A Novel Class of Prostate Cancer Therapeutic Targets and Their Application in Prostate Cancer Treatment

Abstract

This invention relates to the field of pharmaceutical technology, specifically to NUDT5 and SEPT8 as novel therapeutic targets for prostate cancer and their application in prostate cancer treatment. This invention provides a novel targeted therapy regimen for prostate cancer, which specifically kills prostate cancer cells in vivo by inhibiting their proliferation through the simultaneous targeting of NUDT5 and SEPT8 proteins, which are highly expressed in high-risk prostate cancer, thus offering a new approach to the clinical treatment of prostate cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a novel therapeutic target for prostate cancer and its application in the treatment of prostate cancer. Background Technology

[0002] Prostate cancer is the second most common solid tumor in men worldwide, and its rising incidence and cancer-related mortality rates pose a serious threat to the health and lives of middle-aged and elderly men.

[0003] For decades, the standard treatment for most locally advanced or metastatic prostate cancers has been androgen deprivation therapy (ADT). Endocrine therapy depletes the patient's own endogenous androgens or directly targets the patient's androgen receptors, taking advantage of the androgen-dependent nature of prostate cancer. Currently, commonly used novel endocrine drugs to achieve androgen blockade include: bicalutamide, flutamide, abiraterone acetate, enzalutamide, darolutamide, and apalutamide.

[0004] However, castration-resistant prostate cancer (CRPC) invariably relapses within a median of 14–20 months after androgen deprivation therapy (ADT) via chemical or surgical castration. Alterations in androgen receptor signaling are a significant factor contributing to androgen deprivation therapy resistance in prostate cancer. Metastatic castration-resistant prostate cancer (mCRPC) typically exhibits alterations in the AR pathway due to amplification or gain-of-function mutations, increased AR transcription, or an increase in AR cells. Approximately 15%–20% of advanced drug-resistant prostate cancers experience AR-dependent loss of signaling and transform into highly refractory castration-resistant neuroendocrine prostate cancer (NEPC).

[0005] Precision gene therapy is a future trend in translational medicine, and gene-editing technologies capable of simultaneously editing multiple sites on the genome already exist. Identifying universally applicable gene combinations in prostate cancer will greatly contribute to the development of precision gene therapy. Simultaneously, the corresponding protein products of these genes also have the potential to become novel therapeutic targets, leading to better efficacy when used in combination with other drugs.

[0006] The SEPTIN family of proteins are GTP-binding proteins. During metaphase of mitosis, SEPTIN knockout leads to chromosome loss on the metaphase plate, lack of chromosome segregation and spindle elongation, and incomplete cell division due to delayed mitotic exit. During mitosis, SEPTIN proteins may form a scaffold structure, allowing checkpoint regulation of centromere-associated protein E (CENP-E) and other effectors to coordinate cell separation and chromosome aggregation / segregation. In this process, its ATP source may be the hydrolysis of ADP-ribose by NUDT5 in the presence of pyrophosphate. Nuclear-derived ATP is crucial for chromatin sufficiency, transcriptional regulation, and cell proliferation. This invention hypothesizes that NUDT5 and SEPT8 proteins work together within the nucleus. NUDT5 provides a critical energy source in DNA replication and mitosis, while SEPT8 provides the scaffold required for mitosis. Therefore, simultaneously knocking out these two proteins could significantly inhibit the proliferation of prostate cancer cells. The application of simultaneously targeting NUDT5 and SEPT8 in prostate cancer treatment has not yet been reported. Summary of the Invention

[0007] The purpose of this invention is to provide a new targeted therapy for prostate cancer. By simultaneously targeting the NUDT5 and SEPT8 proteins, which are highly expressed in high-risk prostate cancer, this therapy specifically kills prostate cancer cells in vivo by inhibiting their proliferation, thus providing a new approach for the clinical treatment of prostate cancer.

