Application of IGFBP5 inhibiting reagent in preparation of medicine for treating malignant progression type recurrent bladder cancer

Abstract

The invention discloses application of a reagent for inhibiting IGFBP5 in preparation of a medicine for treating malignant progression type recurrent bladder cancer, and relates to the technical field of biological medicine. The invention screens insulin-like growth factor binding protein 5 (IGFBP5) as a potential target of recurrent bladder cancer accompanied by malignant progression based on the recurrence evolution trajectory of bladder cancer. A further experiment shows that the target IGFBP5 can overcome the drug resistance of the target IGFBP5 to conventional chemotherapy and can also inhibit the malignant progression of the target IGFBP5. Therefore, the IGFBP5 can be used as a new therapeutic target of the recurrent bladder cancer accompanied by malignant progression, and a reagent for inhibiting the IGFBP5 can be applied to preparation of a medicine for treating the malignant progression type recurrent bladder cancer and possibly has important value in wider epithelial cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of an agent for inhibiting IGFBP5 in preparation of a drug for treating malignant progression type recurrent bladder cancer. BACKGROUND

[0002] Effective treatment of bladder cancer is severely hindered due to its high recurrence rate. Up to 70% of patients who receive postoperative bladder perfusion chemotherapy will eventually experience tumor recurrence, and 20% to 30% of the recurrent tumors will be malignant progression and even metastasis and eventually lead to the death of the patients. The current first-line treatment strategy (such as chemotherapy) has limited effect on recurrent bladder cancer, especially on recurrent bladder cancer with malignant progression, and there is no clear treatment plan. This predicament is largely due to the limited biological understanding of cancer recurrence and the lack of corresponding treatment targets.

[0003] Therefore, the prior art needs to be improved and developed. SUMMARY

[0004] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide application of an agent for inhibiting IGFBP5 in preparation of a drug for treating malignant progression type recurrent bladder cancer, aiming to take insulin-like growth factor binding protein 5 (IGFBP5) as a new target for recurrent bladder cancer with malignant progression and provide a new strategy for its treatment.

[0005] The technical scheme of the present application is as follows: In a first aspect, application of an agent for inhibiting IGFBP5 in preparation of a drug for treating malignant progression type recurrent bladder cancer is provided.

[0006] Optionally, the malignant progression type recurrent bladder cancer has the pathological characteristics of squamous differentiation and sarcoma.

[0007] Optionally, the agent for inhibiting IGFBP5 inhibits the expression level of IGFBP5 gene or protein.

[0008] Optionally, the agent for inhibiting IGFBP5 is selected from one or more of small molecules, nucleic acids, gene editing systems, proteins and polypeptides.

[0009] Optionally, the agent for inhibiting IGFBP5 is a gene editing system, comprising: a Cas protein and an sgRNA for guiding the Cas protein to recognize and bind to the IGFBP5 gene.

[0010] Optionally, the Cas protein is a Cas9 protein; and the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 6.

[0011] In a second aspect, a drug for treating malignant progressive recurrent bladder cancer is provided, comprising: an agent for inhibiting IGFBP5.

[0012] Optionally, the drug further comprises a pharmaceutically acceptable excipient.

[0013] Optionally, the pharmaceutically acceptable excipient comprises one or more of a pharmaceutically acceptable excipient, an additional agent and an adjuvant.

[0014] Optionally, the administration route of the drug comprises a combination of one or more of oral administration, intravenous injection, intramuscular injection, subcutaneous injection and bladder perfusion.

