Carmen D'Angelo

EVALUATION OF MULTIPLE BIO-PATHOLOGICAL FACTORS IN COLORECTAL ADENOCARCINOMAS: INDEPENDENT PROGNOSTIC ROLE OF p53 AND Bcl-2

About 40% of patients with colorectal carcinoma will develop local or distant tumour recurrences. Integrated analyses of bio‐pathological markers, predictive of tumour aggressiveness, may offer a more rational approach to planning adjuvant therapy. To this end, we analysed the correlation between p53 accumulation, Bcl‐2 expression, DNA ploidy, cell proliferation and conventional clinico‐pathological parameters by testing the prognostic significance of these variables in a series of 171 colorectal carcinoma patients with long‐term follow‐up. The relationships among the various bio‐pathological parameters, analysed by multiple correspondence analysis, showed 2 different clinico‐biological profiles. The first, characterised by p53 negativity, Bcl‐2 positivity, diploidy, low percentage of cells in S‐phase (%S‐phase), a low Ki‐67 score, is associated with Dukes A‐B stage, well differentiated tumours and lack of relapse. The second, defined by p53 positivity, Bcl‐2 negativity, aneuploidy, high %S‐phase and elevated Ki‐67 score, correlates with Dukes C‐D stage, poorly differentiated tumours and presence of relapse. When these parameters were examined according to Kaplan‐Meiers method, significantly shorter disease‐free (DFS) and overall survival (OS) were also observed in patients bearing p53 positive and Bcl‐2 negative tumours, in Dukes B stage. In multivariate analysis, p53 accumulation and Bcl‐2 expression emerged as independent predictors of a worse and better clinical outcome, respectively. Our results indicate that, in colorectal adenocarcinomas, a biological profile, based on the combined evaluation of p53 and Bcl‐2, may be useful for identifying high risk patients to be enrolled in an adjuvant setting, mainly in an early stage of the disease. Int. J. Cancer (Pred. Oncol.) 84:545–552, 1999.

N-cyclic bay-substituted perylene G-quadruplex ligands have selective antiproliferative effects on cancer cells and induce telomere damage.

A series of bay-substituted perylene derivatives is reported as a new class of G-quadruplex ligands. The synthesized compounds have differing N-cyclic substituents on the bay area and differing side chains on the perylene major axis. ESI-MS and FRET measurements highlighted the strongest quadruplex binders in this series and those showing the highest quadruplex/duplex selectivity. Several biological assays were performed on these compounds, which showed that compound 5 (PPL3C) triggered a DNA damage response in transformed cells with the formation of telomeric foci containing phosphorylated γ-H2AX and 53BP1. This effect mainly occurred in replicating cells and was consistent with Pot1 dissociation. Compound 5 does not induce telomere damage in normal cells, which are unaffected by treatment with the compound, suggesting that this agent preferentially kills cancer cells. These results reinforce the notion that G-quadruplex binding compounds can act as broad inhibitors of telomere-related processes and have potential as selective antineoplastic drugs.

Antisense clusterin oligodeoxynucleotides increase the response of HER-2 gene amplified breast cancer cells to Trastuzumab.

Clusterin (CLU) is a heterodimeric secreted glycoprotein implicated in several physiological and pathological processes including cancer. Although recent data showed that overexpression of CLU is closely associated with disease progression in patients with breast tumor, the functional role of CLU expression in this tumor hystotype remains to be determined. The objectives in this study were to evaluate CLU expression levels after treatment with Trastuzumab, a HER2‐targeted monoclonal antibody used in the clinical management of advanced breast cancer patients, and to test the usefulness of combined treatment with OGX‐011, the second generation 2′‐methoxyethyl gapmer oligonucleotides targeting the CLU gene, and Trastuzumab in this tumor hystotype. By using the HER‐2 gene amplified‐BT474 human breast cancer cells, we found Trastuzumab decreased HER‐2 expression and inhibited cell proliferation without affecting apoptosis. Interestingly, Trastuzumab treatment up‐regulated CLU protein expression in a dose‐dependent fashion. We therefore hypothesized that the treatment with OGX‐011, by blocking Trastuzumab‐induced CLU expression, might potentiate the growth‐inhibitory effect of Trastuzumab alone. Although OGX‐011 had no effect on the behavior of the BT474 cells when used alone, it significantly enhanced the sensitivity of cells to Trastuzumab. A significant increase in the percentage of apoptotic cells, analyzed in terms of annexin V positivity and cleavage of poly(ADP‐ribose) polymerase, was observed after combined treatment with OGX‐011 plus Trastuzumab but not with either agent alone. Altogether our findings suggest that combined targeting of HER‐2 and CLU may represent a novel, rational approach to breast cancer therapy.

