KR20210018635A - Composition for anticancer using slc1a5 transcript variant, screening method for anticancer drug, and method for diagnosing cancer - Google Patents

Abstract

The present invention relates to an anticancer composition using a SLC1A5 transcript variant, an anticancer drug screening method, a cancer diagnostic composition, and a method for diagnosing cancer. The SLC1A5 transcript variant of the present invention can be used as a novel cancer diagnostic marker and a target for anticancer treatment.

Description

Anticancer composition using SLC1A5 transcript variant, anticancer agent screening method, and cancer diagnosis method {COMPOSITION FOR ANTICANCER USING SLC1A5 TRANSCRIPT VARIANT, SCREENING METHOD FOR ANTICANCER DRUG, AND METHOD FOR DIAGNOSING CANCER}

The present invention relates to an anticancer composition, an anticancer agent screening method, a cancer diagnosis composition, and a diagnosis method using a SLC1A5 transcript variant.

Glutamine is the most abundant amino acid in the blood, and it performs cellular functions such as synthesis of metabolites that maintain mitochondrial function in cells, production of antioxidants, synthesis of non-essential amino acids, nucleotides and fatty acids, and activation of growth signaling. It plays a very important role.

In particular, since the mitochondrial glutamine metabolism pathway is a very important supplemental pathway in the energy and lipid production of rapidly proliferating cells, it is reported that the dependence of glutamine on the growth of rapidly proliferating cancer cells is particularly high, and its importance is being emphasized.

Specifically, cancer cells use glutamine instead of glucose through a reductive carboxylation pathway to generate acetyl-CoA for lipid synthesis in hypoxia, and pancreatic cancer cells are known to generate reducing power from glutamine in the form of NADPH.

Glutamine has been reported as a major source of high-energy electrons for oxidative phosphorylation reactions, especially in response to Ras activation, and its role as a signaling molecule in vivo is also important in that glutamine activates the mTORC1 pathway and promotes cell growth.

As such, glutamine is an essential element in the metabolic process of cancer cells, and the fact that sometimes cancer cells are addicted to glutamine indicates its importance.

Cancer cells absorb glutamine through several glutamine transporters belonging to the protein groups SLC1, 6, 7, and 38. Among them, SLC1A5 (or ASCT2) is an essential sodium-dependent transporter for neutral amino acids such as alanine, serine, threonine, asparagine and glutamine. SLC1A5 is known to be involved in various cancers such as lung cancer, colon cancer, stomach cancer, kidney cancer, ovarian cancer, breast cancer, tongue cancer, and pancreatic cancer.

When the expression of SLC1A5 is inhibited, it interferes with glutamine uptake, leading to impairment of mTORC1 signaling and activation of autophagy. Inhibition of SLC1A5 expression in lung cancer cells has been reported to reduce glutamine uptake and cell proliferation, and this effect has been reported in acute myelogenous leukemia and multiple solid cancers in addition to lung cancer.

As such, mitochondrial glutamine metabolism is very important in the process of cancer cell metabolism, and in the mitochondrial glutamine metabolism process, it is essential that the cytoplasmic glutamine passes through the mitochondrial membrane through the mitochondrial glutamine transporter, and in-depth research on glutamine transporters. Is required.

Korean Patent Publication No. 10-2008-0110810

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cancer comprising an expression inhibitor or a protein activity inhibitor of the SLC1A5 transcript variant.

Another object of the present invention is to provide a method for screening an anticancer agent in which cells are treated with a test agent to compare the expression level or activity of the SLC1A5 transcript variant.

Another object of the present invention is to provide a composition for cancer diagnosis including an agent for measuring the expression level or protein activity level of the SLC1A5 transcript variant and a method for diagnosing cancer using the same.

Another object of the present invention is to provide a composition for inhibiting anticancer drug resistance, including an expression inhibitor or protein activity inhibitor of the SLC1A5 transcript variant.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of cancer comprising an expression inhibitor or a protein activity inhibitor of the SLC1A5 transcript variant.

In the present specification, “SLC1A5” refers to a neutral amino acid transporter protein whose official name is solute carrier family 1 member 5. Besides SLC1A5, it is also called AAAT, ASCT2, M7V1, M7VS1, R16, RDRC, etc.

The SLC1A5 gene encoded at the 19q13.32 position of the human chromosome consists of eight exons, and three types of isoforms due to transcriptional mutations have been reported to date. The longest 541 amino acid transcript variant (NCBI genebank mRNA sequence NM_005628.2; protein sequence NP_005619.1) lacks a portion corresponding to exon 2. In the present specification, it was used as SLC1A5 or SLC1A5/ASCT2 without any indication, and it may be referred to as a wild type (WT) or normal type of SLC1A5, not a transcript variant in the sense that it is mainly expressed in normal cells.

In the present invention, "SLC1A5 transcript variant" may mean a protein having a mitochondrial targeting sequence and glutamine transporter activity essential for the activity of a glutamine transporter in mitochondria. The wild type (WT) of SLC1A5 or the transcript variant of SLC1A5 shorter than the normal type consists of 339 amino acids (NCBI genebank mRNA sequence NM_001145145.1; protein sequence NP_001138617.1), and the part corresponding to exon 1 is lacking. In the specification, it was referred to as SLC1A5_var.

According to an embodiment of the present invention, the short SLC1A5_var transcript variant lacking exon 1 of SLC1A5 contains a mitochondrial targeting sequence (MTS) at the N-terminal region of the protein. . Thanks to this, it is expressed as a protein and is targeted to the inner membrane of mitochondria, and functions as a major transporter (mitochondrial glutamine transporter) responsible for the transport of glutamine in the mitochondria. Accordingly, it was confirmed that if MTS does not function normally due to mutations, etc., SLC1A5_var protein cannot be targeted to mitochondria, and as a result, glutamine absorption in mitochondria does not occur (FIG. 6 ).

According to another embodiment of the present invention, SLC1A5_var is overexpressed in various types of cancer cells, and in particular, pancreatic cancer, lung cancer, and colon cancer cells have significantly higher expression than normal cells.

Glutamine metabolism in mitochondria is particularly essential for the proliferation of rapidly cell-dividing cancer cells.When SLC1A5_var protein in mitochondria is absent due to suppression of SLC1A5_var protein expression, glutamine is not transported to mitochondria and the proliferation of cancer cells is significantly reduced. Confirmed.

On the other hand, since SLC1A5_var transports alanine and serine to mitochondria in addition to glutamine, suppressing the expression of SLC1A5_var inhibits the transport of glutamine as well as alanine and serine. SLC1A5_var protein plays a very important role in cancer cell metabolism and proliferation as a mitochondrial glutamine transporter, and inducing cancer cell-specific death by lowering the expression level or protein activity of SLC1A5_var protein in mitochondria can be a new and effective anticancer treatment strategy. Is to suggest.

Accordingly, it was confirmed that a substance or agent that inhibits the expression level or protein activity of the SLC1A5_var protein can be a candidate group for the development of cancer therapeutic agents that inhibit glutamine metabolism in mitochondria.

The mitochondrial targeting sequence (MTS) is composed of 10 to 70 amino acids, and refers to an amino acid sequence for targeting a new protein to a mitochondria. It is mainly found at the N-terminus of a protein, and is composed of a pattern in which a sequence of a hydrophobic amino acid and a sequence of an amino acid having a charge are intersected to form an amphipathic helix. The mitochondrial targeting sequence additionally includes a sequence for retargeting to a structure such as an inner membrane or an outer membrane matrix in the mitochondria, and the signal sequence is cut off when targeting is completed.