[0015] Beneficial effects: Compared with the prior art, the present application provides a new therapeutic target of malignant progressive recurrent bladder cancer, IGFBP5. Experiments show that targeting IGFBP5 can overcome its resistance to conventional chemotherapy and also inhibit its malignant progression. Therefore, IGFBP5 can be used as a new therapeutic target for recurrent bladder cancer with malignant progression, and an agent for inhibiting IGFBP5 can be used for preparing a drug for treating malignant progressive recurrent bladder cancer, and may have important value in a wider range of epithelial cancers. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1Figure 1 is a schematic and results chart related to the construction of a non-muscle invasive bladder cancer cell line with lineage tracing function; wherein a is a schematic of the CRISPR / Cas9 lineage tracing system, luciferase (Luc) is used to monitor tumor growth and treatment response, Cas9 and sgRNAs attach and cut specific sequences on the integrated CRISPR target in the genome, producing various indels (rectangles of different colors) of genetic lineages, and a unique static barcode can distinguish multiple copies of each cell target site; b is a flow chart of establishing an engineered non-muscle invasive bladder cancer (NMIBC) cell line, lineage tracing system elements are sequentially labeled to produce two human-derived NMIBC cell lines; c is an immunoblot analysis of MGHU3 and RT4 cell lysates expressing lenti vector control ("CTR" indicates blank control) or Cas9 ("OE" indicates Cas9 overexpression) using antibodies against Cas9 and β-actin (loading control); d and e are the results after continuous Targetsite-GFP transfection in MGHU3-LT cell line (d) and RT4-LT cell line (e), cells show GFP fluorescence shift, indicating an increase in the number of copies of the target site; f and g are MGHU3-LT (f) and RT4-LT (g) cancer cells sorted by fluorescence-activated cell sorting (FACS) for three positives (mCherry+; GFP+; BFP+).

[0017] Figure 2 Figure 2 is a schematic and results chart related to phenotypic changes of chemotherapy-induced recurrent tumors; wherein a is a schematic diagram of the experimental scheme for simulating the recurrence of bladder tumors after chemotherapy treatment; b is a summary of the treatment response and tumor phenotype of MGHU3-LT model mice; c is a summary of the treatment response and tumor phenotype of RT4-LT model mice; d is a summary of the treatment response and tumor phenotype of genetically engineered bladder cancer mice.

[0018] Figure 3 Figure 3 is an evolutionary dynamic chart of bladder cancer natural progression and recurrence; wherein a is a summary of the main pathways of MGHU3-LT tumor evolution; b is a summary of the main pathways of RT4-LT tumor evolution.

[0019] Figure 4is a related result plot showing IGFBP5 as a common regulator of cancer progression and squamous differentiation; a Venn diagram (left) showing 3 out of 25 genes commonly upregulated in bladder cancer genetically engineered mouse model (GEMM) recurrent tumors, MP tumor progenitor-like cells, and post-chemotherapy patient bladder tumors, with 25 genes associated with epidermal development (right); b UMAP plot showing gene expression of IGFBP5 in 4 types of MGHU3-LT tumors (CTR-MGHU3 for control tumors, UCSD-MGHU3 for urothelial tumors with squamous differentiation, Mets-MGHU3 for metastatic lesions, and MP-MGHU3 for mixed phenotype tumors), with arrowed curves indicating evolutionary trajectories; c immunofluorescence analysis of MGHU3-LT tumors (CTR-MGHU3, UCSD-MGHU3, and MP-MGHU3) confirming high expression of IGFBP5 in MP-MGHU3 tumors, with scale bar at 100 microns; d expression of IGFBP5 in control and recurrent bladder cancer genetically engineered mouse cell lines (MHUC and mUCMP), with *** representing p<0.01, two-tailed t-test for statistical analysis, and data presented as mean ± standard error (SEM); e statistical plot showing IGFBP5 expression in 8 pairs of matched patient primary non-muscle invasive bladder cancer (P-NMIBC) and recurrent muscle invasive bladder cancer (R-MIBC), with IGFBP5 expression level quantified by IHC staining; f Kaplan-Meier curve showing overall survival probability of TCGA-MIBC cohort (n=322) stratified by IGFBP5 expression level, with log-rank test for statistical analysis.

