RICERCA ITALIANA     

STUDY OF THE EXPRESSION, ACTIVITY AND INTRACELLULAR COMPARTIMENTALIZATION OF SIGNALLING PROTEINS IN TUMOUR CELLS EXPOSED OR NOT TO BIOLOGICAL AND CYTOTOXIC AGENTS: A GENOMIC AND PROTEOMIC APPROACH.

Seconda Università degli Studi di Napoli

Abstract

The medical therapy of neoplasms is presently based on the use of conventional cytotoxic agents that mainly act through the induction of an irreversible and lethal damage of tumour cell DNA. Beside the lethal hit to DNA, one of the mechanisms of action of conventional chemotherapy is the triggering of apoptotic program in cancer cells. However, tumour cells can develop mechanisms of escape from the apoptosis involving regulatory loops of the mitogenic signalling and of the cell cycle. To-day it is emerging the need of finding newly synthesized molecules able to inhibit the proliferation of tumour cells and with anticancer activity in vivo. Moreover, it is becoming even more evident that, for the clinical application, it is necessary the definitioon of the effects of these agents on the intracellular genomic and proteic expression profile. Therefore our project is intended to detect new anti-tumor molecules and to define their mechanism of action through the study of their effects on signal transduction pathway, proteic and genomic expression profile in human tumour cells. <<<

Principal Investigator

Ettore BISMUTO Seconda Università degli Studi di NAPOLI

Research Objectives

The work of the Bismuto research unit will develop along two directions: the first one utilizing FLIM microscopy as main investigation technique, and oriented to the intracellular localization of fluorescent ras-FGP (ras chimeric protein with Fluorescent Green Protein)in normal and tumoral cells, treated or not with proper molecules supposed potential drugs; the second one oriented to in vitro structural and functional featuring of the fluorescent ras constructs and of its complexes with other cellular proteins involved in the transduction pathway. Such studies will avail in particular of time-resolved fluorescence techniques and fluorescence correlation spectroscopy provided with two-photon excitation, which allows to observe interactions as well as dynamic and diffusion processes at single molecule level.
The maín goal of Beninati project is the identification of an effective mechanism, to reduce the growth of cancer cell and their metastatic potential by inductíon of cell differentiation. We have enough evidences to believe that this target can be reached by an indirect influence on the metabolism of polyamines, obtained inducing Tgase activity in cancer cells. Since it is addressed only toward cell wíth a negligible Tgase activity, the novelty of thís approach is the improvement of the selectivity of the intervention. Indeed, cancer cells are characterízed by a very low Tgase activíty, usually unsuitable for the índuction of cell differentiation; the treatment with Tgase activators would select them for differentiation and apoptosis. We are planning to collect further information on the role of Tgase on cell growth and differenfiation. In particular, the fruitful collaboration with the team of Prof A. Abbruzzese from the University of Naples, we bring more data about the correlation among Tgases, polyamines and the eukaryotic proteín initiation factor 5A, as highlighted by our recent publication. Preliminary results obtained in our laboratory, have highlighted that Tgase activation induces not only a drastic reduction of cell proliferation but also a sígnificant decline, of the metastatic power of melanoma cells. As a preliminary approach, we are planning to investigate the role of Tgase ín the modulation of cell growth, differentiation and apoptosís using primary cultures of melanocytes. The unit of Prof. Lupoli will define the molecular bases of the growth inhibition and apoptosis induced by SS analogues on medulalry thyroid carcinoma cell lines and will investigate on the interaction between the SS-dependent signalling and the EGF-dependent signal transduction pathway. In details it will study the possible interaction with the ras-erk-dependent signalling studying the specific signal transduction pathway elicited by the different SS analogues. The survival mechanisms that protect tumour cells from apoptosis and growth arrest induced by SS analogues will be defined and the interaction between SS analogues and pharmacological inhibitors of the EGFR-associated tyrosine kinase (ZD1839) or ras function inhibitors (zoledronic acid and farnesyltransferase inhibitor R115777) will be studied. . Baldi unit will evaluate the cDNA microarrays of normal and transformed bronchial mucosa obtained from patients with non small cell lung cancer (NSCLC) or MTC cells (In cooperation with Lupoli). Particular attention will be given to cell cycle regulating genes (CDK, p21, p27, p53, pRB etc.), and to growth factors and the expression of their related receptors. On the basis of the results of these studies, the effects of molecular targeted agents (ZD1839, R115777), alone or in comination with other cytotoxic agents, will be tested on proliferation and apoptosis of primary cultured tumour bronchial cells. At the same time we will also study the expression of p53, bcl-2 gene family members, APAF-1 and survivin on paraffin-embedded tissues of non small cell lung cancer stage I in order to identify the role of these genes in lung cancer pathogenesis, and to evaluate any potential prognostic implication of these findings. <<<