In an embodiment of the present invention, it was confirmed that MTS is present at the N-terminus of the SLC1A5_var protein, and in particular, arginine at the 44th and lysine at the 45th of the SLC1A5_var protein amino acid sequence are important. Among the basic amino acids of the N-terminal region of the SLC1A5_var protein, triple mutations such as R9A/R15A/K17A (NT_3A) had no effect on mitochondrial targeting, but proteins containing the double mutation of R44A/K45A (NT_2A) were converted to mitochondria. It was confirmed that it was not targeted and was distributed in the cytoplasm. That is, the mitochondrial targeting sequence of the SLC1A5_var protein according to the present invention may include arginine and lysine at positions 44 and 45 of the amino acid sequence, respectively.

The form of “(one amino acid) (amino acid position) (one amino acid position)” as used herein means that the amino acid indicated in front is substituted with the amino acid indicated in the rear at the corresponding amino acid position of the wild-type polypeptide. For example, R44A represents a point mutation in which arginine at the 44th amino acid sequence is substituted with alanine.

One letter (three letters) of amino acids as used herein means the following amino acids according to standard abbreviation regulations in the field of biochemistry: A(Ala), alanine; C(Cys), cysteine; D(Asp), aspartic acid; E(Glu), glutamic acid; F(Phe), phenylalanine; G(Gly), glycine; H(His), histidine; I(IIe), isoleucine; K(Lys), lysine; L(Leu), leucine; M(Met), methionine; N(Asn), asparagine; O(Ply), pyrrolysine; P(Pro), proline; Q(Gin), glutamine; R(Arg), arginine; S(Ser), serine; T(Thr), threonine; U(Sec), selenocysteine, V(Val), valine; W(Trp), tryptophan; Y(Tyr), tyrosine.

Specifically, in the present invention, the “expression inhibitor” decreases the expression level of the SLC1A5 transcript variant by a mechanism such as promoting the degradation of the mRNA by specifically binding to the mRNA of the SLC1A5 transcript variant or inhibiting the translation into a protein. As meant to be, it may be one or more selected from the group consisting of antisense RNA, siRNA, shRNA, and miRNA that specifically binds to the mRNA of the SLC1A5 transcript variant, but is not limited thereto.

More specifically, the mRNA includes the nucleotide sequence of SEQ ID NO: 2, the siRNA includes the nucleotide sequence of SEQ ID NO: 7, and the shRNA may include the nucleotide sequence of SEQ ID NO: 10, but is not limited thereto. Does not.

In the present invention, "expression" may mean that a protein or nucleic acid, particularly an mRNA encoding a protein, is generated in a cell, but is not limited thereto and may be appropriately changed and applied as necessary.

Specifically, in the present invention, the "protein activity inhibitor" is not limited to the type or size of the substance as long as it inhibits the protein activity of the SLC1A5 transcript variant having glutamine transport activity in the mitochondria, and is specific to the SLC1A5 transcript variant protein. It may be one or more selected from the group consisting of peptides, antibodies, and aptamers having domains that bind specifically, but is not limited thereto.

In one embodiment of the present invention, substances such as 1-γ-glutamyl-p-nitroanilide (GPNA), benzylserine, and HgCl ₂ known to inhibit glutamine transporter activity of wild-type SLC1A5 protein are mitochondria of the SLC1A5_var transcript variant. It was confirmed to inhibit my glutamine transporter activity.

More specifically, the protein activity inhibitor of the SLC1A5 transcript variant according to the present invention inhibits the protein activity of the SLC1A5 transcript variant having glutamine transport ability in mitochondria, and the SLC1A5 transcript variant is responsible for transporting glutamine in the cytoplasm, not mitochondria. Protein activity means that it can be preserved as it is. The protein activity inhibitor of the SLC1A5 transcript variant meeting this purpose may be an SLC1A5 transcript variant, for example, an antibody or aptamer that specifically binds to the SLC1A5_var transcript variant protein.

In the present invention, the "aptamer" refers to an RNA molecule that specifically binds to a specific target molecule such as a protein or carbohydrate. By specifically binding the antibody or aptamer to the SLC1A5_var transcript variant protein, the amino acid sequence of a portion of the protein is masked to prevent appropriate targeting to the mitochondria or posttranslational modification necessary for activity, or Protein activity can be inhibited by various mechanisms, such as altering the structure of some or all of the protein, or blocking a protein site that is important for activity, for example, a transporter pore.

In addition, specifically, the pharmaceutical composition of the present invention may further include a sugar metabolism inhibitor. More specifically, the sugar metabolism inhibitor is in the group consisting of 2-deoxyglucose, 3-bromopyruvate (3-bp), WZB117, phloretin, and STF-31. One or more may be selected, and most specifically, 2-deoxyglucose, but is not limited thereto.

In an embodiment of the present invention, treatment of a pancreatic cancer cell line with 2-deoxyglucose (2-deoxyglucose or 2-deoxy-D-glucose; 2-DG) with siRNA specific to SLC1A5_var almost completely inhibits cancer cell survival. Confirmed. That is, since glutamine metabolism and sugar metabolism in mitochondria are closely related, it was confirmed that the anti-cancer effect was further doubled when suppressing the expression or activity of SLC1A5_var and simultaneously inhibiting sugar metabolism with 2-DG or the like (FIG. 8 ). .

Specifically, the types of cancer targeted by the present invention include leukemia, lymphoma, hematopoietic malignancy, cervical cancer, sarcoma, testicular cancer, and malignant melanoma. (malignant melanoma), endocrine tumor, bone cancer, prostate cancer, uterus cancer, breast cancer, bladder cancer, brain cancer, Liver cancer, stomach cancer, pancreas cancer, skin cancer, lung cancer, larynx cancer, head and neck cancer, esophageal cancer , It may be one or more selected from the group consisting of colorectal cancer and ovarian cancer. More specifically, the cancer may be one or more selected from lung cancer, colon cancer, or pancreatic cancer, in which the SLC1A5_var transcript variant protein is confirmed to be overexpressed in normal cells, and most specifically, pancreatic cancer, but is not limited thereto.

Another aspect of the present invention comprises the steps of: (a) treating a cell expressing the SLC1A5 transcript variant with a test agent; (b) measuring the expression level or protein activity of the SLC1A5 transcript variant in the cells treated with the test agent; And (c) comparing the expression level of the SLC1A5 transcript variant expressed in cells not treated with the test agent or the activity of the protein to determine whether the test agent inhibits the expression level or activity of the SLC1A5 transcript variant; It provides a cancer treatment screening method comprising a.

In the present invention, the term "agent" or "test agent" means any substance, molecule, element, compound, entity, or It may include a combination of. Specifically, the substances selected through the method of the present invention are, for example, methionine amino acid analogs, siRNA, shRNA, miRNA, ribozyme, DNAzyme, peptide nucleic acid (PNA), antisense oligonucleotides, antibodies, aptamers, peptides, natural extracts. And chemical substances, but is not limited thereto.

The step of treating the test agent in step (a) refers to a process of inducing contact of the test agent with a factor affecting the mRNA or protein expression or protein activity of the SLC1A5 transcript variant. For example, the test agent may be processed outside the cell membrane and introduced into the cell in vitro , or the test agent may be expressed in cells by known standard recombinant DNA and molecular cloning techniques. It may be, but is not limited to the example.

