Luca Esposito

ATP-noncompetitive CDK inhibitors for cancer therapy: an overview

Introduction: Cyclin-dependent kinases (CDKs) are the key drivers of cell cycle progression and are often deregulated in cancer, therefore, targeting CDKs has long been pursued as a therapeutic strategy to tackle cancer. Unfortunately, however, none of the first-generation CDK inhibitors has yielded the expected efficacy to be successfully translated to the clinic mostly because, by targeting the very conserved kinase ATP-binding site resulted to be poorly specific and quite toxic. Areas covered: Here, the authors review recent approaches aimed at developing more specific CDK inhibitors mostly through the aid of computational drug design studies and report various small molecules and peptides, which resulted in promising CDK ATP-noncompetitive inhibitors. Expert opinion: Despite few successes, these new approaches still need additional considerations to generate effective antitumoral agents. The authors discuss some of the hurdles to overcome for a successful clinical translation.

Anticancer Therapeutic Strategies Based on CDK Inhibitors

Normal cell cycle progression is controlled by the sequential action of cyclin-dependent kinases (CDKs), the activity of which depends on their binding to regulatory partners (cyclins). Deregulation of cell cycle is one of the first steps that transform normal cells into tumor cells. Indeed, most cancer cells bear mutations in members of the pathways that control the CDK activity. For this reason, this kinase family is a crucial target for the development of new drugs for cancer therapy. Recently, both ATP-competitive CDK inhibitors and the last generation of non-ATP-competitive inhibitors are emerging as promising agents for targeted therapies. Many clinical trials are in progress, using CDK inhibitors both as single agents and in combination with traditional cytotoxic agents. In this review, we will discuss new therapeutic strategies based on the use of CDK inhibitors in cancer.

RB gene family: Genome-wide ChIP approaches could open undiscovered roads

Many in vitro and reporter assays have helped to clarify how transcription factors regulate gene transcription. Today, it is important to decode the map of all transcription factor binding sites in the genome context. Chromatin immunoprecipitation followed by genome‐wide analyses have tremendously opened new ways to analyze the mechanisms of action of DNA binding factors, cofactors and epigenetic modifications. It is now possible to correlate these regulatory mechanisms with genomic features such as the promoter, enhancer, silencer, intragenic, and intergenic DNA sequences. These approaches help to clarify the complex rules that govern many biological processes. In this review we discuss the genome‐wide approaches applied to the retinoblastoma gene family (RBF), the central player of cell cycle control. There are also new, possible directions that are suggested within the review that can be followed to further explore the role of each pRb members in the transcriptional networks of the cell. J. Cell. Biochem. 109: 839–843, 2010.

Androgen receptor serine 81 mediates Pin1 interaction and activity

Hormone-dependent tumors are characterized by deregulated activity of specific steroid receptors, allowing aberrant expression of many genes involved in cancer initiation, progression and metastasis. In prostate cancer, the androgen receptor (AR) protein has pivotal functions, and over the years it has been the target of different drugs. AR is a nuclear receptor whose activity is regulated by a phosphorylation mechanism controlled by hormone and growth factors. Following phosphorylation, AR interacts with many cofactors that closely control its function. Among such cofactors, Pin1 is a peptidyl-prolyl isomerase that is involved in the control of protein phosphorylation and has a prognostic value in prostate cancer. In the present study, we demonstrate that ARSer81 is involved in the interaction with Pin1, and that this interaction is important for the transcriptional activity of AR. Since Pin1 expression positively correlates with tumor grade, our results suggest that Pin1 can participate in this process by modulating AR function.

SRC Family Kinase Inhibition Through a New Pyrazolo[3,4‐d]Pyrimidine Derivative as a Feasible Approach for Glioblastoma Treatment

