ES2794149A1 - PCSK9 INHIBITORS FOR THE PREVENTION AND/OR TREATMENT OF CANCER (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

PCSK9 inhibitors for the prevention and/or treatment of cancer. The present invention relates to the use of a proprotein convertase subtilisin/kexin type 9 or PCSK9 inhibitor agent for the prevention and/or treatment of metastases in a subject suffering from cancer who is subjected, or will be subjected, to ventilation mechanics. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

PCSK9 INHIBITORS FOR THE PREVENTION AND / OR TREATMENT OF

CANCER

The present invention falls within the field of molecular biology applied to medicine, pharmacology and oncology. Specifically, the invention relates to the use of a proprotein convertase subtilisin / kexin type 9 or PCSK9 inhibitor agent for the prevention and / or treatment of metastases in a subject suffering from cancer and is subjected, or will be subjected, to mechanical ventilation. .

BACKGROUND OF THE INVENTION

Survival to tumors, more specifically lung tumors, is determined by different factors, including both the characteristics of the tumor and the patient. One of the key events in tumor progression that has the greatest impact on the prognosis of patients is the appearance of metastases in organs other than the lung. Although different molecular mechanisms responsible for this metastasis process have been characterized in detail, there is currently no approved drug that acts specifically to inhibit said metastatic process.

Lung tissue is constantly subjected to mechanical stress caused by changes in intrathoracic pressure during the respiratory cycle. Under normal conditions, during spontaneous ventilation, these pressures are low and are evenly distributed throughout the lung parenchyma, causing an isotropic deformation of the parenchyma. When a patient requires mechanical ventilation, either due to respiratory failure or the need for ventilatory support during a procedure (such as general anesthesia with muscle paralysis), the ventilation pressures used are positive, of greater magnitude, and with a heterogeneous distribution to along the parenchyma. This condition can cause an increase in mechanical load in different areas of the lung, especially at the interfaces between aerated and non-aerated tissue.

Thus, the mechanical stress to which cells are subjected is one of the main determinants of their behavior. Cells settled on a rigid extracellular matrix exhibit a different behavior from those settled on a soft matrix. The mechanical load is transmitted to the cell interior through a mechanotransduction process, which culminates in a change in gene expression and, therefore, in cell behavior. Thus, mechanical stress can induce changes in cell proliferation, migration or differentiation.

Lung cancer patients are subjected to mechanical ventilation during the general anesthesia required for surgery on their tumor. The comorbidities (i.e. the existence of two or more diseases in the same individual, generally related) that these patients present mean that they may also require mechanical ventilation when being operated on for any other pathology, or in cases of respiratory failure. The impact of the mechanical stress caused by said mechanical ventilation on the evolution of tumors, in particular, on the evolution of lung tumors is not known.

Therefore, in the state of the art there is a need to know the effects of mechanical stress, preferably as a consequence of mechanical ventilation, on the evolution of the tumor and its metastasis, and to provide the appropriate therapies for the treatment of said tumors of the most efficient way possible.

DESCRIPTION OF THE INVENTION

The authors of the present invention have studied the effects of mechanical stress on the development and evolution of tumors, preferably lung tumors, finding that those tumors that are subjected to mechanical stress show greater invasiveness, thus favoring tumor metastasis.

To reach this conclusion, the inventors subjected tumor cells to mechanical stress by means of equibiaxial stretching using a cell deformation equipment, and studied their cell migration by means of a matrigel assay. The result showed that the area of the well covered by migrating cells was greater in the group of cells that had undergone mechanical stress (see Figures 1A and 1B) than in the group of cells that had not undergone mechanical stress. Then the inventors proceeded to perform in vivo tests in mice that caused lung tumors, and observed that the animals subjected to mechanical ventilation showed a higher proportion of brain, kidney and liver metastases than those that were maintained with spontaneous respiration (Figure 2).

Likewise, the inventors have also observed that tumor cells that suffer mechanical stress and, therefore, are more prone to evolve metastasis, have a high expression of genes related to the cellular processing of cholesterol (Figure 3A), especially PCSK9 (acronym derived from its English name proprotein convertase subtilisin / Kexin 9), compared to those tumors that are not subjected to mechanical stress (see Figure 3B), and that the inhibition of the function of said enzyme prevents the increase of said invasiveness / aggressiveness (see Figure 4).

For the purposes of the present invention, mechanical ventilation is understood as the use of a device to generate cyclical positive pressures in the airway or cyclical negative pressures in the pleural space in order to facilitate alveolar ventilation in a subject, either because it is incapable of generate these pressures by itself or because they are insufficient.

In a first aspect, the present invention relates to a PCSK9 inhibitor agent, hereinafter "inhibitor of the invention", for use in the prevention and / or treatment of metastasis in a subject suffering from cancer and undergoing, or will be subjected to mechanical ventilation.

As used in the present invention, the term "PCSK9 inhibitor agent" refers to any molecule capable of totally or partially inhibiting both the expression of the PCSK9 gene and the activity of the protein encoded by said PCSK9 gene , that is, the PCSK9 protein. Thus, the inhibitory agent induces the suppression or reduction of the transmission of biochemical signals through PCSK9. As explained above, the activity of the PCSK9 protein comprises promoting cell proliferation and metastasis. Therefore, a PCSK9 inhibitor will be characterized by preventing or decreasing cell proliferation and metastasis. The activity and expression of PCSK9 can be easily assayed by any method known in the art. This definition also includes those compounds that prevent or they decrease gene transcription or expression, mRNA maturation, mRNA translation and post-translational modification of the protein. In a more preferred embodiment, the inhibitory agent can be an antibody that recognizes the PCSK9 protein, or a polynucleotide that encodes an antisense sequence of specific nucleotides against the PCSK9 sequence , for example, using RNAi, antisense, ribozyme or aptamers. Inhibitory activity can be tested by measuring the expression level of PCSK9, at the level of the protein or at the level of RNA.

Inhibitors of the expression and / or activity of PCSK9 can be identified using procedures such as a polymerase chain reaction (PCR), such as for example, a real-time PCR, nested PCR, multiplex PCR, a DNA array or RNA, a hybridization, fluorescence energy transfer in time (TR-FRET) assays, Western Blot blotting assay, immunohistochemical techniques, ELISA, a protein array, immunoprecipitation or radioimmunoassay, among others. The protocols and kits for carrying out such techniques are well known to those of skill in the art and are commercially available.

The PCSK9 gene encodes an enzymatic protein present in the blood with endoprotease activity. The name is an acronym for its name in English Proprotein Convertase Subtilisin Kexin 9, that is, proprotein convertase subtilisin / kexin type 9, or PCSK9 protein. Other names by which this gene is known are FH3, HCHOLA3, LDLCQ1, NARC-1, NARC1, or PC9. The PCSK9 gene is conserved in a large number of species, such as the chimpanzee, Rhesus monkeys, the mouse, the rat, the chicken, the zebrafish, S. cerevisiae, S. pombe and the frog, among others. In humans, the PCSK9 gene comprises a nucleotide sequence SEQ ID NO: 1 (NCBI, accession number NG_009061.1) which, when transcribed, gives rise to a messenger RNA comprising the sequence SEQ ID NO: 2 (NCBI, accession number NM_174936.3).

