Use of lsd1 gene and related products

Abstract

The application belongs to the field of biological medicine research, and discloses the use of LSD1 gene and related products. The LSD1 gene is used as a target in the preparation of a drug for resisting lung adenocarcinoma deterioration and / or lung adenocarcinoma drug resistance. The application finds that the deletion of the LSD1 gene can inhibit the transdifferentiation of lung adenocarcinoma and adenosquamous carcinoma into lung squamous carcinoma, and simultaneously inhibit the occurrence of adenosquamous carcinoma and lung squamous carcinoma. Further, the LSD1 inhibitor can also inhibit the transdifferentiation of adenosquamous carcinoma and lung adenocarcinoma into lung squamous carcinoma, and simultaneously inhibit the occurrence of adenosquamous carcinoma and lung squamous carcinoma. Further, the LSD1 inhibitor can inhibit the occurrence of lung squamous carcinoma. Therefore, the LSD1 gene can be used as a potential therapeutic target for resisting lung adenocarcinoma deterioration, and simultaneously, the LSD1 gene can be used as a potential therapeutic target for overcoming drug resistance in lung adenocarcinoma targeted therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical research, specifically relating to the uses of the LSD1 gene and related products. Background Technology

[0002] Lung cancer includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC accounting for approximately 85% of all lung cancers. NSCLC differs from small cell lung cancer (SCLC). SCLC is a neuroendocrine tumor with a very rapid proliferation rate, far exceeding that of normal cells. Therefore, SCLC is highly sensitive to radiotherapy and chemotherapy, exhibiting excellent treatment outcomes and remission rates significantly higher than other types of lung cancer. Consequently, radiotherapy and chemotherapy have become powerful clinical treatments for SCLC (Rudin, CM et al., 2019).

[0003] Non-small cell lung cancer (NSCLC) includes squamous cell carcinoma (SCC), adenocarcinoma (ADC), large cell carcinoma, and adenosquamous carcinoma (AdSCC). SCC accounts for 20% of NSCLC, while AdSCC accounts for 1-10%. Compared to other types of NSCLC, AdSCC has a worse prognosis.

[0004] Adenosquamous carcinoma (AdSCC) is a unique subtype characterized by high malignancy and plasticity (Cooke et al., 2010; Fitzgibbons and Kern, 1985; Jia and Chen, 2011; Naunheim et al., 1987; Takamoriet et al., 1991; Zhang et al., 2017). Unlike lung adenocarcinoma (ADC) or lung squamous cell carcinoma (SCC), AdSCC contains both adenoma and squamous cell pathological components, making it prone to developing drug resistance (Hou et al., 2016; Travis et al., 2015). Furthermore, patients with AdSCC often have a poor prognosis (Maeda et al., 2012). The adenomatous and squamous components of AdSCC often share the same genetic alterations (Jia and Chen, 2011; Kang et al., 2007; Tochigi et al., 2011), suggesting that AdSCC may initially originate from a single pathology and gradually transform into other pathologies as malignancy progresses or treatment fails (Haratani et al., 2016; Hou et al., 2016; Jukna et al., 2016).

[0005] For patients with adenosquamous carcinoma, surgery is currently the most effective treatment. Due to the similarity in driver mutation genes between adenosquamous and lung adenocarcinoma patients, inhibitors targeting EGFR and ALK have also shown some efficacy in patients with advanced adenosquamous carcinoma (Li and Lu, 2018). However, for lung squamous cell carcinoma patients, there is currently a lack of targeted mutations, preventing the application of targeted therapies. Nevertheless, the emergence of cancer immunotherapy has provided a new breakthrough in the treatment of non-small cell lung cancer patients without targeted mutations. Immunotherapy enhances the body's own immune system to fight cancer. Recent studies have shown that combining immune checkpoint inhibitors with chemotherapy can significantly improve the survival time of non-small cell lung cancer patients with high PD-L1 expression (Chen et al., 2020). Furthermore, adenosquamous and lung squamous cell carcinomas lack the well-established drug treatments available for small cell lung cancer, making the development of targeted drugs for these two types of carcinoma particularly important.

[0006] Genetically engineered mouse models (GEMMs) have shown that Kras / Lkb1 is a mouse model capable of spontaneously undergoing adenosquamous carcinoma transdifferentiation. Lkb1 deficiency promotes the transdifferentiation of established ADCs into SCCs by activating the Hippo pathway and inhibiting the pentose phosphate pathway, and alterations in corresponding signaling pathways and metabolic reprogramming have also been found in sequencing with human AdSCCs (Gao et al., 2014; Han et al., 2014; Ji et al., 2007; Li et al., 2015; Zhang et al., 2017). Furthermore, animal model studies have shown that inhibitors effective against ADC therapy can develop resistance due to adenosquamous carcinoma transdifferentiation (Li et al., 2015). Multiple clinical case studies have demonstrated that adenosquamous carcinoma transdifferentiation is one of the mechanisms by which ADC patients develop resistance to targeted therapy (Hou et al., 2016; Lin et al., 2020; Schoenfeld et al., 2020). Therefore, how to intervene in drug resistance caused by the metadifferentiation of adenosquamous carcinoma is an urgent clinical problem to be solved.

[0007] Transdifferentiation in adenosquamous carcinoma is primarily driven by transcriptional reprogramming and epigenetic regulation, rather than mutational events (Quintanal-Villalonga et al., 2021). The histone demethyltransferase LSD1 is significantly elevated in SOX2-expressing SCC, and LSD1 can directly regulate SOX2 through H3K4 and H3K9 modification (Zhang et al., 2013). Proteomic analysis of human SCC has also identified LSD1 as a potential therapeutic target for SOX2-positive SCC (Satpathy et al., 2021).

[0008] Previous studies have shown that inhibiting the epigenetic regulator LSD1 can enhance the efficacy of immunotherapy (Sheng et al., 2018; Sheng et al., 2021) and also inhibit the growth of small cell lung cancer (Mohammad et al., 2015) and squamous cell carcinoma of the skin (Egolf et al., 2019).

[0009] LSD1, short for lysine-specific demethylase 1 (LSD1) (Shi et al., 2004), primarily regulates gene expression through the demethylation and dimethylation of lysine residues, particularly H3K4 and H3K9 (Jostes et al., 2019). Several reversible and irreversible LSD1 inhibitors have been developed to date (including tranylcypromine, IMG-7289, INCB059872, and GSK2879552) (Duan et al., 2017; Fang et al., 2019; Song et al., 2016; Zheng et al., 2016), and clinical trials are currently validating their efficacy and safety in treating depression, myeloma, and solid tumors (Zhang et al., 2021).

