Application of CLN6 inhibitor in treatment and prognosis evaluation of prostate cancer

Abstract

The invention relates to application of a CLN6 inhibitor in prostate cancer treatment and prognosis evaluation. Through deep research, CLN6 can be used as a key factor for generation and development of prostate cancer and generation of treatment drug resistance of enzirulamine, and by inhibiting CLN6 or reducing the biological activity of CLN6, proliferation, metastasis and other activities of prostate cancer can be obviously inhibited, the drug resistance of enzirulamine is reversed, the treatment sensitivity of enzirulamine is improved, and the survival rate of patients is prolonged; the prognosis is improved. According to the invention, related mechanisms of drug resistance generation and regulation in the process of treating prostatic cancer by the enzheutamine are enriched, sufficient scientific basis and theoretical basis are provided for exploring new prostatic cancer diagnosis, prognosis judgment and treatment molecular targets and developing new targeted drugs, accurate treatment can be better realized, and the application of enzheutamine in treatment of prostatic cancer is promoted. The method has important social value and scientific significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of CLN6 inhibitors in the treatment and prognostic assessment of prostate cancer. Background Technology

[0002] Prostate cancer is the second most common malignant tumor among men worldwide, accounting for more than 10% of cancer-related deaths and ranking as the fifth leading cause of cancer death globally. In its localized stage, prostate cancer typically grows slowly, and patients usually achieve good outcomes with surgical resection or radiation therapy. In recent decades, advances in diagnostic techniques and treatment strategies have significantly improved prognostic management for lethal prostate cancer, particularly through androgen deprivation therapy (ADT) and AR signaling inhibitors (ARSIs), including abiraterone, enzalutamide, apatamide, and dalotamide, which have demonstrated significant survival benefits.

[0003] However, to further optimize patient outcomes, a deeper understanding of the complex interactions between molecular drivers and dysregulation mechanisms promoting the progression of lethal prostate cancer is essential. The significant heterogeneity of lethal prostate cancer and the diverse clinical presentations observed in patients present significant challenges to such research. However, by focusing on ubiquitous gene mutations and regulatory pathways, research findings are more likely to be effectively translated into clinical practice, providing broader applicability and impact.

[0004] Metabolic reprogramming is a hallmark of tumor heterogeneity and treatment resistance, with glycolysis playing a crucial role. Normal cells primarily rely on oxidative phosphorylation (OXPHOS) for energy production, while tumor cells exhibit a marked preference for glycolysis. Although each glucose molecule produces less ATP, glycolysis provides a faster rate of energy production, enabling tumor cells to meet the higher metabolic demands of proliferation and survival. For example, in pancreatic cancer, neurotransmitter activation of the GFRA2-RET signaling axis promotes glycolysis via phosphorylation of hexokinase 2, thereby driving tumor progression. Similarly, in colorectal cancer, ATF4 promotes tumor growth by upregulating glycolytic activity. Recent studies in lethal prostate cancer have highlighted the role of glycolysis in mediating treatment resistance. However, the complex regulatory network of glycolysis requires further investigation to fully elucidate its mechanistic contributions to cancer progression and drug resistance. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the treatment of lethal prostate cancer in the prior art, and to conduct an in-depth study on its glycolytic regulation mechanism. It has been clarified that CLN6 is a key regulatory gene in the glycolytic pathway of lethal prostate cancer, and it has been confirmed that CLN6 can serve as a potential therapeutic target for intervening in glycolysis-driven lethal prostate cancer, providing a new strategy for the treatment and prognostic assessment of prostate cancer.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0007] The first aspect of this invention provides the use of CLN6 inhibitors in the preparation of medicaments for the prevention and / or treatment of prostate cancer.

[0008] Preferably, the CLN6 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the CLN6 gene.

[0009] Preferably, the CLN6 inhibitor is selected from shRNA designed based on the CLN6 gene; the shRNA sequence is shown in SEQ ID NO: 1 (5'-GCTGCTTTACTGCCTCTAAAG-3').

[0010] Preferably, the prostate cancer is a lethal prostate cancer.

[0011] The second aspect of this invention provides the use of CLN6 inhibitors in the preparation of medicaments that enhance the sensitivity of enzalutamide to prostate cancer treatment.

[0012] Preferably, the CLN6 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the CLN6 gene.

[0013] Preferably, the CLN6 inhibitor is selected from shRNA designed based on the CLN6 gene; the shRNA sequence is shown in SEQ ID NO: 1.

[0014] Preferably, the prostate cancer is a lethal prostate cancer.

[0015] A third aspect of the present invention provides the use of a reagent for detecting CLN6 expression levels in the preparation of a product for evaluating the sensitivity of enzalutamide to prostate cancer treatment.

[0016] Preferably, the reagent for detecting CLN6 expression level includes primers for detecting CLN6 gene expression level and / or reagents for detecting CLN6 protein content.

