CN118121690A - Use of inhibitors of tyrosine phosphorylation of prostata specific acid phosphatase or/and ATG14 in prostate cancer - Google Patents

Abstract

The application belongs to the technical field of anti-tumor, and particularly relates to application of a tyrosine phosphorylation inhibitor of prostatic specific acid phosphatase or/and ATG14 in prostatic cancer. Use of an inhibitor of tyrosine phosphorylation of prostate specific acid phosphatase and/or ATG14 at a site of at least one of Y279, Y357 and Y488 in the manufacture of a medicament for preventing or treating prostate cancer or castration-resistant prostate cancer. In the application, after all 13 tyrosine (Y) sites simulate dephosphorylation (F) mutation, three sites Y279/Y357/Y488 show that ATG14 is attenuated in combination with Beclin 1. cPAP was shown to affect binding to Beclin1 by dephosphorylating ATG14, thereby inhibiting autophagy initiation, and three possible tyrosine phosphorylation sites were found.

Description

Application of tyrosine phosphorylation inhibitors of prostatic isotype acid phosphatase and/or ATG14 in prostate cancer

Technical Field

The present invention belongs to the field of anti-tumor technology, and specifically relates to the application of a tyrosine phosphorylation inhibitor of prostatic isotropic acid phosphatase or/and ATG14 in prostate cancer.

Background technique

Prostate cancer (PCa) refers to an epithelial malignant tumor that occurs in the prostate gland. This type of tumor is hormone-dependent. The incidence of prostate cancer in my country shows an increasing trend every year, and the rate of increase in incidence is also increasing year by year, and most cases are diagnosed in the middle and late stages.

Currently, the treatment of prostate cancer includes surgical treatment and non-surgical treatment. Since PCa is a hormone-dependent tumor, androgen deprivation therapy (ADT) is usually recommended for patients who are not suitable for surgical treatment. ADT is a drug that reduces the level of androgen in the human body and inhibits the growth of PCa. For advanced prostate cancer, androgen deprivation therapy is effective in the early stages, but most patients will develop castration-resistant prostate cancer (CRPC) with a very poor prognosis and short survival.

Phosphorylation modification is crucial in the process of autophagy, and phosphorylation disorders can lead to inhibition or overactivation of autophagy. Human prostate-specific acid phosphatase (ACPP or PAP) is a tyrosine phosphatase. There are cellular forms (cPAP) and secretory forms (sPAP) in the human body. The functions of the two are completely different. sPAP is mainly present in serum and has been used as a prostate cancer marker before the widespread use of PSA (prostate specific antigen). cPAP is highly expressed in normal adult prostate epithelial cells, while its expression in prostate cancer cells is greatly reduced, which is negatively correlated with the progression of prostate cancer and is a tumor suppressor. However, the molecular mechanism of cPAP regulating autophagy is still unclear.

Summary of the invention

The purpose of the present invention is to provide a use of a tyrosine phosphorylation inhibitor of prostatic isotropic acid phosphatase or/and ATG14 in prostate cancer, so as to clarify the molecular mechanism of cPAP regulating autophagy.

The use of a tyrosine phosphorylation inhibitor of prostatic isotype acid phosphatase or/and ATG14 in prostate cancer of the present invention adopts the following technical scheme:

Use of a tyrosine phosphorylation inhibitor of prostatic isotype acid phosphatase or/and ATG14 in the preparation of a drug for preventing or treating prostate cancer or castration-resistant prostate cancer, wherein the tyrosine phosphorylation site is at least one of Y279, Y357 and Y488.

Use of a drug that inhibits at least one of the tyrosine phosphorylation sites Y279, Y357 and Y488 of ATG14 in the preparation of a drug for preventing or treating prostate cancer or castration-resistant prostate cancer.

A drug for preventing, alleviating and/or treating prostate cancer or castration-resistant prostate cancer, wherein the drug contains a tyrosine phosphorylation inhibitor of prostatic isotype acid phosphatase and/or ATG14, wherein the phosphorylation site is at least one of Y279, Y357 and Y488.

Preferably, the drug also contains an autophagy inhibitor.

More preferably, the drug also contains a Beclin1 protein inhibitor.

More preferably, the prostate cancer or castration-resistant prostate cancer cells are 22RV1 and PC32.

Beneficial effects:

After all 13 tyrosine (Y) sites were simulated to be dephosphorylated (F) mutated in this application, three sites Y279/Y357/Y488 showed that the binding of ATG14 to Beclin1 was weakened, indicating that cPAP affects the binding of ATG14 to Beclin1 by dephosphorylating ATG14, thereby inhibiting the initiation of autophagy. This application adopted three tyrosine phosphorylation sites.

