WO2009074707A1

WO2009074707A1 - Grk2 antitumour therapy

Info

Publication number: WO2009074707A1
Application number: PCT/ES2008/070231
Authority: WO
Inventors: Federico MAYOR MÉNDEZ; Petronila PENELA MÁRQUEZ; Alicia Salcedo Haro; Ana RUIZ GÓMEZ; María Helena HOLGUÍN ASENSIO
Original assignee: Universidad Autónoma de Madrid
Priority date: 2007-12-11
Filing date: 2008-12-11
Publication date: 2009-06-18
Also published as: ES2332558B1; ES2332558A1

Abstract

The invention relates to the use of siRNA in order to inhibit the expression of GRK2 as antitumour therapy in cancers in which AKT co-operates with GRK2 in the acquisition of tumour characteristics and to the use of said siRNA as a therapeutic agent in the treatment of melanoma, breast cancer, ovarian cancer, prostate cancer, gliomas and thyroid cancer.

Description

GRK2 ANTITUMORAL THERAPY

The present invention belongs to the field of biology, molecular biology and biochemistry. It is directed to the regulation of the expression of GRK2 for the treatment of melanoma, breast cancer, ovarian cancer, prostate cancer, gliomas and thyroid cancer.

BACKGROUND OF THE INVENTION

G protein-coupled receptor kinases (GRKs - G protein-coupled receptor kinases) participate, together with arrests, in the processes of regulation of multiple G protein-coupled receptors (GPCRs) of great physiological and pharmacological relevance. These proteins form a family of seven members (GRK1-GRK7) that specifically phosphorylate agonist-activated receptors in serine / threonine residues, promoting internalization, recycling and / or degradation processes of GPCRs in combination with β-arrestinas (Fig. 1 ). GRK proteins also have other non-GPCR membrane substrates, as well as cytosolic substrates that diversify the functional consequences of the activity of these kinases. Likewise, numerous molecules have been identified with which the GRKs interact independently of the kinase activity (Pao, 2002; Ribas, 2007), which demonstrates the existence of scaffold or scaffolding functions for this family of proteins .

Since GPCRs modulate basic cellular processes

(differentiation, migration, cell cycle, antiapoptotic responses) frequently its activity is involved in the origin and development of tumors of different nature (Dorsam, 2007). Thus, potent mitogens such as thrombin, S1 P, LPA, sonic hedgehod or endothelin regulate Ia Proliferation in tumor cells and their receptors are overexpressed in several types of cancer (colon, breast, prostate, melanoma). Likewise, chemokine receptors such as CXCR4 or CXCR2 play a central role in tumor metastasis. Tumor cells that overexpress these receptors are activated by chemokines released by stromal cells, macrophages and leukocytes infiltrated in the tumor, stimulating the motility and survival of cancer cells.

Since the signaling of GPCRs receptors in tumor contexts frequently exhibits changes, either as a result of an amplification in their expression levels, or as a result of alterations in the mechanisms of regulation of their functionality leading to a gain in function, It is of general interest to analyze the processes that in turn modulate the activity of the regulatory molecules of these receptors. In this sense, the GRK2 protein, which is the most ubiquitous and best characterized isoform of the family of GRKs, is emerging as a key molecule in cell signaling with great pathophysiological potential in tumor contexts, since it would not only regulate the activity of different GPCRs involved in cancer, but also of cytosolic proteins involved in proliferative and survival signaling pathways (p38, Smads, among others), as well as non GPCRs membrane proteins with oncogenic potential (EGF receptor). Likewise, GRK2 is able to modulate various proteins such as PI3K, ERK or GIT1 independently of phosphorylation, suggesting that it has properties of adapter protein or "scaffold" (Fig. 2). Given the complex functional diversity of GRK2, there are sophisticated mechanisms in the cell that regulate both the kinase activity and its expression levels, whose alteration is associated with pathological situations. Thus, the study of the processes that regulate the stability and functionality of GRK2, has revealed that changes in the stability of the protein and / or its functionality have repercussions. Direct in cell physiology, showing the participation of GRK2 in cell migration and in neoplastic transformation processes.

DESCRIPTION OF THE INVENTION

The present invention faces the problem of inhibiting the action of the GRK2, not only at the level of its serine-threonine kinase activity, but of its "scaffold" functions. Thus, the suppression of the expression of GRK2 in cancerous diseases in which this protein is overexpressed, and in which it is contributing or cooperating with AKT in the acquisition and maintenance of tumor characteristics, would be an appropriate therapy.