MDM4 (MDMX) overexpression enhances stabilization of stress-induced p53 and promotes apoptosis.

Rescue of embryonic lethality in MDM4-/- mice through concomitant loss of p53 has revealed a functional partnership between the two proteins. Biochemical studies have suggested that MDM4 may act as a negative regulator of p53 levels and activity. On the other hand, MDM4 overexpression has been reported to stabilize p53 levels and to counteract MDM2-degradative activity. We have investigated the functional role of MDM4 overexpression on cell behavior. In both established and primary cells cultured under stress conditions, overexpression of MDM4 significantly increased p53-dependent cell death, in correlation with enhanced induction of the endogenous p53 protein levels. This phenomenon was associated with induced p53 transcriptional activity and increased levels of the proapoptotic protein, Bax. Further, p53 stabilization was accompanied by decreased association of the protein to its negative regulator, MDM2. These findings reveal a novel role for MDM4 by demonstrating that in non-tumor cells under stress conditions it may act as a positive regulator of p53 activity, mainly by controlling p53 levels. They also indicate a major distinction between the biological consequences of MDM4 and MDM2 overexpression.

Exploring the Chemical Space of G‑Quadruplex Binders: Discovery of a Novel Chemotype Targeting the Human Telomeric Sequence

Recent findings have unambiguously demonstrated that DNA G-rich sequences can adopt a G-quadruplex folding in living cells, thus further validating them as crucial targets for anticancer therapy. Herein, to identify new potent G4 binders as antitumor drug candidates, we have targeted a 24-nt G4-forming telomeric sequence employing a receptor-based virtual screening approach. Among the best candidates, in vitro binding experiments allowed identification of three novel G4 ligands. Among them, the best compound features an unprecedented binding selectivity for the human telomeric DNA G-quadruplex with no detectable binding for other G4-forming sequences present at different genomic sites. This behavior correlates with the detected ability to generate DNA damage response in tumor cells at the telomeric level and efficient antiproliferative effect on different tumor cell lines at low micromolar concentrations.

DNA Damage Persistence as Determinant of Tumor Sensitivity to the Combination of Topo I Inhibitors and Telomere-Targeting Agents

Purpose: We previously reported that the G-quadruplex (G4) ligand RHPS4 potentiates the antitumor activity of camptothecins both in vitro and in tumor xenografts. The present study aims at investigating the mechanisms involved in this specific drug interaction. Experimental Design: Combination index test was used to evaluate the interaction between G4 ligands and standard or novel Topo I inhibitors. Chromatin immunoprecipitation was performed to study the presence at telomeres of various types of topisomerase, while immunolabeling experiments were performed to measure the activation of DNA damage both in vitro and in tumor xenografts. Results: We report that integration of the Topo I inhibitor SN-38, but not the Topo II poison doxorubicin with telomere-based therapy is strongly effective and the sequence of drug administration is critical in determining the synergistic interaction, impairing the cell ability to recover from drug-induced cytotoxicity. The synergistic effect of this combination was also observed by using novel camptothecins and, more interestingly, mice treated with ST1481/RHPS4 combination showed an inhibition and delay of tumor growth as well as an increased survival. The study of the mechanism(s) revealed that treatment with G4 ligands increased Topo I at the telomeres and the functional relevance of this observation was directly assessed by showing that standard and novel camptothecins stabilized DNA damage both in vitro and in xenografts. Conclusions: Our results demonstrate an outstanding efficacy of Topo I inhibitors/G4 ligands combination, which likely reflects an enhanced and persistent activation of DNA damage response as a critical determinant of the therapeutic improvement. Clin Cancer Res; 17(8); 2227–36. ©2011 AACR.

