Publications by authors named "Manuela Porru"

41 Publications

Loss of HER2 and decreased T-DM1 efficacy in HER2 positive advanced breast cancer treated with dual HER2 blockade: the SePHER Study.

J Exp Clin Cancer Res 2020 Dec 10;39(1):279. Epub 2020 Dec 10.

Division of Medical Oncology 2, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Background: HER2-targeting agents have dramatically changed the therapeutic landscape of HER2+ advanced breast cancer (ABC). Within a short time frame, the rapid introduction of new therapeutics has led to the approval of pertuzumab combined with trastuzumab and a taxane in first-line, and trastuzumab emtansine (T-DM1) in second-line. Thereby, evidence of T-DM1 efficacy following trastuzumab/pertuzumab combination is limited, with data from some retrospective reports suggesting lower activity. The purpose of the present study is to investigate T-DM1 efficacy in pertuzumab-pretreated and pertuzumab naïve HER2 positive ABC patients. We also aimed to provide evidence on the exposure to different drugs sequences including pertuzumab and T-DM1 in HER2 positive cell lines.

Methods: The biology of HER2 was investigated in vitro through sequential exposure of resistant HER2 + breast cancer cell lines to trastuzumab, pertuzumab, and their combination. In vitro experiments were paralleled by the analysis of data from 555 HER2 + ABC patients treated with T-DM1 and evaluation of T-DM1 efficacy in the 371 patients who received it in second line. Survival estimates were graphically displayed in Kaplan Meier curves, compared by log rank test and, when possibile, confirmed in multivariate models.

Results: We herein show evidence of lower activity of T-DM1 in two HER2+ breast cancer cell lines resistant to trastuzumab+pertuzumab, as compared to trastuzumab-resistant cells. Lower T-DM1 efficacy was associated with a marked reduction of HER2 expression on the cell membrane and its nuclear translocation. HER2 downregulation at the membrane level was confirmed in biopsies of four trastuzumab/pertuzumab-pretreated patients. Among the 371 patients treated with second-line T-DM1, median overall survival (mOS) from diagnosis of advanced disease and median progression-free survival to second-line treatment (mPFS2) were 52 and 6 months in 177 patients who received trastuzumab/pertuzumab in first-line, and 74 and 10 months in 194 pertuzumab-naïve patients (p = 0.0006 and 0.03 for OS and PFS2, respectively).

Conclusions: Our data support the hypothesis that the addition of pertuzumab to trastuzumab reduces the amount of available plasma membrane HER2 receptor, limiting the binding of T-DM1 in cancer cells. This may help interpret the less favorable outcomes of second-line T-DM1 in trastuzumab/pertuzumab pre-treated patients compared to their pertuzumab-naïve counterpart.
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http://dx.doi.org/10.1186/s13046-020-01797-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731769PMC
December 2020

Hybrid lipid self-assembling nanoparticles for brain delivery of microRNA.

Int J Pharm 2020 Oct 2;588:119693. Epub 2020 Aug 2.

Department of Pharmacy, University Federico II of Naples, Via Domenico Montesano, 49, 80131 Naples, Italy. Electronic address:

Hybrid self-assembling nanoparticles (SANPs) have been previously designed as novel drug delivery system that overcomes stability issues following long-term storage and with an easy scale-up. This system has been successfully used to deliver anionic-charged agents, e.g. bisphosphonates, in different types of tumors, such glioblastoma (GBM). Here, SANPs were tested and optimized for the delivery of nucleic acids, in particular of a specific microRNA, e.g. miR603, used for its potential role in controlling the chemoresistance in different forms of cancer, e.g. (GBM). To this aim, SANPs with different lipids were prepared and characterized, in terms of size, polydispersity index, zeta potential, miRNA encapsulation, stability in BSA, serum and hemolytic activity. Then, SANPs were tested in vitro on two different cell lines of GBM. Finally, miRNA biodistribution was tested in vivo in an orthotopic model of GBM. The majority of the formulations showed good technological characteristics and were stable in BSA and serum with a low hemolytic activity. The intracellular uptake studies on GBM cell lines showed that SANPs allow to achieve a higher miRNA delivery compared to others transfection agents, e.g. lipofectamine. Finally, in vivo biodistribution studies in an orthotopic of GBM demonstrated that the optimized SANP formulations, were able to deliver miRNA in different organs, e.g. the brain.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119693DOI Listing
October 2020

Harnessing Omics Approaches on Advanced Preclinical Models to Discovery Novel Therapeutic Targets for the Treatment of Metastatic Colorectal Cancer.