In this sense, the present invention provides a sequence of ribonucleotides or RNA that belongs to the so-called siRNA (small interfering RNA), small interfering RNA or silencing RNA, capable of inhibiting the genetic expression of the GRK2 protein (hereinafter siRNA of the invention), providing new effective techniques against cancer proliferation and cell migration. The solution provided by this invention is based on the fact that the inventors have observed that the inhibition of cell proliferation and migration in certain types of cancer is possible, through the use of a specific RNA sequence molecule that binds to the messenger RNA that encodes for the GRK2 protein preventing its translation and therefore inhibiting its expression. Thus, the use of siRNA, as described herein, allows the inhibition of GRK2 expression in tumor pathologies in which the GRK2 protein is overexpressed as a result of a functional amplification of the dependent signaling pathway. of PI3K, contributing to tumor transformation and cooperating - with Akt. Therefore, in accordance with a first aspect of the present invention, there is provided a composition (hereinafter composition of the invention) comprising a siRNA capable of inhibiting cell proliferation and migration based on the inhibition of Ia expression GRK2 protein, for use in those pathological contexts in which GRK2 is contributing or cooperating with AKT in the acquisition or maintenance of tumor characteristics.

A preferred embodiment of the invention, constitutes a composition of the invention comprising an RNA construct (RNA construct of the invention) that contains at least any one of the possible nucleotide sequences of siRNA capable of inhibiting the expression of GRK2, and notwithstanding that, in addition, any of the RNA sequences and constructions of the invention described above that are subject to modifications, preferably chemical, that lead to greater stability against the action of ribonucleases and thereby to be part of the present invention Greater efficiency Without these modifications, this involves the alteration of its mechanism of action, which is the specific binding to the RISC complex (RNA-induced silencing complex), activating it and manifesting a helicase activity that separates the two strands leaving only the antisense strand associated with the complex. The resulting ribonucleoproteal complex binds to the target mRNA (GRK2 messenger RNA). If the complementarity is not perfect, RISC is associated with the messenger and the translation is attenuated. But if it is perfect, RISC acts as RNasa, cutting the messenger and being free to repeat the process.

The composition of the present invention is used in the treatment of those types of cancer, in which it has been observed that the protein

GRK2 is cooperating with AKT, such as melanoma, breast cancer, ovarian cancer, prostate cancer, gliomas and thyroid cancer. The preparation of the siRNA sequence of the invention or of the RNA construction of the invention would be evident to one skilled in the art, and could be carried out by chemical synthesis, which also allows the incorporation of chemical modifications both in the different nucleotides of the product such as the incorporation of other chemical compounds at any of the ends. On the other hand, the synthesis could also be carried out enzymatically using any of the available RNA polymerases. Enzymatic synthesis also allows some chemical modification of inhibitor products or RNAs.

The design of the nucleotide sequence of the siRNA of the invention would also be evident to one skilled in the art. Thus, it could be done through a random design in which 19-21 bases of the target mRNA are selected without taking into account the sequence or positional information it has in the transcript. Another non-limiting alternative of the present invention would be the conventional design using simple parameters developed by the pioneers of the technique (Calipel, A. et al., 2003) completed with a BLAST nucleotide analysis. Another possibility could be a rational design, in which a computer procedure is used to identify the optimal siRNA targets in an mRNA. The target sequences are analyzed in groups of 19 nucleotides at the same time and those with the best characteristics are identified based on an algorithm that incorporates a large number of thermodynamic and sequence parameters.

A second aspect of the invention constitutes a composition (hereinafter the second composition of the invention) comprising a genetic construction of DNA, hereinafter genetic construction of DNA of the invention, which would direct the in vitro or intracellular transcription of Ia siRNA sequence or RNA construct of the invention, and comprising at least one of the following types of sequences: a) DNA nucleotide sequence, preferably double stranded, comprising, at least, the sequence encoding the siRNA of the invention or the RNA construct of the invention for in vitro transcription, or, b) DNA nucleotide sequence, preferably double stranded, corresponding to a gene expression system or vector comprising the sequence coding for the RNA sequence of the invention operably linked with at least one promoter that directs the transcription of said nucleotide sequence of interest, and with other sequences necessary or appropriate for the transcription and its adequate regulation in time and place, for example, start and end signals, cutoff sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc .. for use in those pathological contexts in which the GRK2 is contributing to the acquisition or maintenance nimiento of tumor characteristics in cooperation with the signaling pathway dependent on PI3K and Akt. Multiple of these expression systems or vectors can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 1989) and are part of the present invention.

The compositions of the present invention allow the transfection of the siRNA of the invention into a cell, in vivo or in vitro. The transfection could be carried out, but not limited to, direct transfection or vectors that facilitate the access of the siRNA inside the cell. Thus, examples of these vectors are, without limitation, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, Herpes simplex viruses, non-viral DNA plasmids, cationic liposomes and molecular conjugates.

Thus, for example, the siRNAs of the present invention, as well as RNA or DNA precursors of these siRNAs, can be conjugated with release peptides or other compounds to favor the transport of these siRNAs. inside the cell. Among these proteins are those known in the state of the art, which have properties that favor cell penetration. As examples, and not limited to these, those described in the patent application US 20040204377 are included.