[0020] Figure 5Figure 5 is a related graph showing that IGFBP5 ablation suppresses invasive progression and inhibits squamous differentiation to overcome chemoresistance; where a is an immunoblot analysis of sgScr ("sgScr" indicates control) or sgIGFBP5 ("sgIGFBP5" indicates IGFBP5 knockdown) MP-MGHU3-LT cell lysates using antibodies against IGFBP5 and β-actin; b is a representative bioluminescence image of mice engrafted with sgScr MP-MGHU3-LT cancer cells, sgIGFBP5-1 MP-MGHU3-LT cancer cells, and sgIGFBP5-2 MP-MGHU3-LT cancer cells (6 mice per group); c is a bar graph of the relative bioluminescence intensity of sgScr MP-MGHU3-LT tumor-bearing mice, sgIGFBP5-1 MP-MGHU3-LT tumor-bearing mice, and sgIGFBP5-2 MP-MGHU3-LT tumor-bearing mice (6 mice per condition), **** represents p<0.0001, two-tailed t-test was used for statistical analysis, data presented as mean ± standard error (SEM); d is a Kaplan-Meier curve showing the probability of survival of mice engrafted with sgScr MP-MGHU3-LT cancer cells (11 mice) and sgIGFBP5 MP-MGHU3-LT cancer cells (11 mice per condition), log-rank test was used for statistical analysis; e is the staining results of sgScr MP-MGHU3-LT tumor, sgIGFBP5-1 MP-MGHU3-LT tumor, and sgIGFBP5-2 MP-MGHU3-LT tumor tissues (representative images of 6 mice), H&E (top), EPCAM (green), Involucrin (IVL; yellow), Vim (red), and DAPI (blue), scale bar is 100 microns (top) and 20 microns (bottom); f is a bar graph showing the proportion of keratin pearl area of sgScr MP-MGHU3-LT tumor, sgIGFBP5-1 MP-MGHU3-LT tumor, and sgIGFBP5-2 MP-MGHU3-LT tumor (independent sections of 6 mice), **** represents p<0.0001, ns = not significant, two-tailed t-test was used for statistical analysis, each dot represents one image (3 images per sample), data presented as median; g is a bar graph showing the proportion of IVL positive tumor cells in sgScr MP-MGHU3-LT tumor, sgIGFBP5-1 MP-MGHU3-LT tumor, and sgIGFBP5-2 MP-MGHU3-LT tumor (independent sections of 6 mice), **** represents p<0.0001, two-tailed t-test was used for statistical analysis.0001, ns = not significant, two-tailed t test was used for statistical analysis, each dot represents one image (3 images per sample), data are presented as median; h is a heatmap showing the relative RNA expression levels of squamous differentiation-related genes in sgScr MP-MGHU3-LT tumors (n = 3) and sgIGFBP5 MP-MGHU3-LT tumors (n = 3) measured by bulk RNA-seq assay; i is an enrichment plot of biological processes of downregulated genes in sgIGFBP5 MP-MGHU3-LT cancer cells compared with sgScr MP-MGHU3-LT cancer cells; j is the experimental scheme to explore whether IGFBP5 interference can overcome the resistance to chemotherapy using sgScr MP-MGHU3-LT cancer cells and sgIGFBP5 MP-MGHU3-LT cancer cells; k is a representative bioluminescence image of sgScr MGHU3-LT tumor-bearing mice and sgIGFBP5 MP-MGHU3-LT tumor-bearing mice treated with normal saline or chemotherapy drugs (gemcitabine hydrochloride) (6 mice in each condition); l is the relative bioluminescence intensity of sgScr MGHU3-LT tumor-bearing mice and sgIGFBP5 MP-MGHU3-LT tumor-bearing mice treated with normal saline or chemotherapy drugs (gemcitabine hydrochloride) (6 mice in each condition), ** represents p < 0.01, **** represents p < 0.001, two-tailed t test was used for statistical analysis, data are presented as mean ± standard error (SEM). DETAILED DESCRIPTION

[0021] The present application provides the use of an agent inhibiting IGFBP5 in the preparation of a drug for treating malignant progressive recurrent bladder cancer. In order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail as follows.

[0022] The present application provides the use of an agent inhibiting IGFBP5 in the preparation of a drug for treating malignant progressive recurrent bladder cancer.