Smad-Interacting Protein-1 and MicroRNA 200 Family Define a Nitric Oxide–Dependent Molecular Circuitry Involved in Embryonic Stem Cell Mesendoderm Differentiation

Objective—Smad-interacting protein-1 (Sip1/ZEB2) is a transcriptional repressor of the telomerase reverse transcriptase catalytic subunit (Tert) and has recently been identified as a key regulator of embryonic cell fate with a phenotypic effect similar, in our opinion, to that reported for nitric oxide (NO). Remarkably, SIP1/ZEB2 is a known target of the microRNA 200 (miR-200) family. In this light, we postulated that Sip1/ZEB2 and the miR-200 family could play a role during the NO-dependent differentiation of mES. Methods and Results—The results of the present study show that Sip1/ZEB2 expression is downregulated during the NO-dependent expression of mesendoderm and early cardiovascular precursor markers, including Flk1 and CXCR4 in mES. Coincidently, members of the miR-200 family, namely miR-429, -200a, -200b, and -200c, were transcriptionally induced in parallel to mouse Tert. This regulation occurred at the level of chromatin. Remarkably, miR-429/miR-200a overexpression or Sip1/ZEB2 knockdown by short hairpin RNA interference elicited a gene expression pattern similar to that of NO regardless of the presence of leukemia inhibitory factor. Conclusion—These results are the first demonstrating that the miR-200 family and Sip1/ZEB2 transcription factor are regulated by NO, indicating an unprecedented molecular circuitry important for telomerase regulation and early differentiation of mES.

In vivo administration of liposomal vincristine sensitizes drug-resistant human solid tumors

Here we evaluated the antitumor efficacy of vincristine (VCR) encapsulated in sphingomyelin/cholesterol liposomes (SM/Chol) on drug‐resistant human solid tumors. We firstly used the M14 human melanoma line and the counterpart resistant derivative, M14/R. The M14/R, selected after doxorubicin exposure, was cross resistant to VCR: the in vitro treatment with free VCR reduced the survival of M14, while M14/R line was completely resistant to VCR. Encapsulation in liposomes improved the efficacy of VCR in M14 cells and sensitized the M14/R line to the drug. Experiments in vivo confirmed these results. The treatment of M14 bearing mice with VCR resulted in marked reduction of tumor growth, while no antitumoral effect was observed in M14/R tumors. The administration of VCR encapsulated in liposomes was able to sensitize M14/R tumors to the drug, the antitumoral effect being comparable to that observed in M14 tumors after the same treatment. By injecting animals with the same dose of liposomal VCR fractionated into 3 daily injections and administering repeated cycles of treatment, to a marked improvement of the antitumor activity of liposomal VCR was observed. TUNEL assay in tumor sections indicated that the improved efficacy of liposomal VCR was related to the induction of massive necrosis and apoptosis. To confirm the efficacy of liposomal VCR on drug‐resistant tumors, MCF7 breast and LoVo colon carcinomas, sensitive and resistant to VCR treatment, were also employed. The results showed that the treatment with liposomal VCR of mice bearing breast or colon resistant tumors reduced the tumor mass and delayed the tumor regrowth to the same extent observed in the sensitive counterpart. Together, these results demonstrate the ability of VCR encapsulated in liposomes in sensitizing drug resistant tumors of different histotypes.

Potentiation of the antitumoral activity of gemcitabine and paclitaxel in combination on human breast cancer cells

The purpose of this study was to evaluate the antitumoral activity of different gemcitabine-based combination on an experimental model of human breast cancer, in order to identify the most effective treatment and to provide a rationale for clinical investigations. To this aim, CG5 breast cancer cells were treated in vitro with gemcitabine followed by epirubicin, doxorubicin, docetaxel or paclitaxel. The reversed sequence was also investigated. Results, analyzed by multiple drug effect/combination index (CI) isobologram, demonstrated that the combination gemcitabine/paclitaxel was the most active showing synergism with a CI of about 0.5 in the two sequences employed. Moreover, the synergistic interaction of gemcitabine and paclitaxel was correlated to a block of the cells in the G0/G1 compartment of cell cycle and to an increase of apoptotic cells compared to each drug. Based on these evidences, the antitumoral efficacy of gemcitabine/paclitaxel combination has been studied in vivo. Mice bearing CG5 human breast xenografts treated with paclitaxel and gemcitabine in combination showed a significant higher inhibition of tumor growth (~ 70%) compared to that with either agent alone (25%). In conclusion, this study suggests that paclitaxel is the most promising agent for combination protocols with gemcitabine and supports the use of gemcitabine/paclitaxel combination in the clinical management of advanced breast cancer.

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

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.