In a particular embodiment of the inhibitor of the invention, the PCSK9 gene comprises a nucleotide sequence that has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 , or 99% identity with the sequence SEQ ID NO: 1 or SEQ ID NO: 2. In another particular embodiment, the PCSK9 gene comprises a nucleotide sequence that has 100% identity with the sequence SEQ ID NO: 1 or SEQ ID NO: 2.

The term "identity", "percentage identity" or "sequence identity" between two sequences (nucleic acids or proteins), is understood to designate a percentage of nucleotides or identical amino acids between the two sequences being compared, obtained after the best alignment, said percentage being purely statistical and the differences between the two sequences being distributed at random and along their entire length. By "best alignment" or "optimal alignment" is meant the alignment by which the percent identity determined as described below is the highest. Comparisons between two nucleotide or amino acid sequences are traditionally carried out: comparing these sequences after they have been optimally aligned, such comparison being carried out by segment or by "comparison window" to identify and compare local regions of similarity sequence. The optimal alignment of these sequences for comparison can be carried out, in particular with the help of one of the following algorithms: the local homology algorithm of Smith and Waterman (1981), the local homology algorithm of Neddleman and Wunsch (1970), the search for similarity procedure of Pearson and Lipman (1988), the computer programs that use these algorithms (GAP, BESTFIT, BLASTP, BLASTN, BLASTX, TBLASTX, FASTA and TFASTA in the software package Wisconsin Genetics, (Genetics Computer Group, 575 Science Dr., Madison, Wl), or the internet servers in particular those of the National Center for Biotechnology Information (NCBI) (http: //www.ncbi.nlm.nih.gov), EMBL (http: // www .embl.org) and the Ensembl project (http: //www.ensembl.org)). In order to obtain the optimum alignment, the BLAST program is preferably used, with the BLOSUM 62 matrix. The PAM or PAM250 matrices can also be used, as well as an identity matrix for the nucleotide sequences.

Examples of inhibitors of PCSK9 gene expression include, but are not limited to, an RNA interference (RNAi), an antisense oligonucleotide, a DNA enzyme, a ribozome molecule, an aptamer, and an interference CRISPR (CRISPRi).

The term "RNAi", "interference RNA" or siRNA, for the purposes of the present invention means any RNA that is capable of down-regulating the expression of PCSK9. It encompasses small interfering RNA (siRNA), double stranded RNA (dsRNA), single stranded RNA (ssRNA), micro RNA (miRNA), and short hairpin RNA (shRNA) molecules. SiRNAs are usually designed against a region 50-100 nucleotides after the downstream end of the translation initiation codon, while the 5'UTR (untranslated region) and 3'UTR are usually avoided.

RNAi are agents that are capable of inhibiting the expression of a target gene through RNA interference. An siRNA can be chemically synthesized, it can be obtained by in vitro transcription, or it can be synthesized in vivo in the target cell. Typically siRNAs consist of an RNA double strand between 15 and 40 nucleotides in length and which may contain a 1 to 6 nucleotide 3 'and / or 5' overhang. The length of the overhang is independent of the total length of the siRNA molecule. SiRNAs act by degradation or post-transcriptional silencing of the target messenger.

The siRNAs of the invention are substantially homologous to the mRNA of the PCSK9 gene (SEQ ID NO: 1) or to the nucleotide sequence (SEQ ID NO: 2) encoding the PCSK9 protein (SEQ ID NO: 3). By "substantially homologous" it is understood that they have a sequence that is sufficiently complementary or similar to the target mRNA, such that the siRNA is capable of causing its degradation by RNA interference. SiRNAs suitable for causing such interference include, but are not limited to, siRNAs made from RNA, as well as siRNAs containing various chemical modifications, including but not limited to:

- siRNAs in which the bonds between nucleotides are different from those that appear in nature, such as phosphorothionate bonds.

- Conjugates of the RNA strand with a functional reagent, such as a fluorophore.

- Modifications of the ends of RNA chains, in particular the 3 'end by modification with different hydroxyl functional groups in position 2'.

- Nucleotides with modified sugars such as 2 'O-alkylated moieties such as 2'-0-methylribose or 2'-0-fluorosibose.

- Nucleotides with modified bases such as halogenated bases (for example 5-bromouracil and 5-iodouracil), alkylated bases (for example 7-methylguanosine).

SiRNAs can be used as is, that is, in the form of a double-stranded RNA with the aforementioned characteristics. However, the use of vectors containing the sequences of the sense and antisense strands of siRNAs under the control of suitable promoters for their expression in the cell of interest is also possible. Suitable vectors for siRNA expression are those in which the two DNA regions that code for the two siRNA strands are arranged in tandem on the same DNA strand separated by a spacer region that, when transcribed, forms a loop and where a single promoter directs the transcription of the DNA molecule that gives rise to hcRNA (in short hairpin RNA or shRNA). SiRNAs can be generated intracellularly from so-called shRNAs, characterized in that the antiparallel chains that make up siRNA are connected by a loop or hairpin region. ShRNAs can be encoded by plasmids or viruses, particularly retroviruses, and be under the control of a promoter. Suitable promoters for the expression of shRNA are the promoters suitable for its expression in the cell of interest, as indicated above.

The siRNAs and shRNAs of the invention can be obtained using a number of techniques known to those skilled in the art. The region of the nucleotide sequence that is taken as the basis for designing siRNAs is not limiting and may contain a region of the coding sequence (between the start codon and the stop codon) or, alternatively, it may contain sequences from the region 5 'or 3' untranslated, preferably between 25 and 50 nucleotides in length and in any position 3 'to the start codon.

In a preferred embodiment of the invention, RNAi capable of inhibiting the PCSK9 gene are selected from the list consisting of: Inclisiran, M-005989-01 (Dharmacon), L-005989-00 (Dharmacon) and E-005989-00 ( Dharmacon).

In the present invention, "antisense oligonucleotide" or "antisense RNA" is understood as the nucleic acid molecule that directly inhibits the expression of the gene by joining the DNA double helix through Hoogsteen type bonds , forming a triple helix that prevents transcription mediated by RNA polymerase II, or when pairing with sense RNAs and forms double-stranded molecules that cannot be translated, so they are digested by cellular RNAse H. In general, these procedures refer to the range of techniques generally employed in the art and include any procedure that relies on specific binding to oligonucleotide sequences.

Such oligonucleotide probes are preferably modified oligonucleotides, which are resistant to endogenous nucleases, eg, exonucleases and / or endonucleases, and which are therefore stable in vivo. Examples of nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phospothothionate, and methylphosphonate DNA analogs.

With regard to obtaining the antisense oligonucleotide, oligodeoxyribonucleotide regions derived from the translation starting location, for example, between -10 and 10 of the target gene are preferred. Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to the mRNA encoding the target protein (PCSK9 protein). The antisense oligonucleotides will bind to the mRNA transcripts and prevent translation.