[0023] Specifically, the previous research of the present application screened insulin-like growth factor binding protein 5 (IGFBP5) as a potential target for recurrent bladder cancer with malignant progression based on the recurrence evolution track of bladder cancer. Further experiments showed that targeting IGFBP5 can overcome its resistance to conventional chemotherapy and also inhibit its malignant progression. Therefore, IGFBP5 can be used as a new therapeutic target for recurrent bladder cancer with malignant progression, and an agent inhibiting IGFBP5 can be used in the preparation of a drug for treating malignant progressive recurrent bladder cancer, which may also have important value in a wider range of epithelial cancers.

[0024] wherein the malignant progression recurrent bladder cancer refers to the malignant degree of the recurrent bladder tumor is improved compared with the primary tumor.

[0025] In some embodiments, the malignant progression recurrent bladder cancer has pathological characteristics of squamous differentiation and sarcoma.

[0026] In some embodiments, the agent for inhibiting IGFBP5 inhibits the expression level of IGFBP5 gene or protein.

[0027] In some embodiments, the agent for inhibiting IGFBP5 is selected from one or more of small molecules, nucleic acids, gene editing systems, proteins and polypeptides.

[0028] In some embodiments, the agent for inhibiting IGFBP5 is a gene editing system, comprising: a Cas protein and an sgRNA for guiding the Cas protein to recognize and bind to the IGFBP5 gene.

[0029] In some embodiments, the Cas protein is a Cas9 protein; and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 6.

[0030] An embodiment of the present application provides a medicine for treating malignant progression recurrent bladder cancer, comprising: an agent for inhibiting IGFBP5.

[0031] In some embodiments, the medicine further comprises a pharmaceutically acceptable excipient.

[0032] The term "pharmaceutically acceptable" refers to a substance that does not change the main pharmacological effect or physicochemical property of the active component in the medicine, and has relative safety, can be applied to the body without causing adverse biological reactions, and does not have adverse interactions with other components in the medicine.

[0033] In some more specific embodiments, the pharmaceutically acceptable excipient comprises one or more of pharmaceutically acceptable excipients, additional agents and adjuvants.

[0034] In some embodiments, the administration route of the medicine comprises a combination of one or more of oral administration, intravenous injection, intramuscular injection, subcutaneous injection and bladder perfusion.

[0035] The present application is further illustrated below through specific embodiments.

[0036] Embodiment 1 To investigate bladder cancer recurrence in vivo in real time, this embodiment attempts to establish a robust lineage tracing model to explore and record the entire recurrence process. In this process, it is expected that bladder cancer cells will first respond to intravesical chemotherapy, then develop resistance, and finally regrow to produce a recurrent tumor. First, following the method described in the reference (DOI: 10.1126 / science.abc1944), two human low-grade non-muscle-invasive bladder cancer (NMIBC) cell lines, MGHU3 and RT4, were modified using a CRISPR / Cas9-driven evolutionary lineage tracing system to obtain the lineage tracing (LT) cell lines MGHU3-LT and RT4-LT. Figure 1 Specifically, the modified cells contain: (i) a luciferase for in vivo imaging to monitor treatment response and tumor growth; (ii) a Cas9-mCherry for generating heritable indels; (iii) a multi-target site containing three cleavage sites for recording lineage information, which can be captured as transcripts from a single-cell intact cDNA library; (iv) a static 14-base-pair random barcode located upstream of the target sites to distinguish individual copies of these target sites; and (v) sgRNA guiding Cas9 to the target sites, thereby initiating lineage recording. Importantly, mismatches between the sgRNA and the target sites are engineered to reduce binding affinity, thereby modulating the lineage recording rate for long-term tracking.