Cancers (Basel) 2020 Jul 8;12(7). Epub 2020 Jul 8.

UOSD SAFU, Department of Research, Advanced Diagnostic, and Technological Innovation, IRCSS-Regina Elena National Cancer Institute, 00144 Rome, Italy.

Metastatic colorectal cancer (mCRC) remains challenging because of the emergence of resistance mechanisms to anti-epidermal growth factor receptor (EGFR) therapeutics, so more effective strategies to improve the patients' outcome are needed. During the last decade, the application of a multi-omics approach has contributed to a deeper understanding of the complex molecular landscape of human CRC, identifying a plethora of drug targets for precision medicine. Target validation relies on the use of experimental models that would retain the molecular and clinical features of human colorectal cancer, thus mirroring the clinical characteristics of patients. In particular, organoids and patient-derived-xenografts (PDXs), as well as genetically engineered mouse models (GEMMs) and patient-derived orthotopic xenografts (PDOXs), should be considered for translational purposes. Overall, omics and advanced mouse models of cancer represent a portfolio of sophisticated biological tools that, if optimized for use in concert with accurate data analysis, could accelerate the anticancer discovery process and provide new weapons against cancer. In this review, we highlight success reached following the integration of omics and experimental models; moreover, results produced by our group in the field of mCRC are also presented.
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http://dx.doi.org/10.3390/cancers12071830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408740PMC
July 2020

Circulating miRNAs in Small Extracellular Vesicles Secreted by a Human Melanoma Xenograft in Mouse Brains.

Cancers (Basel) 2020 Jun 19;12(6). Epub 2020 Jun 19.

Institute for Biomedical Technologies (ITB), CNR, 20090 Segrate, Italy.

The identification of liquid biomarkers remains a major challenge to improve the diagnosis of melanoma patients with brain metastases. Circulating miRNAs packaged into tumor-secreted small extracellular vesicles (sEVs) contribute to tumor progression. To investigate the release of tumor-secreted miRNAs by brain metastasis, we developed a xenograft model where human metastatic melanoma cells were injected intracranially in nude mice. The comprehensive profiles of both free miRNAs and those packaged in sEVs secreted by the melanoma cells in the plasma demonstrated that most (80%) of the sEV-associated miRNAs were also present in serum EVs from a cohort of metastatic melanomas, included in a publicly available dataset. Remarkably, among them, we found three miRNAs (miR-224-5p, miR-130a-3p and miR-21-5p) in sEVs showing a trend of upregulation during melanoma progression. Our model is proven to be valuable for identifying miRNAs in EVs that are unequivocally secreted by melanoma cells in the brain and could be associated to disease progression.
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http://dx.doi.org/10.3390/cancers12061635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352810PMC
June 2020

TRF2 and VEGF-A: an unknown relationship with prognostic impact on survival of colorectal cancer patients.

J Exp Clin Cancer Res 2020 Jun 15;39(1):111. Epub 2020 Jun 15.

Oncogenomic and Epigenetic Unit, IRCCS - Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Background: Colorectal cancer is one of most common tumors in developed countries and, despite improvements in treatment and diagnosis, mortality rate of patients remains high, evidencing the urgent need of novel biomarkers to properly identify colorectal cancer high-risk patients that would benefit of specific treatments. Recent works have demonstrated that the telomeric protein TRF2 is over-expressed in colorectal cancer and it promotes tumor formation and progression through extra-telomeric functions. Moreover, we and other groups evidenced, both in vitro on established cell lines and in vivo on tumor bearing mice, that TRF2 regulates the vascularization mediated by VEGF-A. In the present paper, our data evidence a tight correlation between TRF2 and VEGF-A with prognostic relevance in colorectal cancer patients.

Methods: For this study we sampled 185 colorectal cancer patients surgically treated and diagnosed at the Regina Elena National Cancer Institute of Rome and investigated the association between the survival outcome and the levels of VEGF-A and TRF2.