In an even more preferred embodiment of the present invention, the transfer to the siRNA tumor cells of the invention could be performed by fusing SIRR-GRK2 constructs to RNA-aptamers. The presence in the SIRR-GRK2 constructions of RNA-aptamers with the capacity of selective binding to tumor antigens allows the siRNA to be directed to tumor cells to inhibit the expression of GRK2. This would allow a selective and efficient transfer to the tumor cells.

Normally aptamers are susceptible to degradation by nucleases in the intracellular medium. In this sense and in order to solve this problem, modifications in the chemical structure can be presented that provide them with a remarkable resistance to degradation. The modifications that involve the 2 'position have been particularly used since it is known that the -OH radical in this carbon is indispensable in the reaction mechanism of the nucleases.

The most common change is the substitution with a group -NH2 that allows the life time of oligoribonucleotides to be extended more than 20 times. Another strategy to prevent the degradation of aptamers is to use the specular configurations of the targets in a conventional selection to generate the normal D-aptamers. Once their sequence is known, they are synthesized in their L-enantiomeric conformation that is specifically recognized by the natural target but is not a substrate for the nucleases present in the cellular medium.

The siRNAs of the invention can also be supplied mixed with releasing agents, such as dendrimers. In a third aspect of the invention it is contemplated that the compositions of the present invention are pharmaceutical compositions, which comprise a therapeutically effective amount of the siRNA sequence of the invention and / or RNA construction of the invention and / or genetic DNA constructs of the invention, together with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles.

In the context of the present specification, "siRNA" (small interfering RNA or small interfering RNA) is understood as a class of double stranded RNA 20 to 25 nucleotides long, and more preferably between 21 and 23 nucleotides, which is involved in the route of RNA interference, where the siRNA interferes with the expression of a specific gene. In the present invention, this specific gene is GRK2. In this sense, the present invention uses illustratively as siRNA Ia

SEQ ID NO: 2, nucleotide sequence capable of inhibiting the expression of

GRK2 in tumor cells, which prevents these malignant cells from developing tumors in vivo (see example 6).

In the context of the present invention, the sequences SEQ ID NO:

3 and SEQ ID NO: 4 represents the sequences that flank the 5 ^' and 3 ^{' region} respectively of SEQ ID NO: 2 to create the hairpin in the structure that will be expressed with the adenovirus. Fig. 3 shows the most likely secondary structure that this siRNA construct would adopt.

As "GRK2", in the context of the present invention, a nucleotide or polynucleotide sequence is defined, which constitutes the coding sequence of the GRK2 protein, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, b) nucleic acid molecules whose complementary chain hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with a homology of 99%, 98%, 95%, 90%, or 80 % with SEQ ID NO: 1.

in which the polypeptide encoded by said nucleic acids possesses the catalytic activity, the scaffold activity and the structural characteristics of GRK2.

In the context of the present invention the term "homology" refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between two or more amino acid sequences.

Since the nucleotide sequences of the GRK2 gene are related in terms of their evolution, and since the function of this depends on the amino acid sequence to which it is transcribed, it can be expected that the overall homology of the genomes at the amino acid level, in different groups of populations and / or individuals, and in different animals, and more specifically at the level of the amino acid sequence that is included in SEQ ID NO: 1, is 80% or greater, and more preferably 90% or greater and more preferably 95, 98 or 99% or greater. The correspondence between the amino acid sequence of SEQ ID NO: 1 and the sequence belonging to another individual or organism can be determined by methods known in the art. In the context of this report, "AKT" means those proteins with kinase activity that phosphorylate various substrates in serines and threonines and are activated by their translocation to the plasma membrane and their binding to the PIP3 phospholipid. These proteins, of which there are several isoforms (Akt1 or PKBα, Akt2 or PKBβ and Akt3 or PKBy), are regulated by numerous growth factors and play a key role in cell survival, proliferation and cell migration.

As used in the present invention the term

"chemical modifications" refers to the introduction of chemically modified nucleotides in the RNA sequence of the invention, for example, and without being limited thereto, S groups replacing O in the phosphodiester chain, or the inclusion of 5 methylcytosines, which allow increasing The efficiency of the same (confer greater resistance against degradation, favor its entry into the cells, etc.), as well as any modification in the pentose or in the nitrogenous base. Other modifications of the siRNA that can increase the stability are those described in Bolcato-Bellemin et al. 2007 in which short sequences of protruding and complementary poly-dA and poly-dT sequences are introduced at the ends of the siRNA thus favoring the aggregation and formation of concatamers.

As used herein, the term "transfection" refers to the introduction or transfer of an exogenous nucleic acid molecule into a eukaryotic cell, including, but not limited to, a ribonucleic or deoxyribonucleic acid molecule (by example,

RNA or naked DNA).