Specifically, the SLC1A5 transcript variant in step (a) may include the amino acid sequence of SEQ ID NO: 1, and may be a protein having mitochondrial targeting sequence and glutamine transporter activity, but is not limited thereto.

The method of measuring the expression level of the SLC1A5 transcript variant in step (b) is to measure the level of the mRNA encoding the SLC1A5 transcript variant or the protein in the mitochondria, and any method commonly used in the relevant technical field is not limited. It can be selected and used, and examples of analysis methods include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, and real-time RT-PCR. ), RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing, hybridization method using nanostring, tissue There are, but are not limited to, a method of in situ hybridization of the fragment. In addition, when checking the expression level at the protein level, the protein level can be confirmed by using an antibody that specifically binds to the SLC1A5 transcript variant protein. Similar to antibodies, aptamers that specifically bind to proteins may be used. On the other hand, depending on the protein measurement method, an antibody may not be required. For example, mass spectrometry involving analysis of a standard substance of the SLC1A5 transcript variant is an example.

Specifically, the activity of the protein in step (b) may mean the activity of a mitochondrial glutamine transporter.

Another aspect of the present invention, (a) obtaining a sample from the subject; (b) measuring the expression level or protein activity of the SLC1A5 transcript variant in the sample; And (c) determining that the subject has cancer when the measured expression level or protein activity level is higher than that of the control group.

Specifically, the sample in step (a) may be one or more selected from the group consisting of cancer tissue, blood, plasma, serum, lymph fluid, and urine, and the expression level of the SLC1A5 transcript variant in step (b) is mRNA or It is the level of protein expression in mitochondria, and the activity of the protein may be that of the mitochondrial glutamine transporter.

Also, specifically, the cancer may be one or more selected from the group consisting of lung cancer, colorectal cancer, and pancreas cancer, but is not limited thereto.

Another aspect of the present invention provides a composition for diagnosis of cancer, including an agent for measuring the expression level or protein activity level of the SLC1A5 transcript variant.

Specifically, the agent for measuring the expression level may be a probe that specifically binds to the SLC1A5 transcript variant mRNA, or a primer consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

In addition, specifically, it may be one or more selected from the group consisting of a peptide domain, an antibody, and an aptamer that specifically binds to the SLC1A5 transcript variant protein.

In the present invention, the "primer" refers to a short single-stranded oligonucleotide that acts as a starting point for DNA synthesis. The primer specifically binds to a polynucleotide that is a template in a suitable buffer and temperature conditions, and DNA polymerase is linked to the primer by adding a nucleoside triphosphate having a base complementary to the template DNA. Is synthesized. Primers generally consist of 15 to 30 nucleotide sequences, and the melting temperature (T _m ) of bonding to the template strand varies depending on the base composition and length.

The sequence of the primer does not need to have a sequence that is completely complementary to some of the base sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template to function unique to the primer. Therefore, the primer for measuring the expression level of the SLC1A5 transcript variant mRNA in the present invention does not need to have a sequence that is completely complementary to the SLC1A5 gene sequence, and by amplifying a specific section of the SLC1A5 transcript variant mRNA or cDNA through DNA synthesis. SLC1A5 transcript variant mRNA is sufficient to have a length and complementarity suitable for the purpose of measuring the amount. Primers for the amplification reaction consist of a set (pair) that complementarily bind to the template (or sense) at both ends of a specific section of the SLC1A5 transcript variant to be amplified and the opposite (antisense, antisense). do.

In the present invention, the “probe” refers to a polynucleotide such as RNA or DNA having a length of several to several hundred base pairs that can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. It means a fragment of, and is labeled, so that the presence or absence of the target mRNA or cDNA to be bound, the amount of expression, etc. can be confirmed. For the purpose of the present invention, a probe complementary to the SLC1A5 transcript variant mRNA may be hybridized with a sample of a subject to measure the amount of mRNA expression. Probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.

In the present invention, the primer or probe that specifically binds to the mRNA of the SLC1A5 transcript variant binds only to the mRNA of the specific SLC1A5 transcript variant to be detected, specifically, the SLC1A5_var transcript variant, and other types of transcript variants or wild-type It does not bind to SLC1A5 mRNA. For example, in the case of the sequence of the SLC1A5_var transcript variant, as shown in Fig. 1, exon 1 (E1) is missing, and the SLC1A5 transcript variant (SLC1A5/ASCT2) lacks exon 2 (E2). , By designing that the binding site of the primer or probe on the mRNA contains part or all of E2, it is possible to construct a primer or probe that specifically binds only to the SLC1A5_var transcript variant without binding to the SLC1A5 transcript variant. . As an example of a primer that specifically binds only to the SLC1A5_var transcript variant, the sequence of the primer used in an embodiment of the present invention is described in SEQ ID NO: 3 (forward; forward primer) and SEQ ID NO: 4 (for reverse primer). Has been.

The primer or probe according to the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, primers or probes can be variously modified according to methods known in the art within a range that does not interfere with hybridization with SLC1A5 transcript variant mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc. ) Or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.), and binding of a labeling material using fluorescence or enzymes.

In the present invention, the "antibody" refers to an immunoglobulin that specifically binds to an antigenic site. The antibody in the present invention does not react to a specific SLC1A5 transcript variant to be detected, for example, SLC1A5 transcript variant protein and other types of proteins other than the SLC1A5_var transcript variant, and is specific only to the SLC1A5_var transcript variant protein. It means an antibody that binds. Specifically, since the SLC1A5 transcript variant does not contain exon 2, an antibody that specifically binds to the exon 2 site or the conjugation part of exon 2 and exon 3 is an antibody that specifically binds only to the SLC1A5_var transcript variant protein. An antibody against the SLC1A5 transcript variant can be prepared by cloning the gene of the SLC1A5 transcript variant into an expression vector to obtain a protein encoded by the gene, and from the obtained protein according to a conventional method in the art. The antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies may be included.

Another aspect of the present invention provides a composition for inhibiting anticancer drug resistance, comprising an expression inhibitor or a protein activity inhibitor of the SLC1A5 transcript variant.

In one embodiment of the present invention, it has been confirmed that the SLC1A5_var variant protein is overexpressed in various types of cancer cells, and when the expression is inhibited, cancer cell proliferation is inhibited and cancer cell death is promoted to exert an anticancer effect. In another example, it was found that the SLC1A5_var mutant protein contributes not only to this direct anticancer effect, but also to anticancer drug resistance of cancer cells. In order for an anticancer drug to exert a successful therapeutic effect, cancer cells must be killed at the blood concentration of the drug that can survive normal tissues. Even after administering a drug capable of reaching the blood concentration determined to have a therapeutic effect, the cancer cells respond. It does not mean that it does not die. The therapeutic effect on cancer cells decreases, and the side effects of anticancer drugs may worsen.

In one embodiment of the present invention, as a result of suppressing the expression of the SLC1A5_var variant in a pancreatic cancer cell line that has already acquired resistance to gemcitabine, the cancer cells regain their sensitivity to gemcitabine, thereby recovering GI50 (half maximal growth). inhibition concentration) remarkably decreased, and it was observed that cancer cell death was promoted (FIG. 9). Accordingly, it was confirmed that it can be used as an anticancer resistance inhibitor that inhibits the development of resistance of existing anticancer drugs by inhibiting the expression or protein activity of the SLC1A5_var variant having glutamine transport activity in the mitochondria, thereby exerting more effective cancer treatment effects. .