[0010] However, there are currently no clinical trials to verify the efficacy of LSD1 inhibitors in patients with non-small cell lung cancer.

[0011] GSK2879552 is a small molecule inhibitor of LSD1 catalytic activity identified after screening 2.5 million compounds (Mohammad et al., 2015). To verify the antitumor activity of GSK2879552, its therapeutic effect was evaluated in 165 cell lines of small cell lung cancer and leukemia. Treatment with GSK2879552 inhibited the growth of 9 / 28 small cell lung cancer cell lines and 20 / 29 leukemia cell lines (Mohammad et al., 2015). GSK2879552 significantly inhibited the growth of small cell lung cancer in a humanized tissue xenograft (PDX) model (Mohammad et al., 2015). GSK2879552 has also been applied in clinical trials for various cancers (Fang et al., 2019).

[0012] Adenosquamous carcinoma has been extensively studied as a new drug resistance mechanism. After targeted therapy, lung adenocarcinoma is prone to transform into lung squamous carcinoma. During the transformation process, adenosquamous carcinoma is formed, which leads to drug resistance.

[0013] However, how to overcome the drug resistance caused by the transformation of lung adenocarcinoma to lung squamous cell carcinoma and how to further target adenosquamous carcinoma remains unclear. Summary of the Invention

[0014] In order to overcome the problems existing in the prior art, the purpose of this invention is to provide the uses of the LSD1 gene and related products.

[0015] To achieve the above-mentioned objectives and other related objectives, the present invention adopts the following technical solution:

[0016] The first aspect of this invention protects the use of the LSD1 gene as a target in the preparation of medicaments for the prevention of lung adenocarcinoma progression and / or for the prevention of lung adenocarcinoma resistance.

[0017] Preferably, the lung adenocarcinoma deterioration refers to the lung adenocarcinoma becoming resistant to targeted drug therapy. One way to develop resistance is through transdifferentiation into adenosquamous carcinoma, which then further transdifferentiates into lung squamous cell carcinoma. Once transdifferentiated into lung squamous cell carcinoma, there is no drug treatment available.

[0018] Preferably, targeting the LSD1 gene specifically means using the LSD1 gene and / or its expression products as targets for drug screening to find drugs that can inhibit the LSD1 gene and / or its expression. Antibody drugs, small molecule drugs, and similar drugs can also target the LSD1 gene.

[0019] Another aspect of the present invention protects the use of LSD1 inhibitors in the preparation of medicaments for the prevention of adenocarcinoma progression and / or for the prevention of drug resistance in lung adenocarcinoma.

[0020] Preferably, the LSD1 inhibitor refers to a molecule that has an inhibitory effect on LSD1.

[0021] More preferably, the inhibitory effect on LSD1 refers to inhibiting the transcription or translation of the LSD1 gene, or molecules that can specifically inhibit the expression or activity of the LSD1 protein, thereby reducing the expression level of the LSD1 gene in lung cancer cells and achieving the purpose of inhibiting the transdifferentiation, proliferation, growth, differentiation, survival and / or drug resistance of lung cancer cells.

[0022] Preferably, the LSD1 inhibitor is the only active ingredient or one of the active ingredients in the product.

[0023] More preferably, the product necessarily includes an LSD1 inhibitor, and uses the LSD1 inhibitor as the active ingredient for the aforementioned effects.

[0024] More preferably, in the product, the effective ingredient that performs the aforementioned function may be only an LSD1 inhibitor, or may include other molecules that can perform the aforementioned function.

[0025] More preferably, the product can be a single-component substance or a multi-component substance.

[0026] More preferably, the form of the product is not particularly limited and can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0027] More preferably, the product is primarily targeted at mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes, or humans.

[0028] More preferably, the product includes, but is not limited to, a drug.

[0029] Preferably, the LSD1 inhibitor is selected from double-stranded RNA, shRNA, antibody, or small molecule compound.

[0030] More preferably, the small molecule compound is selected from one or both of transphenylcyclopropylamine and cyclopropylamine LSD1 inhibitors.

[0031] More preferably, the small molecule compound is selected from one or more of transphenylcyclopropylamine, IMG-7289, INCB059872, and GSK2879552. Even more preferably, the inhibitor is selected from GSK2879552.

[0032] Another aspect of the invention also protects a composition comprising the LSD1 inhibitor described above, and a pharmaceutically acceptable carrier.

[0033] Preferably, the composition can be a pharmaceutical composition.

[0034] Preferably, the carrier includes a diluent or an excipient.

[0035] Preferably, when the composition is used for prevention or treatment of a subject, an effective dose of the composition needs to be applied to the subject.

[0036] Preferably, the composition is not particularly limited in form and can be in various forms such as solid, liquid, gel, semi-fluid, or aerosol.

[0037] Preferably, the composition is primarily intended for mammals. The mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes, or humans.

[0038] Preferably, in the uses or compositions described above, the anti-progression of lung adenocarcinoma refers to inhibiting the metadifferentiation of adenosquamous carcinoma.

[0039] Preferably, transdifferentiation refers to the transformation of one type of differentiated cell into another type of differentiated cell.

[0040] More preferably, the inhibition of adenosquamous carcinoma transdifferentiation is to inhibit the transformation of lung adenocarcinoma into adenosquamous carcinoma.

[0041] More preferably, the inhibition of adenosquamous carcinoma transdifferentiation is to inhibit the transformation of adenosquamous carcinoma into lung squamous carcinoma.

[0042] More preferably, the inhibition of adenosquamous carcinoma transdifferentiation is to inhibit the transformation of lung adenocarcinoma into lung squamous carcinoma.

[0043] More preferably, it inhibits the proliferation of lung squamous cell carcinoma.

[0044] More preferably, it promotes apoptosis in lung squamous cell carcinoma.

[0045] More preferably, it inhibits the occurrence of adenosquamous carcinoma.

[0046] More preferably, for the prevention and / or treatment of non-small cell lung cancer, wherein the non-small cell lung cancer is selected from adenosquamous carcinoma and / or squamous cell carcinoma of the lung.