First Results

Bismuto
- Construction of plasmids in eukaryotic vectors for the transfection of molecular constructs between ras and GFP in human normal and tumour cells before and after the treatment with molecules of biological interest: 5 months.
- Characterization of proteic expression in normal and cancer cells after appropriate treatments: 2 months.
- Expression of the constructs in E.Coli and successive extraction and purification: 2 months.

Beninati
1. Identification and synthesis of intracellular Tgase activators (Tg-activators). This part of the program will be performed in collaboration with the laboratory of Dr. J.E.Folk, National Institutes of Health, Oral and Pharyngeal Cancer Branch, NIDR, Bethesda, MD,USA. The synthesis procedures are based on experimental protocols of Dr. Folk. The modified molecules are methylxanthines and retinoic acid analogues. It will be estimated the rate of Tgase activation in vitro and in vivo , with the discrimination between protein activation and gene expression. Determination of the parameters, affected by the Tg-activators, correlated with cell growth, differentiation and apoptosis (p53, c-myc and bcl-2 expression and FACS analysis of the cell cycle). Investigation of the action of Tg-activators on normal (primary culture of melanocytes) and neoplastic cells (human and murine melanoma cells). Preparation of plasmids in order to increase the expression of Tgase on experimental animals (with the collaboration of Dr. F. Facchiano, ISS, Rome, Italy). Induction of experimental melanomas in the transfected animal.
2. Identification of the most active Tg-activators by a statistical screening (Prof. E. Capucci, University of Rome "Tor Vergata"). Evaluation of the metastatic power of melanoma cell lines and variation induced by Tg-activators. Characterization of the type of Tgase involved in cell differentiation focused on cultured enterocytes (with the collaboration of Dr. C. Bergamini, Univ. of Ferrara, Italy). Evaluation of the effects of Tg-activators on: tumor cell adhesiveness, invasiveness, proteolytic activity and migration (both in vivo and in Boyden chambers).
3. In vivo experiments I. preliminary treatments of B16-F10 cells with Tg-activators and inoculation in syngeneic animals. Statistical evaluation of the reduction of metastasis and survival. Selection of cancer cells unaffected by the treatments. This part of the program will be performed with an ongoing collaboration with Dr. H.K. Kleinman, National Institutes of Health, Bethesda, MD, USA.
Lupoli
1. Definition of pharmacological and biochemical interaction between different SS analogues in medullary thyroid carcinoma TT cells.
2. Definition of the molecular mechanisms on the basis of apoptosis and growth inhibition induced by biological agents.
Baldi
Definition of molecular prognostic markers for the prediction of survival and response of stage I-II NSCLC with DNA microarrays techniques.Bismuto
Summarizing, the chronological development of this phase of the project is the following:
- Characterization by means of basic spectroscopic techniques: 2 months
- Fluorescence emission decay studies: 4 months
- Single molecules fluorescence studies (FCS): 4 months
Beninati
ADVANCED STUDY
1. Evaluation of possible toxic effects and identification of the in vivo dosage of Tg-activators.
2. Evaluation of biochemical markers of growth, differentiation and cell death in melanocytes and melanoma cells (ornithine decarboxylase, tyrosinase, melanin, bcl-2, etc.). Evaluation of the body growth of the Tg-activator-treated animals. Biochemical study on the reaction leading to the formation of cross-links protein-polyamine and identification of the protein substrates (in collaboration with Prof. C.Esposito, Univ. of Salerno, Italy). Synthesis and purification of the standards for the analytical assay (HPLC). Purification of Tgase from melanoma cells and its biochemical characterization. Identification of the different types of Tgases by SDS-PAGE and Western blotting procedures. Purification of rat liver and melanoma PAO. Modification of protein and synthetic peptides, using purified Tgase, in the presence of radiolabelled polyamines. Identification by SDS-PAGE of the oxidative prducts of PAO. Inhibition of PAO activity in melanoma cells with MDL 72527 (generous gift from Merrel Dow, USA), lowering of intracelluar endogenous polyamines (DFMO, MGBG) and increase of the synthesis of protein polymers with conjugated radiolabelled polyamine.
3. In vivo experiments II. Treatment of animals with PAO inhibitors and Tg-activators (oral administration of the drugs). Experiments of human melanomas (SKMEL) using nude mice (with the collaboration of Dr. F.Facchiano, ISS, Rome, Italy). Statistical evaluation of the results (responsible Prof. E. Capucci, University of Rome "Tor Vergata").
4. Preliminary protocol for clinical trials.
Lupoli
1. Definition and overcome of survival mechanisms from the antiproliferative stimuli induced by SS analogues or inhibitors of Ras activity.
2. Determination of the molecular level of interaction of the different combinations.
Baldi
3. Study of the in vivo expression of signal transduction molecules and SSRs and their correlation with clinical outcome.
Baldi
Concenring the structural and functional characterization in vitro of ras fluorescent constructs (see research unit of prof Bismuto), our research group will be deeply involved in the construction of chimeric proteins where at the NH2 terminal of ras will be positioned Fluorescent Green Proteins. These sequences will be subcloned in expression vectors in order to express in vivo such fluorescent proteins in mammalian cells. Moreover, these sequences will be subcloned also in vectors that will allow the production in great amount of these proteins by procariotic systems. <<<