Specifically, the anticancer agent may be a platin-based anticancer agent cisplatin and oxaliplatin, a PD-1 inhibitor, a CTLA-4 inhibitor, and a pyrimidine nucleoside prodrug series anticancer agent, and further Specifically, it may be gemcitabine or 5-FU, which are anticancer drugs of the pyrimidine nucleoside prodrug family, and most specifically, gemcitabine, but is not limited thereto.

The gemcitabine is an anticancer chemotherapeutic agent used for various cancers such as breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, and bladder cancer. Gemcitabine is a nucleoside analog compound that prevents the generation of new DNA and ultimately induces apoptosis. More specifically, gemcitabine is a pyrimidine nucleoside prodrug, and is a nucleoside analog in which hydrogen (H) at the 2'carbon position of deoxycytidine is substituted with fluorine (F).

In addition, specifically, the expression inhibitor may be one or more selected from the group consisting of antisense RNA, siRNA, shRNA and miRNA that specifically binds to the mRNA of the SLC1A5 transcript variant, and more specifically, the mRNA is of SEQ ID NO: 2 Including a nucleotide sequence, the siRNA includes the nucleotide sequence of SEQ ID NO: 7, and the shRNA may include the nucleotide sequence of SEQ ID NO: 10, but is not limited thereto.

The protein activity inhibitor may be one or more selected from the group consisting of a peptide domain, an antibody, and an aptamer that specifically binds to the SLC1A5 transcript variant protein, but is not limited thereto.

The pharmaceutical composition according to the present invention may contain the SLC1A5 transcript variant expression inhibitor or protein activity inhibitor alone or may be formulated in a suitable form with a pharmaceutically acceptable carrier, and may further contain an excipient or diluent. have. In the above, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness when administered to humans.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery substances used for oral administration of the peptide preparation. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and formulations may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration Can be

The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the route of administration as described above.

In the case of a formulation for oral administration, the composition of the present invention may be formulated using a method known in the art as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. I can. For example, in oral preparations, tablets or dragees can be obtained by mixing the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing into a granule mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In the case of a formulation for parenteral administration, it can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a formula generally known for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per 1 kg of patient body weight per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the age, weight, health condition, sex, disease severity, diet and excretion rate of the patient, as well as the formulation method, route of administration and number of treatments. Therefore, considering these points, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

The present invention specifically confirmed that the SLC1A5 transcript variant is a mitochondrial glutamine transporter essential for cancer cell metabolism, and furthermore, the SLC1A5 transcript variant is overexpressed in various cancer cells and is also involved in anticancer drug resistance, and when the expression of the SLC1A5 transcript variant is inhibited, cancer cells It has been confirmed that proliferation is significantly inhibited, and the SLC1A5 transcript variant can be utilized as a novel cancer diagnostic marker and a target for anticancer therapy.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the two novel SLC1A5 transcript variants, SLC1A5 and SLC1A5_var, identified in the present invention (A: the constitution of the exon and intron of the human SLC1A5 gene, and the two transcript variants SLC1A5 (NM_005628. 2) and SLC1A5_var (NM_001145145.1) B: As the exon structure of the mRNA transcripts of SLC1A5 (blue) and SLC1A5_var (red), the binding site of siRNA and the RT-PCR amplification product are indicated).
2 shows the results of analyzing the expression pattern of SLC1A5_var in various cancer cells (A, B: pancreatic cancer cell line; C, D: colon cancer cell line; E, F: lung cancer cell line).
3 shows the results of confirming the distribution of SLC1A5_var in the mitochondria in the cells by immunofluorescence (A: results of confirming HeLA cells transformed with HA-taggged SLC1A5_var or HA-taggged SLC1A5 by immunofluorescence. B: SLC1A5_var and cells Quantitative analysis of the presence of organelle markers using Zen colocalization analysis).
Figure 4 shows the results of confirming the distribution of SLC1A5_var in the mitochondria in the cell through the cell organelle fractionation experiment (A: immunoblot result of the cell organelle fractionation experiment of SLC1A5_var obtained from MiaPaCa2 cells. B: Mitochondria of MiaPaCa2 against SLC1A5_var Organelle fractionation test results).
5 shows the experimental results confirming that the SLC1A5 transcript variant is a mitochondrial glutamine transporter for mitochondria isolated from MiaPaCa2 cells expressing the control vector, SLC1A5, SLC1A5_var, or SLC1A5_var D186A mutation (A: time course Degree of glutamine uptake (Gln uptake) according to B: degree of amino acid uptake C: control vector, SLC1A5, SLC1A5_var, or SLC1A5_var The degree of glutamine uptake in the case of treatment with siRNA for the D186A mutation D: siRNA The degree of amino acid absorption in the case of treatment E: Experimental results showing that the inhibitor of the SLC1A5 transcript variant inhibits the glutamine transporter activity of the SLC1A5 transcript variant).
Figure 6 relates to the experimental results of analyzing the mitochondrial targeting signal using the point mutation of the SLC1A5 transcript variant, the wild type (wild type) is WT, the point mutation (R9A/R15A/K17A) of 3 amino acids is 3A. , Point mutation (R44A/K45A) of two amino acids is represented by 2A (A: Schematic of the N-terminus (NT) and C-terminus (CT) of SLC1A5_var. B: At the N-terminus of EGFP (green)) As a result of confocal microscopy observation of HeLa cells transformed to express the conjugated SLC1A5_var fragment, mitochondria were stained with MitoTracker (red)).
7 shows the results of confirming the anticancer effect when the expression of the SLC1A5 transcript variant was suppressed using siRNA (A: relative cell growth of pancreatic cancer cell lines and patient-derived pancreatic cancer cells. B: pancreatic cancer cell line) Degree of apoptosis induced by knockdown of SLC1A5_var C: Xenograft test result of MiaPaCa2 cells expressing control vector or SLC1A5_var shRNA).
8 shows the results of confirming the effect of suppressing the expression of the SLC1A5 transcript variant using siRNA and the combined treatment of 2-DG on cancer cell growth.
9 shows the results of confirming the relationship between the SLC1A5 transcript variant and the drug resistance effect on gemcitable, an anticancer agent, by measuring the cell viability of cancer cells using the WST-1 assay (A , B: Effect of SLC1A5_var overexpression on gemcitamine sensitivity C, D: Effect of knockdown of SLC1A5_var on gemcitamine sensitivity).

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Confirmation of the expression pattern of SLC1A5 transcript variant (SLC1A5_var) in cancer cells

In order to find a mitochondrial glutamine transporter that has not been identified so far, transcript variants of the glutamine transporter were investigated.

The human SLC1A5 gene consists of eight exons, and there are two transcript variants with different transcription start sites (NM_005628.2 and NM_001145145.1; Fig. 1A). Long-length transcript variants (SLC1A5/ASCT2, NM_005628.2) lack exon 2 and consist of 541 amino acids, and short transcript variants (SLC1A5_var, NM_001145145.1) lack exon 1, 339 It was confirmed that it consists of dog amino acids (Fig. 1B). The SLC1A5 transcript variant was named SLC1A5_var.

In order to confirm the expression pattern of the SLC1A5 transcript variant in various cancer cell lines, the mRNA level of each transcript variant was analyzed using RT-PCR.

Specifically, RNA for the RT-PCR was isolated using an RNA extraction kit (MiniBEST Universal RNA Extraction Kit, Takara) and synthesized into cDNA using a cDNA synthesis kit (PrimeScript™ 1st strand cDNA Synthesis Kit, Takara). The synthesized cDNA was performed by reverse transcription polymer chain reaction (RT-PCR) under 26 cycle conditions at 95°C, 45°C and 72°C for 30 seconds each using a primer set consisting of primers having nucleotide sequences of SEQ ID NOs: 3 and 4 After amplification, the reaction results were confirmed through 1% agarose gel electrophoresis. The results were quantitatively analyzed using ImageJ software and GAPDH was used as a quantification criterion.