Both oligonucleotides that are complementary to the 5 'and 3' untranslated regions of the mRNA function effectively to inhibit translation. In the event that oligonucleotides complementary to the 5 'untranslated region of the mRNA are designed, these should include the complement of the AUG initiation codon. Oligonucleotides complementary to the coding regions of mRNA are less effective translation inhibitors, but could also be used according to the invention. If designed to hybridize to the 5 ', 3' or coding region of the mRNA, the antisense nucleic acids should be at least six nucleotides in length and preferably less than about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length. The ability of antisense oligonucleotides to inhibit gene expression can be verified by in vitro studies that are routine practice for those skilled in the art. The results obtained in said studies can be compared with those obtained using a control oligonucleotide which, preferably, will be approximately the same length as the oligonucleotide to be tested and differing from the antisense sequence no more than is necessary to prevent nonspecific hybridization.

The antisense oligonucleotides can be single or double stranded DNA or RNA or chimeric mixtures or derivatives or modified versions thereof. The oligonucleotide can be modified into the base group, the sugar group (a modified sugar group selected from the group that includes, but is not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose), or the phosphate backbone [oligomers peptide nucleic acid or ANP (in English Peptide Nucleic Acid or PNA)], for example, to improve the stability of the molecule, its capacity of hybridization etc. The oligonucleotide can include other linked groups, such as peptides (eg, to target host cell receptors) or agents to facilitate transport across the cell membrane or blood-brain barrier. For this purpose, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a carrier agent, hybridization-triggered cleavage agent, etc. In the context of the present invention, it is also possible that the antisense oligonucleotide is an alpha-anomeric oligonucleotide. While antisense oligonucleotides complementary to the coding region of the mRNA target sequence can be used, those complementary to the non-translated transcribed region can also be used. In some cases, it can be difficult to achieve intracellular antisense oligonucleotide concentrations sufficient to suppress translation of endogenous mRNAs. Therefore, when introducing the antisense oligonucleotide into cells, it can go in a recombinant DNA construct in which the antisense oligonucleotide is located under the control of a strong promoter. Alternatively, the expression of the target gene can be reduced by directing complementary deoxyribonucleotide sequences to the regulatory region of the gene (i.e., the promoter and / or enhancers) to form triple helix structures that prevent transcription of the gene in the target cells. in the body. In certain embodiments, the antisense oligonucleotides are antisense morpholinos.

On the other hand, the invention also contemplates the use of DNA enzymes to inhibit the activity of the PCSK9 protein. DNA enzymes are capable of inhibiting the expression of the gene encoding the PCSK9 protein thanks to the incorporation of antisense technology such as ribozymes. DNA enzymes are designed in so that they recognize a particular nucleic acid target sequence, similar to an antisense oligonucleotide, but are also capable of specifically cutting the target nucleic acid, that is, they also have catalytic capacity.

Additionally, the present invention also contemplates the use of ribozymes to inhibit the expression of the PCSK9 gene . In the present invention, “ribozymes” are understood to be small RNA molecules with catalytic activity designed to recognize a specific mRNA and degrade it thanks to its specific endonucleolytic activity. The mechanism of action of ribozyme involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA, followed by an endonucleolytic cleavage event. The composition of the ribozyme molecules preferably includes one or more sequences complementary to the target mRNA, and the well-known sequence responsible for cutting the mRNA or a functionally equivalent sequence. Ribozymes used in the present invention include hammerhead ribozymes, RNA endoribonucleases (hereinafter "Cech-type ribozymes"). Ribozymes can be composed of modified oligonucleotides (eg, to improve stability, targeting, etc.) and should be delivered to cells that express the target gene in vivo. A preferred method of delivery involves using a DNA construct that "encodes" the ribozyme under the control of a strong constitutive promoter, such that the transfected cells will produce sufficient amounts of the ribozyme to destroy endogenous target messengers and inhibit translation. Since ribozymes, unlike other antisense molecules, are catalytic, a lower intracellular concentration is required for their efficacy.

In the context of the present invention "aptamers" are understood to be nucleic acid molecules that are capable of binding to another molecule of particular interest with high affinity and specificity. In the context of the present invention, this target molecule of particular interest would be PCSK9. An aptamer is typically made by in vitro selection for binding to a target molecule. However, in vivo selection is also possible. It is typically between 10 and 300 nucleotides in length. Most commonly, an aptamer must be between 30-100 nucleotides in length.

In a preferred embodiment, it is also possible to use ZFNs, TALENs and CRISPRs as methods for genomic editing, preferably for inhibition of the PCSK9 gene . Thus, an agent capable of induced altering PCSK9 expression or activity may comprise: a nuclease capable of modifying the endogenous PCSK9 gene, such as to down-regulate or abolish PCSK9 expression, or a heterologous repressor protein capable to repress the transcription of the endogenous PCSK9 gene, such as the heterologous repressor protein. The agent can comprise more than one nuclease. In certain embodiments, the agent comprises more than one TALE or zinc finger protein, whereby a TALE or zinc finger targets PCSK9. In other embodiments, the agent comprises more than two nucleases, capable of targeting multiple genes. In certain embodiments, a CRISPR-Cas system is used and multiple guide RNAs are used to direct the CRISPR enzyme to multiple gene targets.

For the purposes of the present invention, "CRISPRi", analogous to RNAi, have the ability to suppress gene transcription, in a reversible way as they are specific, but without making cuts.

Thus, in a particular embodiment, the inhibitor agent for the expression of the PCSK9 gene is an RNAi selected from the list consisting of: siRNA, dsRNA, ssRNA, miRNA, shRNA molecules, or any combination thereof, an antisense oligonucleotide, a DNA enzyme, a ribozyme, an aptamer or a CRISPRi. In a more preferred embodiment, RNAi capable of inhibiting the PCSK9 gene are selected from the list consisting of: Inclisiran, M-005989-01 (Dharmacon), L-005989-00 (Dharmacon) and E-005989-00 (Dharmacon) .

Additionally, the total or partial inhibition of the activity of the protein encoded by said PCSK9 gene , that is, the PCSK9 protein, can be carried out through the use of agents that specifically bind to, and block, the PCSK9 protein. In humans, the PCSK9 protein comprises the sequence SEQ ID NO: 3 (NCBI, accession number NP_777596.2). The PCSK9 protein has a very important role in the regulation of cholesterol metabolism.

Within the context of the present invention, functionally equivalent variants of the PCSK9 protein are also included. By "functionally equivalent variant of the PCSK9 protein" is understood to be that protein that (i) comprises a sequence in which one or more amino acids are substituted by a conserved or non-conserved amino acid residue (preferably a conserved amino acid), or (ii) comprises a sequence that comprises an insertion or a deletion of one or more amino acids, and which It has the same function as the PCSK9 protein, that is, to regulate the metabolism of cholesterol.

PCSK9 is a serine protease that plays a very important role during the metabolism of LDL ( low density lipoproteins), inhibiting the recycling of the LDL receptor. Physiologically, circulating LDL particles bind to LDL receptors (LDLR) found on the cell surface. Once the LDL-LDLR complex is formed, its internalization occurs. Inside the cell, the LDL particle dissociates from the receptor and is eliminated by the lysosomes, allowing the receptor to recycle back to the cell surface. The binding of PCSK9 to the LDLR receptor prevents its recycling. The PCSK9-LDLR complex is driven to lysosomes for degradation. There is a reduction in the expression of the receptor on the cell surface and this results in a decreased ability to eliminate circulating LDL particles.