[0017] Preferably, the primers for detecting CLN6 gene expression levels are selected from the following primer pairs:

[0018] The upstream sequence of the primer pair is shown in SEQ ID NO: 2 (5'-GGCCAGAGACACTGGGA-3'), and the downstream sequence is shown in SEQ ID NO: 3 (5'-AACCAGAGGTCGAGGTGGA-3').

[0019] Preferably, the reagent for detecting CLN6 protein content is selected from anti-CLN6 antibody; for example, it can be selected from ab272678 (Abcam).

[0020] Preferably, the prostate cancer is a lethal prostate cancer.

[0021] A fourth aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of prostate cancer, comprising a CLN6 inhibitor and enzalutamide.

[0022] Preferably, the CLN6 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the CLN6 gene.

[0023] Preferably, the CLN6 inhibitor is selected from shRNA designed based on the CLN6 gene; the shRNA sequence is shown in SEQ ID NO: 1.

[0024] Preferably, the pharmaceutical composition may optionally contain a pharmaceutically acceptable carrier.

[0025] Preferably, the pharmaceutically acceptable pharmaceutical excipient is selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants, and colorants.

[0026] Preferably, the prostate cancer is a lethal prostate cancer.

[0027] The fifth aspect of the present invention provides a kit for evaluating the sensitivity of enzalutamide to prostate cancer treatment, including reagents for detecting CLN6 expression levels.

[0028] Preferably, the reagent for detecting CLN6 expression level includes primers for detecting CLN6 gene expression level and / or reagents for detecting CLN6 protein content.

[0029] Preferably, the primers for detecting CLN6 gene expression levels are selected from the following primer pairs:

[0030] The upstream sequence of the primer pair is shown in SEQ ID NO: 2, and the downstream sequence is shown in SEQ ID NO: 3.

[0031] Preferably, the reagent for detecting CLN6 protein content is selected from anti-CLN6 Antibody.

[0032] Preferably, the kit further includes one or more of PCR enzyme, PCR buffer, dNTPs, and fluorescent substrate.

[0033] Preferably, the fluorescent substrate is selected from Syber Green or fluorescently labeled probes.

[0034] Preferably, the prostate cancer is a lethal prostate cancer.

[0035] It should be understood that, unless otherwise specified, in the context of this invention, CLN6 includes CLN6 nucleotides and the CLN6 protein encoded by those nucleotides. The CLN6 inhibitor refers to a substance capable of specifically downregulating CLN6 expression levels and / or the transcriptional level of its mature mRNA and / or the expression level or activity of the CLN6 protein. For example, methods such as antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA erasers, target masking, and / or multi-target methods can be used to downregulate CLN6 expression levels and / or activity; any method that can reduce CLN6 levels and / or activity is acceptable. The primers and / or primer pairs refer to PCR primers used to synthesize the CLN6 gene cDNA strand in PCR, thereby detecting the expression level of the CLN6 gene mRNA. In addition to the primers and / or primers listed in this invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the CLN6 gene sequence using conventional methods and techniques in the art, including but not limited to molecular biology, and screening the designed primers and / or primer pairs using conventional experimental methods, as long as they can specifically detect the CLN6 expression level; conventional reagents and methods in the art can also be used to detect the CLN6 protein expression level; the same applies to other genes / proteins.

[0036] Androgen receptor (AR) signaling is a key driver in the development and progression of prostate cancer. The combined use of adrenergic agonists (ADTs) and antirogen inhibitors (ARSIs) is a fundamental approach to treating lethal prostate cancer. Although many patients initially respond well to ARSIs, tumor heterogeneity leads to varying treatment outcomes, posing a significant challenge to clinical management. Furthermore, patients who develop resistance to long-term ARSI therapy often have limited or no viable alternative treatment options. Therefore, identifying novel therapeutic targets to overcome ARSI resistance in lethal prostate cancer is a crucial strategy for improving patient prognosis.

[0037] In prostate cancer, glycolysis exhibits a dynamic, stage-specific pattern that significantly influences disease progression and metastatic spread. Unlike many solid tumors, glycolysis is downregulated in the early stages of prostate cancer development. Conversely, malignant transformation is characterized by a metabolic shift towards oxidative phosphorus and fatty acid oxidation (FAO), resulting in a more energy-efficient cell phenotype. However, in lethal prostate cancer, glycolysis is reactivated. This metabolic reprogramming is coordinated through interactions with the tumor microenvironment, particularly bone marrow adipocytes, triggering hypoxia-inducible factor-1α (HIF-1α) signaling, upregulating glucose transporters and key glycolytic enzymes such as hexokinase-2 (HK2). Increased glycolytic flux supports tumor cell survival in hypoxic environments, accelerates ATP production, promotes immune evasion through lactate accumulation, and facilitates extracellular matrix remodeling and angiogenesis, thereby driving aggressive disease behavior.