As a result, the expression of SLC1A5_var in all pancreatic cancer cell lines was increased compared to that of normal pancreatic ductal epithelial cells (HPDE), and more particularly, more overexpressed in Panc-1, MiaPaCa-2, AsPC1, and Panc10.05 cell lines. It was confirmed (A, B in Fig. 2).

In addition, even in various colon cancer cell lines, the expression level of SLC1A5_var was measured higher than that of human colon epithelial cells (FHC) (FIG. 2C and D).

On the other hand, unlike SLC1A5_var, SLC1A5 did not increase its expression in pancreatic or colon cancer cell lines.

In lung cancer cell lines excluding NCI-H358, it was confirmed that expression of both SLC1A5 and SLC1A5_var was increased compared to human fibroblast (BJ) or normal bronchial epithelial cells (16HBE) (E in FIG. 2 ). , F).

Similarly, it was confirmed that the mRNA expression patterns of SLC1A5 and SLC1A5_var in cancer cells were also consistent with the protein expression levels confirmed using immunoblot.

Example 2. Confirmation of intracellular distribution pattern of SLC1A5 transcript variant (SLC1A5_var) using immunofluorescence

The intracellular distribution pattern of the SLC1A5 transcript variant protein, which was confirmed to have a high expression level in cancer cells, was confirmed. HeLa cells were transformed by conjugating HA-tag to the cDNA of the SLC1A5 transcript variant, and colocalization with organelle markers was analyzed.

Specifically, the cells used in the experiment were HeLa cells and were labeled with anti-Cox4 antibody, anti-Na ⁺ -K ⁺ ATPase antibody, anti-ERp72 antibody, anti-GM130 antibody, and anti-LAMP2 antibody, respectively, as primary antibodies after fixation with methanol. Thereafter, labeling was performed using an antibody labeled with Alexa-488 or Alexa-594 fluorescence as a secondary antibody. Cell nuclei were stained with DAPI and then observed under a confocal microscope. The degree of image overlap was analyzed by using Zen imaging software on 10 or more images for each sample.

As a result, as shown in Figure 3, the SLC1A5 transcript variant protein containing the HA-tag was observed to coexist with the mitochondrial marker (COX4), but the cell membrane (Na, K-ATPase), endoplasmic reticulum (ERp72), Golgi apparatus (GM130 ) Or a marker such as lysosome (LAMP2), it was confirmed that the SLC1A5 transcript variant protein was present in the mitochondria (Fig. 3A, B).

In contrast, it was confirmed that the SLC1A5 protein coexisted with Na,K-ATPase and existed in the cell membrane.

Example 3. Confirmation of distribution pattern in mitochondria of SLC1A5 transcript variant (SLC1A5_var) protein through organelle fractionation experiment

The SLC1A5_var protein is present in the mitochondria, and an organelle fractionation experiment was performed in order to examine the distribution pattern in more detail (FIG. 4).

Specifically, all procedures of the organelle fractionation experiment were performed at a cold temperature of 4° C. or less, and mitochondria were isolated using KPBS buffer (136 mM KCl, 10 mM KH ₂ PO ₄ , pH 7.2). Cells were first harvested using KPBS buffer, and the cell suspension was centrifuged at 900 g for 3 min. The supernatant was discarded and only the pellet was resuspended with KPBS containing 5mg/ml of aprotinin, 10mg/ml of leupeptin, and 250mM of PMSF. Thereafter, the cells were broken using a Dounce homogenizer, and the broken cells were centrifuged at 600 g for 5 minutes. Again, the supernatant was discarded and the pellet was centrifuged at 7000 g twice and once at 10000 g for 10 minutes, and the resulting pellet was used as a mitochondrial fraction.

Separation of the inner membrane of mitochondria was performed under low osmotic pressure conditions. The mitochondrial fraction was put in a swelling buffer (10M KH ₂ PO ₂ , digitonin 2 mg/ml, pH 7.4) and then stored on ice for 1 hour. And the same volume of iso-osmotic solution (32% sucrose, 30% glycerol, and 10mM MgCl ₂ ) was added. Thereafter, centrifugation was performed at 10000 g and 10 min, and the resulting supernatant was used as the mitochondrial outer membrane fraction, and the pellet was used as the mitochondrial inner membrane and matrix fraction. The pellet was resuspended again using a swelling buffer without digitonin, stored on ice for 1 hour, and the same volume of iso-osmotic solution was added again, followed by centrifugation at 17000 g for 1 hour. After separation, the supernatant was used as a matrix fraction and the pellet was used as a mitochondrial inner membrane fraction.

Immunoblotting was performed on the mitochondrial organelle fraction. For immunoblot, cells were crushed using lysis buffer (40mM HEPES at pH 7.4, 0.5% Triton X-100, 10mM β-glycerol phosphate, 10mM pyrophosphate, 2.5mM MgCl ₂ ) and ultrasonic waves. PNGase F was treated for observation of SLC1A5_var. Unlike conventional immunoblotting, the sample was not boiled, and at least about 30 ug of protein per sample was separated by SDS-PAGE. After the process of transferring to the PVDF membrane, the primary antibody corresponding to each protein is treated at 4°C for 8 hours, and the secondary antibody with HRP that recognizes each primary antibody is attached to each existing membrane. Protein was identified.

As a result, similar to the results of the immunofluorescence experiment of Example 2, SLC1A5 protein was observed in the cell membrane fraction in which Na,K-ATPase was intensively distributed, but most of the SLC1A5_var protein was confirmed in the mitochondrial fraction in which COX4 was concentrated (Fig. 4A). .

Further, the SLC1A5_var protein was isolated together with Tim23, a marker of the mitochondrial inner membrane, but was fractionated independently from Tom20, a marker of the outer mitochondrial membrane, and MnSOD2, a marker of the mitochondrial matrix (FIG. 4B). Therefore, it was confirmed that the SLC1A5_var protein was distributed in the mitochondria and the SLC1A5 protein was distributed in the cell membrane.

Example 4. Check whether SLC1A5_var is a mitochondrial glutamine transporter

To find out whether SLC1A5_var is involved in glutamine uptake in mitochondria, a mutation (D186A) in which aspartic acid at the site where sodium ions bind in the conserved site of the NMDG motif of SLC1A5_var was changed to alanine was constructed.

SLC1A5_var or SLC1A5_var D186A were stably expressed in MiaPaCa2 cells, which are pancreatic cancer cell lines, respectively, and glutamine uptake activity in mitochondria was measured.

Specifically, after obtaining a mitochondrial fraction by the method of Example 3, resuspended in a buffer containing 10 mM NaCl, 100 mM glutamine, 100 mM serine, 100 mM alanine or 100 mM glutamic acid in KPBS buffer, and then stored at 37°C. Started amino acid absorption. Thereafter, the reaction was terminated by adding 20mM HgCl2, and after completion, each sample was centrifuged for 10000g and 5min. Thereafter, the supernatant was taken and the remaining amino acids consumed compared to the first were measured using an amino acid measurement kit. The measured value was corrected by measuring the amount of mitochondrial protein in each sample and using it as a reference value for quantification.