Therefore, in another particular embodiment, the PCSK9 protein comprises an amino acid sequence that has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence SEQ ID NO: 3. In another particular embodiment, the PCSK9 protein comprises an amino acid sequence that has 100% identity with the sequence SEQ ID NO: 3.

Examples of inhibitors of the PCSK9 protein include, but are not limited to, a molecule that specifically recognizes and binds to the PCSK9 protein, or a fragment of said protein, or a functionally equivalent variant of said protein as previously defined. , and blocks its enzymatic action on the LDL-R receptor. Thus, in a particular embodiment, the inhibitory agent is a molecule that specifically recognizes the PCSK9 protein. In another still more particular embodiment, the molecule that specifically recognizes the protein encoded by the PCSK9 gene is an antibody that specifically binds to PCSK9, preferably a monoclonal antibody that specifically binds to PCSK9.

In the present invention, "PCSK9 protein specific antibodies" are understood to be those antibodies or peptides with binding capacity that specifically recognize the PCSK9 protein, binding to it and preventing it from performing one or more of its functions. In the context of the present invention, the antibody prevents the PCSK9 protein from promoting cell proliferation and metastasis.

By "antibody" is understood a glycoprotein of the gamma globulin type that is part of the humoral immune system that binds specifically to an antigen. The term "antibody" as used herein includes polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab 'and F (ab') 2, scFv, diabodies, tribodies, tetrabodies, and humanized antibodies. Antibodies can be prepared using any of the procedures that are known to those skilled in the art. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. The means for preparing and characterizing antibodies are also known in the state of the art. Once the antibodies with the ability to bind to the PCSK9 protein have been identified, those capable of inhibiting the activity of this protein will be selected using an inhibitory assay identification assay. In a particular embodiment, the antibody with the ability to inhibit the PCSK9 protein is a monoclonal antibody. More preferably, the monoclonal antibody is selected from the list consisting of: Evolocumab (Amgen, USA), Alirocumab (Sanofi, USA), Bococizumab (Pfizer, USA), 1D05-lgG2 (Merck, USA), RG-7652 (Genentech , USA) and LY3015014 (Eli Lilly, USA).

Assays to identify whether a given compound is an inhibitor of the PCSK9 protein include, but are not limited to: immunoprecipitation, radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay, and enzyme-linked immunosorbent assay (ELISA). ). Additionally, the above immunoassays can be used in combination, such as immunoprecipitation followed by Western blotting. Such assays can be direct, indirect, competitive, or non-competitive immunoassays as described in the art.

In the present invention "cancer" is understood as a wide range of conditions caused by an excessive proliferation of malignant cells (also known as cancerous or cancerous), with typical behavioral traits and uncontrolled growth (growth and division beyond normal limits , invasion of the surrounding tissue and sometimes metastasis). It comprises any disease of an organ or tissue in a mammal, preferably man, characterized by a poorly controlled, or uncontrolled, multiplication of normal or abnormal cells in said tissue, and its effect on the entire body. The term cancer, within this definition, includes benign neoplasms, dysplasias, hyperplasias, as well as neoplasms that show metastasis, or any other transformation such as, for example, leukoplakia that often precede the outbreak of cancer. Cells and tissues are cancerous when they grow and replicate faster than normal, moving or dispersing in the surrounding healthy tissue or any other tissue in the body, which is known as metastasis, it assumes abnormal shapes and sizes, shows changes in its nucleocytoplasmic ratio, nuclear polychromasia, and finally ceases. Cancer cells and tissues can affect the body as a whole causing paraneoplastic syndromes, or if cancer occurs in a vital organ or tissue, its normal function being interrupted or damaged, with possible fatal results. The end result of the evolution of a cancer that involves a vital organ, whether primary or metastatic, is the death of the affected mammal. Cancer tends to spread, and its extent is usually related to changes in survival from the disease.

Cancer is generally said to be in one of three growth stages: early or localized, when the tumor is still confined to the tissue of origin, or in its primary location; direct extension, when tumor cancer cells have invaded adjacent tissue or spread only to regional lymph nodes; or metastasis, when cancer cells have migrated to distant parts of the body from the primary site, via the circulatory or lymphatic system, and established in secondary sites.

Cancer is said to be malignant because of its tendency to cause death if it is not treated. Benign tumors do not usually cause death, although they can do so if they interfere with the normal function of the body due to their characteristics or location, size or paraneoplastic effects. Here malignant tumors fall within the definition of cancer within the scope of this definition as well. In general, cancer cells divide at a higher rate than normal cells, but the distinction between the growth of cancerous and normal tissues is not so much that cell division is much faster, as the partial or complete loss of stopping its growth and differentiation into useful and limited tissue, of the type that characterizes the functional balance of normal tissue growth. In a preferred embodiment of this aspect of the invention, cancer is selected from the list consisting of: This term includes, without limitation, cancer of the lung, breast, heart, small intestine, colon, spleen, kidney, bladder, head, neck , ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, hepatobiliary and liver; as well as tumors such as, without limitation, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatolastoblastoma, rhabdomyosarcoma cancer, retinal-biliary cancer , sarcoma and teratoma. Also, this term includes acrolentiginous melanoma, actinic adenocarcinoma, keratosis, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin's gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, carcinoma, capillary carcinoma, carcinoma, carcinoma Cholangiocarcinoma, cystadenoma, endodermal breast tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hepatic adenoma, hemangioendothelinoma, hepatic adenoma, endothelialgioma , hepatocellular carcinoma, hepatobiliary cancer, insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloep itelioma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, serous papillary adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, carcinoma of carcinoma, carcinoma of microbes soft tissue, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, warty carcinoma, vipoma, Wilm's tumor, intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma, glioblastoma, small cell and non-small cell cancer, metastatic melanoma, androgen independent metastatic prostate cancer, cancer androgen-dependent metastatic prostate cancer and breast cancer. In a particular embodiment of the present invention, the cancer is lung, breast, colon or kidney cancer. In yet another more preferred embodiment, the lung cancer is a primary lung cancer or a metastatic lung cancer.

As used herein, the term "subject", "individual" or "patient", used interchangeably throughout this document, refers to a mammal, and includes, but is not restricted to, domestic animals and farm animals, primates, and humans, eg, humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a human male or female of any age or race.

For the purposes of the present invention, it is understood that a subject is subjected to mechanical ventilation, when they require general anesthesia during any surgical procedure, as well as in cases of respiratory failure.

Lung cancer patients are subjected to mechanical ventilation during the general anesthesia required for surgery on their tumor. The comorbidities (i.e. the existence of two or more diseases in the same individual, generally related) that these patients present mean that they may also require mechanical ventilation when being operated on for any other pathology, or in cases of respiratory failure. The impact of the mechanical stress caused by said mechanical ventilation on the evolution of tumors, in particular, on the evolution of lung tumors is not known.

The term "treatment", as used in the present invention, refers to combating the effects caused as a consequence of the disease or pathological condition of interest in a subject (preferably a mammal, and more preferably a human) including:

(i) inhibit the disease or pathological condition, that is to say stop the development thereof;

(ii) alleviating the disease or pathological condition, that is, causing the disease or pathological condition or its symptoms to regress;

(iii) stabilize the disease or pathological condition.