Results: Tissue microarray immunohistochemical analyses revealed that TRF2 positively correlates with VEGF-A expression in our cohort of patients. Moreover, analysis of patients' survival, confirmed in a larger dataset of patients from TCGA, demonstrated that co-expression of TRF2 and VEGF-A correlate with a poor clinical outcome in stage I-III colorectal cancer patients, regardless the mutational state of driver oncogenes.

Conclusions: Our results permitted to identify the positive correlation between high levels of TRF2 and VEGF-A as a novel prognostic biomarker for identifying the subset of high-risk colorectal cancer patients that could benefit of specific therapeutic regimens.
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http://dx.doi.org/10.1186/s13046-020-01612-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294609PMC
June 2020

BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors.

Nat Commun 2019 07 17;10(1):3143. Epub 2019 Jul 17.

Genome Stability and Tumourigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.

Heterozygous germline mutations in BRCA2 predispose to breast and ovarian cancer. Contrary to non-cancerous cells, where BRCA2 deletion causes cell cycle arrest or cell death, tumors carrying BRCA2 inactivation continue to proliferate. Here we set out to investigate adaptation to loss of BRCA2 focusing on genome-wide transcriptome alterations. Human cells in which BRCA2 expression is inhibited for 4 or 28 days are subjected to RNA-seq analyses revealing a biphasic response to BRCA2 abrogation. The early, acute response consists of downregulation of genes involved in cell cycle progression, DNA replication and repair and is associated with cell cycle arrest in G1. Surprisingly, the late, chronic response consists predominantly of upregulation of interferon-stimulated genes (ISGs). Activation of the cGAS-STING-STAT pathway detected in these cells further substantiates the concept that BRCA2 abrogation triggers cell-intrinsic immune signaling. Importantly, we find that treatment with PARP inhibitors stimulates the interferon response in cells and tumors lacking BRCA2.
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http://dx.doi.org/10.1038/s41467-019-11048-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637138PMC
July 2019

Chlorambucil targets BRCA1/2-deficient tumours and counteracts PARP inhibitor resistance.

EMBO Mol Med 2019 07 24;11(7):e9982. Epub 2019 May 24.

Genome Stability and Tumorigenesis Group, Department of Oncology, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK.

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.
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http://dx.doi.org/10.15252/emmm.201809982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609913PMC
July 2019

TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment.

Nucleic Acids Res 2019 04;47(7):3365-3382

Oncogenomic and Epigenetic Unit, IRCCS - Regina Elena National Cancer Institute, Via Elio Chianesi 53, Rome 00144, Italy.

The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.
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http://dx.doi.org/10.1093/nar/gkz041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468246PMC
April 2019

Chitosan-Based Polyelectrolyte Complexes for Doxorubicin and Zoledronic Acid Combined Therapy to Overcome Multidrug Resistance.

Pharmaceutics 2018 Oct 9;10(4). Epub 2018 Oct 9.

Department of Pharmacy, University of Naples Federico II, D. Montesano 49, 80131 Naples, Italy.

This study aimed to develop nanovectors co-encapsulating doxorubicin (Doxo) and zoledronic acid (Zol) for a combined therapy against Doxo-resistant tumors. Chitosan (CHI)-based polyelectrolyte complexes (PECs) prepared by ionotropic gelation technique were proposed. The influence of some experimental parameters was evaluated in order to optimize the PECs in terms of size and polydispersity index (PI). PEC stability was studied by monitoring size and zeta potential over time. In vitro studies were carried out on wild-type and Doxo-resistant cell lines, to assess both the synergism between Doxo and Zol, as well as the restoring of Doxo sensitivity. Polymer concentration, incubation time, and use of a surfactant were found to be crucial to achieving small size and monodisperse PECs. Doxo and Zol, only when encapsulated in PECs, showed a synergistic antiproliferative effect in all the tested cell lines. Importantly, the incubation of Doxo-resistant cell lines with Doxo/Zol co-encapsulating PECs resulted in the restoration of Doxo sensitivity.
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http://dx.doi.org/10.3390/pharmaceutics10040180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321278PMC
October 2018

Adipose-derived stem cell-mediated paclitaxel delivery inhibits breast cancer growth.

PLoS One 2018 7;13(9):e0203426. Epub 2018 Sep 7.

Anatomic Pathology, Department of Biomedicine and Prevention, Tor Vergata University, Rome, Italy.