[0037] Next, in this embodiment, the MGHU3-LT and RT4-LT described above were used in an orthotopic xenograft model of bladder cancer to investigate tumor recurrence. Figure 1 (a). In short, MGHU3-LT and RT4-LT were surgically injected into the bladder cavity of immunodeficient mice (NCG) to construct MGHU3-LT and RT4-LT models, respectively. A small number of cells colonized the bladder mucosa within the bladder cavity, thus mimicking undetectable residual tumors in human diseases (Reference DOI: 10.1038 / s41596-018-0112-8). On day 3 after tumor cell injection, mice began receiving instillation of either saline (50 μL) or chemotherapy (240 mg / kg gemcitabine hydrochloride, Sigma-Aldrich, 122111-03-9). Treatment was administered weekly for four cycles. Afterward, the tumors were allowed to grow freely, and finally, tumors with different pathological phenotypes were collected for scRNA-seq and target site amplicon sequencing to collect pedigree information. Figure 2In the chemotherapy group of the MGHU3-LT model, two different urothelial phenotypes were found in tumors compared to the control group, urothelial carcinoma with squamous differentiation (UCSD) and mixed phenotypes (MP) (with features of sarcoma and urothelial carcinoma with squamous differentiation) Figure 2 In some MP tumors, metastasis was also found Figure 2 Similarly, in the chemotherapy group of the RT4-LT model, UCSD tumors were found that were different from the urothelial phenotype of the control group Figure 2 In addition, this example delivered an adenovirus expressing Cre recombinase driven by the KRT5 promoter (AdenoKRT5-Cre; Hanheng Biotechnology Co., Ltd (Shanghai, China), ref. DOI: 10.1038 / s41591-019-0499-y) to the bladder of Trp53 flox / flox ; Pten flox / flox ; Akaluc-IRES-tdTomato genetically engineered mice (from commercially available Akaluc-IRES-tdTomato mice (The Jackson Laboratory, ref. 024829) and C57BL / 6J mice (The Jackson Laboratory, ref. 000664) Trp53 flox / flox Mice, Pten flox / flox were crossed to obtain C57BL / 6J mice, and Akaluc-IRES-tdTomato mice were inbred to obtain Akaluc-IRES-tdTomato mice (The Jackson Laboratory, ref. 024829) and C57BL / 6J mice (The Jackson Laboratory, ref. 000664) Trp53 and Pten, Akaluciferase and tdTomato were simultaneously knocked in to obtain bladder cancer genetically engineered mice (Akaluc-IRES-tdTomato; AdenoKRT5-Cre) Trp53 flox / flox ; Pten flox / flox ; Akaluc-IRES-tdTomato; AdenoKRT5-Cre). The same procedures as described above for the orthotopic xenograft model were performed, and MP tumors were found in the chemotherapy group of mice that were different from the urothelial phenotype of the control group Figure 2 In d.

[0038] Next, to explore the evolutionary trajectories of tumor relapse, the present embodiment uses the “evolution-coupled” algorithm (Ref. DOI: 10.1016 / j.cell.2022.04.015) to quantify the phylogenetic distance between pairs of cell states to infer evolutionary trajectories. It is found that both MGHU3-LT and RT4-LT tumors give rise to luminal-like cells and cells in the Epidermoid-like cell state (ELCS) during the natural progression of bladder cancer. During tumor relapse, ELCs (Epidermoid-like cells) as seeds of relapse give rise to tumors of different phenotypes through different evolutionary modes. Specifically, in the RT4-LT model, ELCs prefer self-renewal over differentiation into other states of cells to generate non-malignant progressing relapsed UCSD tumors. While in the MGHU3-LT model, ELCs favor differentiation and adopt a branching evolutionary mode: one forms UCSD tumors and the other forms MP tumors Figure 3 a and b in the middle).