As a result, as shown in FIG. 5, it was confirmed that only the mitochondria isolated from the cells overexpressing SLC1A5_var increased glutamine levels above the control vector over time. On the other hand, it was confirmed that glutamine was not absorbed in mitochondria isolated from cells overexpressing SLC1A5 or SLC1A5_var D186A (FIG. 5A).

In addition, it was found that in mitochondria isolated from cells overexpressing SLC1A5_var, not only glutamine, but also alanine and serine, other known substrates of SLC1A5, were absorbed (FIG. 5B).

On the other hand, the uptake of glutamine was inhibited in mitochondria isolated from cells that suppressed SLC1A5_var overexpression using si-RNA, but this phenomenon was not observed in mitochondria isolated from cells that suppressed SLC1A5 overexpression (Fig. 5C). The same results were observed for alanine and serine (FIG. 5D).

Furthermore, as a result of confirming whether the previously known inhibitors of SLC1A5, GPNA (l-γ-glutamyl-p-nitroanilide) and benzylserine, can inhibit mitochondrial glutamine transport by SLC1A5_var, the inhibitors are at a baseline level. (basal level) and SLC1A5_var were shown to inhibit the absorption of glutamine mediated by mitochondria (Fig. 5E). In addition, HgCl _2, known to terminate the transport reaction by SLC1A5 in the proteoliposome, also inhibited mitochondrial glutamine absorption.

When considering the above results comprehensively, it was clearly confirmed that SLC1A5_var is a mitochondrial glutamine transporter.

Example 5. Confirmation of mitochondrial targeting signal of SLC1A5_var

As confirmed in the above example, since the SLC1A5_var protein exists in the mitochondrial inner membrane, it was confirmed whether SLC1A5_var contains a mitochondrial-specific targeting signal.

In order for a protein to be transported to the mitochondria, the newly produced protein must have a mitochondrial targeting sequence (MTS) recognized by a molecular chaperone. Molecular chaperones are responsible for delivering the protein to be targeted to the mitochondrial surface where the mitochondrial translocase is located.

MTS is located in the N-terminal region of the protein, and is composed of a hydrophobic alpha-helical transmembrane region (a-helical transmembrane region) consisting of 17 to 23 amino acids and one or two basic amino acids linked thereto. Accordingly, the N- or C-terminus of the SLC1A5_var protein was conjugated to an Enhanced green fluorescent protein (EGFP; GenBank: AFA52654.1) to confirm the presence of MTS. By introducing a point mutation into the basic amino acid of the SLC1A5_var protein related to MTS, it was confirmed whether EGFP targets mitochondria (FIG. 6A).

Specifically, plasmids capable of expressing NT_WT, NT_3A, NT_2A, CT and EGFP, which are a control vector or a part of SLC1A5_var, were transfected for each cell while culturing the cells in a cell culture dish dedicated to a confocal microscope. After 48 hours, mitochondria were labeled with MitoTracker Red, a reagent capable of staining mitochondria, and observed with a confocal microscope.

As a result, as shown in FIG. 6, EGFP bound with wild-type N-terminal (referred to as NT_WT) and R9A/R15A/K17A N-terminal mutation (referred to as NT_3A) was targeted to mitochondria, but R44A/K45A N-terminal mutation ( NT_2A) and bound EGFP could not be targeted to mitochondria and were dispersed in the cytoplasm.

Similarly, in the cell fractionation experiment using differential centrifugation, EGFP conjugated to NT_WT or NT_3A was detected with COX4 in the fraction from which mitochondria are separated, whereas EGFP conjugated to NT_2A or control EGFP was detected in the cytoplasm or cells. The endomembrane was observed in the separated fraction.

Finally, SLC1A5_var (SLC1A5_var_2A), which was constructed to contain a mutation such as NT_2A, could not be targeted as mitochondria in cells, and mitochondria isolated from cells overexpressing SLC1A5_var_2A were also found to be unable to absorb glutamine.

The above results suggest that SLC1A5_var contains a functional MTS for mitochondrial targeting at the N-terminal region.

Example 6. Inhibition of expression of SLC1A5_var and confirmation of anticancer effect

From the above examples, it was confirmed that SLC1A5_var is overexpressed in various cancer cell lines. Since glutamine is essential for cancer cell metabolism, in order to confirm the function of SLC1A5_var in cancer cells, siRNA was used to suppress the expression of SLC1A5_var in pancreatic cancer cells and confirmed its effect on tumor growth.

As a result of suppressing the expression of SLC1A5_var using si-RNA that specifically binds to SLC1A5_var only, 8 types of pancreatic cancer cell lines to be analyzed (AsPC1, BxPC3, SU.86.86, Panc10.05, Panc1, MiaPaCa2, CFPAC-1, HPAF) -II) and two types of patient-derived pancreatic cancer cells (YPAC-16, YPAC-26) showed that the growth of cancer cells was significantly inhibited (Fig. 7A).

In addition, it was also confirmed whether or not apoptosis in cancer cells in which the expression of SLC1A5_var was suppressed. Specifically, SLC1A5 and SLC1A5_var were knocked down, respectively, using siRNA to cells being cultured in a 96-well cell culture plate. Thereafter, the growth of cells was observed using an Incucyte, a real-time cell culture microscope. Thereafter, apoptotic cells were analyzed by FACS using an Annexin V probe attached with FITC. The Annexin V specifically binds strongly to phosphatidyl serine exposed to the cell membrane of apoptotic cells, which can be used to label and quantify apoptotic cells.

As a result of the apoptosis experiment, it was confirmed that apoptosis was also significantly increased in cancer cells in which the expression of LC1A5_var was suppressed (FIG. 7B).

On the other hand, siRNA against SLC1A5 did not show this cancer cell proliferation inhibitory effect at all, and it was confirmed that the reintroduction of the cDNA of SLC1A5_var into the pancreatic cancer cell line (MiaPaCa2) whose growth was inhibited with siRNA specific to SLC1A5_var restored the growth rate of cancer cells. .

Furthermore, the anticancer effect of suppressing the expression of SLC1A5_var observed in the cell experiment was also observed in the in vivo experiment using a mouse as a model.

Specifically, the MiaPaCa2 pancreatic cancer cell line (MiaPaCa2, 1.0×10 ⁷ cells) transformed with control DNA or SLC1A5_var-specific shRNA was injected subcutaneously into the scapula region of nude mice, and tumor growth in mice was observed once every two days.

As a result, in mice injected with cells in which the expression of SLC1A5_var was suppressed, tumors hardly grew (Fig. 7C). These results suggest that glutamine transport and glutamine metabolism in mitochondria by SLC1A5_var may be a novel and effective therapeutic target for cancer treatment.

Example 7. Inhibition of the expression of SLC1A5_var and confirmation of the effect of combination therapy with an anticancer agent

The present inventors confirmed that when siRNA for inhibiting the expression of SLC1A5_var and 2-deoxyglucose (2-DG) for inhibiting sugar metabolism were used in combination, the effect of pancreatic cancer growth inhibition was maximized (FIG. 8).

There was no significant change in the cell viability of the pancreatic cancer cell line (MiaPaCa2) when treated with siRNA (si-con and si-1A5, respectively) for the control or SLC1A5, and the survival rate was reduced to a half level only when 2-DG was added.

On the other hand, when SLC1A5_var-specific siRNA (si-1A5_var) was treated, the cancer cell survival rate decreased to less than half, and it was confirmed that the addition of 2-DG almost completely suppressed the cancer cell survival rate.