Breast cancer represents the main malignancy in women and autologous fat grafting is a diffuse procedure in the management of post-surgical breast defects causing patients' psychosocial problems, with high costs for the public health. Recently, beneficial effects of fat grafting during post-surgical breast reconstruction have been amplified from the enrichment with human adipose-derived stem cells (ASCs) present in the stromal vascular fraction (SVF) of adult adipose tissue isolated during intraoperatory procedures. The major concern about the ASC enrichment during post-surgery breast reconstruction depends on their potential ability to release growth factors and hormones that can promote proliferation of residual or quiescent cancer cells, with the risk of de novo cancer development or recurrence. The recent description that adult stem cells primed in vitro may be vehicle for anti-cancer drug delivery offers a new vision concerning the role of ASCs in breast reconstruction after cancer surgery. Paclitaxel (PTX) is a chemotherapeutic agent acting as a microtubule-stabilizing drug inhibiting cancer cell mitotic activity. We optimized PTX loading and release in cultured ASCs and then analyzed the effects of PTX-loaded ASCs and their conditioned medium on CG5 breast cancer survival, proliferation and apoptosis in vitro, and inCG5 xenograft in vivo. We documented that ASCs can uptake and release PTX in vitro, with slight cytotoxic effects. Interestingly, PTX-loaded ASCs in co-culture, as well as conditioned medium alone, inhibited CG5 cell proliferation and survival in vitro and xenograft tumor growth in vivo. The antitumor effect of PTX-loaded ASCs may offer a new perspective concerning the use of ASCs during breast reconstruction becoming an additional local preventive chemotherapeutic agent against tumor recurrence. However, further experiments in vitro and in vivo are needed to collect more evidence confirming the efficacy and safety in cancer patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0203426PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128546PMC
February 2019

HSP90 Inhibition Drives Degradation of FGFR2 Fusion Proteins: Implications for Treatment of Cholangiocarcinoma.

Hepatology 2019 01 14;69(1):131-142. Epub 2018 Dec 14.

Unit of Oncogenomics and Epigenetics, IRCCS Regina Elena National Cancer Institute, Rome, Italy.

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F-TKIs) can elicit meaningful objective clinical responses in patients carrying FF-positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F-TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2-TACC3 (transforming acidic coiled-coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2-TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF-driven ICC.
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http://dx.doi.org/10.1002/hep.30127DOI Listing
January 2019

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

J Exp Clin Cancer Res 2018 Mar 13;37(1):57. Epub 2018 Mar 13.

UOSD SAFU, Regina Elena National Cancer Institute, Rome, Italy.

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.
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http://dx.doi.org/10.1186/s13046-018-0719-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850913PMC
March 2018

Xenograft as In Vivo Experimental Model.

Methods Mol Biol 2018 ;1692:97-105

Experimental Chemotherapy Laboratory, UOSD SAFU, Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

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.
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http://dx.doi.org/10.1007/978-1-4939-7401-6_9DOI Listing
June 2018

BRCA1 and BRCA2 tumor suppressors protect against endogenous acetaldehyde toxicity.

EMBO Mol Med 2017 10;9(10):1398-1414

Department of Oncology, Genome Stability and Tumorigenesis Group, The CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits the growth of BRCA1/2-deficient tumors and in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.
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http://dx.doi.org/10.15252/emmm.201607446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623864PMC
October 2017

Focal adhesion kinase depletion reduces human hepatocellular carcinoma growth by repressing enhancer of zeste homolog 2.

Cell Death Differ 2017 05 24;24(5):889-902. Epub 2017 Mar 24.