In the context of the present invention, "aptamers" means nucleic acid molecules that are capable of binding to another molecule of particular interest with great affinity and specificity (Tuerk and GoId, 1990; Ellington and Szostak, 1990). In the context of the present invention, this target molecule of particular interest would be a specific tumor antigen or an indicative marker that the cell has acquired tumor characteristics. An aptamer is typically obtained by in vitro selection of the binding to a target molecule. However, in vivo selection is also possible. It is typically between 10 and 300 nucleotides in length. More commonly, an aptamer must be between 30 and 100 nucleotides in length.

In this report, "dendrimers" means highly branched polymers with a well-defined architecture, capable of releasing other compounds to the cell.

In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of the siRNA sequence of the invention or to the amount of an RNA or DNA gene construct that allows its calculated intracellular expression to produce the desired effect. and, in general, will be determined, among other causes, by the characteristics of said sequences and constructions and the therapeutic effect to be achieved. The pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art. The pharmaceutical composition provided by this invention may be provided by any route of administration, for which said composition will be formulated in the pharmaceutical form suitable to the route of administration chosen. In a particular embodiment, the administration of the composition provided by this invention is carried out subcutaneously, other routes being possible, such as, for example, intraperitoneal, parenteral, etc. Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Regulation of G-protein coupled receptors (GPCRs) by GRKs and β-arrestin proteins. Homologous desensitization begins with the phosphorylation of the receptor activated by the GRKs and concludes with the recruitment of the β-arrestins that functionally decouple the receptor from the G proteins. Likewise, the β-arrests interact directly with the endocytic machinery necessary to lead to carried out the internalization of receptors. Once present in endosomes, the scaffolding capacity of β-arrestin allows the coupling of desensitized receptors with various signaling proteins such as c-Src and MAPK. Subsequently, the dephosphorylated receptor is recycled and located again in the plasma membrane or it degrades in lysosomes.

Fig. 2. Functional interactome of GRK2. Numerous proteins involved in signal transduction interact with GRK2 and are regulated by this protein. Thus, the kinase activity of GRK2 not only determines the functional state of a large number of GPCR receptors but also that of various cytosolic substrates, which allows modulating different cellular processes. On the other hand, new functions of GRK2 arise due to its ability to interact with a multitude of proteins, regardless of their kinase activity. Fig. 3. Construction of siRNA that represents the most probable secondary structure, and that includes SEQ ID NO: 2, and the flanking sequences that form the hairpin, SEQ ID NO: 3 and SEQ ID NO: 4. It is the construction of siRNA with which the examples of the invention have been carried out.

Fig. 4 Analysis of GRK2 levels and the activation status of AKT in samples of patients with breast carcinoma. Samples of infiltrating ductal carcinoma of the breast as well as normal tissue from them were processed to quantify, by means of immunodetection techniques with specific antibodies, the levels of the GRK2 protein and phosphorylation of AKT. The densitometric data obtained were normalized with the levels of actin and total AKT, respectively. A and B) The means of each individual from 4-5 determinations are represented taking as reference the values of a randomized control sample. The average value (-) is indicated in each population (control and tumor). C) Stratified representation of positive and negative AKT tumor samples in relation to the degree of GRK2 expression, taking as reference the average value of the control population. Positive or negative samples are considered to be those that differ at least 15% from the average control value taking into account the standard deviation of the mean. D) Histogram representation of previous GRK2 levels corresponding to tumor samples, showing relevant clinical data.

Fig. 5 The union of growth factors such as IGF1 to its receptors promotes the stimulation of PI3K and the production of PIP3 phospholipid, which induces the activation of AKT kinase. The Mdm2 ligase, responsible for the degradation of GRK2, is phosphorylated by AKT and said modification directs the ligase to the nucleus preventing its interaction with GRK2 and favoring the stabilization of the protein and its overexpression. Fig. 6 GRK2 levels affect the ability to migrate MCF-7 and MCF10A cells in response to fibronectin. Chemotactic migration experiments in fibronectin response were performed in MCF7 (panel A) and MCF10A (panel B) cells transfected with the GRK2 expression vector, the GRK2 siRNA construct expression vector or the empty vector in combination with the CD-8 vector and selected by the dynabeads method. The ± SEM of 3-4 experiments performed in duplicates are represented

Fig. 7 GRK2 modulation of the migration induced by different diffusible chemotactic signals. The MCF7 cells (panel A) transiently transfected with an empty vector, the GRK2 expression vector or the SÍRNA-GRK2 construct, were selected as above for chemotactic migration assays at the indicated signals. Similar tests were carried out in Hs578T cells (panel B) infected or not with the control adenovirus and the construction of SÍRNA-GRK2 at MOI 100. The means ± SEM of 3-4 experiments performed in duplicate were represented where ^* p < 0.05 and ^** p <0.01, when compared with the transfection controls of the empty vector or with cells infected with Adcontrol (Hs578T), the motility of the latter does not differ with respect to the uninfected cells.