[0038] This invention further clarifies that activation of the glycolytic pathway is a significant predictor of poor prognosis in patients with lethal prostate cancer treated with ARSI, and identifies CLN6 as a key regulatory gene in the glycolytic pathway of lethal prostate cancer. Enzalutamide-resistant prostate cancer cells exhibit increased glycolytic activity, and inhibition of glycolysis with 2-DG effectively reversed this resistance. CLN6 is a member of the neuronal lipofuscin (NCL) gene family, which is associated with a group of inherited neurodegenerative lysosomal storage diseases. The CLN6 gene encodes a highly conserved protein containing 311 amino acids, with 7 transmembrane domains, a molecular weight of approximately 36 kDa, and is ubiquitous in vertebrate species. This protein is located in the endoplasmic reticulum and is an integral part of lysosomal function. CLN6 deficiency impairs the transport of lysosomal enzymes from the endoplasmic reticulum, leading to reduced lysosomal enzyme levels, thus highlighting the crucial role of CLN6 in lysosomal biogenesis.

[0039] Current research on CLN6 in cancer is very limited. This invention, through in-depth research, reveals that CLN6 expression is upregulated in enzalutamide-resistant prostate cancer cell lines. Silencing CLN6 attenuates the proliferation of these resistant cells and inhibits glycolytic activity. The mechanism by which CLN6 regulates glycolysis is still unclear. As a member of the HALLMARK_GLYCOLYSIS gene set, CLN6 functions as a regulator of atypical glycolysis. Considering its crucial role in the endoplasmic reticulum-lysosome metabolic axis, CLN6 may affect glycolysis through endoplasmic reticulum stress or metabolic reprogramming pathways.

[0040] In summary, this invention emphasizes the crucial role of the glycolysis pathway in lethal prostate cancer and identifies CLN6 as a pivotal gene in overcoming treatment resistance. It clarifies that CLN6 serves as a key factor in the development and progression of prostate cancer and the emergence of enzalutamide resistance. Inhibiting CLN6 or reducing its biological activity can significantly suppress prostate cancer proliferation and metastasis, reverse enzalutamide resistance, improve treatment sensitivity, prolong patient survival, and improve prognosis. This invention enriches the understanding of the mechanisms underlying the development and regulation of enzalutamide resistance in prostate cancer treatment, providing ample scientific evidence and theoretical foundation for exploring new molecular targets for prostate cancer diagnosis, prognosis, and treatment, and developing new targeted drugs. It contributes to achieving better precision medicine and has significant social and scientific value. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the Cox regression analysis results for the progression-free survival (PFS) pathway enrichment scores in the SU2C cohort.

[0042] Figure 2 This is a schematic diagram of the SU2C cohort risk map analysis results based on pathway enrichment scores for progression-free survival.

[0043] Figure 3 This is a schematic diagram of the SU2C cohort risk map analysis results based on the overall survival pathway enrichment score.

[0044] Figure 4 This is a schematic diagram illustrating the effects of high and low glycolytic activity on progression-free survival in prostate cancer patients in a Kaplan-Meier survival analysis.

[0045] Figure 5 This is a schematic diagram illustrating the effects of high and low glycolytic activity on overall survival in prostate cancer patients in a Kaplan-Meier survival analysis.

[0046] Figure 6 This is a schematic diagram showing the effect of enzalutamide on the proliferation of wild-type and drug-resistant prostate cancer cells.

[0047] Figure 7 This is a schematic diagram showing the effect of enzalutamide on the colony formation of wild-type and drug-resistant prostate cancer.

[0048] Figure 8 This is a schematic diagram showing the results of glucose uptake experiments on wild-type and drug-resistant prostate cancer cells.

[0049] Figure 9 This is a schematic diagram showing the experimental results of lactate production in wild-type and drug-resistant prostate cancer cells.

[0050] Figure 10This is a schematic diagram showing the effect of 2-DG treatment on the proliferation of drug-resistant prostate cancer cells.

[0051] Figure 11 This is a schematic diagram illustrating the effect of co-treatment with enzalutamide and 2-DG on the proliferation of drug-resistant prostate cancer cells.

[0052] Figure 12 A schematic diagram illustrating the effect of co-treatment with enzalutamide and 2-DG on the colony formation of drug-resistant prostate cancer cells.

[0053] Figure 13 This is a schematic diagram showing the differences in CLN6 expression levels between normal prostate cells and prostate cancer cells.

[0054] Figure 14 This is a schematic diagram showing the inhibitory activity of shRNC on CLN6 expression levels in prostate cancer cells.

[0055] Figure 15 A schematic diagram illustrating the effect of knocking down CLN6 on glucose uptake in prostate cancer cells.

[0056] Figure 16 A schematic diagram illustrating the effect of knocking down CLN6 on lactate production in prostate cancer cells.

[0057] Figure 17 A schematic diagram illustrating the effect of knocking down CLN6 on the extracellular acidification rate of prostate cancer cells.

[0058] Figure 18 A schematic diagram illustrating the effect of CLN6 knockdown on prostate cancer cell proliferation.