[0047] Another aspect of the present invention also protects the use of a substance for detecting LSD1 in the preparation of a product, said product having at least one of the following functions:

[0048] 1-1) Diagnosis of adenosquamous carcinoma and / or pulmonary squamous cell carcinoma;

[0049] 1-2) Assess whether lung adenocarcinoma has transformed into adenosquamous carcinoma or lung squamous cell carcinoma;

[0050] 1-3) Assess drug resistance in non-small cell lung cancer, wherein the non-small cell lung cancer is selected from at least one of lung adenocarcinoma, adenosquamous carcinoma, and lung squamous cell carcinoma.

[0051] Preferably, the product is selected from one or more of reagent kits, membrane strips, and chips.

[0052] Another aspect of the present invention also protects a method for preventing the progression of lung adenocarcinoma and / or for preventing lung adenocarcinoma drug resistance, comprising the step of administering an LSD1 inhibitor to a subject.

[0053] Preferably, the object can be a mammal or a mammalian cell. The mammal is preferably a rodent, even-toed ungulate, perissodactyl, lagomorph, or primate. The primate is preferably a monkey, ape, or human. The cell can be an isolated cell.

[0054] Preferably, the subject may be a patient suffering from adenosquamous carcinoma and / or lung adenocarcinoma cells, or an individual expecting treatment. Alternatively, the subject may be ex vivo cancer cells from an individual with adenosquamous carcinoma and / or lung adenocarcinoma, or an individual expecting treatment for adenosquamous carcinoma and / or lung adenocarcinoma.

[0055] The LSD1 inhibitor can be administered to subjects before, during, and after lung cancer treatment.

[0056] Furthermore, the LSD1 gene is derived from humans.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1) This invention, through adenovirus-mediated knockout of LSD1 in Kras / Lkb1 / Lsd1 model mice, found that knockout of LSD1 significantly reduced tumor burden, especially the tumor burden of lung squamous cell carcinoma, and reduced the number of tumors, especially the number of lung squamous cell carcinomas. After knocking out the LSD1 gene, the overall tumors in the mice exhibited lung adenocarcinoma characteristics, proving that the LSD1 gene is related to the transdifferentiation of adenosquamous carcinoma. Knocking out the LSD1 gene can inhibit the transdifferentiation of lung adenocarcinoma into adenosquamous carcinoma, and further, the transdifferentiation of adenosquamous carcinoma into lung squamous cell carcinoma.

[0059] 2) This invention uses an LSD1 inhibitor, such as GSK2879552, to treat Kras / Lkb1 model mice, further demonstrating...

[0060] LSD1 inhibitors can effectively reduce the tumor burden and number of squamous cell carcinomas in the lungs. After LSD1 knockout, the tumors in mice exhibit the characteristics of lung adenocarcinoma, indicating that LSD1 inhibitors can significantly inhibit the occurrence of adenocarcinoma, inhibit the transdifferentiation of adenocarcinoma, and thus the transdifferentiation of adenocarcinoma into squamous cell carcinoma in the lungs.

[0061] 3) This invention found that LSD1 inhibitor treatment of mice can significantly inhibit the proliferation of lung squamous cell carcinoma and promote apoptosis of lung squamous cell carcinoma, further confirming that LSD1 inhibitor can significantly inhibit the development of lung squamous cell carcinoma and can effectively inhibit the occurrence and deterioration of lung adenocarcinoma.

[0062] 4) Transdifferentiation of lung adenosquamous carcinoma is a drug resistance mechanism of targeted therapy for adenocarcinoma. Inhibiting transdifferentiation of adenosquamous carcinoma is of great significance for overcoming drug resistance to targeted therapy for adenocarcinoma. This invention found that treating mice with LSD1 gene knockout and LSD1 inhibitors can inhibit the occurrence of transdifferentiation of adenosquamous carcinoma. Therefore, LSD1 can be a potential therapeutic target for drug resistance caused by transdifferentiation of adenosquamous carcinoma. Attached Figure Description

[0063] Figure 1 The diagram shown is a flowchart of the construction process of the Kras / Lkb1 / Lsd1 mouse model in Example 1 of the present invention.

[0064] Figure 2 The graph shows the tumor burden statistics of KL and KLL model mice after the LSD1 gene was knocked out in Example 1 of this invention. ** indicates P < 0.01.

[0065] Figure 3The graph shows the tumor count in KL and KLL model mice after the LSD1 gene was knocked out in Example 1 of this invention. Here, ns represents P>0.05.

[0066] Figure 4 The graph shows the incidence of adenosquamous carcinoma in KL and KLL model mice after the LSD1 gene was knocked out in Example 1 of this invention. AdSCC represents adenosquamous carcinoma.

[0067] Figure 5 The image shows the tumor burden statistics of lung adenocarcinoma and lung squamous cell carcinoma tissues in KL and KLL model mice after LSD1 gene knockout in Example 1 of this invention. In the image, ADC represents lung adenocarcinoma, SCC represents lung squamous cell carcinoma; ** represents P<0.01, and ns represents P>0.05.

[0068] Figure 6 The graph shows the number of adenocarcinoma and squamous cell carcinoma tumors in KL and KLL model mice after the LSD1 gene was knocked out in Example 1 of this invention. In the graph, ADC represents lung adenocarcinoma, SCC represents lung squamous cell carcinoma; ** represents P < 0.01, and ns represents P > 0.05.

[0069] Figure 7 The images shown are H&E staining and immunohistochemical staining images of KL and KLL model mice after the LSD1 gene was knocked out in Example 1 of this invention. In these images, ADC represents lung adenocarcinoma and SCC represents lung squamous cell carcinoma.

[0070] Figure 8 This is a schematic diagram of GSK2879552 treatment of KL model mice in Example 2 of the present invention.

[0071] Figure 9 The image shows the tumor burden statistics of KL model mice after treatment with GSK2879552 and saline, respectively, in Example 2 of this invention. ** indicates P < 0.01.

[0072] Figure 10 The graph shown is a statistical chart of tumor numbers in KL model mice after treatment with GSK2879552 and saline, respectively, in Example 2 of this invention. Here, ns represents P > 0.05.

[0073] Figure 11 The graph shown is a statistical chart of the incidence of adenosquamous carcinoma in KL model mice after treatment with GSK2879552 and saline, respectively, in Example 2 of the present invention. AdSCC represents adenosquamous carcinoma.