Liver Research Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer in humans. The focal adhesion tyrosine kinase (FAK) is often over-expressed in human HCC and FAK inhibition may reduce HCC cell invasiveness. However, the anti-oncogenic effect of FAK knockdown in HCC cells remains to be clarified. We found that FAK depletion in HCC cells reduced in vitro and in vivo tumorigenicity, by inducing G2/M arrest and apoptosis, decreasing anchorage-independent growth, and modulating the expression of several cancer-related genes. Among these genes, we showed that FAK silencing decreased transcription and nuclear localization of enhancer of zeste homolog 2 (EZH2) and its tri-methylation activity on lysine 27 of histone H3 (H3K27me3). Accordingly, FAK, EZH2 and H3K27me3 were concomitantly upregulated in human HCCs compared to non-tumor livers. In vitro experiments demonstrated that FAK affected EZH2 expression and function by modulating, at least in part, p53 and E2F2/3 transcriptional activity. Moreover, FAK silencing downregulated both EZH2 binding and histone H3K27me3 levels at the promoter of its target gene NOTCH2. Finally, we found that pharmacological inhibition of FAK activity resembled these effects although milder. In summary, we demonstrate that FAK depletion reduces HCC cell growth by affecting cancer-promoting genes including the pro-oncogene EZH2. Furthermore, we unveil a novel unprecedented FAK/EZH2 crosstalk in HCC cells, thus identifying a targetable network paving the way for new anticancer therapies.
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http://dx.doi.org/10.1038/cdd.2017.34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423113PMC
May 2017

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

J Exp Clin Cancer Res 2016 12 5;35(1):189. Epub 2016 Dec 5.

UOSD SAFU, Department of Research, Diagnosis and Innovative Technologies, Traslational Research Area, Regina Elena National Cancer Institute, Via Elio Chianesi 53, Rome, 00144, Italy.

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.
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http://dx.doi.org/10.1186/s13046-016-0462-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139018PMC
December 2016

EMICORON: A multi-targeting G4 ligand with a promising preclinical profile.

Biochim Biophys Acta Gen Subj 2017 May 9;1861(5 Pt B):1362-1370. Epub 2016 Nov 9.

Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Italy. Electronic address:

Background: During the last decade, guanine G-rich sequences folding into G-quadruplex (G4) structures have received a lot of attention and their biological role is now a matter of large debate. Rising amounts of experimental evidence have validated several G-rich motifs as molecular targets in cancer treatment. Despite that an increasing number of small molecules has been reported to possess excellent G4 stabilizing properties, none of them has progressed through the drug-development pipeline due to their poor drug-like properties. In this context, the identification of G4 ligands with more favorable pharmacological properties and with a well-defined target activity could be fruitful for anticancer therapy application.

Scope Of Review: This manuscript outlines the current state of knowledge regarding EMICORON, a G4-interactive molecule structurally and biologically similar, on the one side, to coronene and, on the other side, to a bay-monosubstituted perylene.

Major Conclusions: Overall this work evidences that EMICORON, a new promising G4 ligand, possesses a marked antitumoral activity both standing alone and in combination with chemotherapeutics. Moreover, EMICORON represents a good example of multimodal class of antitumoral drug, able to simultaneously affect multiple targets participating in several distinct signaling pathways, thus simplifying the treatment modalities and improving the selectivity against cancer cells.

General Significance: Due to the importance of G4 forming sequences in crucial biological processes participating in tumor progression, their successful targeting with small molecules could represent a very important innovation in the development of effective therapeutic strategies against cancer. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
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http://dx.doi.org/10.1016/j.bbagen.2016.11.010DOI Listing
May 2017

Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds.

Mol Cell 2016 Feb 31;61(3):449-460. Epub 2015 Dec 31.

Genome Stability and Tumourigenesis Group, CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK. Electronic address:

G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.
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http://dx.doi.org/10.1016/j.molcel.2015.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747901PMC
February 2016

Intragenic G-quadruplex structure formed in the human CD133 and its biological and translational relevance.

Nucleic Acids Res 2016 Feb 27;44(4):1579-90. Epub 2015 Oct 27.

Area of Translational Research, Regina Elena National Cancer Institute, via E. Chianesi 53, 00144 Rome, Italy

Cancer stem cells (CSCs) have been identified in several solid malignancies and are now emerging as a plausible target for drug discovery. Beside the questionable existence of CSCs specific markers, the expression of CD133 was reported to be responsible for conferring CSC aggressiveness. Here, we identified two G-rich sequences localized within the introns 3 and 7 of the CD133 gene able to form G-quadruplex (G4) structures, bound and stabilized by small molecules. We further showed that treatment of patient-derived colon CSCs with G4-interacting agents triggers alternative splicing that dramatically impairs the expression of CD133. Interestingly, this is strongly associated with a loss of CSC properties, including self-renewing, motility, tumor initiation and metastases dissemination. Notably, the effects of G4 stabilization on some of these CSC properties are uncoupled from DNA damage response and are fully recapitulated by the selective interference of the CD133 expression.In conclusion, we provided the first proof of the existence of G4 structures within the CD133 gene that can be pharmacologically targeted to impair CSC aggressiveness. This discloses a class of potential antitumoral agents capable of targeting the CSC subpopulation within the tumoral bulk.
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http://dx.doi.org/10.1093/nar/gkv1122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770210PMC
February 2016

Down-regulation of the Lamin A/C in neuroblastoma triggers the expansion of tumor initiating cells.

