CN117100747A - Application of FOXO protein inhibitor in preparation of medicines for treating gastric adenocarcinoma tumor - Google Patents
Application of FOXO protein inhibitor in preparation of medicines for treating gastric adenocarcinoma tumor Download PDFInfo
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- CN117100747A CN117100747A CN202311179716.8A CN202311179716A CN117100747A CN 117100747 A CN117100747 A CN 117100747A CN 202311179716 A CN202311179716 A CN 202311179716A CN 117100747 A CN117100747 A CN 117100747A
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- cckbr
- foxo
- gastric adenocarcinoma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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- A61K31/47—Quinolines; Isoquinolines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
The application belongs to the technical fields of molecular biology and biological medicine, and particularly relates to application of a FOXO protein inhibitor in preparation of a medicine for treating gastric adenocarcinoma tumor. The application discovers CCKBR (Cholecystokinin B Receptor) positive stomach Dou Gan cell-like tumor cells in gastric adenocarcinoma patients for the first time, specifically exists FOXO (Forkheadbox O) family (FOXO 1, FOXO3 and FOXO 4) high expression in CCKBR+ gastric adenocarcinoma tumor patients, and the high expression of the FOXO is closely related to clinical prognosis. The application provides a novel treatment target of gastric adenocarcinoma of CCKBR+, and the FOXO protein inhibitor AS1842856 can be effectively used for the selection and prognosis evaluation of a treatment scheme of gastric adenocarcinoma, can inhibit the growth of CCKBR positive poorly differentiated gastric Dou Gan cell-like tumor cells and provides a novel possibility for the subsequent targeted treatment of gastric adenocarcinoma.
Description
Technical Field
The application belongs to the technical fields of molecular biology and biological medicine, and particularly relates to application of a FOXO protein inhibitor in preparation of a medicine for treating gastric adenocarcinoma tumor.
Background
Gastric adenocarcinoma is a type of gastric cancer, accounting for 95% of the total gastric cancer, and is malignant by gastric gland cells, and is called gastric adenocarcinoma. Gastric adenocarcinoma still has the problem of difficult treatment and easy recurrence at present, and the reason is mainly because no effective therapeutic target is targeted for killing, however, because clinical gastric adenocarcinoma is extremely heterogeneous, a broad-spectrum effective therapeutic target cannot be selected, so that the selection of the effective therapeutic target is important for gastric adenocarcinoma subpopulations, especially the low-differentiation gastric adenocarcinoma with higher malignancy.
Early studies demonstrated that stem cells can undergo malignant transformation to form stem Cell type gastric adenocarcinoma, such as LGR5 and AQP5 positive stem Cell-like gastric adenocarcinoma, have been clinically demonstrated and have different tumor Cell characteristics (Nat Cell biol.2021dec;23 (12): 1299-1313., J Exp Clin Cancer res.2022nov 14;41 (1): 322, cell Physiol biochem.2015;36 (6): 2447-55). CCKBR (Cholecystokinin B Receptor) positive antral cells belong to +4 Stem cells and are considered tumor initiating cells in mice (Gut.2015 Apr;64 (4): 544-53, cell Stem cell.2020May7;26 (5): 739-754.e8.), but have not been reported in clinical patients. The analysis of CCKBR expression in clinical gastric adenocarcinoma shows that CCKBR+ tumor belongs to stem cell malignant transformation tumor, has extremely poor survival rate, and indicates that CCKBR+ patients are patients with higher malignant degree, and the tumors often show low differentiation and refractory characteristics.
The FOXO family is a class of transcription factors encoded by FOXO genes, mainly comprising three protein family members FOXO1, FOXO3 and FOXO4, which have highly conserved DNA binding domains and thus have similar functions, and FOXO proteins whose activity is tightly regulated in cell homeostasis and plays a key role are often considered life-span genes (particularly FOXO 3), which are mainly associated with aging, autophagy, and stem cell maintenance.
At present, the conventional treatment method of gastric adenocarcinoma is still adopted when the gastric adenocarcinoma tumor of CCKBR+ is treated, and the treatment of the gastric adenocarcinoma tumor of the CCKBR+ is difficult to treat and easy to relapse due to lack of pertinence. Therefore, the treatment of the tumor aiming at CCKBR+ is a difficult problem to be solved in the field, and the research on an effective treatment target of the gastric adenocarcinoma tumor aiming at CCKBR+ and the targeted killing have important clinical significance and practical value. There is no product application of the FOXO protein in treating gastric adenocarcinoma tumor of cckbr+.
Disclosure of Invention
In order to solve the problem that the prior art does not have the purpose of products for treating gastric adenocarcinoma tumor of CCKBR+ through regulating and controlling the FOXO protein, the application discovers that AS1842856 can obviously inhibit the growth of gastric adenocarcinoma tumor cells of CCKBR+ through inhibiting the FOXO protein, and provides the purpose of the FOXO protein inhibitor in preparing medicaments for treating gastric adenocarcinoma tumor.
In order to achieve the above purpose, the application adopts the following technical scheme:
the application provides an application of a FOXO protein inhibitor in preparing a medicine for treating gastric adenocarcinoma tumor.
Preferably, the gastric adenocarcinoma tumor is a cckbr+ gastric adenocarcinoma tumor.
Preferably, the FOXO protein inhibitor is AS1842856, and the AS1842856 is targeted to inhibit the function of FOXO protein; the structure of the AS1842856 is AS follows:
preferably, the AS1842856 inhibits the growth of gastric adenocarcinoma tumor cells of cckbr+, which are gastric Dou Gan cell-like tumor cells of cckbr+.