Fig. 8 The ability to invade matrigel in MCF7 cells is modulated by GRK2 levels. A) Transfected cells under the same conditions mentioned above were used to perform matrigel invasion experiments in response to 20% serum. B) Uninfected MCF7 cells and infected with the control adenovirus or with the SÍRNA-GRK2 to MOI 100 construct were used to perform matrigel invasion experiments in response to the indicated stimuli. The ± SEM of 3-4 experiments performed in duplicate are represented where ^* p <0.05 ^** p <0.01 and ^*** p <0.001, compared with Transfection controls of empty vector or those of cells infected with the control adenovirus. Representative autoradiographs of changes included in GRK2 levels are shown.

Fig. 9 GRK2 levels affect the invasive capacity of cell lines from metastatic tumors. Hs578T breast carcinoma cells (panel A) and B16F10 melanoma (panel B) without infecting and infected with the control adenovirus or with the SÍRNA-GRK2 to MOI 100 construct were used for serum invasion tests in matrigel. ^* p <0.05, ^** p <0.01. The reduction of GRK2 levels was confirmed by immunodetection.

Fig. 10. Effect of GRK2 levels on cell proliferation. MCF-7 cells were infected as indicated above with the adenoviral vector of the siRNA construct of GRK2 or with the control adenovirus. Cell proliferation was determined by the CelITiter 96® method

AQueous (promise) in multiwell plates-96 at the indicated times. The ± SEM of 4 experiments performed in quadruplicate are shown. * p <0.05 and ^** p <0.01.

Fig. 11. Inhibition of the expression of GRK2 in malignant cells prevents the formation of orthotopic tumors in mice. MCF7 breast carcinoma cells infected with adenovirus control or with the adenovirus of the SYRNA-GRK2 construct were inoculated subcutaneously in the respective flanks of immunocompromised mice as indicated in the scheme. Tumor growth was analyzed for 19 days and the size recorded by calipers was plotted. DETAILED EXHIBITION OF REALIZATION MODES

Next, the invention will be illustrated by tests carried out by the inventors, which show the specificity and effectiveness of the inhibition of the expression of GRK2 by means of siRNA in the treatment of cancer that occurs with the overexpression of GRK2 and the functional amplification of the PI3K / Akt signaling pathway, and more specifically, in breast cancer, where GRK2 would cooperate with Akt in the acquisition of tumor characteristics.

EXAMPLE 1. Analysis of GRK2 expression levels and the degree of AKT activation in a population of samples of 27 patients affected by breast cancer.

For this study, infiltrating ductal breast carcinomas with different degrees of differentiation were analyzed. As controls, 12 samples of non-tumor breast tissue from the examined patients were analyzed. Although the expression of GRK2 in the mammary gland of the analyzed individuals generally exhibits a certain degree of variability, it can be observed that the levels detected by "immunoblot" are, on average, significantly lower in the control samples compared to the tumor samples ( Fig. 4A). Likewise, the activation of AKT in non-tumor samples is almost imperceptible, being frequently below the detection limits of our test conditions (Fig. 4B). Of the tumor samples analyzed, 40% show high levels of activation of AKT (positive Akt samples) while 60% show equal or less activation of AKT (negative Akt samples and no change) than the average value observed in non-tumor samples. . Within the samples it is possible to stratify the population based on the degree of expression of GRK2 in terms of greater, lesser or no change compared to the average observed in The population control. Thus, in positive AKT samples it is observed that the distribution stratified by GRK2 levels is: High levels of GRK2 69%, low levels of GRK2 18%. However, in the negative AKT samples the distribution is: High levels of GRK2 40%, low levels 65%. These data indicate a certain positive correlation between the degree of activation of AKT and the levels of the GRK2 protein in coherence with the PI3K / AKT / Mdm2 / GRK2 regulatory axis that we have previously described in the cellular systems of transformed breast epithelium (Salcedo et al., 2006). Fig. 5 shows a scheme of these results. Moreover, within the control samples there are two patients with high levels of GRK2 that move away from the average of the control population and who also show a high activation of AKT.

Based on the results obtained, which show the upward regulation of GRK2 levels after the activation of AKT in breast carcinoma cells and in samples of patients with breast carcinomas positive for the activation of AKT, the following examples show It will try to determine if GRK2 was a simple passive marker of the activation of AKT or an "active" factor in the processes of cellular transformation caused by the overstimulation of the PI3K / AKT pathway.