In other words, these results suggest that glutamine metabolism and sugar metabolism through SLC1A5_var in cancer cells have the effect of doubling each other, so inhibiting glutamine transport in mitochondria in anticancer treatment strategies would be very effective. Is to do.

Example 8. Confirmation of anticancer drug resistance inhibitory effect of SLC1A5_var

In addition to the effect of directly inhibiting the proliferation of cancer cells, SLC1A5_var identified in the above example was confirmed to be involved in the development of resistance to previously known anticancer drugs.

Since it is known that hypoxia is a major determinant of drug resistance of pancreatic cancer cells, this example also induces a hypoxic state in pancreatic cancer cell lines and patient-derived pancreatic cancer cells, resulting in resistance to gemcitabine, an anticancer drug. Confirmed.

The correlation between gemcitabine resistance and the expression of SLC1A5_var in pancreatic cancer cell lines was confirmed by measuring cell viability of cancer cells using the WST-1 assay. Specifically, activity of living cells was measured as a method of measuring that tetrazolium salt, which is well soluble in water, becomes a colored formazan dye when reduced by mitochondrial dehydrogenase of living cells. After adding the WST-1 solution provided by the WST-1 kit to the cells, the absorbance at 450 nm was measured 1 hour later.

As a result, as shown in FIG. 9A, in MiaPaCa2 cells overexpressing SLC1A5, GI _{50, which} is the concentration at which gemcitabine can inhibit cell proliferation, is 27.7±8.1 nM and 30.9±9.4 nM, respectively, whereas SLC1A5_var overexpresses SLC1A5_var. GI ₅₀ in MiaPaCa2 cells was measured to be 247.5±47 nM, confirming that gemcitabine's cancer cell proliferation inhibitory efficiency was significantly reduced. In the Panc-1 cell line, which is another pancreatic cancer cell line, the efficacy of inhibiting cancer cell proliferation of gemcitabine was greatly reduced, and the same result was shown (FIG. 9B).

On the other hand, when si-RNA was used to suppress the expression of SLC1A5_var, it was confirmed that the pancreatic cancer cell line again restored the sensitivity of gemcitabine, and the GI ₅₀ of gemcitabine was significantly reduced, inhibiting the proliferation of cancer cells and increasing apoptosis ( Fig. 9C, D).

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> COMPOSITION FOR ANTICANCER USING SLC1A5 TRANSCRIPT VARIANT, SCREENING METHOD FOR ANTICANCER DRUG, AND METHOD FOR DIAGNOSING CANCER <130> 19PP30506 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 339 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens SLC1A5 transcript variant (SLC1A5_var) protein <400> 1 Met Tyr Ser Thr Thr Tyr Glu Glu Arg Asn Ile Thr Gly Thr Arg Val 1 5 10 15 Lys Val Pro Val Gly Gln Glu Val Glu Gly Met Asn Ile Leu Gly Leu 20 25 30 Val Val Phe Ala Ile Val Phe Gly Val Ala Leu Arg Lys Leu Gly Pro 35 40 45 Glu Gly Glu Leu Leu Ile Arg Phe Phe Asn Ser Phe Asn Glu Ala Thr 50 55 60 Met Val Leu Val Ser Trp Ile Met Trp Tyr Ala Pro Val Gly Ile Met 65 70 75 80 Phe Leu Val Ala Gly Lys Ile Val Glu Met Glu Asp Val Gly Leu Leu 85 90 95 Phe Ala Arg Leu Gly Lys Tyr Ile Leu Cys Cys Leu Leu Gly His Ala 100 105 110 Ile His Gly Leu Leu Val Leu Pro Leu Ile Tyr Phe Leu Phe Thr Arg 115 120 125 Lys Asn Pro Tyr Arg Phe Leu Trp Gly Ile Val Thr Pro Leu Ala Thr 130 135 140 Ala Phe Gly Thr Ser Ser Ser Ser Ala Thr Leu Pro Leu Met Met Lys 145 150 155 160 Cys Val Glu Glu Asn Asn Gly Val Ala Lys His Ile Ser Arg Phe Ile 165 170 175 Leu Pro Ile Gly Ala Thr Val Asn Met Asp Gly Ala Ala Leu Phe Gln 180 185 190 Cys Val Ala Ala Val Phe Ile Ala Gln Leu Ser Gln Gln Ser Leu Asp 195 200 205 Phe Val Lys Ile Ile Thr Ile Leu Val Thr Ala Thr Ala Ser Ser Val 210 215 220 Gly Ala Ala Gly Ile Pro Ala Gly Gly Val Leu Thr Leu Ala Ile Ile 225 230 235 240 Leu Glu Ala Val Asn Leu Pro Val Asp His Ile Ser Leu Ile Leu Ala 245 250 255 Val Asp Trp Leu Val Asp Arg Ser Cys Thr Val Leu Asn Val Glu Gly 260 265 270 Asp Ala Leu Gly Ala Gly Leu Leu Gln Asn Tyr Val Asp Arg Thr Glu 275 280 285 Ser Arg Ser Thr Glu Pro Glu Leu Ile Gln Val Lys Ser Glu Leu Pro 290 295 300 Leu Asp Pro Leu Pro Val Pro Thr Glu Glu Gly Asn Pro Leu Leu Lys 305 310 315 320 His Tyr Arg Gly Pro Ala Gly Asp Ala Thr Val Ala Ser Glu Lys Glu 325 330 335 Ser Val Met <210> 2 <211> 1927 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens SLC1A5 transcript variant (SLC1A5_var) mRNA <400> 2 atggggtgga gctgaggccc ggggcgacct gccctggcct gccgtgggag gccagcattc 60 ctcaccccga acccacaatg cctcagggct agcacgccag cctcttgggg ctgaaggagg 120 ctgactcccc aagtcctgca aaggggtgca agcctagttc caaccctgcc gggctgaggc 180 atgagtaggg gctgtatttg cgtgcaggcg ggcccccaaa gcttccactc gctgccttaa 240 aacgccaacc cagcctctca gacaatgctg ccctcccact atgtactcta ccacctatga 300 agagaggaat atcaccggaa ccagggtgaa ggtgcccgtg gggcaggagg tggaggggat 360 gaacatcctg ggcttggtag tgtttgccat cgtctttggt gtggcgctgc ggaagctggg 420 gcctgaaggg gagctgctta tccgcttctt caactccttc aatgaggcca ccatggttct 480 ggtctcctgg atcatgtggt acgcccctgt gggcatcatg ttcctggtgg ctggcaagat 540 cgtggagatg gaggatgtgg gtttactctt tgcccgcctt ggcaagtaca ttctgtgctg 600 cctgctgggt cacgccatcc atgggctcct ggtactgccc ctcatctact tcctcttcac 660 ccgcaaaaac ccctaccgct tcctgtgggg catcgtgacg ccgctggcca ctgcctttgg 720 gacctcttcc agttccgcca cgctgccgct gatgatgaag tgcgtggagg agaataatgg 780 cgtggccaag cacatcagcc gtttcatcct gcccatcggc gccaccgtca acatggacgg 840 tgccgcgctc ttccagtgcg tggccgcagt gttcattgca cagctcagcc agcagtcctt 900 ggacttcgta aagatcatca ccatcctggt cacggccaca gcgtccagcg tgggggcagc 960 gggcatccct gctggaggtg tcctcactct ggccatcatc ctcgaagcag tcaacctccc 1020 ggtcgaccat atctccttga tcctggctgt ggactggcta gtcgaccggt cctgtaccgt 1080 cctcaatgta gaaggtgacg ctctgggggc aggactcctc caaaattacg tggaccgtac 1140 ggagtcgaga agcacagagc ctgagttgat acaagtgaag agtgagctgc ccctggatcc 1200 gctgccagtc cccactgagg aaggaaaccc cctcctcaaa cactatcggg ggcccgcagg 1260 ggatgccacg gtcgcctctg agaaggaatc agtcatgtaa accccgggag ggaccttccc 1320 tgccctgctg ggggtgctct ttggacactg gattatgagg aatggataaa tggatgagct 1380 agggctctgg gggtctgcct gcacactctg gggagccagg ggccccagca ccctccagga 1440 caggagatct gggatgcctg gctgctggag tacatgtgtt cacaagggtt actcctcaaa 1500 acccccagtt ctcactcatg tccccaactc aaggctagaa aacagcaaga tggagaaata 1560 atgttctgct gcgtccccac cgtgacctgc ctggcctccc ctgtctcagg gagcaggtca 1620 caggtcacca tggggaattc tagcccccac tggggggatg ttacaacacc atgctggtta 1680 ttttggcggc tgtagttgtg gggggatgtg tgtgtgcacg tgtgtgtgtg tgtgtgtgtg 1740 tgtgtgtgtg tgtgtgttct gtgacctcct gtccccatgg tacgtcccac cctgtcccca 1800 gatcccctat tccctccaca ataacagaaa cactcccagg gactctgggg agaggctgag 1860 gacaaatacc tgctgtcact ccagaggaca ttttttttag caataaaatt gagtgtcaac 1920 tatttaa 1927 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SLC1A5 transcript variant (SLC1A5_var) specific primer (forward) <400> 3 cactatgtac tctaccac 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SLC1A5 transcript variant (SLC1A5_var) specific primer (reverse) <400> 4 ctcatctact tcctcttc 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SLC1A5 primer (forward) <400> 5 gcggatgatc atcttgcc 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SLC1A5 primer (reverse) <400> 6 ctcatctact tcctcttc 18 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SLC1A5 transcript variant (SLC1A5_var) specific siRNA <400> 7 gcugcccucc cacuaugua 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> SLC1A5 siRNA <400> 8 ccagagaaac ucucguauu 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> control siRNA <400> 9 acaacagcca caacgucua 19 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SLC1A5 transcript variant (SLC1A5_var) specific shRNA <400> 10 ctatgtactc taccacctat g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> control shRNA <400> 11 acaacagcca caacgtctat a 21