[0039] The core challenge in studying tumor relapse is to capture key intermediate states and reconstruct the continuous process from micro-residual lesions to relapse longitudinally. The lineage tracing method of the present embodiment can map the relapse dynamics in real time with high resolution in a unique way without any irrefutable assumptions (e.g., transcriptional similarity), thus revealing key evolutionary bifurcation points and evolutionary rules associated with relapse. Therefore, the present embodiment further seeks potential targets for treating relapsed tumors based on the above revealed evolutionary dynamics of relapsed tumors. The inventors observed that cells evolved from a key bifurcation point (progenitor-like cells) are highly malignant and even cause patient death because they develop into sarcomatoid carcinoma and even trigger metastasis Figure 3 a in the middle). In addition, drug-resistant ELCs are a key factor for the squamous features of relapsed tumors Figure 3 a and b in the middle). Therefore, these results highlight targeting progenitor-like cells to suppress aggressive progression and simultaneously overcome chemotherapy resistance of squamous features, which might be able to treat malignant progressing relapsed bladder cancer.

[0040] To elucidate potential co-regulatory factors of squamous cell characteristics and invasive progression at the molecular level, this embodiment identified 25 co-upregulated genes in progenitor-like cells of MP tumors, recurrent tumors in genetically engineered mice, and tumors from patients after chemotherapy (data from GEO, GSE124035; GSE48075; GSE32894, respectively). In addition to PTGS2 and CEBPD, two factors known to be associated with chemotherapy resistance in bladder cancer, candidate genes also included THBS2, IGFBP5, VCAN, ID1, and PFKFB3, which are known to be associated with stem cell characteristics, invasiveness, angiogenesis, and poor prognosis in bladder cancer. Notably, among these 25 genes associated with invasive progression, three specifically participated in squamous differentiation (…). Figure 4 (a). The inventors also observed that IGFBP5 is upregulated along the evolutionary trajectory of MP tumors, reaching peak expression in the mesenchymal cell population regardless of its evolutionary path. Figure 4 (b)

[0041] Immunofluorescence analysis showed that, compared with the control group (CTR-MGHU3) and urothelial carcinoma with squamous differentiation (UCSD-MGHU3), IGFBP5 was upregulated in the mixed phenotype tumor (MP-MGHU3). Figure 4 (c). RT-qPCR also confirmed that, compared with the primary tumor cells MHUC (Mouse Heterogeneous Urothelial Carcinoma), mUCMP (Mouse Urothelial Carcinoma of Mixed Phenotypes) cells derived from recurrent tumors in genetically engineered bladder cancer mice highly expressed IGFBP5 (…). Figure 4 (d). Furthermore, in the TCGA MIBC cohort, patients with high IGFBP5 expression had significantly shorter overall survival (OS) than those with low expression. Figure 5 (e).

[0042] To further validate the function of IGFBP5 in in vivo experiments, this embodiment utilizes CRISPR / Cas9 technology to transfect sgIGFBP5 (sgIGFBP5-1 or sgIGFBP5-2) into MP-MGHU3-LT cells derived from the MGHU3-LT model MP tumor (fluorescently activated cell sorting showing triple positivity (GFP+; mCherry+; BFP+)) to knock out IGFBP5, obtaining IGFBP5-KO MP-MGHU3-LT cells (or denoted as sgIGFBP5-1 MP-MGHU3-LT cancer cells / sgIGFBP5-2 MP-MGHU3-LT cancer cells); the control group was transfected with scrambled sgRNA (sgScr) to obtain IGFBP5-sgScr MP-MGHU3-LT cells (or denoted as sgScr MP-MGHU3-LT cancer cells). Figure 5 (a) Compared with the control group (IGFBP5-sgScr) cancer cells, the growth of IGFBP5 knockout (IGFBP5-KO) cancer cells was significantly inhibited, which was confirmed by a weaker bioluminescent signal. Figure 5 (b and c). The survival benefit of IGFBP5-KO tumor mice was also superior to that of IGFBP5-sgScr control tumor mice. Figure 5 (d). This embodiment then evaluated the number of keratin pearls and the expression of IVL (Involucrin) to explore the role of IGFBP5 in regulating squamous characteristics. Histopathological analysis showed that the number of keratin pearls in IGFBP5-KO tumors was significantly reduced compared with IGFBP5-sgScr control tumors (d). Figure 5 (e and f). IVL is persistently expressed in most IGFBP5-sgScr tumor cells, but is significantly reduced in IGFBP5-KO tumors (e and f). Figure 5 (e and g).