Timescale

24 months

National and international background

From the pioneering work with acute transforming retroviruses to the current post-genomic era, ras genes have always been at the leading edge of signal transduction and molecular oncology (1,2). Yet, a complete understanding of ras function and dysfunction — mainly in human cancer — is still to come (3). The knowledge of such cellular mechanisms is fundamental for developping efficacious drugs for the treatment of different tumor types (4). The Ras proteins are members of a large superfamily of low molecular-weight GTP-binding proteins (5). In vitro purified Ras possess low rated GTP-ase activity due to which they slowly convert the GTP molecule bound to them. The GDP may afterwards be exchanged to GTP again. However, these processes are catalyzed and regulated inside the cell by means of different effector proteins (guanine nucleotide exchange factors GEFs e GTPases activating proteins GAPs). Proteins belonging to the Ras family monitor cell growth processes and play a role in apoptosis and senescence processes (6,7). They exist in either activated and inhibited mutated forms in different human tumors. The normal function of Ras proteins requires post-translational modifications whose main aim is to localize them to the correct subcellular compartment — principally the inner face of the plasma membrane, for the sake of efficiently interacting with the proper molecules of the signal transduction pathway (2,8).
The aim of this research unit's project is to state the intracellular localization of Ras proteins and other molecules interacting with them, in normal cells and in tumoral cells from human epidermoid carcinoma and medullary thyroid carcinoma. This study will be also carried out in presence of certain substances, considered as potential anti-tumoral drugs (somatostatine analogues, biphosphonates and conventional cytotoxic agents as docetaxel) (9). Fluorescence microscopy techniques play a basic role in the cell function studies, because they often do not request cell destruction to isolate components of interest. In particular, the availability of intrisically fluorescent proteins (green fluorescent protein, GFP) whose gene may be fused with a gene of interest (in this case Ras proteins and others related to them, from normal and tumoral cells) and re-introduced in the cell where it expresses the fluorescent chimeric product, revolutionized the study perspectives of cellular processes in vivo (10). We intend to utilize in our research an advanced microscopy technique (Fluorescence Lifetime Imaging Microscopy, FLIM) (11). The FLIM microscopy advantages are manifold and its potentialities explored only in part. Usually fluorescence microscope reconstructs images starting from fluorescence intensity measurements. This signal has several intrinsic complications which reduce sensibility and image resolution: a) biological samples show an intrinsic self-fluorescence; b) due to high amplitude of emission bands, there is overlapping of fluorescence signals if in presence of more fluorophore species; c) observed signal depends on fluorescent molecules concentration. Lifetime, instead, does not depend on signal intensity, and so on the number of molecules as well, and is better resolved in different fluorescent species. Consequently, images obtained by FLIM microscopy are at high-resolution and signals coming from different fluorophores may be observed simultaneously. Thanks to the advanced instruments of specialized laboratories such as the Laboratory for Fluorescence Dynamics of Illinois University at Urbana-Champain, which our research unit for over a decade works together to, it will be possible to obtain images by means of the same FLIM technique but with two-photon excitation, which allows to get better resolved images because of the Raman signals cut-off and lower impact section of multiple excitation. The possibility of forming a construct between signalling components such as ras and GFP (fluorescent molecules) will allow the study of the compartimentalization of these molecules after adequate stimuli.