The PI3K / AKT pathway is amplified in many types of cancer, such as ovarian, pancreas, breast or thyroid. The activation of AKT confers oncogenic properties as it regulates the progression of the cell cycle, promotes survival, inhibits apoptosis, and stimulates cell migration and invasiveness through the secretion of metalloproteases. The presence of GRK2 could cooperate with AKT in the regulation of these cellular functions or even mediate some of the effects of AKT in them. Therefore, we analyze the proliferative capacity of cells, their mobility and their invasive migration after manipulating the expression of GRK2, either by transiently increasing their levels by transfection, either by decreasing them with siRNA constructs or by infection with GRK2 siRNA adenovirus, both in transformed HS578T, MCF-7 and BL16F10 cells, as in non-transformed MCF-10A cells.

EXAMPLE 2. Participation of GRK2 in the cell migration of MCF-7 in response to fibronectin.

Cellular migration is a complex process that requires coordinating changes in the cytoskeleton of the cell (necessary to generate the precise tensile forces for locomotion), with pro-migratory extracellular stimuli, transmitted to the cell interior by different membrane receptors, between those that stand out GPCRs and receptors with kinase activity. The interactions of integrin receptors and their extracellular matrix ligands are not only important in the adhesion of the cells to the substrate, determining their mobility, but also in providing migration routes acting as guides. This role is critical in the process of invasion of tumor cells, which must degrade the basal lamina and the extracellular matrix of the tissue of origin and recognize certain components of the extracellular matrix as physical support for locomotion. In this context, fibronectin plays an important role in tumor progression. This glycoprotein can be presented as a soluble molecule or plasma fibronectin, the result of the action of the plasmin protease, or as a component of the extracellular matrix in its unproteolyzed form. A characteristic common to numerous tumors is the increase in the activity of the UPAR / plasmin system and the greater presence of plasma fibronectin. Also, numerous transformed cells with invasive potential express specific receptors for this type of fibronectin). Therefore, we characterized the role of GRK2 in the migratory response to fibronectin of transformed MCF-7 breast epithelial cells. The cells were transfected transiently with a GRK2 expression plasmid or with a specific siRNA construct, and were magnetically selected with the "dynabeads" method to enrich the proportion of transfected cells. The increased expression of GRK2 in MCF-7 cells causes a greater migration to fibronectin (Fig. 6A). On the contrary, the inhibition of the expression of the kinase causes a sharp decrease in migration compared to the control cells that have been transfected with an empty plasmid (pcDNA3) (Fig. 6A).

These results suggested a positive correlation between GRK2 levels and cell motility. In order to generalize this correlation, we analyzed whether the presence of GRK2 regulated migration in cells normally with low motility such as the non-transformed MCF-10A cell line, which also has low levels of GRK2 expression. As seen in Figure 6B, the overexpression of the kinase stimulates the migratory phenotype of these cells to fibronectin signals. In this case, the number of MCF-10A cells that migrate with increasing levels of GRK2 is similar to the number of migrating cells registered for MCF-7 (MCF-7 = 44; MCF-10A + GRK2 = 47.4, average of cells that migrate by field in both cases). Therefore, these results suggest that potential oncogenic signals that converge on the activation of AKT, would increase GRK2 levels in normal epithelial cells and this increase would facilitate cell migration in a tumor context.

EXAMPLE 3. The migratory response of transformed breast epithelial cells is affected by variations in GRK2 levels in response to different signals that stimulate AKT activity.

The aberrant migration of cancer cells responds to various chemotactic gradients that stimulate their evasion. of the primary tumor focus. Some of these chemotactic signals, such as S1 P and IGF-1, often come from the stroma "reactive" to the tumor, produced by fibroblasts and immune system cells. Both S1 P and IGF-1 are the factors that regulate not only the migration of breast epithelial cells, but also their proliferation and survival. These signals converge in the activation of AKT, which would trigger the accumulation of GRK2 and the potentiation of cell migration. Therefore, we wanted to determine if the migration mediated by this type of stimuli was modulated by GRK2 expression levels.

Figure 7A shows that the increase in GRK2 levels in transiently transfected MCF-7 cells induces greater migration against S1 P and IGF-1 stimuli. Again, and similar to what was observed in fibronectin-dependent migration, the presence of a specific interference RNA of GRK2 attenuates the migratory response of MCF-7 to these soluble signals, reducing 50% compared to control cells. .

Likewise, the GRK2-dependent regulation of the migration to chemotactic signals also extends to other transformed cell types of breast epithelium with a different degree of differentiation from that of MCF-7, as observed in the Hs578T cell line, derived from a little differentiated infiltrating ductal carcinoma. After the infection of these cells with the adenoviral vector siRNA of GRK2, the migration in response to IGF-1 and S1 P is strongly inhibited, reaching a

45.5 ± 10% of the normal migratory response to IGF-1 and 26.2 ± 5% with

S1 P, while the cells with the control adenovirus do not show alterations in motility compared to the untreated cells (Fig. 7B).