Oncotarget 2015 Oct;6(32):32821-40

Institute of Cell Biology and Neurobiology-CNR, Monterotondo, Rome, Italy.

Tumor-initiating cells constitute a population within a tumor mass that shares properties with normal stem cells and is considered responsible for therapy failure in many cancers. We have previously demonstrated that knockdown of the nuclear envelope component Lamin A/C in human neuroblastoma cells inhibits retinoic acid-mediated differentiation and results in a more aggressive phenotype. In addition, Lamin A/C is often lost in advanced tumors and changes in the nuclear envelope composition occur during tumor progression. Based on our previous data and considering that Lamin A/C is expressed in differentiated tissues, we hypothesize that the lack of Lamin A/C could predispose cells toward a stem-like phenotype, thus influencing the development of tumor-initiating cells in neuroblastoma. This paper demonstrates that knockdown of Lamin A/C triggers the development of a tumor-initiating cell population with self-renewing features in human neuroblastoma cells. We also demonstrates that the development of TICs is due to an increased expression of MYCN gene and that in neuroblastoma exists an inverse relationship between LMNA and MYCN expression.
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http://dx.doi.org/10.18632/oncotarget.5104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741732PMC
October 2015

Targeting G-Quadruplex DNA Structures by EMICORON Has a Strong Antitumor Efficacy against Advanced Models of Human Colon Cancer.

Mol Cancer Ther 2015 Nov 24;14(11):2541-51. Epub 2015 Aug 24.

Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy.

We previously identified EMICORON as a novel G-quadruplex (G4) ligand showing high selectivity for G4 structures over the duplex DNA, causing telomere damage and inhibition of cell proliferation in transformed and tumor cells. Here, we evaluated the antitumoral effect of EMICORON on advanced models of human colon cancer that could adequately predict human clinical outcomes. Our results showed that EMICORON was well tolerated in mice, as no adverse effects were reported, and a low ratio of sensitivity across human and mouse bone marrow cells was observed, indicating a good potential for reaching similar blood levels in humans. Moreover, EMICORON showed a marked therapeutic efficacy, as it inhibited the growth of patient-derived xenografts (PDX) and orthotopic colon cancer and strongly reduced the dissemination of tumor cells to lymph nodes, intestine, stomach, and liver. Finally, activation of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies, demonstrated that EMICORON had an unprecedented antitumor activity warranting further studies of EMICORON-based combination treatments.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0253DOI Listing
November 2015

Erratum: identification of novel RHPS4-derivative ligands with improved toxicological profiles and telomere-targeting activities.

J Exp Clin Cancer Res 2015 Feb 5;34. Epub 2015 Feb 5.

Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Nottingham, NG7 2RD, UK.

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http://dx.doi.org/10.1186/s13046-014-0101-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326507PMC
February 2015

A basal level of DNA damage and telomere deprotection increases the sensitivity of cancer cells to G-quadruplex interactive compounds.

Nucleic Acids Res 2015 Feb 23;43(3):1759-69. Epub 2015 Jan 23.

Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy

Here, with the aim of obtaining insight into the intriguing selectivity of G-quadruplex (G4) ligands toward cancer compared to normal cells, a genetically controlled system of progressive transformation in human BJ fibroblasts was analyzed. Among the different comparative evaluations, we found a progressive increase of DNA damage response (DDR) markers throughout the genome from normal toward immortalized and transformed cells. More interestingly, sensitivity to G4 ligands strongly correlated with the presence of a basal level of DNA damage, including at the telomeres, where the chromosome ends were exposed to the DDR without concurrent induction of DNA repair activity, as revealed by the lack of 53BP1 recruitment and telomere aberrations. The link between telomere uncapping and the response to G4 stabilization was directly assessed by showing that a partial TRF2 depletion, causing a basal level of telomere localized DDR, rendered telomerized fibroblasts prone to G4-induced telomere damage and anti-proliferative defects. Taken together these data strongly indicate that the presence of a basal level of telomere-associated DDR is a determinant of susceptibility to G4 stabilization.
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http://dx.doi.org/10.1093/nar/gkv006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330372PMC
February 2015

Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells.