[0074] Figure 12The image shows the tumor burden statistics of lung adenocarcinoma and lung squamous cell carcinoma in KL model mice after treatment with GSK2879552 and saline, respectively, in Example 2 of this invention. In the image, ADC represents lung adenocarcinoma, SCC represents lung squamous cell carcinoma; ** represents P < 0.01, and ns represents P > 0.05.

[0075] Figure 13 The image shows the tumor counts of adenocarcinoma and squamous cell carcinoma in KL model mice after treatment with GSK2879552 and saline, respectively, in Example 2 of this invention. In the graph, ADC represents lung adenocarcinoma, and SCC represents lung squamous cell carcinoma; ** represents P<0.01, and * represents P<0.05.

[0076] Figure 14 The images shown are H&E staining and immunohistochemical staining images of mice in the control and experimental groups in Example 2 of this invention. Ctrl represents the control group, and GSK2879552 represents the experimental group.

[0077] Figure 15 The image shown is a representative Ki67 immunofluorescence image of KL model mice treated with GSK2879552 in Example 3 of this invention. Ctrl represents the control group, and GSK2879552 represents the experimental group.

[0078] Figure 16 The image shows the Ki67 immunofluorescence statistics of KL model mice treated with GSK2879552 in Example 3 of this invention. Ctrl represents the control group, GSK2879552 represents the experimental group, and **** represents P < 0.0001.

[0079] Figure 17 The image shown is a representative immunohistochemical image of cleaved Caspase 3 in KL model mice after treatment with GSK2879552 in Example 3 of this invention. Ctrl represents the control group, and GSK2879552 represents the experimental group.

[0080] Figure 18 The image shows the immunohistochemical statistics of cleaved Caspase 3 in KL model mice after treatment with GSK2879552 in Example 3 of this invention. Ctrl represents the control group, and GSK2879552 represents the experimental group; *** indicates P < 0.001.

[0081] Figure 19The image shows the results of RNA seq gene expression analysis performed on normal lung tissue, lung adenocarcinoma tumors, and lung squamous cell carcinoma tumors from KL model mice according to this invention. In the figure, Normal represents normal tissue, KL ADC represents lung adenocarcinoma tumors from KL model mice, and KL SCC represents lung squamous cell carcinoma tumors from KL model mice. Detailed Implementation

[0082] The inventors collected normal lung tissue, lung adenocarcinoma tumors, and lung squamous cell carcinoma tumors from KL model mice and analyzed gene expression differences using RNA-seq. Each column represents one sample. The results showed that the LSD1 gene expression was highest in lung squamous cell carcinoma. The RNA-seq analysis results are shown below. Figure 19 The inventors later constructed a Kras / Lkb1 / Lsd1 mouse model and found that LSD1 gene deletion completely inhibited the transdifferentiation of adenosquamous carcinoma. Furthermore, administration of an LSD1 inhibitor (such as GSK2879552) to the Kras / Lkb1 mouse model showed that the LSD1 inhibitor significantly inhibited adenosquamous carcinoma transdifferentiation. In addition, the LSD1 inhibitor also inhibited the proliferation of lung squamous cell carcinoma and promoted apoptosis. Therefore, the LSD1 gene can serve as a potential therapeutic target for inhibiting both lung squamous cell carcinoma and adenosquamous carcinoma, and also provides a clinical basis for overcoming adenosquamous carcinoma transdifferentiation and drug resistance caused by transdifferentiation through targeting LSD1.

[0083] Based on the above research results, further exploration and development of new diagnostic and therapeutic methods targeting this gene could provide more options for the diagnosis, treatment, drugs, and products of lung adenocarcinoma and / or adenosquamous carcinoma and / or lung squamous cell carcinoma.

[0084] LSD1 gene

[0085] The terms "LSD1 gene" and "lysine-specific demethylase 1" are used interchangeably. Inhibition of their expression can improve drug resistance in tumor treatment. In one embodiment of the invention, the LSD1 gene is derived from humans or other eukaryotes, such as mice, rats, cattle, or monkeys, and is highly conserved among them.

[0086] This invention reveals for the first time that LSD1 is associated with the transdifferentiation of adenosquamous carcinoma. Inhibiting LSD1 expression inhibits the transformation of lung adenocarcinoma into adenosquamous carcinoma; inhibiting LSD1 expression inhibits the transformation of adenosquamous carcinoma into lung squamous cell carcinoma; inhibiting LSD1 expression inhibits the transformation of lung adenocarcinoma into lung squamous cell carcinoma; inhibiting LSD1 expression inhibits the proliferation of lung squamous cell carcinoma; inhibiting LSD1 expression inhibits the apoptosis of lung squamous cell carcinoma; and inhibiting LSD1 expression inhibits the occurrence of adenosquamous carcinoma.

[0087] This invention reveals for the first time that LSD1 is associated with drug resistance caused by transdifferentiation of adenosquamous carcinoma. If LSD1 is highly expressed after targeted drug treatment of lung adenocarcinoma, it indicates that the targeted drug for lung adenocarcinoma has developed resistance.

[0088] Based on the above findings of this invention, those skilled in the art can use LSD1 as a target to design or screen tumor drugs that inhibit the progression of lung adenocarcinoma and / or drug resistance in lung adenocarcinoma. For example, those skilled in the art can design or screen LSD1 inhibitors based on the known LSD1 sequence and administer them to cancer patients in combination with chemotherapy drugs, thereby improving tumor resistance to therapeutic drugs, increasing the success rate of tumor treatment, reducing the dosage of tumor therapeutic drugs, and alleviating patient suffering.

[0089] LSD1 inhibitors

[0090] The LSD1 inhibitor of this invention refers to a molecule that has an inhibitory effect on LSD1.

[0091] As used in this article, "has an inhibitory effect on LSD1" includes, but is not limited to, inhibiting the expression or activity of LSD1.

[0092] As used herein, “inhibiting LSD1 activity” means reducing LSD1 activity. Preferably, LSD1 activity is reduced by at least 10% compared to before inhibition, more preferably by at least 30%, even more preferably by at least 50%, more preferably by at least 70%, and most preferably by at least 90%.

[0093] As used in this article, "inhibiting LSD1 expression" can specifically mean inhibiting the transcription or translation of the LSD1 gene. Specifically, it can mean preventing the LSD1 gene from being transcribed, reducing the transcriptional activity of the LSD1 gene, preventing the LSD1 gene from being translated, or reducing the translation level of the LSD1 gene.

[0094] Those skilled in the art can use conventional methods to regulate LSD1 gene expression, such as gene knockout, homologous recombination, and interfering RNA.