Glioblastoma (GB) is the most common and aggressive primary tumor of the central nervous system. The current standard of care for GB consists of surgical resection, followed by radiotherapy combined with temozolomide chemotherapy. However, despite this intensive treatment, the prognosis remains extremely poor. Therefore, more effective therapies are urgently required. Recent studies indicate that SRC family kinases (SFKs) could represent promising molecular targets for GB therapy. Here, we challenged four GB cell lines with a new selective pyrazolo[3,4‐d]pyrimidine derivative SFK inhibitor, called SI221. This compound exerted a significant cytotoxic effect on GB cells, without significantly affecting non‐tumor cells (primary human skin fibroblasts), as evaluated by MTS assay. We also observed that SI221 was more effective than the well‐known SFK inhibitor PP2 in GB cells. Notably, despite the high intrinsic resistance to apoptosis of GB cells, SI221 was able to induce this cell death process in all the GB cell lines, as observed through cytofluorimetric analysis and caspase‐3 assay. SI221 also exerted a long‐term inhibition of GB cell growth and was able to reduce GB cell migration, as shown by clonogenic assay and scratch test, respectively. Moreover, through in vitro pharmacokinetic assays, SI221 proved to have a high metabolic stability and a good potential to cross the blood brain barrier, which is an essential requirement for a drug intended to treat brain tumors. Therefore, despite the need of developing strategies to improve SI221 solubility, our results suggest a potential application of this selective SFK inhibitor in GB therapy. J. Cell. Biochem. 116: 856–863, 2015.

Rapid maxillary expansion in growing patients. Hyrax versus transverse sagittal maxillary expander: a cephalometric investigation

The aim of this retrospective study was to cephalometrically evaluate and compare the skeletal and dental effects of a transverse sagittal maxillary expander (TSME) and a Hyrax-type expander (RME) in children with maxillary hypoplasia. Fifty subjects (26 males and 24 females), aged from 6 to 15 years, with a maxillary crossbite caused by basal apical narrowness, were divided into two equal groups. Twenty-five were treated with a TSME and the other 25 with a RME. For each patient, a lateral cephalogram was obtained before treatment (T0) and at the end of the retention period (T1). Changes in the two groups during the observation period were calculated, compared, and statistically analysed with a paired samples t -test. In the TSME group, SNP-A, I SN, and I FH and in the RME group SN-SNP.SNA, N-Me, and U6.PP displayed a statistically significant increase (P < 0.05). The increase in SNP-A, I SN, and I FH in the TSME group was significantly greater following treatment than in the RME group. The results support the use of the TSME to produce skeletal changes and dentoalveolar modification and to correct maxillary hypoplasia. It was also demonstrated that in patients with an anterior open bite, the use of the TSME is not contraindicated as the anterior vertical dimension did not increase significantly.

Blood-Derived Stem Cells (BDSCs) Plasticity: In Vitro Hepatic Differentiation

The limited availability of hepatic tissue suitable for the treatment of liver disease and drug research encourages the generation of hepatic‐like cells from alternative sources as support for the regenerative medicine. Human blood derived stem cells (BDSCs) express surface markers and genes characteristic of pluripotent stem cells and have the ability to differentiate into different cell types, including tissues of endodermal origin (i.e., liver). Therefore they can represent a valuable source of hepatocytes for medicine. In this investigation, we exploited a fast hepatic differentiation protocol to generate hepatocyte‐like cells from human BDSCs using only hepatocyte growth factor (HGF) and fibroblast growth factor‐4 (FGF‐4) as growth factors. The resulting cell population exhibited hepatic cell‐like morphology and it was characterized with a variety of biological endpoint analyses. Here, we demonstrate how human BDSCs can be reprogrammed in hepatocyte‐like cells by morphological, functional analysis, reverse transcriptase (RT)‐PCR, and Western Blot assay. This study defines a fast and easy reprogramming strategy that facilitates the differentiation of human BDSCs along a hepatic lineage and provides a framework for a helpful source in the stem cells therapy and liver disorders. J. Cell. Physiol. 228: 1249–1254, 2013.

RBL2/p130 is a direct AKT target and is required to induce apoptosis upon AKT inhibition in lung cancer and mesothelioma cell lines

The retinoblastoma (RB) protein family includes RB1/p105, RBL1/p107, and RBL2/p130, which are key factors in cell-cycle regulation and stand at the crossroads of multiple pathways dictating cell fate decisions. The role of RB proteins in apoptosis is controversial because they can inhibit or promote apoptosis depending on the context, on the apoptotic stimuli and on their intrinsic status, impacting on the response to antitumoral treatments. Here we identified RBL2/p130 as a direct substrate of the AKT kinase, a key antiapoptotic factor hyperactive in multiple cancer types. We showed that RBL2/p130 and AKT1 physically interact and AKT phosphorylates RBL2/p130 Ser941, located in the pocket domain, but not when this residue is mutated into Ala. We found that pharmacological inhibition of AKT, through the highly selective AKT inhibitor VIII (AKTiVIII), impairs RBL2/p130 Ser941 phosphorylation and increases RBL2/p130 stability, mRNA expression and nuclear levels in both lung cancer and mesothelioma cell lines, mirroring the more extensively studied effects on the p27 cell-cycle inhibitor. Consistently, AKT inhibition reduced cell viability, induced cell accumulation in G0/G1, and triggered apoptosis, which proved to be largely dependent on RBL2/p130 itself, as shown upon RBL2/p130 silencing. AKT inhibition induced RBL2/p130-dependent apoptosis also in HEK-293 cells, in which re-expression of a short hairpin-resistant RBL2/p130 was able to rescue AKTiVIII-induced apoptosis upon RBL2/p130 silencing. Our data also showed that the combination of AKT and cyclin-dependent kinases (CDK) inhibitors, which converge on the re-activation of RBL2/p130 antitumoral potential, could be a promising anticancer strategy.