Based on these results we can conclude that the levels of

GRK2 are critical in the migration of breast epithelial cells in response to certain stimuli involved in tumor progression. In this context, ligands of the ErbB receptor family are especially relevant in the development of breast tumors. The ErbB2 protocol is overexpressed in about 30% of this type of tumors. Since the stimulation of these receptors is a potent signal for the induction of cell motility and survival, their presence constituting a prognostic marker of the malignancy of the tumor and its metastatic capacity, we analyze whether the expression of GRK2 modulated the chemotactic response induced by these receptors Since a specific ligand for ErbB2 has not been identified, MCF-7 cells were stimulated with heregulin, which promotes the heterodimerization of ErbB2 receptors with ErbB3 and ErbB4 receptors, and their activation. As can be seen in Figure 7A, the overexpression of GRK2 significantly increases the migration of MCF-7 cells to heregulin. These results reinforce the functional implication of this kinase in the development and / or acquisition of the tumor characteristics of the transformed cells.

EXAMPLE 4. GRK2 enhances the invasive capacity of various breast tumor cells with varying degrees of transformation.

As we have already mentioned, one of the key characteristics of tumor progression is the ability to metastasize or invade other tissues for which the transformed cells acquire the ability to degrade the basal lamina and the extracellular matrix through which they migrate in response to chemotactic gradients. Since GRK2 modulated the migration to these gradients, we considered whether the presence of GRK2 also had any effect on the invasion capacity of breast epithelial cells stimulated by relevant chemotactic signals in breast tumors. Thus, the levels of GRK2 were modified in the slightly invasive cell line MCF-7, transfecting as It has already been described with the kinase expression vector or with the siRNA-GRK2 construct, and the invasive response of these cells to stimuli present in the serum was analyzed by matrigel invasion assays (Fig. 8A). These tests simulate to some extent the conditions of invasion "in vivo", since the matrigel, extracellular matrix obtained from isolated mouse tumors and composed of laminin, collagenolV and growth factors among others, mimics the basal lamina of tissues .

In these experimental conditions a clear correlation is observed between the levels of GRK2 expression and cell invasiveness. Thus, the overexpression of the kinase promotes 75% more invasion with respect to the control cells, while the decrease in GRK2 reduces the invasion capacity of MCF-7 cells by 50% in response to serum (Fig. 8A). Similarly, the invasion of the matrigel in response to IGF-1, S1 P and heregulin is also significantly inhibited (more than 60%) by blocking the expression of GRK2 in MCF-7 cells (Fig. 8B).

The next question was to determine if the effects of GRK2 on the invasion capacity were also extensible to cell lines with a markedly invasive phenotype. To do this, we use breast cells

HS578T and B16 F10 melanoma cells, capable of inducing tumors in nude mice and metastasizing, as well as penetrating collagen matrices with high efficiency in response to serum. As can be seen in Figure 9, the decrease in GRK2 levels, by infection with the siRNA-GRK2 adenoviral vector, promotes a notable reduction in the ability to invade matrigel Hs578T and B16F10 cells.

This set of results shows for the first time that GRK2 is a key molecule in the modulation of migration and tumor invasion to different stimuli that signal through membrane receptors of a very diverse nature. This also suggests that GRK2 must play a basic and general functional role in the migration and invasion process. In line with this concept, the regulation of GRK2 invasiveness is observed not only in tumor lines of very different origin, such as breast carcinomas and melanomas, but also in non-transformed cells. Therefore, GRK2 must modulate fundamental activities in migration, perhaps related to changes in the cytoskeleton, adhesion and / or integration of diffusible migratory signals in these processes.

In this context, the regulation of GRK2 expression levels by AKT would represent a key mechanism by which cells would acquire a greater migratory potential and would have facilitated the process of tumor transformation. Thus, cells with high levels of GRK2 could present and acquire a transformed phenotype. Indeed, the analysis of the endogenous levels of GRK2 in several cell lines shows that non-transformed and reduced motility cells such as MCF10A and 184B5 express lower levels of GRK2 than transformed and mobile cells such as MCF-7 or MDA-MB-468 (Fig .10).

EXAMPLE 5. GRK2 levels affect the rate of cell proliferation.

Another of the functions of AKT in various types of tumors mentioned above is their participation in cell survival processes, promoting mechanisms to escape apoptosis and cell proliferation. Since GRK2 is subject to upward regulation mediated by AKT, we wonder if GRK2 could be modulating, in addition to the migration / invasion of breast epithelial cells, processes such as cell proliferation. To do this, we analyze the growth rate in serum response of MCF-7 cells treated with a control adenovirus or with the viral construction of the GRK2 siRNA after 24, 48, 72 and 96 hours of culture. The decrease in GRK2 levels affects the proliferative capacity of the cells after 72 hours. In the time interval of 72 to 96 hours, cells that have been infected with the SÍRNA-GRK2 adenovirus show lower growth (163.5 ± 36% and 167 ± 33% proliferation at 72 and 96 hours, respectively , compared with 24h) than the control cells (253 ± 34% proliferation at 72 hours and 331 ± 60% at 96 hours).