[0095] The inhibition of LSD1 gene expression can be verified by detecting expression levels using PCR and Western Blot. Preferably, compared with wild type, LSD1 gene expression is reduced by at least 10%, more preferably by at least 30%, even more preferably by at least 50%, more preferably by at least 70%, even more preferably by at least 90%, and most preferably by no expression of LSD1 gene at all.

[0096] The small molecule compounds of this invention refer to compounds composed of several or dozens of atoms with a molecular mass of less than 1000.

[0097] Preferably, the LSD1 inhibitor is one or both of tranylcyclopropane and cyclopropylamine LSD1 inhibitors. More preferably, the LSD1 inhibitor is selected from one or more of tranylcyclopropane, IMG-7289, INCB059872, and GSK2879552. Specifically, it is GSK2879552, a cyclopropylamine LSD1 inhibitor.

[0098] Composition

[0099] The compositions of the present invention can utilize LSD1 inhibitors, such as nucleic acid molecules that reduce LSD1 gene expression; and / or LSD1 gene interference nucleic acid constructs; and / or LSD1 gene interference lentiviruses; and / or antibodies that reduce LSD1 gene expression; and / or small molecule compounds that inhibit LSD1 gene expression, as active ingredients to prepare drugs for treating lung adenocarcinoma progression and / or lung adenocarcinoma resistance. Typically, in addition to the active ingredient, the drug may also include one or more pharmaceutically acceptable carriers, depending on the requirements of different dosage forms.

[0100] The present invention provides a composition comprising an effective amount (e.g., 0.000001–50 wt%; preferably 0.00001–20 wt%; more preferably 0.0001–10 wt%) of the LSD1 inhibitor, and a pharmaceutically acceptable carrier.

[0101] The compositions of the present invention can be used to improve the deterioration of lung adenocarcinoma tumors by inhibiting the expression of LSD1, prevent lung adenocarcinoma from transforming into adenosquamous carcinoma, and further transform from adenosquamous carcinoma into lung squamous carcinoma; they can also prevent and / or treat lung adenocarcinoma; they can also prevent and / or treat lung squamous carcinoma; they can also improve the drug resistance of lung adenocarcinoma tumors and improve the effectiveness of lung adenocarcinoma treatment drugs in the treatment of lung adenocarcinoma.

[0102] As used herein, "composition" is preferably a pharmaceutical composition.

[0103] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0104] The pharmaceutically acceptable carrier of the present invention should be compatible with the active ingredient, i.e., miscible with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0105] The compositions of this invention can also be formulated into preparations. Unless otherwise specified, the preparations are not particularly limited and can be formulated into dosage forms such as injections, oral solutions, tablets, capsules, pellets, sprays, and sustained-release formulations, which can be prepared by conventional methods. Generally, the preparation should be matched to the route of administration. The compositions of this invention can be formulated into injections, for example, prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The compositions are preferably manufactured under aseptic conditions.

[0106] Substances, reagent kits, membrane strips, and chips for detecting LSD1

[0107] In another aspect, the present invention provides the use of a substance for detecting LSD1 in the preparation of a product having at least one of the following functions: 1-1) diagnosing adenosquamous carcinoma and / or pulmonary squamous cell carcinoma; 1-2) assessing whether pulmonary adenocarcinoma has transformed into adenosquamous carcinoma and / or pulmonary squamous cell carcinoma; 1-3) assessing drug resistance in non-small cell lung cancer, wherein the non-small cell lung cancer is selected from at least one of pulmonary adenocarcinoma, adenosquamous carcinoma, and pulmonary squamous cell carcinoma. If LSD1 is highly expressed, the diagnosis is adenosquamous carcinoma and / or pulmonary squamous cell carcinoma; if LSD1 is highly expressed, the assessment is that pulmonary adenocarcinoma has transformed into adenosquamous carcinoma and / or pulmonary squamous cell carcinoma; if LSD1 is highly expressed, the assessment is that non-small cell lung cancer has developed drug resistance.

[0108] In this invention, the product includes a reagent kit, a chip, and a membrane strip.

[0109] In some non-limiting embodiments, the kit, chip, or membrane strip contains a substance for detecting LSD1. The kit, chip, or membrane strip also includes instructions or supporting materials describing the potential anticancer effect of using the kit to determine LSD1 in preventing lung adenocarcinoma progression and / or lung adenocarcinoma drug resistance, and / or references to websites or publications describing such effects.

[0110] The types of kits, chips, and membrane strips include, but are not limited to, packaged probes and primer sets (e.g., TaqMan probes / primers), arrays / microarrays, biomarker-specific antibodies, biomarker-specific beads, and may also contain one or more probes, primers, or other reagents for detecting one or more biomarkers of the present invention.

[0111] In some embodiments, the kit may include containers (including microtiter plates suitable for use in automated implementation of the method), each container having one or more of the various reagents (typically in concentrated form) used in the method, including, for example, pre-made microarrays, buffers, suitable nucleoside triphosphates (e.g., dATP, dCTP, dGTP, and dTTP, or rATP, rCTP, rGTP, and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more probes and primers of the present invention (e.g., poly(T) or random primers of appropriate length linked to a promoter that reacts with RNA polymerase).

[0112] Methods to combat the progression of lung adenocarcinoma and / or drug resistance in lung adenocarcinoma

[0113] The present invention also provides a method for targeting LSD1 to prevent the progression of lung adenocarcinoma and / or to prevent drug resistance in lung adenocarcinoma, the method comprising: administering an LSD1 inhibitor to a subject.

[0114] The target organism can be a mammal or a lung adenocarcinoma cell line from a mammal. The mammal is preferably a rodent, even-toed ungulate, perissodactyl, lagomorph, or primate. The primate is preferably a monkey, ape, or human. The lung adenocarcinoma cells can be isolated lung adenocarcinoma cells.

[0115] The subject can be a patient with lung adenocarcinoma or an individual with lung adenocarcinoma who is seeking treatment. Alternatively, the subject can be ex vivo cells from a patient with lung adenocarcinoma or an individual with lung adenocarcinoma who is seeking treatment.

[0116] The subject can be a patient suffering from adenosquamous carcinoma or an individual with adenosquamous carcinoma expecting treatment. Alternatively, the subject can be ex vivo cells from a patient with adenosquamous carcinoma or an individual with adenosquamous carcinoma expecting treatment.