[0059] Figure 19 A schematic diagram illustrating the effect of CLN6 knockdown on prostate cancer cell colony formation. Detailed Implementation

[0060] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0061] Unless otherwise specified, the cell lines listed in this invention, including PC-3, DU145, 22Rv1, LNCaP, C4-2, and C4-2B, were all purchased from the Manassas Culture Collection (USA) and cultured according to existing techniques. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). They were also cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available.

[0062] In this invention, informed consent was obtained from all patients using clinical samples, and the relevant procedures and methods were approved by the ethics committee, complying with medical ethics requirements and the Good Clinical Practice (GCP) guidelines for drug clinical trials. All experimental procedures adhered to the Declaration of Helsinki. The experimental methods used in this invention, such as bioinformatics analysis, molecular biology experiments, and cell biology experiments, are all conventional methods and techniques in the field. Bioinformatics analysis was performed using R software version 4.3, with the "limma" package used for differential analysis and the "survival" and "survminer" packages used for KM survival curve plotting. Representative results from biological experimental replicates are presented in the contextual figures, and data are displayed as mean ± SD and mean ± SEM as specified in the figures. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 8.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance were used to compare the differences in means between two or more groups. *p < 0.05 was considered a significant difference.

[0063] Example 1

[0064] First, the pathway activity of SU2C lethal prostate cancer samples was assessed using ssGSEA collected based on the Hallmark pathway, with progression-free survival (PFS) as the primary endpoint. Comprehensive Cox regression analysis showed that among the top five pathways, glycolysis had the most statistically significant p-value, with a hazard ratio (HR) greater than 1 (see [link to Cox regression analysis]). Figure 1 ).

[0065] Subsequently, the samples in the SU2C cohort were divided into high-risk and low-risk groups for Kaplan-Meier survival analysis to investigate the factors influencing progression-free survival (PFS) and overall survival (OS) in lethal prostate cancer. Figure 2 and Figure 3 The results of the SU2C cohort risk map analysis based on pathway enrichment scores for progression-free survival and overall survival are presented respectively. The heatmap shows the expression profiles of the top five pathways.

[0066] Furthermore, the samples were divided into high-activity and low-activity groups according to glycolytic activity scores, and KM survival analysis was performed to investigate the impact of glycolytic pathway activity on progression-free survival and overall survival in lethal prostate cancer. The results are as follows: Figure 4-5 As shown in the figure, higher glycolytic pathway activity was consistently associated with poorer progression-free survival (PFS) and overall survival (OS) in lethal prostate cancer. These results highlight the crucial role of glycolysis as a key determinant of poor prognosis in lethal prostate cancer.

[0067] Example 2

[0068] Enzalutamide is one of the first drugs used to treat lethal prostate cancer. To investigate the relationship between glycolysis and treatment response, an enzalutamide-resistant prostate cancer cell line model was established. The resistant cell lines were constructed as follows: C4-2B or LNCaP cells were stimulated with 25 μM or 10 μM enzalutamide for 6 months to induce enzalutamide-resistant cell lines (LNCaP_ENZR, C4-2B_ENZR). Normal C4-2B and LNCaP cells, as well as the enzalutamide-resistant cells (LNCaP_ENZR, C4-2B_ENZR), were used for cell proliferation and colony formation experiments to evaluate the effectiveness of the enzalutamide-resistant prostate cancer cell line construction. The specific steps of the cell proliferation experiment are as follows:

[0069] (1) When the cells reach the logarithmic growth phase, trypsin digestion and cell counting are performed. Appropriate cell densities are selected based on the doubling time of various cell types, and the cells are seeded into 96-well plates (3 replicates). Different concentrations (e.g., 10⁻⁶) are used in each group. 0 10 0.5 10 1 10 1.5 10 2 The sample was treated with enzalutamide (μM).

[0070] (2) The cells were cultured in an incubator at 37°C and collected after 48 hours of culture. 10 μL of CCK-8 was added to each well, and the culture plate was incubated in the incubator for 2 hours. The absorbance at 450 nm was measured to assess the cell proliferation status.

[0071] Experimental results are as follows Figure 6 As shown in the figure. The results showed that, compared with normal cells (C4-2B and LNCaP cells), drug-resistant cell lines (LNCaP_ENZR and C4-2B_ENZR cells) had significantly reduced sensitivity to enzalutamide and significantly increased proliferation activity.

[0072] The specific experimental steps for cell clone formation are as follows:

[0073] (1) When the cells grow to the logarithmic phase, trypsin digest and count them. Select an appropriate cell density (about 1000 cells / well) according to the doubling time of various cells. Seed the cells into a 6-well plate containing 2 mL of 37℃ pre-warmed culture medium and gently rotate to disperse the cells evenly. Treat the cells with enzalutamide (C4-2B: 25 μM, LNCaP_ENZ: 10 μM) and culture them in a cell culture incubator at 37℃, 5% CO2 and saturated humidity.

[0074] (2) When visible clones appear in the culture dish, stop the culture, discard the supernatant, carefully wash twice with PBS, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol and wash the residual methanol with water; cell clones can then be observed; under a microscope, a number of cells > 50 is considered a valid clone.