Preferably, the AS1842856 targets binding to FOXO protein, inhibiting the transcriptional activity of FOXO protein.
Preferably, the degree of infiltration of the cckbr+ gastric adenocarcinoma tumor is significantly positively correlated with FOXO expression, and the clinical prognosis of the cckbr+ gastric adenocarcinoma tumor is significantly positively correlated with FOXO expression.
Preferably, the expression of FOXO comprises expression of FOXO1, FOXO3 and FOXO4 in the FOXO family.
The application also provides a medicine for treating and preventing and/or treating gastric adenocarcinoma tumor of CCKBR+.
Preferably, the medicament comprises AS1842856 AS claimed in claim 3.
Preferably, the drug is a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, isotopically labeled derivative, stereoisomer or prodrug of AS1842856.
Compared with the prior art, the application has the following beneficial effects:
the application provides an application of a FOXO protein inhibitor in preparing a medicine for treating gastric adenocarcinoma tumor. The application obtains a single cell transcriptome library of gastric adenocarcinoma tumor patients of CCKBR+ for the first time, and discovers that the tumors show low differentiation and refractory property and have no effective treatment targets. In order to identify genes related to pathogenesis of gastric adenocarcinoma tumor of CCKBR+ and find some new therapeutic targets, the application screens the potential therapeutic targets of the low-differentiation tumor formed by malignant transformation of gastric Dou Gan cells of the CCKBR+ by using single cell sequencing technology and in-vitro and in-vivo experiments. The application provides a treatment target for gastric adenocarcinoma tumor aiming at CCKBR+, and discovers that a FOXO protein inhibitor AS1842856 is a potential treatment target for gastric adenocarcinoma transformed by gastric Dou Gan cells aiming at CCKBR+, and AS1842856 can target and bind to the FOXO protein, inhibit the transcriptional activity of the FOXO protein and inhibit gastric adenocarcinoma tumor of CCKBR+, thereby providing a basis for the subsequent gastric adenocarcinoma targeted treatment.
Drawings
FIG. 1 is a single cell transcriptome library of gastric adenocarcinoma tumors that established and identified stem cell transformed CCKBR+ in example 1 of the present application, wherein A is a marker that identifies CCKBR as a subset of epithelial cells Epi8 using single cell sequencing; b is a gastric adenocarcinoma tumor for identifying CCKBR+ and widely expresses a gastric Dou Nianye glandular cell marker, and the tumor cells are proved to be formed by malignant transformation of gastric antrum basal cells; c is identifying that such cells express the antral basal stem cell markers LGR5, AQP5, LRIG1 and A4GNT; d is the cell molecular characteristics of gastric adenocarcinoma tumor cells identified as CCKBR+; e is a transcription factor that enriches for such cell-related factors, including FOXO3 and FOXO4; f is quasi-sequential identification of gastric adenocarcinoma tumor of CCKBR+ as tumor of antral fundus origin; g is the obvious up-regulation of the gastric adenocarcinoma tumor FOXO signal pathway for identifying CCKBR+; h is a FOXO activation level that also identified a higher tumor phenotype of CCKBR+ using an exogenous single cell library GSE 183904.
FIG. 2 shows that the gastric adenocarcinoma tumor found in example 1 of the present application has a poor prognostic power and survival, wherein A is the expression and occupancy of CCKBR in the tumor detected by immunohistochemistry; b is the clinical tumor ratio, tumor position and differentiation of the tumor of the statistical CCKBR+; c is immunofluorescence result to detect the co-expression of CCKBR and CD133, CD44 in gastric adenocarcinoma; gastric adenocarcinoma tumors with D cckbr+ have a poor prognosis in gastric adenocarcinoma; stem cell-like tumor cells with E being cckbr+ exhibit poor prognostic characteristics;
FIG. 3 shows very high expression of FOXO1, FOXO3 and FOXO4 in CCKBR+ gastric adenocarcinoma tumor of example 1 of the present application, wherein A is the FOXO family expression level in the gastric adenocarcinoma tumor detected as CCKBR positive by RT-qPCR (Quantitative Real-time PCR); b is the change of the expression level of FOXO in CCKBR positive and negative tumors; changes in FOXO expression levels of CCKBR positive tumors relative to paracancerous tissues; d is the analysis of the expression correlation of CCKBR and FOXO in tumors;
FIG. 4 shows that very high levels of tumor expression, FOXO1, FOXO3 and FOXO4, and that FOXOS and gastric adenocarcinoma tumor patients in the TCGA database of example 1 of the application are associated with a worse prognosis, wherein A is the detection of changes in the expression level of CCKBR in the TCGA database relative to that of patients with low expression; b is the relationship of prognosis of FOXOS in patients with gastric adenocarcinoma;
FIG. 5 shows that the FOXO protein inhibitor AS1842856 of example 1 of the present application can inhibit the stem cell stem property and inhibit the generation of tumor balls. Wherein A is that when gastric adenocarcinoma cell line is subjected to tumor ball culture to enrich stem cells, the expression of CCKBR, FOXO1, FOXO3 and FOXO4 is detected by RT-qPCR; b is CCKBR and FOXO expression elevation has correlation; c is a bright field picture of the growth state of the tumor cell sphere inhibited by AS1842856 with different concentrations when the tumor cell sphere is treated by FOXO protein inhibitor AS 1842856; d is a statistical plot showing the change in tumor sphere diameter when AS1842856 treats tumor spheres; e is immunofluorescence display AS1842856 for changes in the expression levels of CCKBR and CD133 in tumor spheres when treated with tumor spheres; f is immunofluorescence display of changes in CCKBR and KI67 expression levels of tumor spheres when AS1842856 treated.