[0117] The subject can be a patient suffering from squamous cell carcinoma of the lung or an individual with squamous cell carcinoma of the lung expecting treatment. Alternatively, the subject can be ex vivo cells from a patient with squamous cell carcinoma of the lung or an individual with squamous cell carcinoma of the lung expecting treatment.

[0118] The LSD1 inhibitor can be administered to subjects before, during, and after treatment for lung adenocarcinoma.

[0119] The method of this invention can be used to screen non-drug-resistant antitumor drugs targeting tumors from a compound library, serving as an important indicator for compound screening and evaluation. This method can also be used in clinical practice for predicting and diagnosing personalized treatment for cancer patients, screening for antitumor drugs with optimal tumor treatment efficacy for each individual.

[0120] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0121] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0122] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0123] In the following embodiments of the present invention:

[0124] 1. Antibody

[0125] TP63 (Cat #ab53039, Abcam), SOX2 (Cat #ab92494, Abcam), FOXA2 (Cat #ab108422, Abcam), NKX2-1 (Cat #ab133638, Abcam), KRT5 (Cat # BS1208, Bioworld), Cleaved Caspase 3 (Cat #9661, CST), Ki67 (Cat #NB500-170, Novus).

[0126] 2. Staining of tissue sections

[0127] Hematoxylin and eosin (H&E) staining: After dehydration, paraffin embedding, and paraffin sectioning, tissue sections were stained with H&E. After hydration, they were stained with hematoxylin and eosin sequentially, dehydrated and dried, mounted with neutral resin, and observed.

[0128] Immunohistochemical staining: After dewaxing and hydration, tissue sections were treated with 3% hydrogen peroxide at room temperature for 15 min, followed by microwave antigen retrieval (sequentially high heat for 5 min, thawing for 2 min, and medium-low temperature for 20 min), blocked with blocking solution, incubated overnight with primary antibody (TP63 (Cat #ab53039, Abcam), SOX2 (Cat #ab92494, Abcam), FOXA2 (Cat #ab108422, Abcam), NKX2-1 (Cat #ab133638, Abcam), KRT5 (Cat # BS1208, Bioworld), Cleaved Caspase 3 (Cat #9661, CST)), incubated with secondary antibody (goat anti-rabbit IgG polymer) at 37℃ for 15 min, developed with DAB for 5 min, counterstained with hematoxylin, dehydrated and dried, mounted with neutral resin, and observed.

[0129] Immunofluorescence staining: After dewaxing and hydration, tissue sections were treated with 3% hydrogen peroxide at room temperature for 15 min, followed by microwave antigen retrieval (sequentially high heat for 5 min, thawing for 2 min, and medium-low temperature for 20 min), blocked with blocking solution, incubated overnight with primary antibody (Ki67 (Cat #NB500-170, Novus)), and incubated with secondary antibody (anti-rabbit IgG-488) at room temperature for 40 min. After drying, the sections were mounted for observation.

[0130] 3. Statistical Analysis

[0131] All experimental data were analyzed using Student's t-test, and P < 0.05 was considered significant.

[0132] Example 1: Knockout of the LSD1 gene

[0133] In this embodiment, a Kras / Lkb1 / Lsd1 mouse model was constructed. After adenovirus-induced knockout of the LSD1 gene in mice, tumor burden, tumor number, H&E staining, and immunohistochemical staining were observed. These included the following:

[0134] 1. Construction of the Kras / Lkb1 / Lsd1 mouse model

[0135] The construction method of the Kras / Lkb1 (KL) mouse model is described in reference 1 (Ji, H., Ramsey, MR, Hayes, DN, Fan, C., McNamara, K., Kozlowski, P., Torrice, C., Wu, MC, Shimamura, T., Perera, SA, et al. LKB1 modulates lung cancer differentiation and metastasis. Nature (2007) 448: 807-810.). The construction method of the Lsd1 mouse model is described in reference 2 (Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, JR, Cole, PA, Casero, RA, and Shi, Y. (2004). Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941-953).

[0136] The Kras / Lkb1 / Lsd1 mouse model is obtained by crossing the Kras / Lkb1 (KL) model mouse with the Lsd1 model mouse. The resulting hybrid offspring are the Kras / Lkb1 / Lsd1 mouse model.

[0137] 2. Knockout of the LSD1 gene

[0138] KL model mice were used as the control group, and KLL model mice were used as the experimental group. Each mouse in both the control and experimental groups received 2×10⁻⁶ mice via nasal inhalation. 6 Pfu Ad-Cre recombinant adenovirus was used. After 10 weeks, mice were sacrificed, dissected, and lung tissue was extracted for histological and pathological analysis. Histological analysis included weighing to analyze tumor burden and counting to analyze tumor number. Pathological analysis included preparing paraffin sections of lung tissue for subsequent H&E staining and immunohistochemical staining. The prepared H&E-stained sections were subjected to routine pathological observation and compared with the results of immunohistochemical staining.

[0139] Figure 1This is a flowchart illustrating the construction process of the Kras / Lkb1 / Lsd1 (KLL) mouse model and the Kras / Lkb1 (KL) mouse model in this embodiment. G12D refers to the mutation of glycine G at position 12 of the Kras gene to aspartic acid D; LSL-G12D stands for Loxp-Stop-Loxp KrasG12D, which means that after adenovirus infection, the Stop element is cleaved, and KrasG12D is expressed.

[0140] Figure 2 This is a statistical graph showing the tumor burden of KL and KLL model mice after LSD1 gene knockout in this embodiment. KL (n=4), KLL (n=6); ** represents P<0.01.

[0141] from Figure 2 It can be seen that knocking out the LSD1 gene significantly reduced the overall tumor burden in KLL model mice (P < 0.05).

[0142] Figure 3 This is a statistical chart showing the number of tumors in KL and KLL model mice after the LSD1 gene was knocked out in this embodiment. KL (n=4), KLL (n=6); ns represents P>0.05.

[0143] from Figure 3 It was found that after knocking out the LSD1 gene, there was no significant difference in the number of tumors between the KL model mice and the KLL model mice (P>0.05).

[0144] The proportion of mice developing adenosquamous carcinoma symptoms in the control and experimental groups was counted to obtain the incidence of adenosquamous carcinoma.

[0145] Figure 4 This is a statistical chart showing the incidence of adenosquamous carcinoma in KL and KLL model mice after LSD1 gene knockout in this embodiment. KL (n=4), KLL (n=6); AdSCC represents adenosquamous carcinoma.