[0008] To achieve the objectives of the invention described above, the present invention adopts the following technical solution:

[0009] This invention first examined the expression levels of NUDT5 and SEPT8 in different prostate cancer cell lines, clarifying the universality of NUDT5 and SEPT8 as therapeutic targets for prostate cancer. Next, the PC3 prostate cancer cell line was selected, and PC3 cell lines with NUDT5 knockout alone, SEPT8 knockout alone, and NUDT5+SEPT8 double knockout were constructed using CRISPR / Cas9 gene editing technology. Cell cycle status of different cell lines was detected by flow cytometry. To further explore the effects of NUDT5 and SEPT8 on prostate cancer cell proliferation, this invention used the ZenCell OWL live-cell imaging system combined with cell proliferation curve plotting and EdU staining to detect cell proliferation rate. Simultaneously, the effects of SEPT8 and NUDT5 on cell migration ability were detected by cell scratch assay, and the effects of SEPT8 and NUDT5 on cell invasion ability were detected by Transwell assay. The reduction in cancer cell proliferation rate, migration, and invasion ability indicates a decrease in the malignancy of cancer cells, suggesting that gene knockout can inhibit cancer cell activity and that this gene could serve as a target for targeted therapy of prostate cancer.

[0010] In a first aspect, the invention provides the use of NUDT5 and SEPT8 as targets in the preparation of a prostate cancer therapeutic agent.

[0011] A second aspect of the invention provides the use of reagents that inhibit the expression of NUDT5 and SEPT8 in the preparation of medicaments for treating prostate cancer.

[0012] Furthermore, the reagents for inhibiting NUDT5 and SEPT8 expression are sgRNA, siRNA, shRNA, miRNA, or antisense nucleotides that specifically interfere with the expression and processing of NUDT5 and SEPT8 genes, or recombinant vectors containing siRNA, shRNA, miRNA, or antisense nucleotides, or inhibitors that specifically inhibit the normal biological function of NUDT5 and SEPT8 proteins.

[0013] Furthermore, the reagent for inhibiting NUDT5 and SEPT8 expression is a reagent for inhibiting NUDT5 and SEPT8 expression using gene knockout technology, specifically CRISPR-Cas9 technology.

[0014] Furthermore, the nucleotide sequences of the sgRNA used in the reagents for inhibiting NUDT5 and SEPT8 expression are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0015] A third aspect of the present invention provides the use of a recombinant vector in the preparation of a prostate cancer therapeutic drug, wherein the recombinant vector contains sgRNA that specifically interferes with the expression of NUDT5 and SEPT8 genes.

[0016] Furthermore, the nucleotide sequences of the sgRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0017] In a fourth aspect, the present invention provides a medicament for treating prostate cancer, wherein the medicament uses an agent that inhibits the expression of NUDT5 and SEPT8 as an active ingredient.

[0018] The advantages of this invention are:

[0019] Compared to existing prostate cancer treatments, this invention, by targeting specific proteins highly expressed in prostate cancer, can reduce the off-target effects of targeted drug delivery. Furthermore, in in vitro experiments, this invention demonstrated that simultaneously knocking out these two target proteins exhibits highly effective inhibition of prostate cancer cell proliferation, migration, and invasion. In addition, combined targeted therapy reduces the required concentration of the targeted drug for each target, resulting in relatively higher safety. Attached Figure Description

[0020] Figure 1 The expression levels of NUDT5 and SEPT8 in different prostate cancer cell lines were detected by Western blotting.

[0021] Figure 2 The study used CRISPR-Cas9 technology to knock out SEPT8 and NUDT5 in the PC3 cell line. A Surveyor Assay was used to detect the gene editing efficiency of the sgRNA of SEPT8 (A) and NUDT5 (B), with lentiCRISPR v2-Scramble as the control group. (C) Western Blot was used to detect the knockout efficiency of SEPT8 or NUDT5 in the PC3 stable cell line; (D) Western Blot was used to detect the knockout efficiency of SEPT8 and NUDT5 in the PC3 SEPT8 and NUDT5 double knockout stable cell line.