FIG. 6 shows that the FOXO protein inhibitor AS1842856 of example 1 of the present application has selective killing effect on stem cell-like CCKBR+ gastric adenocarcinoma tumors, wherein A is HE morphological staining of gastric adenocarcinoma organoids and paired tumor tissues; b is immunohistochemical staining of organoids and paired tumor tissue; c is a bright field picture after organoid treatment by AS 1842856; d is the change of cell viability after organoid treatment by AS 1842856; e is a photograph of a tumor of a mouse 17 days after treatment of MKN45 cell line (CCKBR positive) loaded severely immunodeficient mice with AS 1842856; f is the result of statistics of mice tumors 17 days after treatment of MKN 45-loaded severely immunodeficient mice with AS 1842856; g is a tumor growth curve for treatment of MKN 45-loaded severely immunodeficient mice with AS1842856.
Detailed Description
The present application will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the application. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Example 1
1. Establishment and identification of a stem cell transformed CCKBR+ gastric adenocarcinoma tumor single cell transcriptome library
5 fresh samples of surgically excised gastric adenocarcinoma tumor were placed in sterile ribonuclease (RNase) -free culture dishes containing calcium-and magnesium-free 1 XPBS (NaCl 8.0g per liter of distilled water; KCl 0.2g Na) 2 HPO4 1.44g；KH 2 PO 4 0.24 g). Tumor tissue was transferred to a petri dish, cut into 0.5 mm-sized pieces of tissue using an ophthalmic scissors and a scalpel, washed with 1x PBS, and excess tissue, such as blood clots, connective tissue, and fat layers, were removed. Then the solution was separated with tissue (per 100mLThe tumor tissue was incubated with 0.35g of collagenase IV,200mg of papain, 12000 units of DNase I) in PBS and shaken for 20 minutes at 100rpm in a water bath at 37℃to give a single cell suspension. The single cell suspension was stopped from digestion with 1 XPBS and filtered through a 70-mesh filter. The filtered suspension was resuspended in 100. Mu.l of dead cell removal microparticles (MAC 130-090-101) and usedDead cell removal kit (MAC 130-090-101) removes dead cells. 13000 cells were captured for each sample using 10X Genomics Chromium Single-Cell 3' kit (V3) and cDNA synthesis and single Cell library construction were performed as follows:
cDNA synthesis:
the Master Mix was formulated on ice and the composition of the Master Mix is shown in table 1.
TABLE 1 composition of Master Mix
| Component (A) | 1x(μl) | 8x(μl) |
| RT Reagent B | 18.8 | 165.0 |
| Template Switch Oligo | 2.4 | 20.8 |
| Reducing Agent B | 2.0 | 17.3 |
| RT Enzyme C | 8.7 | 76.8 |
| Total | 31.8 | 279.8 |
31.8 μl Master Mix was dispensed for use in eight consecutive rows (on ice). Mixing Master Mix and the dissociated cells to 70 μl, adding into the well, shaking Gel Beads (-80deg.C, taking out, standing at room temperature for 30 min), attaching 10×Gasket seal, and running 10×machine.
100 μl of water-in-oil was pipetted into a new pre-chilled eight-row for PCR reactions. The reaction procedure is shown in table 2:
TABLE 2 Single cell transcriptome PCR reaction procedure
| Lid Temperature | Reaction Volume | Run Time |
| 53℃ | 125μl | 55min |
| Step | Temperature | Time |
| 1 | 53℃ | 45min |
| 2 | 85℃ | 5min |
| 3 | 4℃ | Hold |
125. Mu.l of Recovery Agent was added to the reaction solution (mixing was not performed) and allowed to stand at room temperature for 2 minutes, and 125. Mu.l of pink Recovery Agent/Partitioning Oil was discarded.
Dynabeads Cleanup Mix was formulated as in table 3; the solution buffer I was prepared according to Table 4; 200 mu l Dynabeads Cleanup Mix is added into the obtained reaction solution, uniformly mixed, kept at room temperature for 10min, placed on a magnetic rack until the reaction solution is clear, the supernatant is discarded, 300 mu l of freshly prepared 80% ethanol is added, and the reaction solution is discarded at room temperature for 30 s. 200 μl of freshly prepared 80% ethanol was added, the supernatant was discarded at room temperature for 30s, centrifuged gently, placed on a magnetic rack, the remaining alcohol removed, and air dried for 1min. Then 35.5 mu l Elution BufferI was added, mixed well, left on a magnetic rack for 1min at room temperature until clear, and 35. Mu.l of supernatant was aspirated into a new tube.