BRIEF DESCRIPTION OF THE DRAWINGS

In Figure 1, A is the target band collected after transfection of FLAG-cPAP in 293T cells for protein spectrum detection; B is the partial results of cPAP binding protein spectrum; C is Co-IP showing the binding of ATG14 to cPAP and Beclin1; D is the pan-tyrosine phosphorylation level of ATG14 after transfection of cPAP plasmid; E is a schematic diagram of the ATG14 gene domain and tyrosine site.

Detailed ways

Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

1. Test methods

1. cPAP was subjected to protein spectrum detection and its binding to autophagy-related protein 14 antibody (ATG14) was verified by co-immunoprecipitation (Co-IP) technology.

2. In 22RV1 and PC3 cell lines overexpressing ACPP (cPAP), Western Blot was used to verify the expression of autophagy-related ATG14, p-ATG14, LC3, Beclin1 and P62 proteins; mRFP-GFP-LC3 was used to verify the autophagy level.

3. Use pan-tyrosine phosphorylation antibody to verify whether cPAP changes the tyrosine phosphorylation level of ATG14.

4. All 13 tyrosine sites of ATG14 were mutated (tyrosine Y was mutated to phenylalanine F to simulate the dephosphorylation state), and Co-IP experiments were performed to verify the binding of ATG14 and Beclin1 and screen for functional action sites.

5. The protein bands of FLAG-ATG14 were collected by SDS-PAGE in 293T cells, and phosphorylation mass spectrometry was performed to analyze the tyrosine phosphorylation sites. Combined with the previous experiment, the action sites were screened out.

6. Customize a specific ATG14 phosphorylation antibody for this site, and verify by Western Blot that the phosphorylation level of this site is reduced/increased after overexpression/sh ACPP (cPAP).

7. After the site Y was mutated to D (simulating the continuous phosphorylation modification state) and F (simulating the dephosphorylation state), Western Blot, mRFP-GFP-LC3 lentivirus transfection and electron microscopy were used to detect autophagy-related indicators to verify the function of this site.

Proteins: p62(18420-1-AP), Beclin-1(11306-1-AP), LC3 antibody(ab128025), ATG14(28021-1-APp-ATG14).

The steps of Western blot are as follows:

1) Clean the glue-filled glass plate (0.1 mm) with deionized water and dry it vertically.

2) Prepare separation glue (as shown in Table 1) and slowly add it to the glass plate, add isopropyl alcohol sealing glue, let it stand for 20 minutes to completely solidify, and pour out the isopropyl alcohol.

Table 1

Reagent (μL) 10% 15% Distilled water 1300 600 30% Acr-Bis 1700 2400 1M Tris-HCl (pH = 8.8) 1900 1900 10% SDS 50 50 10% APS 50 50 TEMED 5 3

3) Add 5% concentrated gel, insert a Teflon comb, and let it stand for more than 20 minutes until it solidifies.

Reagent (μL) 5%

Distilled water 1500

1M Tris-HCl (pH = 6.8) 250

10% SDS20

10% APS20

TEMED 2

4) Place the gel in the electrophoresis tank, add electrophoresis buffer, minimize bubbles, and remove the comb.

5) Add 40 μg of sample to each well, add 20 μL of prestained protein marker to one well, fill with electrophoresis buffer, turn on the power, first use 80V constant voltage mode, and switch to 100V constant voltage when the indicator bromofinin front enters the separation gel. Stop when bromofinin reaches about 500 nm from the bottom of the gel and take out the gel plate.

6) Transfer, block and develop: ① Cut the PVDF membrane according to the size of the gel in advance and soak it in methanol for 15 seconds, then wash it with _ddH2O for 3 minutes, and then soak it in transfer buffer; ② Pry open the glass plate in the transfer buffer, trim it and soak it for 15 minutes; ③ Assemble the transfer membrane, press negative electrode → sponge → filter paper → gel

→PVDF membrane→filter paper→sponge→positive electrode in order, drive away bubbles, put it into the electrotransfer instrument, add transfer buffer, install the ice box, turn on the power, and transfer the membrane at 300mA constant current for 120min; ④ Take out the PVDF membrane, put it into 10% BSA blocking solution, and gently shake and incubate it on a shaker for 1-2h; ⑤ Incubate with primary antibody, wash the membrane 3 times with TBST, put it into the antibody incubation box, add the appropriately diluted primary antibody, and shake it at 4℃ overnight; ⑥ Incubate with secondary antibody: take it out the next day, wash the membrane 3 times with TBST, each time for 10min, then put the PVDF membrane in the appropriately diluted secondary antibody, and incubate it on a shaker at room temperature for 1-2h; take out the membrane, wash the membrane 3 times with TBST, each time for 10min; ⑦ Development: prepare horseradish peroxidase substrate in a 1:1 ratio, evenly and fully add it to the strip, quickly place it in a gel imager for exposure and imaging, and save the picture.