The term "prevention", as used in the present invention, consists of preventing the onset of the disease, that is, avoiding the disease or pathological condition in a subject (preferably a mammal and more preferably a human), in in particular, when said subject has a predisposition to the pathological condition but has not yet been diagnosed.

In another particular embodiment, the PCSK9 expression inhibitor agent for use as described herein is comprised within a pharmaceutical composition. In yet another more particular embodiment, the inhibitor of PCSK9 expression is in a therapeutically effective amount, and optionally said pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant and / or excipient.

In the sense used in the description, the term "therapeutically effective amount" refers to the amount of inhibitor of the expression and / or activity of PCSK9 according to the present invention, calculated to produce the required effect and, in general, will be determined, among other factors, by the inherent properties of the compounds and of the subjects to be treated, including age, patient's condition, severity of the alteration or disorder, and the route and frequency of administration.

The pharmaceutically acceptable adjuvants and carriers that can be used in such compositions are the adjuvants and carriers known to those skilled in the art and commonly used in the production of therapeutic compositions. The term "pharmaceutically acceptable carrier" is a substance used in the composition to dilute any of the components comprised therein to a certain volume or weight. The pharmaceutically acceptable carrier is an inert substance or one of identical action to any of the elements comprised in the composition of the present invention. The function of the vehicle is to facilitate the incorporation of other elements, to allow a better dosage and administration or to provide the composition with consistency and shape. Further, the pharmaceutical composition of the invention may comprise one or more adjuvants and / or excipients. The term "excipient" refers to a substance that helps the absorption of the elements of the composition of the invention, stabilizes said elements, activates or helps the preparation of the composition in the sense of providing it with consistency or providing it with flavors that make it more enjoyable. Therefore, the excipients could have the function of keeping the ingredients bound together, such as, for example, in the case of starches, sugars or celluloses, the function of sweetening, the function of giving color, the function of protecting the composition, such as such as, for example, isolating it from air and / or humidity, the function of filling a tablet, capsule or any other form of presentation, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

Examples of pharmaceutically acceptable carriers are known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, different types of wetting agents, sterile solutions, etc. Compositions comprising said carriers can be formulated by conventional procedures known in the state of the art. As used herein, the terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, as they relate to compositions, carriers, diluents, and reagents, are used interchangeably and indicate that the materials they are capable of being administered to a subject without producing undesirable physiological effects.

In a particular embodiment, said pharmaceutical composition is prepared in solid form or in aqueous suspension, in a pharmaceutically acceptable diluent. The composition provided by the present invention can be administered through any appropriate route of administration, whereby said composition will be formulated in the appropriate pharmaceutical form for the selected route of administration. In a particular embodiment, the administration of the composition provided by the invention is carried out parenterally, orally, intraperitoneally, subcutaneously, intracranially, etc. A review of the different pharmaceutical forms of drug administration and the excipients required to obtain them can be found, for example, in the "Treaty of Galenic Pharmacy", C. Faulii Trillo, 1993, Luzán 5, SA Ediciones, Madrid.

In another preferred embodiment, the pharmaceutical composition for use as described herein may further comprise other drugs or agents, preferably chemotherapeutics, used in the treatment of cancer, more specifically in the treatment of lung cancer, with a view to acting in a complementary manner. or as a reinforcement. In yet another more particular embodiment, the chemotherapeutic agents are selected from the list consisting of: cisplatin, carboplatin, docetaxel, gemcitabine, paclitaxel, vinorelbine, etoposide, vinblastine, pemetrexed and irinotecan.

In another aspect, the present invention relates to an in vitro method for designing a personalized therapy for a subject suffering from cancer and who is, or will be subjected to mechanical ventilation, comprising

a) quantify the level of expression and / or activity of PCSK9 in a biological sample isolated from the subject, and

b) comparing the level of expression and / or activity obtained in step (a) with the expression and / or activity value of PCSK9 in a control sample,

wherein if the expression and / or activity value obtained in step (a) is greater than the expression and / or activity value of the control sample, then the therapy comprises the administration to the subject of a PCSK9 inhibitor agent, according to It has been described throughout this document.

For the purposes of the present invention, the term "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, such as blood, serum, urine, saliva, cerebrospinal fluid, or to a solid sample or semisolid, such as tissues, feces, and the like, or alternatively, a solid tissue such as those commonly used in histological diagnosis, obtained by any method known to a person skilled in the art. In a more preferred embodiment, the isolated biological sample is a tumor tissue sample.

"Tumor tissue sample" refers to a tissue sample derived from the tumor, either the primary tumor or the metastatic tumor, in particular breast cancer, colon cancer, lung cancer, kidney cancer or thyroid cancer, more particularly primary lung cancer or metastatic lung cancer. Bliss Sample can be obtained by conventional methods, eg, biopsy, using methods well known to those skilled in the related medical arts. Methods for obtaining a biopsy sample include coarse partitioning of a tumor, or microdissection or other cell separation methods known in the art. Tumor cells can be further obtained by fine needle aspiration cytology. To simplify storage and handling of samples, samples can be fixed in formalin and embedded in paraffin or first frozen and then embedded in a cryosetting medium, such as OCT compound, by immersion in a highly cryogenic medium that allows rapid freezing. The sample according to the present invention also comprises any body biofluid containing tissue from the tumor, RNA from the tumor, DNA from the tumor, or protein from the tumor including, without being limited to, plasma or serum, such as plasma or serum. with the presence of exosomes or DNA of tumor origin.

The determination of PCSK9 expression and / or activity levels needs to be correlated with values from a control sample or reference sample. Depending on the type of tumor being analyzed, the exact nature of the control sample may vary. Thus, in the case of designing a personalized therapy, then the reference sample is a biological sample from a subject with lung cancer who is going to undergo, or has already undergone mechanical ventilation, or that correspond to the median value of PCSK9 expression and / or activity levels measured in a collection of tumor tissues in biopsy samples, or samples of biological fluids such as blood or plasma, from subjects with lung cancer who have not undergone mechanical ventilation .

The collection of samples from which the reference level is derived will preferably be made up of subjects suffering from the same type of cancer as the patient under study.

Thus, the expression "control sample" refers to an isolated biological sample, as previously defined, from a healthy subject or from a subject suffering from cancer, preferably lung cancer, but with the particular characteristic that it has not been or will not be subjected to mechanical ventilation.

The term "reference value" refers to a laboratory value used as a reference for values / data obtained by using samples obtained from patients. The reference value or reference level can be an absolute value, a relative value, a value that has an upper and / or lower limit, a series of values, an average value, a median, a mean value, or an expressed value by reference to a control or reference value. A reference value can be based on the value obtained from an individual sample, such as a value obtained from a sample of the patient under study but obtained at an earlier point in time. The reference value can be based on a high number of samples, such as the values obtained in a population of subjects in the chronological age group coinciding with that of the patient under study or based on a set of samples of inclusion or exclusion of the sample to be analyzed.

In another particular embodiment, the in vitro method of the invention is characterized in that the quantification of the expression level of the PCSK9 gene is carried out by quantifying the cDNA, mRNA or the protein encoded by the PCSK9 gene or a fragment of said protein. More preferably, the quantification of the cDNA or of the mRNA is carried out by any technique for the quantification of said compounds, which is known to the person skilled in the art. More preferably, said quantification is carried out by any of the techniques selected from the list consisting of: a polymerase chain reaction, a DNA or RNA array, or hybridization.