Table 3 composition of 3 Dynabeads Cleanup Mix
| Dynabeads Cleanup Mix | 1x(μl) | 8x(μl) |
| Clearup Buffer | 182 | 1602 |
| Dynabeads MyOne SLANE | 8 | 70 |
| Reducing Agent B | 5 | 44 |
| Nulear-free Water | 5 | 44 |
Table 4 composition of 4 Elution Buffer I
Configuration cDNA Amplification Reaction Mix PCR reactions were performed: mu.l of Amp Mix obtained in the above step and 15. Mu.l of cDNA Primers were mixed to obtain cDNA Amplification Reaction Mix. cDNA Amplification Reaction Mix and 35 mu l purified GEM RT were mixed and subjected to PCR. The reaction procedure is shown in table 5:
TABLE 5 cDNA amplification PCR reaction procedure
| Lid Temperature | Reaction Volume | Run Time |
| 105℃ | 100μl | 45min |
| Step | Temperature | Time |
| 1 | 98℃ | 3min |
| 2 | 98℃ | 15sec |
| 3 | 63℃ | 20sec |
| 4 | 72℃ | 1min |
| 5 | Go to step 2 for11 cycles | / |
| 6 | 42℃ | 1min |
| 7 | 4℃ | Hold |
The cDNA quality control and quantification were performed as follows: and adding 60 mu l SPRIselect Reagent into the product after the reaction is finished, uniformly mixing, standing at room temperature for 5min, placing on a magnetic rack until the product is clear, and discarding the supernatant. 200 μl of freshly prepared 80% ethanol was added, the supernatant was discarded for 30s at room temperature, repeated once, and the precipitate was dried. Add 40.5. Mu.l Buffer EB, mix well, place on a magnetic rack for 1min at room temperature until clear, pipette 40. Mu.l supernatant into new tube, use Qubit quality and Agilent 2100 quality, and record the concentration.
Single cell library construction was performed next:
formulation Fragmentation Mix (reagents and samples must be formulated on ice throughout): each sample was added 5. Mu. l Fragmentation Buffer and 10. Mu. l Fragmentation Enzyme and mixed well.
Each sample was taken with 10. Mu.l of cDNA obtained above, 25. Mu.l of Buffer EB, 15. Mu. l Fragmentation Mix was added, and then mixed well. Next, PCR reactions were performed, and the reaction procedures are shown in Table 6:
TABLE 6 cDNA fragmentation PCR reaction procedure
| Lid Temperature | Reaction Volume | Run Time |
| 65℃ | 50μl | 35min |
| Step | Temperature | Time |
| Pre-cool block | 4℃ | Hold |
| Fragmentation | 32℃ | 5min |
| End Repair and A tailing | 65℃ | 30min |
| Hold | 4℃ | Hold |
Then adding 30 mu l SPRIselect Reagent of people, uniformly mixing, standing for 5min at room temperature, and setting on a magnetic rack until the mixture is clear. Adding 75 μl of supernatant into new tube, adding 10 μ l SPRIselect Reagent, mixing, standing at room temperature for 5min, standing on magnetic rack until clear, and removing supernatant. 125 μl of 80% ethanol was added, the supernatant was discarded at room temperature for 30s, and the sample was dried. Add 50.5. Mu.l Buffer EB and mix well, room temperature for 1min, magnetic rack until clear, pipette 50. Mu.l supernatant into new tube.
Adaptor Ligation Mix was prepared according to Table 7, and 50. Mu. lAdaptor Ligation Mix and 50. Mu.l of the product obtained by the above reaction were mixed and incubated at 20℃for 15 minutes.
Adding 80 mu l SPRIselect Reagent, mixing, standing at room temperature for 5min, standing on a magnetic rack until the mixture is clear, and discarding the supernatant. 200 μl of freshly prepared 80% ethanol was then added, the supernatant was discarded for 30s at room temperature, and repeated once, air-dried. Add 30.5. Mu.l Buffer EB, mix well, room temperature for 2min, place the magnetic rack until the solution is clear, suck 30. Mu.l supernatant into new tube. To the tube, 50. Mu.l of Amp Mix, 10. Mu.l of SIPrimer, 60. Mu. l Sample Index PCR Mix, 10. Mu. l individual Chromium i7 Sample Index was added, and after mixing well, the PCR reaction was performed, and the reaction steps are shown in Table 8.
Table 7 composition of 7 Adaptor Ligation Mix
| Adaptor Ligation Mix | 1x(μl) | 8x(μl) |
| Ligation Buffer | 20 | 176 |
| DNA Ligase | 10 | 88 |
| Adaptor Oligos | 20 | 176 |
| Total | 50 | 440 |
TABLE 8 library construction PCR reaction procedure
| Lid Temperature | Reaction Volume | Run Time |
| 105℃ | 100μl | 40min |
| Step | Temperature | Time |
| 1 | 98℃ | 45sec |
| 2 | 98℃ | 20sec |
| 3 | 54℃ | 30sec |
| 4 | 72℃ | 20sec |
| 5 | Go to step 2 for 16 cycles | |
| 6 | 72℃ | 1min |
| 7 | 4℃ | Hold |
Adding 60 mu l SPRIselect Reagent of the product after the reaction, fully and uniformly mixing, standing for 5min at room temperature, and placing on a magnetic rack until the mixture is clear. 150 μl of the supernatant was placed in a fresh tube, 20 μ l SPRIselect Reagent was added, mixed well, left at room temperature for 5min, placed on a magnetic rack until the solution was clear, and then the supernatant was removed. 200 μl of 80% ethanol was added, the supernatant was discarded at room temperature for 30s, repeated once, and the precipitate was dried sufficiently. Add 35.5. Mu.l Buffer EB, mix well, room temperature for 2min, place the magnetic rack until the solution is clear, draw 35. Mu.l supernatant into new tube. The constructed single cell library was subjected to high throughput sequencing using an Illumina Nova 6000 pe150 machine.