[0022] Figure 3 The effect of SEPT8 and NUDT5 knockout on the PC3 cell cycle was detected by flow cytometry.

[0023] Figure 4 The effects of knocking out SEPT8 or NUDT5 alone on the proliferation rate of PC3 cells are shown in Figure (A). The figure shows the cell state recorded in real time by ZenCell OWL. The figure (B) shows the statistical graph of cell proliferation rate.

[0024] Figure 5 The effect of simultaneously knocking out SEPT8 and NUDT5 on the proliferation rate of PC3 cells was investigated. (A) The figure shows the cell state recorded in real time by ZenCell OWL, and (B) The figure shows the statistical graph of cell proliferation rate.

[0025] Figure 6 This is an example of using the EdU assay to detect the effect of SEPT8 and NUDT5 knockout on the proliferation rate of PC3 cells. (A) The figure shows the EdU staining results in cells with SEPT8 or NUDT5 knockout alone, or with both SEPT8 and NUDT5 knockout simultaneously, as well as control cells. The fluorescent signal in the Azide-488 channel represents EdU-positive cells, i.e., cells in the process of DNA replication. The more fluorescent signal, the faster the cell proliferation rate. Hoechst 33342 is a nuclear dye. (B) The figure shows a statistical chart of the proportion of EdU-positive cells in the corresponding cell lines.

[0026] Figure 7 Is it knocking out SEPT8 and / or NUDT5?

[0027] Effects on PC3 cell migration ability. (A) Knocking out SEPT8 or NUDT5 alone in PC-3 cells had no effect on PC3 cell migration ability. (B) Simultaneous knockout of SEPT8 and NUDT5 reduced PC3 cell migration ability. The two images on the left are real-time cell images, and the two images on the right are statistical graphs of PC3 cell migration rate.

[0028] Figure 8 The Transwell assay results show that simultaneous knockout of SEPT8 and NUDT5 can inhibit the cell invasion ability of PC3 cell line. (A) Transwell assay of simultaneous knockout of SEPT8 and NUDT5 in PC-3 cells shows that the invasion ability of PC-3 cells with simultaneous knockout of SEPT8 and NUDT5 is significantly reduced. (B) The figure shows its statistical graph. Detailed Implementation

[0029] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0030] Example:

[0031] 1. Experimental Method:

[0032] Cell culture: All cell lines used in this invention were cultured at a constant temperature of 37°C with 5% CO2. The culture medium was RPMI 1640 medium (10% FBS + 1% penicillin / streptomycin antibiotics).

[0033] Western Blot: Equal amounts of protein (approximately 40 μg) were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane with a pore size of 0.22 μm. The primary antibody was diluted with TBST at a ratio of 1:1000 and incubated overnight at 4°C. The HRP-labeled secondary antibody was diluted with TBST at a ratio of 1:5000, and the WB results were detected by chemiluminescence colorimetric reaction.

[0034] Primary resistance:

[0035]

[0036] Secondary antibody:

[0037]

[0038] CRISPR-Cas9 gene knockout:

[0039]

[0040] The guide RNA was constructed on the lentiCRISR v2 plasmid and packaged into lentivirus in 293T cells. The cleavage efficiency of the guide RNA was detected by a surveyor assay. Prostate cancer cell lines were infected with the lentivirus using the polybrene method, and cells were selected by puromycin 1.0 ng / μL. The knockout efficiency was identified by Western blotting.

[0041] ZenCell OWL cell proliferation assay: Prostate cancer cells were seeded at a rate of 5000 cells / well in a 24-well plate, and cell proliferation was recorded in real time using the ZenCell OWL live cell assay system. The cell proliferation rate was calculated using GraphPad.