Claims (27)

A pharmaceutical composition for the prevention or treatment of cancer comprising an expression inhibitor or a protein activity inhibitor of the SLC1A5 transcript variant. The method of claim 1,
The expression inhibitor is one or more selected from the group consisting of antisense RNA, siRNA, shRNA, and miRNA specifically binding to the mRNA of the SLC1A5 transcript variant. The method of claim 2,
The mRNA includes the nucleotide sequence of SEQ ID NO: 2, the siRNA includes the nucleotide sequence of SEQ ID NO: 7, and the shRNA comprises the nucleotide sequence of SEQ ID NO: 10. The method of claim 1,
The protein activity inhibitor is one or more selected from the group consisting of peptides, antibodies, and aptamers having a domain that specifically binds to the SLC1A5 transcript variant protein. The method of claim 1,
The composition further comprises a sugar metabolism inhibitor, the composition. The method of claim 5,
The sugar metabolism inhibitor is one selected from the group consisting of 2-deoxyglucose, 3-bromopyruvate (3-bp), WZB117, phloretin, and STF-31. That is, the composition. The method of claim 1,
The cancer is leukemia, lymphoma, hematopoietic malignancy, cervical cancer, sarcoma, testicular cancer, malignant melanoma, and endocrine tumor. ), bone cancer, prostate cancer, uterus cancer, breast cancer, bladder cancer, brain cancer, liver cancer, stomach cancer ), pancreas cancer, skin cancer, lung cancer, larynx cancer, head and neck cancer, esophageal cancer, colorectal cancer, and ovarian cancer (ovarian cancer) that is one or more selected from the group consisting of, the composition. The method of claim 7,
The cancer is one or more selected from the group consisting of lung cancer, colorectal cancer, and pancreas cancer. (a) treating cells expressing the SLC1A5 transcript variant with the test agent;
(b) measuring the expression level or protein activity of the SLC1A5 transcript variant in the cells treated with the test agent; And
(c) comparing the expression level of the SLC1A5 transcript variant expressed in cells not treated with the test agent or the activity of the protein to determine whether the test agent inhibits the expression level or activity of the SLC1A5 transcript variant; Cancer treatment screening method comprising. The method of claim 9,
The SLC1A5 transcript variant in step (a) comprises the amino acid sequence of SEQ ID NO: 1, and is a protein having mitochondrial targeting sequence and glutamine transporter activity. The method of claim 9,
The measurement of the expression level of the SLC1A5 transcript variant in step (b) is to measure the mRNA expression level or the protein expression level in the mitochondria. The method of claim 9,
The activity of the protein in step (b) is that of the mitochondrial glutamine transporter activity. The method of claim 9,
The cancer is one or more selected from the group consisting of lung cancer, colorectal cancer, and pancreas cancer. (a) obtaining a sample from the subject;
(b) measuring the expression level or protein activity of the SLC1A5 transcript variant in the sample; And
(c) determining that the subject has cancer when the measured expression level or protein activity level is higher than that of the control group. The method of claim 14,
The sample of step (a) is one or more selected from the group consisting of cancer tissue, blood, plasma, serum, lymph fluid, and urine. The method of claim 14,
The expression level of the SLC1A5 transcript variant in step (b) is the level of protein expression in mRNA or mitochondria. The method of claim 14,
The activity of the protein in step (b) is that of the mitochondrial glutamine transporter. The method of claim 14,
The cancer is one or more selected from the group consisting of lung cancer, colorectal cancer, and pancreas cancer. A composition for diagnosis of cancer comprising an agent for measuring the expression level or protein activity level of the SLC1A5 transcript variant. The method of claim 19,
The agent for measuring the expression level is a probe that specifically binds to the SLC1A5 transcript variant mRNA, or a primer consisting of a nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, a composition for cancer diagnosis. The method of claim 19,
The agent for measuring the expression level is one or more selected from the group consisting of a peptide domain, an antibody, and an aptamer that specifically binds to the SLC1A5 transcript variant protein. A composition for inhibiting anticancer drug resistance, comprising an expression inhibitor or a protein activity inhibitor of the SLC1A5 transcript variant. The method of claim 22,
The anticancer agent is a pyrimidine nucleoside prodrug, a composition for inhibiting anticancer drug resistance. The method of claim 23,
The pyrimidine nucleoside precursor is gemcitabine (gemcitabine), the composition for inhibiting anticancer drug resistance. The method of claim 22,
The expression inhibitor is one or more selected from the group consisting of antisense RNA, siRNA, shRNA, and miRNA that specifically binds to the mRNA of the SLC1A5 transcript variant, anticancer agent resistance inhibition composition. The method of claim 25,
The mRNA includes the nucleotide sequence of SEQ ID NO: 2, the siRNA includes the nucleotide sequence of SEQ ID NO: 7, and the shRNA comprises the nucleotide sequence of SEQ ID NO: 10, anticancer agent resistance inhibition composition. The method of claim 22,
The protein activity inhibitor is one or more selected from the group consisting of a peptide domain, an antibody, and an aptamer that specifically binds to the SLC1A5 transcript variant protein.

Images