The sequenced data were subjected to data format conversion by normalization procedure, reference genome markers and quality control (Cell Ranger (vision 5.0.0), seurat (vision 3.1.1), doubletFinder package (vision 2.0.2)). All cells were divided into 12 cell subsets by dimension-reduction clustering, and each cell subset marker was examined to find that the gastric adenocarcinoma tumor cell population of cckbr+ was epithelial cell subset 8 (Epi 8). Analysis by examination of gastric myxocervical cells and basal stem cell markers in Epi8 showed that Epi8 originated from the fundus of stomach Dou Ji. And cell mass analysis was performed by selecting the first 2000 characteristic protein-encoding genes and using a high geometry distribution test to calculate the p-value representing whether the GO (Gene Ontology) functional set is significantly enriched in the list of differential protein-encoding genes. Then, P values were subjected to Benjamini & Hochberg multiplex tests for Q value significance analysis.
With respect to TF (Transcription Factor) enrichment, we entered multiple gene lists (https:// metarecipe. Org /), and the gene list enrichment was determined in the ontological TRRUST script. All genes in the genome were used as enrichment background. p-value <0.01, lowest count 3, enrichment factor >1.5 (enrichment factor is the ratio between observed count and occasional expected count) is collected and grouped into clusters according to their member similarity. The algorithm is the same as that used for pathway and process enrichment analysis. The same operation is performed on the GSE183904 except for sample collection and library establishment, and finally, the activation sequence of related transcription factors and signal paths is obtained. FOXO was found to exhibit abnormal activation in both detection results.
As shown in FIG. 1, the application establishes and identifies a single cell library of gastric adenocarcinoma formed by malignant transformation of stem cells of CCKBR+, and analyzes that FOXO is a key transcription factor and signal path of gastric adenocarcinoma tumor for maintaining CCKBR+.
2. Identification of CCKBR+ gastric adenocarcinoma tumor and hypodifferentiation type and poor patient prognosis correlation
We used immunohistochemical techniques to identify and analyze CCKBR expression in gastric adenocarcinoma, and studied specifically as follows:
paraffin sections were immersed in xylene I for 20min and xylene II for 20min sequentially. Immersing in absolute ethanol I for 10min, absolute ethanol II for 10min,95% ethanol for 5min,80% ethanol for 5min, and 70% ethanol for 5min, and washing with double distilled water for 2min each time. Then placing paraffin tissue slice into repairing box, then adding proper quantity of sodium citrate buffer solution (Zhongsejin bridge, ZLI-9064), immersing the tissue into liquid surface, placing repairing box into microwave oven, repairing with microwave high fire for 8min, stopping for 2min, and heating with high fire for 2min, so that the tissue is not dried. Taking out the repairing box from the microwave oven, naturally cooling, taking out the glass slide after the repairing liquid is cooled to room temperature, and flushing with PBS for 3 times for 3min each time. The prepared hydrogen peroxide (diluted to 3% concentration with double distilled water) was added dropwise to the slice tissue to block endogenous catalase to reduce background staining, incubated for 15min at room temperature, and washed three times with pbs for 3min each. Approximately 50. Mu.l of goat serum was added dropwise to the slide and incubated at 37℃for one hour to reduce nonspecific staining. The liquid surrounding the slide was blotted with blotter paper, circled around the tissue with an immunohistochemical pen, and 100 μl of diluted primary antibody (wherein CCKBR antibodies were purchased from Boster, diluted 1:300, CD133, KI67 and CD44 antibodies were purchased from Biolegend, diluted 1:100) was then added dropwise. After the dripping of the primary antibody, the sections are placed in a wet box at 4 ℃ overnight (more than or equal to 12 h).
Taking out the slice from the refrigerator, incubating at room temperature for 15min, rewarming, washing the slice with PBS for 3 times, each time for 3min, drying the slice with absorbent paper, and dripping biotin-labeled secondary antibody (Zhongsejin bridge, SAP-9100), and incubating at 37deg.C for 30min. The sections were washed 3 times with PBS for 3min each, and horseradish enzyme-labeled streptavidin working solution (Zhonghua gold bridge, SAP-9100) was added dropwise and incubated for 15min at room temperature. The sections were washed 3 times with PBS for 3min each, dried with water absorbing paper after PBS was spun dry, 100. Mu.l of freshly prepared DAB chromogenic solution (Zhongsequoia gold bridge, ZLI-9017) was added dropwise to each section, and after 3min staining, the development was terminated by washing with tap water. Then counterstained with 100 μl hematoxylin (Zhonghua gold bridge, BSBA-4021) for 10 seconds, washed with water, differentiated with 1% ethanol hydrochloride for 1 minute, and rinsed with tap water back for 15 minutes. The slices were then placed in sequence: the materials are dehydrated and transparent in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, absolute ethanol I, absolute ethanol II, xylene I and xylene II, each reagent is placed for a plurality of seconds, and finally the dry slice is air-dried in a fume hood. The sections were air-dried and observed under a microscope using a neutral resin sealer.
Finally, 59 clinical sections (the samples taken from the 59 clinical sections are stomach adenocarcinoma pathological tissue sections from the first affiliated hospital of the university of Anhui medical science, and the preparation method is as described above) are subjected to statistical analysis, and the statistical method adopts chi-square test. In the survival related index, a Kaplan-Meier graph is analyzed by using a Kaplan-Meier Plotter website, and a violin graph is drawn by using Cloupe software.