[0042] EdU assay for cell proliferation: Prostate cancer cells were divided into groups of 2×10⁻⁶ cells. 5 Cells were seeded per well in 6-well plates and cultured for 24 hours. After 24 hours, the cells were fixed and stained using the Beyotime EdU-488 cell proliferation assay kit. The cells were then photographed using a Mica laser confocal system, and the proportion of EdU-positive cells was counted using GraphPad.

[0043] Flow cytometry analysis of cell cycle: Prostate cancer cells were analyzed at a ratio of 2×10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured for 24 hours. The cells were then fixed and stained using a Linko Bio Cell Cycle Detection Kit. The number and proportion of cells in different cell cycles were then detected using MACSQuant.

[0044] Cell scratch healing experiment: Prostate cancer cells were injected at a rate of 1×10⁻⁶. 5Cells were seeded per well in 12-well plates and cultured for 24 hours. Then, straight, appropriate-width scratches were made on the bottom of the culture dish using a pipette tip. After washing the cells with 1×PBS, the culture medium was replaced with FBS-free RPMI 1640 medium and the cells were cultured for another 24 hours. Photos were taken at 0 hours, 24 hours and 48 hours. The scratch width was calculated using Image Pro Plus software and the cell migration rate was calculated using GraphPad.

[0045] Transwell assay: Matrigel diluted 1:8 with RPMI 1640 medium was added to the Transwell chamber and incubated for 3 h at 37°C and 5% CO2. Prostate cancer cells resuspended in FBS-free RPMI 1640 medium (5 × 10⁶ cells / mL) were added to the upper chamber. 5 Add 0.5 mL of RPMI 1640 medium containing 30% FBS to the lower chamber and continue culturing for 24-48 hours. Remove the chamber, fix the cells with 4% paraformaldehyde, stain with 1% crystal violet, take photos, and use GraphPad to calculate the percentage of cells that successfully penetrated the membrane.

[0046] 2. Experimental Results

[0047] First, in four common prostate cancer cell lines—PC3, DU145, LNCap, and C4-2—and the benign prostatic hyperplasia (BPH) epithelial cell line, this invention detected the expression levels of NUDT5 and SEPT8 using Western blotting. Figure 1 As shown, NUDT5 and SEPT8 were expressed at relatively high levels in almost all cell lines, so subsequent validation experiments were conducted on the PC3 cell line.

[0048] Using CRISPR-Cas9 technology, this invention constructed stable cell lines in the PC3 cell line with NUDT5 knockout alone, SEPT8 knockout alone, and NUDT5+SEPT8 double knockout, respectively. Figure 2 The detailed process is as follows: This invention designs a series of sgRNAs and screens for active sgRNAs using a Surveyor Assay, such as... Figure 2 A and 2B. Subsequently, the two sgRNAs SEPT8-g1 and NUDT5-g1 were selected and gene knocked out in PC3 using the CRISPR-Cas9 system. The results are as follows... Figure 2As shown in Figure C, the SEPT8 and NUDT5 genes were successfully knocked out in the PC3 cell line (stable cell lines were selected using 1.0 μg / mL puromycin). Subsequently, based on the two stable cell lines PC3-SEPT8 KO and PC3-NUDT5 KO, the NUDT5 and SEPT8 genes were further knocked out (stable cell lines were selected using 10 μg / mL blasticidin). The results are shown in Figure C. Figure 2 As shown in D, two SEPT8 and NUDT5 double knockout cell lines, namely PC3-S+N KO and PC3-N+S KO stable cell lines, were successfully constructed in the PC3 cell line.

[0049] Next, flow cytometry was used to examine the cell cycle changes in PC3 cell lines that knocked out SEPT8 and NUDT5, respectively. Figure 3 Knocking out SEPT8 or NUDT5 alone in PC3 cells had no effect on the cell cycle of PC3 cells.