[0075] Test results as follows Figure 7 As shown in the figure. The results showed that, compared with normal cells (C4-2B and LNCaP cells), the drug-resistant cell lines (LNCaP_ENZR and C4-2B_ENZR cells) showed significantly increased sensitivity to enzalutamide and significantly improved colony-forming ability. The above experiments demonstrate that the enzalutamide-resistant cell lines constructed in this invention were successfully established.

[0076] After successfully inducing drug resistance, the glucose uptake and lactate production rates of the constructed drug-resistant cells were analyzed. The glucose uptake assay was performed using the glo™ Assay kit (J1341, Promega, Madison, USA), and the specific steps are as follows:

[0077] (1) The drug-resistant cell lines (LNCaP_ENZR and C4-2B_ENZR cells) were seeded at a density of 2000 cells / well in 96-well plates and washed twice with phosphate-buffered saline (PBS); normal cells (C4-2B and LNCaP cells) were used as controls (WT group).

[0078] (2) Add 50 μL of 1 mM 2-deoxyglucose (2-DG) solution to each well, mix gently, and incubate at room temperature for 10 minutes.

[0079] (3) Add 25 μL of stop buffer and mix gently, then add 25 μL of neutralization buffer and mix.

[0080] (4) Add 100 μL of 2DG-6P detection reagent and incubate at room temperature for 30-60 min; use a photometer to detect and record the luminescence signal.

[0081] The lactate production experiment was conducted using a lactate assay kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute). The specific steps are as follows:

[0082] (1) Collect 100 μL of cell culture supernatant; dilute the enzyme stock solution and enzyme diluent at a ratio of 1:100 to prepare the enzyme working solution.

[0083] (2) Dissolve the colorimetric powder in 6 mL of colorimetric diluent to prepare the colorimetric working solution.

[0084] (3) Divide the 12-well plate into 3 groups, add 20 μL of distilled water, 3 mM standard solution or sample to each group, and repeat 3 times for each group.

[0085] (4) Take 1 mL of enzyme working solution and 200 μL of colorimetric working solution for each well, mix them thoroughly, and incubate in a 37°C water bath for 10 minutes.

[0086] (5) Add 2 mL of stop solution to each well to terminate the reaction, and measure the absorbance at 530 nm using a full-wavelength microplate apparatus.

[0087] Experimental results are as follows Figure 8-9 As shown in the figure. The results showed that, compared with the WT group, both glucose uptake and lactate production rates of drug-resistant cells were significantly increased.

[0088] To further verify the role of glycolysis, the glycolysis inhibitor 2-DG was used in cell proliferation and colony formation assays to study its effects on cell function.

[0089] The specific steps of the cell proliferation experiment are as follows:

[0090] (1) When the drug-resistant cell lines (LNCaP_ENZR, C4-2B_ENZR cells) reach the logarithmic growth phase, they are digested with trypsin and counted. Appropriate cell densities are selected based on the doubling time of each cell type, and the cells are seeded into 96-well plates (3 replicates). Different concentrations (e.g., 10⁻⁶) are used in each group. -2 10 -1 10 0 10 1 10 2 , mM) of 2-DG were processed.

[0091] (2) The cells were cultured in a 37°C incubator and collected after 24h, 48h and 72h of culture. 10μL of CCK-8 was added to each well and the culture plate was incubated in the incubator for 2h. The absorbance at 450nm was measured to assess the cell proliferation status.

[0092] The results showed that 2-DG treatment had a dose-dependent and significant inhibitory effect on the proliferation of LNCaP_ENZR and C4-2B_ENZR cells, and the inhibitory effect became more pronounced over time, reaching a peak at 72 hours (see [link to relevant documentation]). Figure 10 ).

[0093] Subsequently, the concentration of 2-DG in the aforementioned drug-resistant cells was controlled at 0.5 mM, and different concentrations of enzalutamide (e.g., 10) were used. 0 10 0.5 10 1 10 1.5 10 2 Cells were treated with 2-DG (μM) and cultured for 72 h. Afterward, they were treated with CCK-8, and absorbance was measured at 450 nm. The results showed that, compared with enzalutamide alone, co-treatment with enzalutamide and 2-DG significantly reduced the proliferation of both drug-resistant cell lines (see [link to study]. Figure 11 ).

[0094] The specific experimental steps for cell clone formation are as follows:

[0095] (1) When the drug-resistant cell lines (LNCaP_ENZR and C4-2B_ENZR cells) grow to the logarithmic phase, they are digested with trypsin and counted. The appropriate cell density (about 1000 cells / well) is selected according to the doubling time of each cell type. The cells are seeded into 6-well plates containing 2 mL of 37℃ pre-warmed culture medium and gently rotated to disperse the cells evenly. Each group is treated with enzalutamide (C4-2B: 25 μM, LNCaP_ENZ: 10 μM) and / or 2-DG (0.5 mM) (DMSO is used as a solvent control). The cells are then cultured in a cell culture incubator at 37℃ with 5% CO2 and saturated humidity.

