNPs) containing miR-182-3p impaired tumor growth in triple-negative breast cancer (TNBC) models, including patient-derived tumor xenografts (PDTXs), without affecting mouse survival or tissue function. Finally, LNPs-miR-182-3p were able to cross the blood-brain barrier and reduce intracranial tumors representing a possible therapeutic option for metastatic brain lesions.

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetic effects.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC. METHODS: The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. RESULTS: TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation. CONCLUSIONS: Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.

Identification of Effective Anticancer G-Quadruplex-Targeting Chemotypes through the Exploration of a High Diversity Library of Natural Compounds.

In the quest for selective G-quadruplex (G4)-targeting chemotypes, natural compounds have been thus far poorly explored, though representing appealing candidates due to the high structural diversity of their scaffolds. In this regard, a unique high diversity in-house library composed of ca. one thousand individual natural products was investigated. The combination of molecular docking-based virtual screening and the G4-CPG experimental screening assay proved to be useful to quickly and effectively identify-out of many natural compounds-five hit binders of telomeric and oncogenic G4s, i.e., Bulbocapnine, Chelidonine, Ibogaine, Rotenone and Vomicine. Biophysical studies unambiguously demonstrated the selective interaction of these compounds with G4s compared to duplex DNA. The rationale behind the G4 selective recognition was suggested by molecular dynamics simulations. Indeed, the selected ligands proved to specifically interact with G4 structures due to peculiar interaction patterns, while they were unable to firmly bind to a DNA duplex. From biological assays, Chelidonine and Rotenone emerged as the most active compounds of the series against cancer cells, also showing good selectivity over normal cells. Notably, the anticancer activity correlated well with the ability of the two compounds to target telomeric G4s.

Targeting the KRAS oncogene: Synthesis, physicochemical and biological evaluation of novel G-Quadruplex DNA binders.

The oncogene KRAS is involved in the pathogenesis of many tumors such as pancreatic, lung and colorectal cancers, thereby representing a relevant target for the treatment of these diseases. The KRAS P1 promoter contains a nuclease hypersensitive, guanine-rich sequence able to fold into a G-quadruplex motif (G4). The stabilization of this G4 structure by small molecules is emerging as a feasible approach to downregulate KRAS expression. Here, a set of novel stabilizing molecules was identified through a virtual screening campaign on the NMR structure of the 22-mer KRAS G4. The most promising hits were then submitted to structure-activity relationships studies which allowed improving their binding affinity and selectivity over double helix DNA and different G4 topologies. The best derivative (19) underwent fluorescence titration experiments and further computational studies to disclose its binding mechanism to KRAS G4. Finally, biological assays showed that this compound is capable to reduce the viability of colorectal cancer cells in which mutated KRAS acts as a driver oncogene. Thus, 19 might represent the prototype of a new class of drugs for the treatment of tumors that, expressing mutated forms of KRAS, are refractory to current therapeutic regimens.

BRCA2 Deletion Induces Alternative Lengthening of Telomeres in Telomerase Positive Colon Cancer Cells.

BRCA1/2 are tumor suppressor genes controlling genomic stability also at telomeric and subtelomeric loci. Their mutation confers a predisposition to different human cancers but also sensitivity to antitumor drugs including poly(ADP-ribose) polymerase (PARP) inhibitors and G-quadruplex stabilizers. Here we demonstrate that BRCA2 deletion triggers TERRA hyperexpression and alternative lengthening mechanisms (ALT) in colon cancer cells in presence of telomerase activity. This finding opens the question if cancer patients bearing BRCA2 germline or sporadic mutation are suitable for anti-telomerase therapies, or how ALT activation could influence the short or long-term response to anti-PARP inhibitors or anti-G-quadruplex therapies.

Dyads of G-Quadruplex Ligands Triggering DNA Damage Response and Tumour Cell Growth Inhibition at Subnanomolar Concentration.

Naphthalene diimide (NDI) dyads exhibiting a different substitution pattern and linker length have been synthesised and evaluated as G-quadruplex (G4) ligands, by investigating their cytotoxicity in selected cell lines. The dyads with the long C7 linker exhibit extremely low IC50 values, below 10 nm, on different cancer cell lines. Contrary, the dyads with the shorter C4 linker were much less effective, with IC values increasing up to 1 µm. Among the three dyads with the longest linker, small differences in the IC50 values emerge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent cross-linking agents.

Targeting KRAS in metastatic colorectal cancer: current strategies and emerging opportunities.

Developing drugs that target KRAS, the most frequently mutated oncogene in cancer, has not been successful despite much concerted efforts dedicated towards it in the last thirty years. Considering the key role this driver oncogene plays, the pharmacological drugging of KRAS remains a key challenge for cancer research. In this review, we highlight the emerging experimental strategies for blocking KRAS function and signaling and its direct targeting. We also report on the results in this field of research produced by our group.

Xenograft as In Vivo Experimental Model.

The identification of experimental models that recapitulate human cancers designed to predict patient clinical response to therapies is a major break in oncology. Cancer stem cells (CSCs) represent a small tumor cell population responsible for drug resistance, where their effective killing may lead to identifying better treatment options. While the CSCs hypothesis highlights the need for a specific tumor target, patient-derived xenografts (PDXs) should also be considered for drug development as they better represent tumor heterogeneity and the environment in which a tumor develops.

Diagnosis and treatment of ALT tumors: is Trabectedin a new therapeutic option?

Telomeres are specialized nucleoprotein structures responsible for protecting chromosome ends in order to prevent the loss of genomic information. Telomere maintenance is required for achieving immortality by neoplastic cells. While most cancer cells rely on telomerase re-activation for linear chromosome maintenance and sustained proliferation, a significant population of cancers (10-15%) employs telomerase-independent strategies, collectively referred to as Alternative Lengthening of Telomeres (ALT). ALT mechanisms involve different types of homology-directed telomere recombination and synthesis. These processes are facilitated by loss of the ATRX or DAXX chromatin-remodeling factors and by abnormalities of the telomere nucleoprotein architecture. Although the functional consequences of telomerase and ALT up-regulation are similar in that they both prevent overall telomere shortening in tumors, these telomere maintenance mechanisms (TMMs) differ in several aspects which may account for their differential prognostic significance and response to therapy in various tumor types. Therefore, reliable methods for detecting telomerase activity and ALT are likely to become an important pre-requisite for the use of treatments targeting one or other of these mechanisms. However, the question whether ALT presence can confer sensitivity to rationally designed anti-cancer therapies is still open. Here we review the latest discoveries in terms of mechanisms of ALT activation and maintenance in human tumors, methods for ALT identification in cell lines and human tissues and biomarkers validation. Then, original results on sensitivity to rational based pre-clinical and clinical anti-tumor drugs in ALT vs hTERT positive cells will be presented.

Patient-derived xenografts: a relevant preclinical model for drug development.

Identifying appropriate preclinical cancer models remains a major challenge in increasing the efficiency of drug development. A potential strategy to improve patient outcomes could be selecting the 'right' treatment in preclinical studies performed in patient-derived xenografts (PDXs) obtained by direct implants of surgically resected tumours in mice. These models maintain morphological similarities and recapitulate molecular profiling of the original tumours, thus representing a useful tool in evaluating anticancer drug response. In this review, we will present the state-of-art use of PDXs as a reliable strategy to predict clinical findings. The main advantages and limitations will also be discussed.