Oncotarget 2015 Feb;6(5):2779-93

Department of Experimental Oncology, Regina Elena National Cancer Institute, Rome, Italy.

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413617PMC
http://dx.doi.org/10.18632/oncotarget.2995DOI Listing
February 2015

Medical treatment of orthotopic glioblastoma with transferrin-conjugated nanoparticles encapsulating zoledronic acid.

Oncotarget 2014 Nov;5(21):10446-59

Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Via Costantinopoli, Naples, Italy.

Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279385PMC
http://dx.doi.org/10.18632/oncotarget.2182DOI Listing
November 2014

Identification of novel RHPS4-derivative ligands with improved toxicological profiles and telomere-targeting activities.

J Exp Clin Cancer Res 2014 Oct 6;33:81. Epub 2014 Oct 6.

Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, via delle Messi d'Oro 156, 00158, Rome, Italy.

The pentacyclic acridinium salt RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate, compound 1) is one of the most interesting DNA G-quadruplex binding molecules due to its high efficacy in tumor cell growth inhibition both in in vitro models and in vivo against human tumor xenografts in combination with conventional chemotherapeutics. Despite compound 1 having desirable chemical and pharmaceutical properties, its potential as a therapeutic agent is compromised by off-target effects on cardiovascular physiology. In this paper we report a new series of structurally-related compounds which were developed in an attempt to minimize its off-target profile, but maintaining the same favorable chemical and pharmacological features of the lead compound. By performing a comparative analysis it was possible to identify which derivatives had the following properties: (i) to show a reduced capacity in respect to compound 1 to inhibit the hERG tail current tested in a patch clamp assay and/or to interact with the human recombinant β2 receptor; (ii) to maintain both a good G4-binding affinity and cancer cell selectivity; and (iii) to trigger DNA damage with specific telomere uncapping. These studies allowed us to identify a novel G4-stabilizing molecule, compound 8, being characterized by reduced off-target effects and potent telomere on-target properties compared to the prototypic compound 1. Moreover, compound 8 shares with compound 1 the same molecular mode of action and an anti-tumour activity specifically restricted to replicating cells, as evident with its particularly efficient activity in combination therapy with a topoisomerase I inhibitor. In conclusion, we have identified a new pentacyclic derivative 8 having suitable properties to be the focus of further investigations as a clinical candidate for cancer therapy.
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http://dx.doi.org/10.1186/s13046-014-0081-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4193996PMC
October 2014

A new avenue toward androgen receptor pan-antagonists: C2 sterically hindered substitution of hydroxy-propanamides.

J Med Chem 2014 Sep 28;57(17):7263-79. Epub 2014 Aug 28.

Institute for the Organic Synthesis and Photoreactivity, Italian National Research Council , Via Gobetti 101, 40129 Bologna, Italy.

The androgen receptor (AR) represents the primary target for prostate cancer (PC) treatment even when the disease progresses toward androgen-independent (AIPC) or castration-resistant (CRPC) forms. Because small chemical changes in the structure of nonsteroidal AR ligands determine the pharmacological responses of AR, we developed a novel stereoselective synthetic strategy that allows sterically hindered C2-substituted bicalutamide analogues to be obtained. Biological and theoretical evaluations demonstrate that C2-substitution with benzyl and phenyl moieties is a new, valuable option toward improving pan-antagonist behavior. Among the synthesized compounds, (R)-16m, when compared to casodex, (R)-bicalutamide, and enzalutamide, displayed very promising in vitro activity toward five different prostate cancer cell lines, all representative of CPRC and AIPC typical mutations. Despite being less active than (R)-bicalutamide, (R)-16m also displayed marked in vivo antitumor activity on VCaP xenografts and thus it may serve as starting point for developing novel AR pan-antagonists.
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http://dx.doi.org/10.1021/jm5005122DOI Listing
September 2014

Human placenta-derived neurospheres are susceptible to transformation after extensive in vitro expansion.