[0096] (2) When visible clones appear in the culture dish, stop the culture, discard the supernatant, carefully wash twice with PBS, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol and wash the residual methanol with water; cell clones can then be observed; under a microscope, a number of cells > 50 is considered a valid clone.

[0097] The results were consistent with the cell proliferation experiments described above, namely that co-treatment with 2-DG and enzalutamide significantly reduced the colony-forming ability of both drug-resistant cell lines (see [link to study]). Figure 12 The above results indicate that prostate cancer cells resistant to enzalutamide exhibit higher glycolytic activity, and that 2-DG-targeted glycolysis can effectively reverse this resistance.

[0098] Example 3

[0099] Having clarified the role of glycolysis in lethal prostate cancer, it is necessary to further identify key pivotal genes in this pathway. Using the SU2C dataset and PFS as the training cohort, LASSO-Cox regression analysis was performed on all genes involved in the glycolysis pathway. CLN6 was found to be highly associated, with a log2 (Hazard Ratio (95% CI)) value of 1.32 (p < 0.001), the highest correlation coefficient among all screened genes. Therefore, it is considered that CLN6 plays a crucial role in the occurrence and progression of lethal prostate cancer through the glycolysis pathway.

[0100] To verify the role of CLN6 in prostate cancer, Western blot analysis was first performed to analyze the expression of CLN6 in various benign prostatic hyperplasia (BPH) and prostate cancer (PCa) cell lines. The specific steps are as follows:

[0101] (1) Take cells in the logarithmic growth phase, digest and collect them, add lysis buffer, and lyse on ice for 1 hour.

[0102] (2) Centrifuge at 15000×g at 4℃ for 15 min, take the supernatant and add loading buffer (to 1×), then incubate at 95℃ for 5 min.

[0103] (3) Take the sample prepared in step (2) and perform protein gel electrophoresis.

[0104] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200mA constant current transfer for 2h).

[0105] (5) After the transfer is completed, the PVDF membrane is removed and placed in the sealing solution and sealed at room temperature for 2 hours on a vertical shaker at 10 rpm.

[0106] (6) After sealing, clean the PVDF membrane, immerse it in the primary antibody (anti-CLN6), and incubate it overnight at 10 rpm and 4°C on a vertical shaker.

[0107] (7) After the primary antibody incubation is complete, wash the PVDF membrane and then immerse it in the secondary antibody (HRP-linked anti-rabbit IgG) and incubate it at room temperature for 2 hours at 10 rpm on a vertical shaker.

[0108] (8) After the secondary antibody incubation is completed, the PVDF membrane is washed and the target protein is detected using a chemiluminescence analyzer (β-actin is used as an internal control).

[0109] Test results as follows Figure 13As shown in the figure. The results showed that compared with BPH, CLN6 expression was significantly increased in PCa (PC-3, DU145, 22Rv1, LNCaP, C4-2, C4-2B), with the highest expression level in DU145 cells; and CLN6 expression was also significantly increased in enzalutamide-resistant PCa cell lines (LNCaP_ENZR and C4-2B_ENZR). Simultaneously, the CLN6 mRNA level in the above cells was detected using primers (upstream sequence as shown in SEQ ID NO: 2, 5'-GGCCAGAGACACTGGGA-3', downstream sequence as shown in SEQ ID NO: 3, 5'-AACCAGAGGTCGAGGTGGA-3'), and the results were consistent with the Western blot results, indicating that CLN6 expression in prostate cancer cells was significantly higher than in BPH (results not shown).

[0110] Subsequently, C4-2_ENZR and LNCaP_ENZR cells (constructed using the method described in Example 2) were transfected with shRNA targeting CLN6 (sh-CLN6), and the expression of CLN6 was detected by Western blotting. The sequence of sh-CLN6 is shown in SEQ ID NO: 1, which is 5'-GCTGCTTTACTGCCTCTAAAG-3'. The specific steps are as follows:

[0111] (1) The shRNA targeting CLN6 (sh-CLN6) was transfected into C4-2_ENZR and LNCaP_ENZR cells, respectively, and cells transfected with blank vector si-NC were set as controls. The cells were cultured under normal culture conditions. Cells in the logarithmic growth phase were digested, collected, and lysed on ice for 1 h.

[0112] (2) Centrifuge at 15000×g at 4℃ for 15 min, take the supernatant and add loading buffer (to 1×), then incubate at 95℃ for 5 min.

[0113] (3) Take the sample prepared in step (2) and perform protein gel electrophoresis.

[0114] (4) After electrophoresis, the protein gel was transferred to a membrane (PVDF membrane, 200mA constant current transfer for 2h).

[0115] (5) After the transfer is completed, the PVDF membrane is removed and placed in the sealing solution and sealed at room temperature for 2 hours on a vertical shaker at 10 rpm.