In another particular embodiment, the in vitro method of the invention is characterized in that the quantification of the PCSK9 protein is carried out by any useful technique for protein quantification known to the person skilled in the art. More preferably, protein quantification is carried out by any of the techniques selected from the list consisting of: Western Blot, immunohistochemical techniques, ELISA or a protein array.

In the present invention, it is understood that "increased expression level" or "higher expression level" is the expression level when it refers to levels of the PCSK9 gene and / or protein greater than those that appear in a reference sample or sample. control. In particular, a sample can be considered to have high levels of expression and / or activity of PCSK9 when the levels of expression and / or activity in the reference sample are at least 1.1 times, 1.5 times, 5 times , 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or even more with respect to the isolated patient sample.

In another particular embodiment, the in vitro method of the invention is characterized in that the PCSK9 inhibitor is comprised within a pharmaceutical composition.

The terms and expressions "subject", "isolated sample" "cancer", "metastasis", "inhibitory agent", "determination of expression levels", "PCSK9 gene", "PCSK9 protein", "increased expression levels" , "Pharmaceutical composition" and "control sample" have been described in detail above and are hereinafter equally applicable to the present aspect of the invention, as well as to the rest of the aspects described below.

Another aspect of the present intention refers to the use of PCSK9 as a pharmacological target for the screening, testing and / or validation of agents, preferably PCSK9 inhibitory agents useful in the prevention and / or treatment of metastasis in a subject suffering from cancer and is or will be undergoing mechanical ventilation.

The term "drug target", as used in the present invention, refers to the PCSK9 gene and / or the protein encoded by it, which is useful for studying the biochemical effect of molecules capable of binding to it. These molecules can be, without limitation, compounds, modulators, or agents that are selected by screening methods, where the inhibition of the expression and / or activity of the PCSK9 gene and / or the protein encoded by it is analyzed.

Another aspect of the invention refers to an in vitro method for the screening and identification of PCSK9 inhibitors, wherein the method comprises:

a) putting an agent in contact with the PCSK9 gene and / or the protein encoded by it,

b) analyze the interaction between the agent and the PCSK9 gene and / or the protein encoded by it, and

c) selecting the agent of step (a) capable of inhibiting the expression and / or activity of the PCSK9 gene and / or the protein encoded by it.

Another aspect of the invention refers to an in vitro method for the screening and identification of agents useful in the prevention and / or treatment of metastases in a subject suffering from cancer and who is subjected, or will be subjected, to mechanical ventilation. , where the method comprises:

a) putting a compound, agent in contact with the PCSK9 gene and / or the protein encoded by it,

b) analyze the interaction between the agent and the PCSK9 gene and / or the protein encoded by it, and

c) selecting the agent of step (a) that has the ability to inhibit the expression and / or activity of the PCSK9 gene and / or the protein encoded by it.

In general, the screening methods mentioned above, in addition to the potential drug molecules (eg, agents, modulators, antagonists, agonists) identified therefrom have applicability in relation to the prevention and / or treatment of metastases. in a subject suffering from cancer and is undergoing, or will be undergoing, mechanical ventilation.

In another embodiment, the present invention provides PCSK9 inhibitory agents, according to the present invention, obtainable by the screening method disclosed herein.

In another aspect, the present invention refers to a method of prevention and / or treatment of metastases in a subject suffering from cancer and is subjected, or will be subjected, to mechanical ventilation, which comprises administering in a therapeutically or prophylactically effective amount an inhibitory agent of PCSK9, or of the pharmaceutical composition comprising it, as described herein, to a subject in need of such treatment.

Another aspect of the present invention refers to a kit or device, hereinafter kit or device of the invention, which comprises the elements necessary to analyze the levels of expression and / or activity of PCSK9, for the prevention and / or treatment of metastasis in a subject suffering from cancer and who is undergoing, or will be undergoing, mechanical ventilation or to design personalized therapy for a subject suffering from cancer and who is undergoing, or will be undergoing, mechanical ventilation.

By "kit" as used in the present invention, it refers to a product that contains the various reagents necessary to carry out the methods of the invention packaged to allow their transport and storage. The kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. Suitable materials for packaging the kit components include glass, plastic (polyethylene, polypropylene, polycarbonate, and the like), bottles, vials, paper, envelopes, and the like. Additionally, the kits of the invention may contain instructions for the simultaneous, sequential or separate use of the various components found in the kit. Said instructions can be in the form of printed material or in the form of an electronic medium capable of storing instructions so that they can be read by a subject, such as electronic storage media (magnetic discs, tapes and the like), optical media (CD- ROM, DVD) and the like. Additionally or alternatively, the media may contain Internet addresses that provide such instructions.

Preferably, the kit or device of the invention comprises a PCSK9 inhibitory agent, as previously described herein.

Another aspect of the present invention relates to the in vitro use of the kit described above for the prevention and / or treatment of metastases in a subject suffering from cancer and is subjected, or will be subjected, to mechanical ventilation, or to design a therapy. personalized to a subject suffering from cancer and who is undergoing, or will be undergoing, mechanical ventilation.

Throughout the description and claims the word "comprises" and its Variations 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 figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph that shows the invasiveness of tumor cells of the B16F10luc (A) and A549 (B) lines, expressed as the area covered by cells after migration through matrigel, depending on whether the cells have been previously subjected to stretching for 24 hours or not, and that have been cultured in a conditioned medium (from the culture of cells subjected to stretching) or conventional medium.

Figure 2 is a graph that shows the proportion of animals with lung tumors that showed metastases in different organs 15 days after the application of mechanical ventilation (MV), compared with those animals that were maintained in spontaneous ventilation (Esp).

Figure 3 shows a panel (A) where overrepresented molecular pathways are observed in an RNA sequencing experiment, comparing B16F10luc cells grown under stretching conditions and cells grown under static conditions. The size of the circles is proportional to the number of genes in each molecular pathway with significant differences between both groups. (B) The graph shows the quantification of PCSK9 expression by polymerase chain reaction in cells grown under stretch conditions and cells grown under static conditions.

Figure 4 is a graph that shows the invasiveness of tumor cells of the lines B16F10luc (A) and A549 (B), expressed as the area covered by cells after migration through matrigel, depending on whether the cells have been previously subjected to stretching for 24 hours or not, and that have been treated with the monoclonal antibody Alirocumab. The light gray bars represent the Control cells treated with an IgG and the dark gray bars represent cells treated with Alirocumab.

EXAMPLES

The invention will now be illustrated by means of tests carried out by the inventors, which show the utility of the claimed matter.

I - MATERIAL AND METHODS

Cell lines.

For the development of the in vitro experiments, the B16F10luc2 cell line ( Mus musculus melanoma line with a stable insertion of the luciferase gene under the human Ubiquitin C promoter) and the A549 line (human lung adenocarcinoma) were used. The cultures were carried out in DMEM medium supplemented with 10% fetal bobbin serum (FBS). Both cell culture and mechanical stress experiments and invasion tests were performed in an incubator at 37 ° C and 5% C02.

Cell culture under conditions of mechanical stress.