[0146] from Figure 4 It is known that LSD1 gene knockout completely inhibits the transdifferentiation of lung adenocarcinoma to adenosquamous carcinoma, and thus can inhibit the transdifferentiation of adenosquamous carcinoma to lung squamous carcinoma.

[0147] Figure 5 This is a tumor burden graph showing the lung adenocarcinoma and lung squamous cell carcinoma tissues of KL and KLL model mice after LSD1 gene knockout in this embodiment. In the graph, ADC represents lung adenocarcinoma, SCC represents lung squamous cell carcinoma; KL (n=4), KLL (n=6), ** represents P<0.01, and ns represents P>0.05.

[0148] from Figure 5It was found that when lung adenocarcinoma and lung squamous cell carcinoma tissues in mouse models were analyzed separately, the tumor burden of both lung adenocarcinoma and lung squamous cell carcinoma was reduced. However, the reduction in tumor burden of lung adenocarcinoma was not significant (P>0.05), while the tumor burden of lung squamous cell carcinoma was significantly reduced (P<0.05). This indicates that knocking out the LSD1 gene can significantly reduce the tumor burden of lung squamous cell carcinoma.

[0149] Figure 6 This is a statistical chart showing the number of lung adenocarcinoma and squamous cell carcinoma tissues in KL and KLL model mice after LSD1 gene knockout in this embodiment. In the chart, ADC represents lung adenocarcinoma, SCC represents lung squamous cell carcinoma; KL (n=4), KLL (n=6), ** represents P<0.01, and ns represents P>0.05.

[0150] from Figure 6 It can be seen that after LSD1 gene injection, there was no significant difference in the number of lung adenocarcinomas between KL and KLL model mice (P>0.05), but the number of lung squamous cell carcinomas was significantly reduced (P<0.05), indicating that lung squamous cell carcinoma was completely suppressed.

[0151] Figure 7 The images show H&E staining and immunohistochemical staining of KL and KLL model mice after the LSD1 gene was knocked out in this embodiment.

[0152] from Figure 7 It was found that in the control group of Kras / Lkb1 (KL) model mice, lung tissue sections stained with H&E showed typical squamous structures, were negative for lung adenocarcinoma markers (NKX2-1 and FOXA2), and were positive for lung squamous cell carcinoma markers (P63, SOX2, and KRT5), indicating that the mouse tumors exhibited characteristics of lung squamous cell carcinoma. In contrast, in the experimental group of Kras / Lkb1 / Lsd1 (KLL) model mice, H&E staining showed typical glandular structures, were positive for lung adenocarcinoma markers (NKX2-1 and FOXA2), and were negative for lung squamous cell carcinoma markers (P63, SOX2, and KRT5), indicating that the mouse tumors exhibited characteristics of lung adenocarcinoma.

[0153] Example 2: Treatment with LSD1 inhibitors

[0154] In this embodiment, the existing LSD1 inhibitor drug GSK2879552 was used to treat KL mouse tumors to investigate whether this drug has a similar effect on inhibiting LSD1 as knocking out LSD1 in mice. This includes the following:

[0155] The KL model group started nasal inhalation of 2×10⁻⁶ oz. from week 0. 6PFU Ad-Cre recombinant adenovirus was used to treat mice with GSK2879552. The mice were administered GSK2879552 via intraperitoneal injection daily for 4 weeks. At week 10, the mice were sacrificed, dissected, and lung tissue was collected for histological and pathological analysis. Histological analysis included weighing to analyze tumor burden and counting tumors. Pathological analysis included preparing paraffin sections of lung tissue for subsequent H&E staining and immunohistochemical staining. The prepared H&E-stained sections were subjected to routine pathological observation, and the results were compared with those obtained from immunohistochemical staining.

[0156] The control group differed from the experimental group in that it did not receive GSK2879552 in week 6, but instead received 1.5 mg / kg of normal saline via intraperitoneal injection daily; otherwise, it was the same as the experimental group.

[0157] Figure 8 This is a schematic diagram of GSK2879552 treatment of KL model mice in this embodiment.

[0158] Figure 9 This is a statistical graph showing the tumor burden of KL model mice after treatment with GSK2879552 and saline, respectively, in this embodiment. Wherein, Ctrl (n=5), GSK2879552 (n=5); ** represents P < 0.01.

[0159] from Figure 9 It can be seen that GSK2879552 treatment significantly reduced the tumor burden in KL model mice.

[0160] Figure 10 This is a statistical chart showing the number of tumors in KL model mice after treatment with GSK2879552 and saline, respectively, in this embodiment. Wherein, Ctrl (n=5), GSK2879552 (n=5); ns represents P>0.05.

[0161] from Figure 10 It can be seen that GSK2879552 treatment significantly reduced the number of tumors in KL model mice.

[0162] Figure 11 This is a statistical chart showing the incidence of adenosquamous carcinoma in KL model mice after treatment with GSK2879552 and saline, respectively, in this embodiment. In the chart, Ctrl (n=5), GSK2879552 (n=5), and AdSCC represent adenosquamous carcinoma.

[0163] from Figure 11 It can be seen that GSK2879552 treatment still significantly inhibited the metadifferentiation of lung adenocarcinoma to adenosquamous carcinoma, and thus could inhibit the metadifferentiation of adenosquamous carcinoma to lung squamous carcinoma.

[0164] Figure 12 This is a statistical graph showing the tumor burden of lung adenocarcinoma and lung squamous cell carcinoma tissues in KL model mice after treatment with GSK2879552 and saline, respectively, in this embodiment. In the graph, ADC represents lung adenocarcinoma, SCC represents lung squamous cell carcinoma; Ctrl (n=5), GSK2879552 (n=5); ** represents P<0.01, ns represents P>0.05.

[0165] from Figure 12 It can be seen that after treatment with GSK2879552, the tumor burden of squamous cell carcinoma of the lung in KL model mice was significantly reduced, while the tumor burden of adenocarcinoma of the lung was reduced, but not significantly. Figure 13 This is a statistical chart showing the number of lung adenocarcinoma and lung squamous cell carcinoma tissues in KL model mice after treatment with GSK2879552 and saline, respectively, in this embodiment. In the chart, ADC represents lung adenocarcinoma, SCC represents lung squamous cell carcinoma; Ctrl (n=5), GSK2879552 (n=5); ** represents P < 0.01, * represents P < 0.05.