[0116] (6) After sealing, clean the PVDF membrane, immerse it in the primary antibody (anti-CLN6), and incubate it overnight at 10 rpm and 4°C on a vertical shaker.

[0117] (7) After the primary antibody incubation is complete, wash the PVDF membrane and then immerse it in the secondary antibody (HRP-linked anti-rabbit IgG) and incubate it at room temperature for 2 hours at 10 rpm on a vertical shaker.

[0118] (8) After the secondary antibody incubation is completed, the PVDF membrane is washed and the target protein is detected using a chemiluminescence analyzer (α-Tubulin is used as an internal control).

[0119] Test results as follows Figure 14 As shown in the figure. The results showed that, compared with the blank vector si-NC group, the expression level of CLN6 in prostate cancer cells transfected with sh-CLN6 was significantly inhibited (**p<0.01, ***p<0.001).

[0120] The glucose uptake and lactate production experiments were repeated using the CLN6 knockdown cell line constructed with sh-CLN6. The results showed that CLN6 knockdown significantly reduced the glucose uptake and lactate production rates of prostate cancer cells (see [link to study]). Figure 15-16 Subsequently, the CLN6 knockdown cell line constructed using sh-CLN6 was used to assess glycolytic activity using the Seahorse XF glycolysis stress test kit (103020-100, Agilent, California, USA). The specific steps are as follows:

[0121] (1) The sensor tube plate was incubated at 37°C overnight with the hydration medium for measurement; the microplate for hippocampal XF cell culture was prepared and the cell confluence was verified under a microscope.

[0122] (2) Prepare a test reagent with 2 mmol / L glutamine, replace the culture medium in the microplate with the test reagent, and incubate in a CO2-free incubator at 37°C for 1 hour.

[0123] (3) Prepare stock solutions of glucose (100 mmol / L), oligomycin (100 μmol / L), and 2-DG (500 mmol / L); load 56 μL, 65 μL, and 69 μL of glucose, oligomycin, and 2-DG solutions at ports A, B, and C, respectively.

[0124] (4) Start the Seahorse XF software, select the glycolysis stress test scheme, and calibrate the sensor box with added compounds on the instrument tray; after calibration, replace the hydration plate with a cell culture microplate for testing.

[0125] Experimental results are as follows Figure 17As shown in the figure. The results showed that the addition of glucose triggered a rapid increase in extracellular acidification rate (ECAR), but the ECAR value in the sh-CLN6 group was consistently lower than that in the sh-NC control group. After oligomycin inhibited ATP synthase, cells became more dependent on glycolysis, leading to an increase in ECAR; however, the peak ECAR value in the sh-CLN6 group was lower than that in the control group. After the addition of 2-DG, the ECAR value in both groups returned to baseline levels.

[0126] Furthermore, the CLN6 knockdown prostate cancer cells constructed above were used to conduct cell proliferation and colony formation experiments. The specific steps of the cell proliferation experiment are as follows:

[0127] (1) shRNA targeting CLN6 (sh-CLN6) was transfected into C4-2_ENZR and LNCaP_ENZR cells (constructed using the aforementioned method), and cells transfected with the blank vector si-NC were set up as controls; different concentrations (e.g., 10) were used in each group. 0 10 0.5 10 1 10 1.5 10 2 The sample was treated with enzalutamide (μM).

[0128] (2) When the cells grow to the logarithmic phase, trypsin digest and count them. Select the appropriate cell density according to the doubling time of various cells and seed them into 96-well plates (3 replicates).

[0129] (3) The cells were cultured in an incubator at 37°C and collected after 48 hours of culture. 10 μL of CCK-8 was added to each well, and the culture plate was incubated in the incubator for 2 hours. The absorbance at 450 nm was measured to assess the cell proliferation status.

[0130] Experimental results are as follows Figure 18 As shown in the figure. The results showed that, compared with the control group (si-NC), inhibiting the expression of CLN6 with CLN6 inhibitor could effectively inhibit the proliferation of prostate cancer cells, improve the inhibitory activity of enzalutamide on drug-resistant cell lines, and reverse their drug resistance.

[0131] The specific experimental steps for cell clone formation are as follows:

[0132] (1) The shRNA targeting CLN6 (sh-CLN6) was transfected into C4-2_ENZR and LNCaP_ENZR cells (constructed using the aforementioned method), and cells transfected with the blank vector si-NC were set as controls.

[0133] (2) When the cells grow to the logarithmic phase, digest them with trypsin and count them. Select an appropriate cell density (about 1000 cells / well) according to the doubling time of various cells. Seed them into a 6-well plate containing 2 mL of 37℃ pre-warmed culture medium and gently rotate it to disperse the cells evenly. Incubate the cells in a cell culture incubator at 37℃ with 5% CO2 and saturated humidity.

[0134] (3) When visible clones appear in the culture dish, stop the culture, discard the supernatant, carefully wash twice with PBS, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol and wash the residual methanol with water; cell clones can then be observed; under a microscope, a number of cells > 50 is considered a valid clone.