[0043] The sgRNA sequences involved in the embodiments are shown in Table 1: Table 1. Specific information about the sgRNA in this embodiment.

[0044] Inspired by these results, this embodiment further performed bulk RNA-seq on IGFBP5-sgScr and IGFBP5-KO cancer cells. Transcriptome analysis showed that IGFBP5 knockout downregulated the expression of squamous differentiation-related genes (IVL, SPRR2A / D / E / G, and TGM4 / 5). Figure 5In addition, genes downregulated by IGFBP5 knockout were significantly enriched not only in squamous differentiation, but also in epithelial cell proliferation, mesenchymal cell differentiation, and amoeboid cell migration, indicating that IGFBP5 intervention can inhibit tumor progression. Figure 5 i).

[0045] Given that cancer cells can acquire chemotherapy resistance through squamous differentiation, this embodiment further evaluated the sensitivity of IGFBP5-KO cancer cells to chemotherapy in vivo. In this embodiment, IGFBP5-sgScr MP-MGHU3-LT cells and IGFBP5-KO MP-MGHU3-LT cells were seeded into the bladder of mice to establish an orthotopic bladder cancer model (method as above). Bladder instillation chemotherapy began on day 3, once a week for two consecutive cycles. Figure 5 (j). Experimental results showed that although bladder instillation chemotherapy delayed the growth of IGFBP5-sgScr MP-MGHU3-LT tumors, its inhibitory effect on tumor proliferation was still weaker than that of IGFBP5-KO. Notably, all IGFBP5-KO tumor mice exhibited a strong chemotherapy response (j). Figure 5 (k and l).

[0046] In summary, the study in this embodiment demonstrates that targeting IGFBP5 has a dual anti-tumor effect: it inhibits malignant progression by regulating key cellular functions (such as epithelial cell proliferation); at the same time, it overcomes chemotherapy resistance by inhibiting squamous cell characteristics.

[0047] In this embodiment, the grouping and nomenclature of cancer cells and tumor-bearing mice include sgIGFBP5 or IGFBP5-KO, indicating IGFBP5 knockout using CRISPR / Cas9 technology; and sgScr or IGFBP5-sgScr, indicating negative controls for IGFBP5 knockout using CRISPR / Cas9 technology. Unless otherwise specified, sgIGFBP5 refers to a combination of sgIGFBP5-1 and sgIGFBP5-2.

[0048] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. Use of an agent inhibiting IGFBP5 in the preparation of a drug for treating malignant progression type recurrent bladder cancer.

2. Use according to claim 1, characterized in that, The malignant progression type recurrent bladder cancer has pathological features of squamous differentiation and sarcoma.

3. Use according to claim 1, characterized in that, The agent inhibiting IGFBP5 inhibits the expression level of IGFBP5 gene or protein.

4. Use according to claim 1, characterized in that, The agent inhibiting IGFBP5 is selected from one or more of small molecules, nucleic acids, gene editing systems, proteins and polypeptides.

5. The use according to claim 1, characterized in that, The agent inhibiting IGFBP5 is a gene editing system, comprising: a Cas protein and an sgRNA guiding the Cas protein to recognize and bind to the IGFBP5 gene.

6. Use according to claim 5, characterized in that, The Cas protein is a Cas9 protein; the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO.

6.

7. A medicament for treating malignant progressive recurrent bladder cancer, characterized by, Comprise: An agent inhibiting IGFBP5.

8. The medicament according to claim 7, characterized in that, The drug further comprises a pharmaceutically acceptable excipient.

9. The medicament according to claim 7, characterized in that, The pharmaceutically acceptable excipient comprises one or more of a pharmaceutically acceptable excipient, an additional agent and an adjuvant.

10. The medicament according to claim 7, characterized in that, The administration route of the drug comprises a combination of one or more of oral administration, intravenous injection, intramuscular injection, subcutaneous injection and bladder perfusion.

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