The results are shown in FIG. 2, and the results of immunohistochemistry show that CCKBR+ gastric adenocarcinoma tumor shows low differentiation and high malignancy, and that the tumor cells show more CD44 and CD133 co-localization and more tumor stem cell characteristics. And Kaplan-Meier and violin images demonstrate that this type of tumor is associated with a worse prognosis for the patient.
3. It was verified that CCKBR+ gastric adenocarcinoma tumor expressed more FOXO protein
The application adopts RT-qPCR to detect whether the clinical CCKBR+ gastric adenocarcinoma tumor expresses FOXO at the same high level. The RNA was extracted using the RNeasy Mini Kit (74104) from QIAGEN. The method comprises the following specific steps:
a proper amount of BufferRLT (500 μl) was added to fresh tumor tissue of gastric adenocarcinoma, and the tissue was ground for 5 minutes to destroy the tissue and release RNA. Then, 500. Mu.l of 70% ethanol was added thereto, and after being blown and mixed uniformly, it was transferred to RNeasy spin column and centrifuged at 12000rpm for 15sec. The centrifuged lower liquid was discarded. Then 700. Mu.l of BufferRW1 was added and covered, and the spin-column membrane was washed by centrifugation at 12000rpm for 15 s. The centrifuged lower liquid was discarded. Then 500. Mu.l Buffer RPE was added to RNeasy spin column. Centrifuge 12000rpm was put into a centrifuge for 15sec. The centrifuged lower liquid was discarded. Then, 500. Mu.l of Buffer RPE was added to the upper layer, and the mixture was centrifuged at 12000rpm for 2 minutes, and the lower layer was discarded. RNeasy spin column was placed in a new 2ml collection tube and centrifuged for 3min to place RNeasy spin column in a new 1.5ml collection tube. Mu.l of RNase-free ddH was added 2 O. The lid was closed and centrifuged at 12000rpm for 1min to elute RNA. Finally obtaining the tissue RNA solution.
The RNA obtained by extraction was reverse transcribed with ToloScript ALL-in-one RT EasyMix for qPCR (22107) of TOLOBIO to obtain the corresponding cDNA. The detailed operation is as follows:
the reaction system was prepared by first melting each reagent component on ice according to table 9:
TABLE 9 reverse transcription reaction System
The reaction procedure is: 50℃for 15min, then 85℃for 5sec.
After completion of the reaction, the obtained cDNA was subjected to RNase-free ddH 2 O advancesRow 5-fold dilution. qPCR reactions were then performed. qPCR was performed using the TOLOBIO 2X Q3 SYBR Qpcr Master Mix (22204) kit. The reaction system is as follows: mix 10 μl, ddH2O 3 μl, primer Forward 1 μl, primer Reverse 1 μl, template 5 μl. Reaction in Bio-Rad CFX96 TM In a Touch machine. The reaction procedure is shown in table 10:
TABLE 10 RT-qPCR reaction procedure
The primer sequences used for the above reactions were:
FOXO1 FORWARD 5’-AAACACCAGTTTGAATTCACCC-3’；REVERSE 5’-TCGACTTATTGTCCTGAAGTGT-3’；
FOXO3 FORWARD 5’-AGCCGAGGAAATGTTCGTC3’；REVERSE 5’-CCTTATCCTTGAAGTAGGGCAC-3’；
FOXO4 FORWARD 5’-CAAGAAGAAACCATCTGTGCTG’；REVERSE 5’-ATATCGGCTTCTTCACGGTTTC-3’。
as shown in figure 3, the immunohistochemistry and RT-qPCR tests prove that the high expression condition of FOXO families (FOXO 1, FOXO3 and FOXO 4) exists in the gastric adenocarcinoma tumor patients with CCKBR+ in a specific mode, and the expression of the FOXO families hardly exists in the gastric adenocarcinoma tumor patients with CCKBR+ in the gastric adenocarcinoma tumor patients with CCKBR, so that the relative results of the gastric adenocarcinoma tumor patients with CCKBR+ and the corresponding paracancerous tissues also show that the FOXO has extremely high expression in the gastric adenocarcinoma tumor with CCKBR+, and the FOXO is hopefully used as a treatment target and is targeted to kill tumor cells.
Tcga database verifies correlated expression profiles of CCKBR and FOXO and clinical prognosis
The application also detects the expression conditions of CCKBR and FOXO from the TCGA STAD database, obtains the original data of TCGA STAD through https:// portal.gdc.cancer.gov/website, distinguishes CCKBR high-expression and low-expression tumors through CCKBR expression, detects the expression conditions related to FOXO, and adopts Kaplan-MeierPlotter website for analysis.
The results are shown in FIG. 4, which demonstrates that similar results are also exhibited in the TCGA STAD database, i.e., the stomach adenocarcinoma tumor tissue with high expression of CCKBR has higher FOXO expression relative to the stomach adenocarcinoma tumor tissue with low expression of CCKBR, and that the FOXO high expression correlates with a poorer prognosis for patients with stomach adenocarcinoma tumor.