The mechanical stress studies on the aforementioned lines were carried out in 6-well plates of Flexcell®, seeding a density of 5x10 5 cells / well. After 24 hours of incubation, the plates were randomized to static conditions, or conditions of equibiaxial stretching using a cell deformation equipment adjusting the pressure to produce a deformation of 15%. The frequency of stretching used was 15 cycles / minute with a 1: 1 stress: relaxation ratio for 24 hours.

Cell migration assay through matrigel.

At the end of 24 hours of stretching or the corresponding time under static conditions, the cells were detached from the flexcell plates using 1 mM EDTA. 5x104 cells were seeded in each matrigel transwell in a volume of 500pl of medium without FBS. 750pL of medium with 10% FBS as chemoattractant was placed in the lower chamber. After 24 hours everyone's medium was aspirated the wells were stained with crystal violet (0.1% crystal violet in 100% methanol), allowing to fix for up to 30 minutes at room temperature. After washing, the matrigel was removed from the transwell and 5 photos of its underside were taken and the stained area was quantified using the Image J program (NIH, USA).

Animal model.

All experiments were carried out in accordance with the guidelines established by the ethics committee of the University of Oviedo. The animals were kept in pathogen-free conditions, with 12-hour light / dark cycles and ad libitum access to both food and water.

The B16F10luc2 cell line was used for the generation of metastases. This line has the luciferase gene integrated into its genome, which made it possible to monitor the evolution of the tumor through bioluminescence, injecting luciferin into the mice intraperitoneally. For the induction of lung metastases, 15,000 cells were injected through the jugular vein into C57BL / 6 mice, resulting in macroscopic tumors at two weeks. These procedures were performed in anesthetized animals (ketamine and xylazine), following the animal care guidelines. 15 days after injection of the melanoma cells, the tumor-bearing animals and their controls were randomized to spontaneous respiration or mechanical ventilation. Mice were intubated using a 22G angiocatheter and connected to a mechanical ventilator (Evita 2 Dura-Neoflow, Drager; Germany) for 2 hours with the following parameters: Pressure-controlled ventilation, peak inspiratory pressure 15 cmH20, PEEP 2 cmH20, rate respiration rate 100 breaths / minute, inspiration: expiration ratio 1: 1, FIO ² 21%.

At the end of the ventilation protocol, the animals were extubated and followed for 15 days to determine the incidence of metastasis. After this period, the mice were sacrificed and the tissues (brain, lung, liver, spleen and kidney) extracted and stored.

Quantitative PCR.

RNA was extracted from stretched and unstretched cell samples on Trizol® (Sigma, Poole, UK) and precipitated by the addition of isopropanol. After centrifuging and washing with ethanol, the RNA pellet was resuspended in water. Complementary DNA was synthesized from 1pg of total RNA using an RT-PCR kit ( Enhanced avian HS RT-PCR kit, Sigma-Aldrich, USA). Quantitative PCR was carried out in triplicate for each sample using 40 ng of cDNA. SYBR Green PCR master mix and specific primers for Luc2 (direct: 5'-AAACGCTTCCACCTACCAGG-3 '(SEQ ID NO: 4); reverse 5'-CCTTAGCCTCGAAGAAGGGC-3' (SEQ ID NO: 5)) were used at a concentration of 10uM .

RNA sequencing.

Gene expression was analyzed by RNA sequencing from RNA extracted from B16F10 Iuc2 cells after mechanical stress experiments. RNA extraction for this analysis was performed using Quiagen's RNeasy Mini Kit, following the manufacturer's instructions. Briefly, after collecting the samples with Trizol®, chloroform is added and the resulting aqueous phase, where the RNA will be found, is introduced into one of the columns of the kit. After treating with DNase and after a series of washes and centrifugations with different buffers, the extracted RNA is collected in RNase-free water. After removal of the ribosomal RNA, the messenger RNA was fragmented for the synthesis of complementary double-stranded DNA using random primers. After binding the adapters to the ends of the chains obtained, they were sequenced in a lllumina kit, generating single-ended reads of 50 nucleotides in length. An average of 30,376,066 readings per sample was obtained. All samples passed the previously established quality control.

The sequences obtained were quantified by pseudo-mapping, using the mouse transcriptome in its mm9 version as a reference, using the SALMON software. The differential expression of genes was performed using the DESEQ2 module for the statistical development environment R. Briefly, the readings for each gene were modeled according to a negative binomial distribution and the estimated differential expression. Genes with a p value adjusted according to the Benjamini-Hochberg method below 0.05 were considered to have differential expression. Those genes with a change in expression greater than 0.5 logarithms were entered in the Ingenuity Pathway Analysis software (Quiagen Inc) for the identification of the cellular pathways involved.

Treatment in cell cultures with alirocumab.

Flexcell plates were seeded at a density of 200,000 cells / well in DMEM medium + 10% FBS. After 4 hours of culture, the medium is changed and alirocumab (final concentration of 7.5pg / mL) or the respective control is added using a nonspecific IgG (final concentration of 7.5pg / mL). The medium was refreshed every 24 hours for the next 72 hours, after which the plates were randomized to static conditions or equibiaxial stretching for 24 hours, deformation of 15%, frequency of 15 cycles / minute and a 1: 1 stress ratio: relaxation.

Statistic analysis.

The results are presented as mean and standard error. Comparisons between groups were made using an analysis of variance, including group and treatment as factors. P-values were adjusted for multiple comparisons using Tukey's HSD technique. The proportions were compared using Fisher's exact test. A p-value <0.05 was considered significant.

II - RESULTS

Mechanical stress increases the invasiveness of tumor cells.

An experiment was first performed using the murine melanoma line B16F10luc2. These cells were seeded and subjected to an equibiaxial deformation of 15% for 24 hours. The same cells were used as a control group, grown under static conditions. After that time, 5x104 cells were seeded in chambers for the study of cell migration through matrigel. At 24 hours the chambers were removed and the area of the well covered by migrating cells was measured. The area was significantly higher in the group of cells cultured under mechanical stress conditions regardless of the culture medium used (conditioned culture medium from the culture of cells subjected to stretching or conventional culture medium), as shown in Figure 1A. The experiment was repeated using lung adenocarcinoma cells of the A549 line, obtaining a similar result (Figure 1B).

To confirm the relevance of this result, an in vivo study was performed . I know injected B16F10luc2 melanoma cells into the jugular vein of male mice of the C57BI / 6 line. The injected cell line contains in its genome a stable luciferase construct under a constitutive promoter. Two weeks after injection, the presence of lung tumors was confirmed by in vivo luminescence after injection of luciferin. The animals were then randomized to receive mechanical ventilation or kept spontaneously breathing. After this time, the mice were returned to their cages and observed for two more weeks. After that time, they were sacrificed and samples of brain, kidney, liver and spleen tissue were obtained. Luciferase expression in these tissues was studied by quantitative PCR, considering a positive result as indicative of the presence of metastasis. Animals subjected to mechanical ventilation showed a higher proportion of brain, kidney and liver metastases than those that were maintained in spontaneous respiration (Figure 2).

These results demonstrate that mechanical stress increases the invasiveness of tumor cells, both in vivo and in vitro.

Mechanical stress increases the expression of PCSK9.