Most of the molecules used as differentiative agents, are capable to increase the expression of intracellular transglutaminase (Tgase E.C.2.3.2.13) (5,6). It is well known that most of these proteins are post-translationally modified by Tgases (8). These enzymes catalyze a calcium-dependent acyl transfer reaction in which the gamma-carboxamide group of a peptide-bound glutamine residue is the acyl donor and the epsilon-amine group of peptide-bound lysine residue is the acyl acceptor (9). This reaction results in the formation of inter- or intramolecular covalent crosslinks leading to a protein potymer. The effects of this reaction on the cellular protein involved in cell adhesion are the stabilization of the linkages between cells and ECM (10). There is a wide body of experimental evidences on the involvement of polyamines in normal and neoplastic growth (5,15). It is well known that in mammalian cells, the transition between the quiescence and the proliferafive state of the cell is paralleled by an íncrease of the key enzyme of polyamine synthesis, ornitine decarboxylase (ODC) and of the intracellutar levels of polyamines (16). This relationship between polyamine levels and cell proliferation has been reported in the tissue regeneration, hypertrophy, normal and neoplastic growth, embryo development and in the stimulation of cells by carcinogens. The possibility of the covalent linkage of polyamines to proteins (17)opened a new investigative field leading to hypothesis on the role of this polyamine-protein conjugates in the modulation of cellular growth and programmed cell death. Polyamines, as second substrates of Tgase, act as regulative agents in the assembly of protein structures. Indeed, the formation of glutamyl-lysine linkages leads to a polymerized protein structure in one metabotic step, while the reaction of a polyamine, which amine group competes with that of lysine, leads to a protein cross-link in two metabolic steps (18).The inítial part of the reaction involves one amine group of polyamine with the formation of a mono(gamma-glutamyl)polyamine derivative, leaving a free amine group ready for the next step of the reaction. The last metabolic passage leads to a bis(gamma-glutamyl)polyamine derivative with a linkage acting as a bridge between the proteins, which length and charge is due to the type of polyamine involved in the catalytic reaction. The possibility of these two metabolic steps in the Tgase reaction with polyamine allows a rough regulation on the base of the intracellular levels of polyamines. Elevated levels of polyamines are able to produce the saturation of the specific glutamyl residues of a protein conducting to the formation of mono-glutamyl-polyamine derivative, avoiding the polymerization of the proteins involved in the reaction. This situation has been observed in cancer cells where the levels of polyamines are usually very elevated (19). On the contrary, lower levels of polyamines are related to a higher protein polymerization, typical of quiescent cells (19). It has been recent1y hypothesized in our laboratory the possibility of more refined modulation of the formation of the polymerized protein complex (20). It is unlike that the variation of the intracellular levels of polyamines is the only regulative mechanism in the Tgase reaction. Moreover, this kind of rough regulation is unable to drive the preferential synthesis of a glutamyl-polyamine derivative. Searching for a new possible metabolic regulation, it has been consídered an enzyme involved in polyamine catabolic oxidation, polyamine oxidase (PAO). This enzyme catalyzes the oxidative breakdown of spermidine and spermine with the formation of a lower molecular weight polyamine,an aminoaldehyde and hydrogen peroxide (22), The role of PAO in cell growth is well known. The possibility that a rat liver purified