Stem Cell Res Ther 2014 Apr 22;5(2):55. Epub 2014 Apr 22.

Introduction: The cancer stem cell model links neoplastic cells with normal stem cell biology, but little is known on how normal stem cells are transformed into cancer stem cells.

Methods: To investigate the processes underlying the transformation of normal stem cells we developed in vitro a cancer stem cell model from human amniotic and chorionic placenta membranes. In this model we studied the expression of specific stem cell molecules by flow cytometry, and genes, by real time RT-PCR. Microscopy immunfluorescence was employed to investigate the proliferative and differentiation patterns. Fluorescence microscopy and FACS were employed to investigate the proliferative and differentiation patterns. To evaluate the tumorigenic potential of our model we injected the cells into NOD.CB17-Prkdcscid/NCrHsd mice.

Results: Normal human stem cells from amniotic and chorionic placenta membranes were converted into neural cell lineages, under specific conditions, to form secondary neurospheres with a capacity for self-renewal. After extensive in vitro culture, these cells underwent spontaneous transformations and acquired a neuroblastoma (NB)-like phenotype with an elevated proliferative potential that is comparable to established neuroblastoma cell lines. The ability of these cells to transform their phenotype was evidenced by increased clonogenic ability in vitro; by augmented expression level of certain proliferation- and transformation-related genes (e.g., CCNA2, MYCN, ENPP2, GRIA3, and KIT); by the presence of multinucleated and hyperdiploid cells. We further demonstrated that the transformed phenotype is an NB by measuring the expression of NB-specific markers, disialoganglioside GD2 and N-Myc proteins.

Conclusions: We have developed a cancer stem cell model starting from normal human stem cells derived from amniotic and chorionic placenta membranes. These cells are able to differentiate into neural cell lineages and to undergo spontaneous transformations and acquire an NB-like phenotype.
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http://dx.doi.org/10.1186/scrt444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055136PMC
April 2014

Evidence for G-quadruplex in the promoter of vegfr-2 and its targeting to inhibit tumor angiogenesis.

Nucleic Acids Res 2014 Mar 11;42(5):2945-57. Epub 2013 Dec 11.

Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy, Laboratory of Molecular Pathology, Department of Pharmacy, University of Naples "Federico II", Naples, Italy, Department of Clinical and Molecular Medicine, University of Rome "La Sapienza", Rome, Italy, Laboratory of Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy, Institute for Research on Cancer and Aging, Nice (IRCAN), Nice University, CNRS UMR7284/INSERM U1081, Faculty of Medicine, Nice, France and Department of Medical Genetics, Archet 2 Hospital, CHU of Nice, France.

Tumor angiogenesis is mainly mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced by cancer cells and active on the endothelium through the VEGF receptor 2 (VEGFR-2). Here we identify a G-rich sequence within the proximal promoter region of vegfr-2, able to form an antiparallel G-quadruplex (G4) structure. This G4 structure can be efficiently stabilized by small molecules with the consequent inhibition of vegfr-2 expression. Functionally, the G4-mediated reduction of VEGFR-2 protein causes a switching off of signaling components that, converging on actin cytoskeleton, regulate the cellular events leading to endothelial cell proliferation, migration and differentiation. As a result of endothelial cell function impairment, angiogenic process is strongly inhibited by G4 ligands both in vitro and in vivo. Interestingly, the G4-mediated antiangiogenic effect seems to recapitulate that observed by using a specific interference RNA against vegfr-2, and it is strongly antagonized by overexpressing the vegfr-2 gene. In conclusion, we describe the evidence for the existence of G4 in the promoter of vegfr-2, whose expression and function can be markedly inhibited by G4 ligands, thereby revealing a new, and so far undescribed, way to block VEGFR-2 as target for anticancer therapy.
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http://dx.doi.org/10.1093/nar/gkt1289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950687PMC
March 2014

On and off-target effects of telomere uncapping G-quadruplex selective ligands based on pentacyclic acridinium salts.

J Exp Clin Cancer Res 2013 Sep 19;32:68. Epub 2013 Sep 19.

Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment. The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant ß2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay. Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with ß2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities. Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.
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http://dx.doi.org/10.1186/1756-9966-32-68DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849007PMC
September 2013