Foxo protein inhibitor AS1842856 inhibits CCKBR positive tumor stem cell stem and cell proliferation
Since gastric adenocarcinoma tumor with high expression of CCKBR shows higher FOXO expression, in order to detect whether the inhibition of FOXO can inhibit the growth of tumor with high expression of CCKBR and to detect whether the FOXO is related to tumor stem property, the application adopts a tumor stem cell balling test to detect whether the self-renewal capacity of tumor stem cells is related to the FOXO, and the specific research is as follows:
6 gastric adenocarcinoma cell lines (MKN 45, AGS, MKN7, HGC27, MKN28, HGC 7901) were cultured in an ultra-low adhesion culture dish in the medium DMEM/F12 (containing 2% B27, 20ng/mL EGF,10 ng/mLbFGF) at a constant temperature of 37℃under 5% CO 2 The concentration was changed every two days, and on the third day (MCE, HY-100596) the cell status was observed by treatment with 1uM and 10uM drug (AS 1842856) at the final concentration for 4 days, and the results are shown in FIG. 5.
As can be seen from fig. 5, the tumor stem cells after the balling culture widely increased the expression of CCKBR, FOXO1, FOXO3 and FOXO4, and the expression of CCKBR and the expression of FOXO family members after balling still showed positive correlation, proving that the gastric adenocarcinoma tumor of cckbr+ and FOXO are correlated, while inhibiting the activity of FOXO inhibits the growth of tumor balls, suggesting that the stem properties of the gastric adenocarcinoma tumor cells of FOXO and cckbr+ are correlated.
6. Establishing a gastric adenocarcinoma organoid model and verifying the growth inhibition effect of AS1842856 on CCKBR positive gastric adenocarcinoma organoids
In order to clinically verify that FOXO and cckbr+ gastric adenocarcinoma tumors are related, the application uses gastric adenocarcinoma organoids as experimental carriers for verification, and the organoids have been widely demonstrated to mimic in vivo tumor characteristics and to be almost completely consistent with drug sensitivity and in vivo primary tumors. The application establishes and identifies gastric adenocarcinoma organoids positive for CCKBR, and carries out a drug sensitivity test of FOXO protein inhibitors on the organoids. The establishment method is as follows:
cutting fresh stomach adenocarcinoma tissue sample from antrum into 1-3mm pieces with ophthalmic scissors 3 DMEM/F12 (containing 1.1 x P/S, 1. Mu.g/uLPrimocin, 2.5%FBS, 0.6mg/mL collage 1, 20. Mu.g/mL hyaluronidase and 10. Mu.MY-27632) was added, and the mixture was placed in an incubator at 37℃and the cells were digested at 300rpm for 2 hours to give a single cell suspension. The dissociated single-cell suspension was centrifuged through a 100-mesh sieve to obtain a cell pellet. Mu.l volumes of cell pellet and 100. Mu.l matrigel were mixed and embedded in 12 well plates, and 1mL of organoid medium (DMEM/F12 medium containing 10mM HEPES,2mM GlutaMax,1X B27,1mM N-actylcysteine, 50ng/mL EGF,100ng/mLNoggin,100ng/mLWnt3a,100ng/mL FGF10, 1. Mu.g/mL R-Spondin1, 10nM Gastrin,500nMA-83-01,1. Mu.g/. Mu.l Primocin, 10. Mu.MY-27632) was added for culture, with fluid changes every 2-3 days. Subsequent experiments were performed after organoids had grown to 14 days.
After 14 days of culture of the organoids described above, the application identified organoids. The organoids grown to 14 days later were selected for embedding, and the specific steps were as follows:
organoids in matrigel were blown using 5mL pre-chilled PBS to disaggregate the matrigel and expose organoids. Placing the mixed solution on ice for cold incubation for 30 minutes, placing the mixed solution into a centrifugal machine at 4 ℃ for centrifugation at 2000rpm for 5 minutes to obtain organoid sediment, fixing the sediment for 1 hour by using a pre-cooled 4% paraformaldehyde solution, and placing the solution into the centrifugal machine for centrifugation at 2000rpm for 5 minutes to obtain fixed organoid sediment. Then, 1g of agarose was added to 50mL of PBS and placed in a microwave oven for 2 minutes with high fire to prepare a 2% agarose solution, 1mL of agarose solution was mixed with the organoids uniformly, and immediately placed in a 50mL centrifuge tube for 3 minutes at 1000rpm to obtain organoid-embedded agarose blocks.
The organoid agarose block is then subjected to conventional dehydration, which is performed as follows: the agarose blocks were immersed in formalin solution for 2 hours, transferred to 80% ethanol solution for 3 hours, 95% ethanol solution for 2 hours, 95% ethanol solution for 1 hour, absolute ethanol for 1 hour, xylene absolute ethanol (1:1) for 30 minutes, xylene for 30 minutes, paraffin wax at 60 ℃ for 3 hours. And (5) paraffin embedding is carried out after dehydration. The obtained sections were subjected to immunohistochemical staining to identify whether the obtained organoids were CCKBR+ or CCKBR-gastric adenocarcinoma organoids, the experimental results are shown in FIGS. 5A-B, and we established and identified CCKBR positive and CCKBR negative gastric adenocarcinoma tumors which highly expressed CCKBR, and which were consistent in CCKBR expression and pathological morphology with primary tumors, with high reproducibility in vivo.
In the drug susceptibility test, organoids were first plated in 96-well plates with 10. Mu.l matrigel per well, 100. Mu.l complete medium containing 1. Mu.M and 101. Mu.M AS1842856 was added, and after 6 days, an equal volume of Promega was added3D Cell Viability Assay (G9681) kit, incubating for 30 minutes at normal temperature, detecting the Lumineancence value in the hole by using an enzyme-labeled instrument, namely detecting the activity of the organoid, drawing a line graph according to the detected activity, and obtaining experimental results shown in figures 5C and D. .