PAO is able to cleave some of the polyamine derivative of a Tgase reaction, has been recently demonstrated in our laboratory (S.Beninati, manuscript in preparation). This information allows the formulation of new working hypothesis on the investigation about the role of Tgase in cell growth and differentiation, with all the possible clinical implication in the control of cancer development. On the sight of this, it is interesting the recent observation on the improvement of the survival of mice inoculated with highly malignant melanoma celis by increasing the intracellular Tgase activity (23).
Medullary thyroid carcinoma (MTC), a malignant neoplasm of parafollicular cells, represents about 5-10% of thyroid tumors. These cells are derived from the neural crest and are calcitonin-producing. Recurrence occurs in about 50% of patients and is often preceded by high serum CT and/or carcinoembryonic antigene (CEA) concentrations. Early detection of secondary disease leads to successful reoperation, in particular in these cases of localized relapse. Adjunctive therapy includes radiotherapy and chemotherapy. MTC is radio- and chemo-resistant. An interesting and innovative therapeutic approach for the chemioresistant MTC is represented by biologic agents, which can act through direct antiproliferative effects and stimulate the antitumor immune response without side-effects. Somatostatin is a cyclic peptid made of 14 amino acids synthesized by several tissues of the body. Several SMS analogues have been synthesized with a biological activity equal or even higher than the native hormone, coupled with enhanced stability and long-lasting action. These analogues have been produced with different affinity for the diverse isoforms of the receptors for SSR. At present, the octapeptide octreotide is the most extensively investigated SMS analogue in preclinical and clinical studies. Clinical studies have demonstrated that octreotide binds to the isoforms SS-R2A, SSR2B, SSR3 E SSR5, while lanreotide is a specific analogue that binds to the isoforms SSR2 and SSR5. However the real impact of different isoforms in the control of cell proliferation has only a clinical use. Moreover, recently a pleiotropic SS analogue, SOM230, has been developed and already used in clinical trials.
The action mechanism of these drugs is complex. In fact they have a direct effect, due to the bind between analogue and receptors on tumoral cell and due to the direct antitumoral action, with inhibition of DNA synthesis and cellular replication by EGF and other growth factors, with consequent cellular block in G1-phase of cellulare cycle. Moreover, they have indirect effects: inhibition of several growth factors, among these insulin growth factor (IGF-1), whose synthesis is blocked and epidermal growth-factor (EGF); modulation of immune response; aptoptosis induction; decrease of angiogenesis; secrection block of hormones involved in regulation of tumoral growth (GH, insulin, secretin, etc).
Our group performed several studies in advanced MTC based on the use of SS analogues and we demonstrated that combination of octreotide or lanreotide with recombinant -2b) is active treatments on advanced MTC, which type 2b (rIFN-interferon- leads to the improvement of tumour associated symptoms and decrease of calcitonin (CT) and carcinoembryonic antigen (CEA) serum levels. On the other hand, these combinations are still poorly effective in inducing clinical responses in MTC patients suggesting the existence of mechanisms of tumour resistance to the growth inhibitory activity of SS analogues and IFN combination.