To identify the mechanisms responsible for the increased invasiveness, RNA was extracted from B16F10luc2 melanoma cells subjected to mechanical stress or cultured under static conditions. RNA was purified and sequenced, and gene expression quantified. Differences were observed in the expression of 5107 genes. The biological processes in which these genes are involved were identified using the Ingenuity Pathway Analysis tool. The most significant processes are shown in Figure 3A. As can be seen, several processes involved in the synthesis and metabolism of cholesterol reached the highest statistical significance. Since PCSK9 plays a central role in cholesterol processing, differences in its expression were confirmed by quantitative PCR. In line with the result of RNA sequencing, the expression of PCSK9 increased with mechanical stress (Figure 3B).

Inhibition of PCSK9 prevents increase in invasiveness mediated by mechanical stress.

To confirm the role of PCSK9 in stress-induced increased invasiveness Mechanically, B16F10luc2 and A549 cells were cultured under equibiaxial stretching conditions in the presence of a monoclonal antibody against PCSK9, alirocumab (PRALUENT, Sanofi-Aventis) or a nonspecific human IgG, for 24 hours. After that time, the cells were transferred to migration chambers with Matrigel. Migration after 24 hours was quantified by the area covered by migrated cells. Alirocumab treatment prevented the increase in invasiveness caused by stretching, as shown in Figure 4, in both murine B16F10luc2 cells (Figure 4A) and human A549 cells (Figure 4B).

III - CONCLUSION

The results shown in the present document demonstrate that mechanical stress, both in an in vitro model and in vivo in animals subjected to mechanical ventilation, produces an increase in the invasiveness of tumor cells. This change in invasiveness is mediated by increased expression of the protein-convertase PCSK9. Inhibition of this enzyme by the monoclonal antibody alirocumab completely blocks the increase in invasiveness caused by mechanical stress. Therefore, treatment with a PCSK9 inhibitor in patients suffering from lung cancer who are undergoing, or who are to undergo mechanical ventilation, decreases the risk of metastasis in such subjects.

Claims (29)

1. A PCSK9 inhibitory agent for use in the prevention and / or treatment of metastases in a subject suffering from cancer and is undergoing or is undergoing mechanical ventilation. 2. Inhibitory agent for use according to claim 1, wherein the cancer is lung cancer. 3. Inhibitory agent for use according to claim 1 or 2, wherein the subject is a human. 4. Inhibitory agent for use according to any of claims 1 to 3, wherein PCSK9 comprises a nucleotide sequence exhibiting at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% identity with the sequence SEQ ID NO: 1o2. 5. Inhibitory agent for use according to any of claims 1 to 3 wherein PCSK9 comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% identity with the sequence SEQ ID NO: 3. 6. Inhibitory agent for use according to any one of claims 1 to 5, wherein the inhibitory agent is selected from the list consisting of: an RNAi, an antisense oligonucleotide, a DNA enzyme, a ribozyme, an aptamer, a CRISPRi , an antibody and a molecule that specifically recognizes the PCSK9 protein or a fragment thereof. 7. Inhibitory agent for use according to claim 6, wherein the inhibitory agent is an antibody. 8. Inhibitory agent for use according to claim 7 wherein the antibody is a monoclonal antibody is selected from the list consisting of: Evolocumab, Alirocumab, Bococizumab, 1D05-IgG2, RG-7652 and LY3015014. 9. Inhibitory agent for use according to any one of claims 1 to 8, wherein the inhibitory agent is comprised within a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier or excipient. 10. Inhibitory agent for use according to claim 9, wherein the composition further comprises a chemotherapeutic agent. 11. In vitro method to design a personalized therapy for a subject suffering from cancer and who is, or will be subjected to mechanical ventilation, comprising: a) quantify the level of expression and / or activity of PCSK9 in an isolated sample from the subject, and b) comparing the level of expression and / or activity obtained in step (a) with the expression value of PCSK9 in a control sample, where if the level of expression and / or activity obtained in step (a) is higher than the expression value of the control sample, then the therapy comprises administering to the subject a PCSK9 inhibitory agent. 12. Method according to claim 11, wherein the cancers lung cancer. 13. Method according to claim 11 or 12, wherein the subject is a human. Method according to any one of claims 11 to 13, wherein the sample isolated from the subject is a sample of a biological fluid or a sample of tumor tissue. 15. Method according to any one of claims 11 to 14, wherein PCSK9 comprises a nucleotide sequence exhibiting at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% identity with the sequence SEQ ID NO: 1o2 16. Method according to any one of claims 11 to 14, wherein PCSK9 comprises an amino acid sequence exhibiting an identity of sequence of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% with the sequence SEQ ID NO: 3. 17. Method according to any one of claims 11 to 16, wherein the PCSK9 inhibitory agent is selected from the list consisting of: an RNAi, an antisense oligonucleotide, a DNA enzyme, a ribozyme, an aptamer, a CRISPRi, an antibody and a molecule that specifically recognizes the PCSK9 protein or a fragment thereof. 18. Method according to claim 17, wherein the inhibitory agent is an antibody. 19. Method according to claim 18, wherein the antibody is a monoclonal antibody selected from the list consisting of: Evolocumab, Alirocumab, Bococizumab, 1D05-IgG2, RG-7652 and LY3015014. 20. Method according to any one of claims 11 to 19, wherein the inhibitory agent is comprised within a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier or excipient. 21. Method according to any one of claims 11 to 20, wherein the quantification of the expression level of the PCSK9 gene is carried out by quantifying the cDNA, mRNA or the protein encoded by the PCSK9 gene or a fragment of said protein. 22. Method according to claim 21, wherein the quantification of the cDNA or mRNA is carried out by means of a polymerase chain reaction, a DNA or RNA array, or a hybridization. 23. Method according to claim 21, wherein the quantification of the protein encoded by the PCSK9 gene , or a fragment of said protein, is carried out by Western Blot, immunohistochemical techniques, ELISA, protein array, immunoprecipitation or radioimmunoassay. 24. In vitro method of screening, testing or validation of agents useful in the prevention and / or treatment of metastases in a subject suffering from cancer and undergoing, or will be undergoing, mechanical ventilation, comprising: a) Contact an agent with PCSK9, b) Analyze the interaction between the agent and PCSK9, and c) Selecting the agent from step (a) with the ability to bind PCSK9 to decrease or inhibit its expression and / or activity. 25. A method according to claim 24, wherein the cancer is lung cancer. 26. A method according to any of claims 24 or 25, wherein the subject is a human. 27. Method according to any one of claims 24 to 26, wherein PCSK9 comprises a nucleotide sequence exhibiting at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% identity with the sequence SEQ ID NO: 1 or 2. 28. Method according to any of claims 24 to 26, wherein PCSK9 comprises an amino acid sequence exhibiting a sequence identity of at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 % identity with the sequence SEQ ID NO: 3 29. In vitro use of a kit comprising a PCSK9 inhibitory agent which is selected from the list consisting of: an RNAi, an antisense oligonucleotide, a DNA enzyme, a ribozyme, an aptamer, a CRISPRi, an antibody and a molecule that specifically recognizes the PCSK9 protein or a fragment thereof, for the prevention and / or treatment of metastasis in a subject suffering from cancer and is, or will be, subjected to mechanical ventilation, or to design a therapy personalized to a subject suffering from cancer and who is, or will be, undergoing mechanical ventilation.