[0135] Test results as follows Figure 19 As shown in the figure. The results showed that, compared with the blank vector si-NC group, silencing the CLN6 gene with shRNA significantly reduced the clonogenic ability of prostate cancer cells, significantly inhibited the clonogenic formation of prostate cancer cells, and improved the sensitivity of drug-resistant cell lines to enzalutamide. The difference was statistically significant.

[0136] As can be clearly seen from the above, this invention first assesses the pathway activity of SU2C lethal prostate cancer samples based on ssGSEA collected from the Hallmark pathway, with progression-free survival (PFS) as the primary endpoint. Comprehensive Cox regression analysis showed that among the first five pathways, glycolysis had the most statistically significant p-value, with a hazard ratio (HR) greater than 1. KM survival analysis was performed in the SU2C cohort, dividing samples into high-risk and low-risk groups. The results showed that higher glycolytic pathway activity was consistently associated with poorer PFS and OS outcomes in lethal prostate cancer. To verify the impact of drug resistance on cellular glycolysis, enzalutamide-resistant prostate cancer cell lines (C4-2B_ENZR and LNCaP_ENZR) were constructed for glucose uptake and lactate production experiments. The results showed that compared with the WT group, both glucose uptake and lactate production were significantly increased in drug-resistant cells. To verify the role of glycolysis, the glycolysis inhibitor 2-DG was used to study its effects on cell function through cell proliferation and colony formation assays. The results showed that, compared with enzalutamide alone, co-treatment with enzalutamide and 2-DG significantly reduced the proliferation and colony formation abilities of two drug-resistant cell lines. After clarifying the role of glycolysis in lethal prostate cancer, using the SU2C dataset and PFS as the training cohort, LASSO-Cox regression analysis was performed on all glycolytic pathway genes, revealing that CLN6 may have a key impact on the occurrence and progression of lethal prostate cancer through the glycolysis pathway. To elucidate the role of CLN6 in prostate cancer, CLN6 expression was first knocked down in C4-2B_ENZR and LNCaP_ENZR cells, and Western blotting confirmed a significant downregulation of its expression efficiency. Glycolysis analysis revealed that knocking down CLN6 significantly reduced glucose uptake and lactate production rates in prostate cancer cells; glucose addition triggered a rapid increase in ECAR, but the ECAR value in the knockdown group was consistently lower than that in the control group. After oligomycin inhibited ATP synthase, cells became more dependent on glycolysis, leading to an increase in ECAR; however, the peak ECAR level in the knockdown group was lower than that in the control group. After the addition of 2-DG, the ECAR levels in both groups decreased to baseline. Cell proliferation and plate colony assays revealed that knockdown of CLN6 expression increased the sensitivity of drug-resistant cell lines to enzalutamide.

[0137] In summary, this invention emphasizes the crucial role of the glycolysis pathway in lethal prostate cancer and identifies CLN6 as a pivotal gene in overcoming treatment resistance. It clarifies that CLN6 serves as a key factor in the development and progression of prostate cancer and the emergence of enzalutamide resistance. Inhibiting CLN6 or reducing its biological activity can significantly suppress prostate cancer proliferation and metastasis, reverse enzalutamide resistance, improve treatment sensitivity, prolong patient survival, and improve prognosis. This invention enriches the understanding of the mechanisms underlying the development and regulation of enzalutamide resistance in prostate cancer treatment, providing ample scientific evidence and theoretical foundation for exploring new molecular targets for prostate cancer diagnosis, prognosis, and treatment, and developing new targeted drugs. It contributes to achieving better precision medicine and has significant social and scientific value.

[0138] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. Use of a CLN6 inhibitor in the preparation of a medicament for preventing and / or treating prostate cancer.

2. Use according to claim 1, characterized in that, The CLN6 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on CLN6 gene.

3. Use of a CLN6 inhibitor in the preparation of a medicament for increasing the sensitivity of enzalutamide to the treatment of prostate cancer.

4. Use according to claim 3, characterized in that, The CLN6 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on CLN6 gene.

5. Use of a reagent for detecting the expression level of CLN6 in the preparation of a product for evaluating the sensitivity of enzalutamide to the treatment of prostate cancer.

6. Use according to claim 5, characterized in that, The reagent for detecting the expression level of CLN6 comprises primers for detecting the expression level of CLN6 gene and / or reagents for detecting the content of CLN6 protein.

7. A pharmaceutical composition for preventing and / or treating prostate cancer, characterized by, The CLN6 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on CLN6 gene.

8. The pharmaceutical composition of claim 7, wherein, The reagent for detecting the expression level of CLN6 comprises primers for detecting the expression level of CLN6 gene and / or reagents for detecting the content of CLN6 protein.

9. A kit for assessing the sensitivity of prostate cancer to treatment with enzalutamide, characterized in that, The CLN6 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on CLN6 gene.

10. The kit of claim 9, wherein The reagent for detecting the expression level of CLN6 comprises primers for detecting the expression level of CLN6 gene and / or reagents for detecting the content of CLN6 protein.

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