Recently, evidence accumulated that bisphosphonates including zoledronate (ZOL) are strong inducers of apoptosis in several cancer cell types such as myeloma, breast cancer and prostate cancer but also in macrophage and epithelial cell lines. These data indicate that the beneficial effect of bisphosphonates may result from a direct anti-tumour activity that may affect a broad range of metastasing tumours. In fact, nitrogen containing bisphosphonates were shown to inhibit the farnesyl diphosphate synthase probably by mimicking the diphosphate moiety and resulting in the inhibition of farnesyl synthesis and also of the formation of higher isoprenoids such as geranyl residues. These data suggest the use of the 2 agents in combination in order to increase their anti-tumour efficacy.
The availability of clinical grade agents that are specific inhibitors of the EGF-Ras-dependent anti-apoptotic and protective pathways of cancer cells encourages the investigation on the combined use of ZD1839/ZOL in combination with SS analogues and cytotoxic agents in the treatment of MTC.
The prognosis for patients with surgically resected lung cancer has not been satisfactory, as the 5-year surival rate is no more than 70% even for patients in stage I (1). This means that approximately 30% of patients have recurrence, generally in distant organs (2). For these patients, surgical resection has not been curative, but multimodality treatment might be beneficial. Because encouraging results have been seen in patients with locally advanced disease, the combined modality approach is now being applied to the patient population with early-stage lung cancer (3,4). Although postoperative adjuvant therapy in patients with completely resected stage I non-small cell lung cancer failed to show prognostic benefit (5), induction (preoperative) treatment may be effective in these patients, as it was in patients with stage III disease (6,7). One of the major problems in this setting is the unnecessary additional treatment given to a large number of patients who could have been cured by surgical resection alone. Therefore, clinical factors to identify the early-stage lung cancer subgroup with a poor prognosis are necessary. A precise regulation of the cell cycle is a fundamental requirement for the homeostasis of the eukaryotic cell. During the last decade, scientists successfully delved into the molecular machinery devoted to the fine regulation of the cell cycle phases, identified and characterized several genes and gene products involved. A key role in this is played by cell cycle kinases (cdk), relatively small proteins with an apparent molecular mass between 33 and 43 kDa, whose activity is regulated by the arrangement in a multimeric complex with larger proteins, called "cyclins" after their cyclical expression and degradation during the cell cycle. Cyclins, a family of proteins, are the regulatory subunits of the multimeric complex whose catalytic activity is provided by the cdk moiety. Different cdk/cyclin complexes, formed with clear-cut timing throughout the cell cycle, together with their phosphorylation/dephosphorylation efficiently regulate the activity of the multimeric holoenzyme. Conversely, cdk/cyclin complexes are negatively modulated by the binding of a family of small proteins called cdk inhibitors (9).
There are numerous reports that investigated the association of cell cycle abnormalities with the prognosis of NSCLC patients. In particular the p16-cyclinD1-CDK4-RB pathway is found not functioning in several neoplasias. p16 functions in the pathway by binding to cyclin-dependent kinase 4 (CDK4) and inhibiting the ability of CDK4 to interact with cyclin D1 and to phosphorylate the product of the retinoblastoma (RB) tumor suppressor gene (10). Mechanisms that affect the balance between p16 and cyclin D1 result in abnormal cell cycling and growth (11). p16 abnormalities are frequently found in NSCLC but are rare in SCLCs (12). The other key component in this pathway is the RB gene (10). It is located in chromosome region 13q14.11 and encodes a 105-kDa protein that is important in regulating the cell cycle during G0/G1 phase (13). Inactivation of either p16 or RB expression, which is found in a great percentage of lung cancers, inactivates the pathway,. However, it is uncommon to have both RB and p16 inactivated in the same tumor. <<<