[0050] This invention further used the ZenCell OWL live-cell imaging system to observe and record the cell proliferation process of different PC3 stable cell lines, and plotted the corresponding cell proliferation curves. The results are as follows: Figure 4 A and 4B. Knocking out SEPT8 or NUDT5 alone did not show any effect on the proliferation rate of the PC3 cell line. Therefore, this invention used the ZenCell OWL live-cell imaging system to observe and record the cell proliferation process in the PC3 cell line with double knockout of SEPT8 and NUDT5, and plotted the corresponding cell proliferation curves, as shown in Figure 4B. Figure 5 As shown in A and 5B, both SEPT8 and NUDT5 double knockout cell lines exhibited a significant slowdown in proliferation rate, suggesting that SEPT8 and NUDT5 have a certain degree of synergistic effect, jointly affecting the cell proliferation process of PC3 cells.

[0051] To more accurately verify the effects of SEPT8 and NUDT5 on the proliferation rate of PC3 cells, this invention used EdU staining to detect four different PC3 cell lines: the control group PC3-Scramble stable cell line, the PC3-SEPT8 KO and PC3-NUDT5 KO single-gene knockout stable cell lines, and the PC3-SEPT8 KO + NUDT5K double-gene knockout stable cell line. Fluorescence images of the four PC3 cell lines were captured using a MICA confocal microscope, and the proportion of EdU-positive cells was calculated using ImageJ software to determine their proliferation rate. Figure 6As shown in the figure, the two stable PC3 cell lines with double knockout of SEPT8 and NUDT5 had significantly fewer EdU-positive cells than the other groups, indicating that simultaneous knockout of SEPT8 and NUDT5 in PC3 cells can inhibit the proliferation rate of PC3 cells. Consistent with previous results, knockout of SEPT8 or NUDT5 alone did not affect the proliferation rate of PC3 cells.

[0052] To investigate the effects of SEPT8 and NUDT5 on the migration ability of the PC3 cell line, this invention used a cell scratch assay to examine the effects of SEPT8 or NUDT5 knockout alone or simultaneously on the migration ability of the PC3 cell line. Figure 7 The results showed that knocking out SEPT8 or NUDT5 alone had no significant effect on the migration ability of the PC3 cell line, while knocking out both SEPT8 and NUDT5 simultaneously significantly slowed down the scratch healing rate of the PC3 cell line. This indicates that the simultaneous loss of SEPT8 and NUDT5 significantly affects cell migration ability, suggesting that SEPT8 and NUDT5 may need to work together in the cell migration process.

[0053] Based on the above experimental results, this invention selected PC3 cell lines with simultaneous knockout of SEPT8 and NUDT5 proteins for Transwell experiments to examine their effect on the invasive ability of PC3 cell lines. Figure 8 After 24 hours of Transwell assay, the number of cells stained with crystal violet was observed under a microscope. It was found that the number of PC3 stable cell lines with simultaneous knockout of SEPT8 and NUDT5 that could successfully cross the cell membrane was much lower than that of the control group, indicating that simultaneous knockout of SEPT8 and NUDT5 in PC3 cell lines can significantly inhibit the cell invasion ability of PC3 cell lines.

[0054] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of reagents for inhibiting NUDT5 and SEPT8 expression in the preparation of drugs for treating prostate cancer; the nucleotide sequences of the sgRNA used in the reagents for inhibiting NUDT5 and SEPT8 expression are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

2. The application of a recombinant vector in the preparation of a prostate cancer therapeutic drug, characterized in that, The recombinant vector contains sgRNA that specifically interferes with the expression of NUDT5 and SEPT8 genes; the nucleotide sequences of the sgRNA are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

3. A drug for treating prostate cancer, characterized in that, The drug uses a reagent that inhibits the expression of NUDT5 and SEPT8 as its active ingredient; the nucleotide sequences of the sgRNA used in the reagent that inhibits the expression of NUDT5 and SEPT8 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

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