Abstract LB-080: Reactivating RBL2/p130 oncosuppressive function as a new, possible antitumoral strategy

Deregulation of cell cycle control is the leading cause of cancer. The retinoblastoma (Rb) family members, including RB1/p105, RBL1/p107 and RBL2/p130, are crucial to restrain cell cycle progression and their inactivation, either direct or indirect, is a hallmark of most human tumors. In particular, RBL2/p130 emerging role in senescence and apoptosis, seems to contribute importantly to its tumor suppressor function. Furthermore, many studies largely contributed to establish RBL2/p130 as an important cancer target, which is inactivated by cell cycle kinases and whose deregulation underlies various cancer types. In this regard, we set out to restore RBL2/p130 function in tumors and exploit its tumor suppressive potential for cancer therapy. In particular, we have identified, through computational chemistry and molecular modeling studies, a small molecule able to act as a specific inhibitor of the CDK2-CycA complex and to reactivate the tumor suppressive function of RBL2/p130 in cancer. We analyzed by MTS assay the cytotoxic effect of our compound on a panel of different tumor cell lines and determined its IC50 values. We evaluated its effects on cell cycle and apoptosis by FACS and dissected its molecular mechanism of action by Western blot and RT-PCR. We found that our compound effectively inhibited proliferation of lung cancer and mesothelioma cell lines by specifically inhibiting CDK2-CycA activity towards RBL2/p130. The decrease in RBL2/p130 phosphorylation led to stabilization of its complex with the E2F4 transcriptional factor and consequent repression of their targets necessary for cell cycle progression, including CDK2 itself. Consistently, our compound arrested cell cycle and triggered apoptosis. Interestingly, apoptosis seemed to be mediated by RBL2/p130 itself because it was repressed in RBL2/p130-silenced cells. Furthermore, our compound proved to be active in an A549 xenograft model of lung cancer. Overall our findings indicate that the pharmacological reactivation of RBL2/p130 induces its cell-cycle restraining and pro-apoptotic functions, the latter being still largely unexplored, and identify a new possible therapeutic strategy for cancer treatment. Citation Format: Francesca Pentimalli, Luca Esposito, Iris Maria Forte, Carmelina Antonella Iannuzzi, Flavio Rizzolio, Tiziano Tuccinardi, Paola Indovina, Silvia Boffo, Antonio Giordano. Reactivating RBL2/p130 oncosuppressive function as a new, possible antitumoral strategy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-080. doi:10.1158/1538-7445.AM2015-LB-080

Evaluation of patients’ compliance in different age groups: Preventive methodology

BACKGROUND The aim of this work was to evaluate the effectiveness of the SSRD Department of University of Milan PREVENTION PROGRAM between subjects of different sex and ages. METHODS Prevention Program is divided into six stages, in which specific and standardized procedures are effected on patient; then checkups are planned after three months. Ninety patients (48 females, 42 males) were included. Subjects were divided into three ages groups: 6-9, 10-12 and over 12 years old. Plaque Index, Bleeding Index, and quantitative and qualitative variations of bacterial plaque were considered. RESULTS Remarkable results were obtained regarding both the effective reduction of bacterial oral flora and patients compliance and learning, especially in the group of patients older than 10 years. The new values of parameters recorded at the end of the study showed that all the subjects included in the sample had an improvement of compliance in oral hygiene, in particular: 1) P.I. level 3, 10-12 age, female; 2) B.I. level 4, males over 10, female 6-9 age; 3) quantitative and qualitative variations of bacterial plaque, level 4, all groups. CONCLUSIONS Patient instruction and motivation allow to obtain optimal results in particular in patients aged more than 10 years.