[0166] from Figure 13 It can be seen that after treatment with GSK2879552, the number of tumors in lung squamous cell carcinoma tissue in the KL model was significantly reduced, while the number of tumors in lung adenocarcinoma tissue increased to some extent, but not significantly.

[0167] Figure 14 The images show H&E staining and immunohistochemical staining of mice in the control and experimental groups in this embodiment. Ctrl represents the control group, and GSK2879552 represents the experimental group.

[0168] from Figure 14 It was found that H&E staining in the control group mice showed typical squamous structures, negative for lung adenocarcinoma markers (NKX2-1 and FOXA2), and positive for lung squamous cell carcinoma markers (P63, SOX2, and KRT5), indicating that the mouse tumors exhibited characteristics of lung squamous cell carcinoma. After treatment with GSK2879552, H&E staining showed typical glandular structures, positive for lung adenocarcinoma markers (NKX2-1 and FOXA2), and negative for lung squamous cell carcinoma markers (P63, SOX2, and KRT5), indicating that the mouse tumors exhibited characteristics of lung adenocarcinoma.

[0169] In this embodiment, the overall results show that GSK2879552 can significantly inhibit the transdifferentiation of adenosquamous carcinoma.

[0170] Example 3: Investigation of the effects of LSD1 inhibitors on apoptosis and proliferation of lung squamous cell carcinoma cells.

[0171] In this embodiment, KL model mice treated with GSK2879552 in Example 2 were used as the research subjects to investigate the effects of LSD1 inhibitors on the proliferation and apoptosis of lung squamous cell carcinoma. The steps included are as follows:

[0172] 1. Investigation of the effect of LSD1 inhibitors on the proliferation of lung squamous cell carcinoma

[0173] After antigen retrieval, the paraffin sections obtained in Example 2 were washed with PBS, blocked with BSA for 1 hour, incubated with Ki67 at 4°C overnight, washed with PBS, incubated with secondary antibody and DAPI at room temperature in the dark for 40 minutes, washed with PBS, mounted with glycerol, and subjected to immunofluorescence microscopy to count the Ki67 positive cell rate (positive cells are marked with red arrows in the figure).

[0174] Figure 15 This is a representative Ki67 immunofluorescence image of KL model mice treated with GSK2879552 in this embodiment.

[0175] from Figure 15 It can be seen that the proliferation ability of lung squamous cell carcinoma cells was weakened after GSK2879552 treatment of KL model mice.

[0176] Figure 16 This is a statistical graph of Ki67 immunofluorescence in KL mice after treatment with GSK2879552 in this embodiment. **** represents P < 0.0001.

[0177] from Figure 16 It was found that after treatment of KL mice with GSK2879552, the number of Ki67 positive cells was significantly reduced (P < 0.0001).

[0178] 2. Investigation on the effect of LSD1 inhibitors on apoptosis in lung squamous cell carcinoma

[0179] Immunohistochemical observation of cleaved caspase 3 was performed and the percentage of cleaved caspase 3 positive cells was counted (positive cells are marked with red arrows in the figure).

[0180] The immunohistochemical steps for Cleaved Caspase 3 are as follows: The paraffin sections obtained in Example 2 were dewaxed, hydrated, treated with 3% hydrogen peroxide at room temperature for 15 min, microwave antigen retrieval (sequentially high heat for 5 min, thawing for 2 min, and medium-low temperature for 20 min), blocked with blocking buffer, incubated with Cleaved Caspase 3 antibody overnight, incubated with secondary antibody at 37°C for 15 min, developed with DAB for 5 min, counterstained with hematoxylin, dehydrated and dried, mounted with neutral resin, and the Cleaved Caspase 3 positive cell rate was observed and counted.

[0181] Figure 17This is a representative immunohistochemical image of cleaned Caspase 3 in KL model mice after treatment with GSK2879552 in this embodiment.

[0182] from Figure 17 It can be seen that GSK2879552 treatment of KL model mice increased apoptosis of lung squamous cell carcinoma cells.

[0183] Figure 18 This is a statistical graph showing the percentage of Cleaved Caspase 3 positive cells in KL model mice after treatment with GSK2879552 in this embodiment. *** represents P < 0.001.

[0184] from Figure 18 It can be seen that after treating KL model mice with GSK2879552, the rate of Cleaved Caspase 3 positive cells increased significantly.

[0185] The results of the examples show that GSK2879552 can inhibit the proliferation of lung squamous cell carcinoma and promote apoptosis in lung squamous cell carcinoma.

[0186] This invention utilizes a mouse model of spontaneous adenosquamous carcinoma transdifferentiation to discover that LSD1 knockout can inhibit adenosquamous carcinoma transdifferentiation. Treatment of KL model mice with the LSD1 inhibitor GSK2879552 also inhibited adenosquamous carcinoma transdifferentiation. Furthermore, the LSD1 inhibitor GSK2879552 also inhibited the proliferation of lung squamous cell carcinoma and promoted apoptosis. These experimental results demonstrate that LSD1 is an important regulator of adenosquamous carcinoma transdifferentiation and can serve as a target for anti-lung adenocarcinoma treatment. Targeting LSD1 may also be a potential therapeutic strategy for adenosquamous carcinoma and drug resistance induced by adenosquamous carcinoma transdifferentiation.

[0187] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The use of LSD1 gene as a target in the preparation of drugs that inhibit the metadifferentiation of lung adenocarcinoma into lung squamous cell carcinoma, wherein knocking out LSD1 gene inhibits the metadifferentiation of lung adenocarcinoma into lung squamous cell carcinoma, wherein the carcinoma is non-small cell lung cancer.

2. Use of an LSD1 inhibitor in the preparation of a drug for inhibiting the metadifferentiation of lung adenocarcinoma into lung squamous cell carcinoma, wherein the LSD1 inhibitor is GSK2879552 and the carcinoma is non-small cell lung cancer.

3. The use as described in claim 2, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

4. The use of a substance that detects LSD1 in the preparation of a product, wherein the product has the following function: assessing whether lung adenocarcinoma has transformed into lung squamous cell carcinoma; if LSD1 is highly expressed, it is assessed as lung adenocarcinoma transforming into lung squamous cell carcinoma, wherein the carcinoma is non-small cell lung cancer.

5. The use as described in claim 4, characterized in that, The product is selected from one or more of the following: reagent kits, membrane strips, and chips.

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