Through drug sensitivity tests, we found that administration of AS1842856 to gastric adenocarcinoma tumor organoids of CCKBR+ significantly inhibited organoid growth.
7. In vivo verification of the growth inhibitory effect of AS1842856 on CCKBR-positive gastric adenocarcinoma tumors
In combination with the above results, the present application uses in vivo treatment to observe whether AS1842856 inhibits the growth of cckbr+ gastric adenocarcinoma tumor in mice. The immunodeficient mouse allogeneic tumor test is adopted for verification, and the detailed steps are as follows:
armpit injection of 10 severely immunodeficiency nude mice of 5 weeks size 5X10 6 The MKN45 cells were randomly divided into two groups of 5 each, drug treated and control groups, respectively. Both groups had the same sex and week-old and were tested in the same environment, and mice were given 50mg/kg of AS1842856 intraperitoneally to the drug-treated group daily from the third day, and the control group was intraperitoneally injected with an equal amount of DMSO. Every other periodOne day of mice were measured for tumor size and v=l×w was used for volume calculation 2 The formula/2 is converted.
As a result, AS1842856 was shown in FIG. 6 at 10 -6 The M concentration has obvious inhibition effect on the gastric adenocarcinoma tumor organoid growth of CCKBR+ and can inhibit the proliferation of tumor cells, but the concentration is 10 -5 M concentration has a certain cytotoxicity to tumor. It was demonstrated that AS1842856 did inhibit gastric adenocarcinoma tumor cell proliferation of cckbr+. And has no obvious cell killing effect on gastric adenocarcinoma tumor organoids of CCKBR-. Meanwhile, after nude mice are subjected to tumor formation on MKN45 cells of CCKBR+, AS1842856 can inhibit tumor growth after AS1842856 treatment cells are given, which indicates that AS1842856 is a potential treatment target of gastric adenocarcinoma tumor of CCKBR+.
The present application provides FOXO protein inhibitors: application of AS1842856 in preparing medicine for treating gastric adenocarcinoma tumor of CCKBR+. According to the application, a gastric adenocarcinoma single-cell library of CCKBR+ is constructed according to a single-cell RNA sequencing technology, and the FOXO is found to be a key transcription factor for maintaining the stem property of gastric adenocarcinoma tumor cells of the CCKBR+ through enrichment analysis. Then, through a tumor balling test, an organoid test and a nude mouse nodulation test, the inhibition of FOXO activity through AS1842856 can inhibit the stem property of tumor cells, and further inhibit the growth of the tumor cells. Therefore, the application discovers that AS1842856 has potential application in preparing the gastric adenocarcinoma tumor treatment medicine for treating and/or preventing CCKBR+, can be used for treating the gastric adenocarcinoma tumor of the CCKBR+, and provides a novel medicine or method for treating the gastric adenocarcinoma tumor of the CCKBR+.
The application screens the potential therapeutic target of the low differentiation tumor formed by the malignant transformation of the stomach Dou Gan cells of the FOXO which is CCKBR+ through a single cell sequencing technology and in vivo and in vitro experiments. The application provides a treatment target for gastric adenocarcinoma tumor of CCKBR+, and discovers that a FOXO protein inhibitor AS1842856 is a potential treatment target for gastric adenocarcinoma tumor of CCKBR+ (gastric adenocarcinoma converted by gastric Dou Gan cells of CCKBR+), and AS1842856 can target and bind to the FOXO protein, inhibit the transcriptional activity of the FOXO protein and inhibit gastric adenocarcinoma tumor of CCKBR+), thereby providing basis for the subsequent gastric adenocarcinoma targeted treatment.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present application describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
- Use of foxo protein inhibitor in the preparation of a medicament for the treatment of gastric adenocarcinoma tumor.
- 2. The use according to claim 1, wherein the gastric adenocarcinoma tumor is a cckbr+ gastric adenocarcinoma tumor.
- 3. The use according to claim 1, wherein the FOXO protein inhibitor is AS1842856 and the AS1842856 is targeted to inhibit the function of FOXO protein;the structure of the AS1842856 is AS follows:
- 4. the use according to claim 3, wherein the AS1842856 inhibits the growth of gastric adenocarcinoma tumor cells of cckbr+, which are gastric Dou Gan cell-like tumor cells of cckbr+.
- 5. The use of claim 4, wherein the AS1842856 is targeted to bind FOXO protein, inhibiting the transcriptional activity of FOXO protein.
- 6. The use according to claim 4, wherein the degree of infiltration of the cckbr+ gastric adenocarcinoma tumor is in clear positive correlation with FOXO expression and the clinical prognosis of the cckbr+ gastric adenocarcinoma tumor is in clear positive correlation with FOXO expression.
- 7. The use according to claim 6, wherein the expression of FOXO comprises expression of FOXO1, FOXO3 and FOXO4 in the FOXO family.
- 8. A medicament for the treatment of gastric adenocarcinoma tumors, which prevent and/or treat cckbr+, characterized in that it comprises AS1842856 according to claim 3.
- 9. The medicament of claim 8, wherein the medicament is a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, isotopically labeled derivative, stereoisomer, or prodrug of AS1842856.
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| WO2023076880A1 (en) * | 2021-10-25 | 2023-05-04 | Board Of Regents, The University Of Texas System | Foxo1-targeted therapy for the treatment of cancer |
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