EXAMPLE 6. In vivo assay of a siRNA capable of inhibiting the expression of GRK2 for use in the treatment of cancers in which GRK2 is cooperating with AKT.

The attenuation of the migratory / invasive and proliferative response of various tumor cell lines (of breast epithelium and melanoma) in the in vitro assays described as a consequence of the inhibition of the expression of the GRK2 protein through the use of siRNA constructs, suggests that This inhibition strategy could be effective in the treatment of certain tumors. In order to provide evidence of this hypothesis, the effect of the suppression of GRK2 on the formation and development of tumors in vivo was analyzed. For this, the induction of orthotopic tumors in immunosuppressed mice (athymic nude fox Nu / Un) was carried out by subcutaneous injection of transformed MCF7 breast epithelial cells (5x106 cells in a glucose / PBS / matrigel suspension per mouse) infected with an adenoviral control construction or with the adenoviral construction of the siRNA-GRK2. The results obtained show that the inhibition of GRK2 in MCF7 cells blocks its ability to form tumors in this model in vivo, not showing signs of tumor at three weeks of inoculation, while in the absence of GRK2 siRNA, the cells Malignant MCF7 do develop the tumor. Therefore, these results indicate the potential of the inhibition of GRK2 with siRNA as an intervention measure in the treatment of breast tumors.

REFERENCES

Dorsam, R. T. and Gutkind, J. S. (2007). G-protein-coupled receptors and cancer. Nat Rev Cancer 7, 79-94

Ellington, A.D. and Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346: 818-22

Pao, C. S. and Benovic, J. L. (2002). Phosphorylation-lndependent Desensitization of G Protein-Coupled Receptors? Sci. STKE. p42.

Ribas C, Pénela P., Murga C, Salcedo A., Garcia-Hoz C, Jury-Pueyo M. et al. (2007). The G protein-coupled kinase receptor (GRK) interactome: Role of GRKs in GPCR regulation and signaling. Biochim Biophys Acta 1768: 913-942

Salcedo, A., Mayor, F. and Pénela, P. (2006). Mdm2 is involved in the ubiquitination and degradation of G-protein-coupled receptor kinase 2. EMBO J 25 (20): 4752 ^ 1762

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed. CoId Spring Harbor Laboratory

Tuerk, C. and GoId, L. (1990). Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-10

Claims

1. Composition comprising a siRNA, capable of inhibiting the expression of GRK2, for use in the treatment of carcinogenic diseases in which GRK2 is cooperating with AKT.

2. Composition comprising a siRNA capable of inhibiting the expression of GRK2 for use in the treatment of melanoma.

3. Composition comprising a siRNA capable of inhibiting the expression of GRK2 for use in the treatment of breast cancer.

4. Composition comprising a siRNA capable of inhibiting the expression of GRK2 for use in the treatment of ovarian cancer.

5. Composition comprising a siRNA capable of inhibiting the expression of GRK2 for use in the treatment of prostate cancer.

6. Composition comprising a siRNA capable of inhibiting the expression of GRK2 for use in the treatment of gliomas.

7. Composition comprising a siRNA capable of inhibiting the expression of GRK2 for use in the treatment of thyroid cancer.

8. Composition according to any of the preceding claims, wherein the siRNA is found as SIRR-GRK2 constructs fused to aptameric RNAs.

9. Composition according to any of claims 1-8, wherein the siRNA is included in a genetic RNA construct.

10. Composition according to any of claims 1-8, wherein the siRNA is included in a genetic DNA construct capable of transcribing in vitro or in vivo to an RNA sequence.

11. Pharmaceutical composition containing a therapeutically effective amount of siRNA capable of inhibiting the expression of GRK2, for use in the treatment of melanoma.

12. Pharmaceutical composition containing a therapeutically effective amount of siRNA, capable of inhibiting the expression of GRK2, for use in the treatment of breast cancer.

13. Pharmaceutical composition containing a therapeutically effective amount of siRNA, capable of inhibiting the expression of GRK2, for use in the treatment of ovarian cancer.

14. Pharmaceutical composition containing a therapeutically effective amount of siRNA, RNA or DNA of the invention, capable of inhibiting the expression of GRK2, for use in the treatment of prostate cancer.

15. Pharmaceutical composition containing a therapeutically effective amount of siRNA, capable of inhibiting the expression of GRK2, for use in the treatment of gliomas.

16. Pharmaceutical composition containing a therapeutically effective amount of siRNA capable of inhibiting the expression of GRK2, for use in the treatment of thyroid cancer.

17. Pharmaceutical composition according to any of claims 11-16, wherein the siRNA is included in a genetic RNA construct.

18. Pharmaceutical composition according to any of claims 11-16, wherein the siRNA is included in a genetic DNA construct capable of transcribing in vitro or in vivo to an RNA sequence.