mRFP-GFP-LC3 adenovirus was obtained from Genomedicech (Shanghai) and used to track autophagosome maturation. Cells were seeded in 24-well plates and reached approximately 60% confluence at the time of infection. For adenoviral infection, cells were infected using polybrene (Obio, Shanghai) according to the manufacturer's instructions. After 48 h of infection, cells were treated with different reagents and then treated with 50 nM RAPA or 50 μM CQ for 1 h. Autophagy was observed under a Leica TCS SP8 confocal microscope (Wetzlar, Germany). Autophagic flux was determined by counting the number of yellow dots (autophagosomes) and red dots (autolysosomes). More than 10 cells per group were analyzed. Images were acquired using a Leica TCS SP8 confocal microscope.

LC-MS/MS detection method: 293T cells in normal growth medium were transfected with Flag-ATG14 plasmid. Transfected cells were collected and lysed 24h after transfection. Flag-ATG14 was immunoprecipitated with Flag-M2 beads, eluted with Flag peptide (Sigma), subjected to SDS-PAGE, and visualized by Coomassie blue staining. The Flag-ATG14 band was excised, distalized, and digested with 12.5ng/mL trypsin in 50mM ammonium bicarbonate. The peptide mixture was analyzed online using a hybrid Q-Exactive mass spectrometer. Mass spectra were searched against the UniProt human database using the SEQUEST algorithm. Peptide matches were filtered and manually verified based on enzyme specificity, mass measurement error, Xcorr and Corr scores.

Electron microscopy: To analyze the formation of autolysosomes, cells were seeded in six-well plates at a density of 5 × 10 ⁵ cells per well and treated as described above. The cells were then collected, fixed in 2.5% glutaraldehyde for 2 h, incubated with 1% OsO ₄ buffer for another 20 min, washed and dehydrated with ethanol solution. The cells were then embedded in epoxy resin. Finally, the samples were cut into ultrathin sections 70-90 nm thick and observed using a CM-120 electron microscope (Philip, Eindhoven, Netherlands).

According to the HPLC-MS/MS test results, Sangon Biotech (Shanghai) designed and synthesized 13 pairs of PCR primers. Using the ATG14 plasmid as a template, the point mutation plasmid was synthesized after ordinary PCR with 2×Phanta-Max Master Mix (Vazyme, Nanjing, China). The product was treated with the restriction endonuclease DpnI (New England Biolabs, Beijing) to eliminate the remaining plasmid. After transformation, screening and transfection in 22RV-1 cells, the point mutation plasmid was successfully constructed for subsequent experiments.

Immunoprecipitation, cultured cells were lysed on ice for 30 min using IP lysis solution containing 1% protease inhibitor cocktail (MCE, USA). After centrifugation for 10 min, 100 μL aliquots containing total cell protein were incubated with 90 μL anti-FLAG M1 magnetic beads (Sigma-Aldrich, St.Louis, MO, USA) at 4 ° C overnight. The precipitate was washed three times with lysis buffer and then boiled with SDS-PAGE loading buffer (NCM Biotech). The supernatant was extracted using appropriate antibodies for immunoblotting.

2. Results and Analysis

In order to clarify the molecular mechanism of cPAP regulating autophagy, the present application performed protein spectrum detection on cPAP, and the results are shown in Figures 1A and 1B. As shown in Figure 1A, FLAG-cPAP was successfully transfected in 293T cells, and the cPAP level was significantly increased. As shown in Figure 1B, among the binding proteins of cPAP, the number of non-redundant peptides of ATG14 is 5, and the number of non-redundant peptides is relatively high.

Among the 274 cPAP-binding proteins, the autophagy-related protein ATG14 (Autophagy-Related Protein 14) with a high number of non-redundant peptides was selected. The co-immunoprecipitation (Co-IP) results (Figure 1C) further confirmed the interaction between cPAP and ATG14. The results are shown in Figures 1A-C.

Since cPAP exhibits tyrosine phosphatase activity in the acidic environment of tumors, it is speculated that it is likely to act by changing the tyrosine phosphorylation level of ATG14.

The present application used a pan-antibody to confirm that cPAP can reduce the tyrosine phosphorylation level of ATG14 (Figure 1D); through a point mutation experiment, all 13 tyrosine sites (Y) of ATG14 were mutated into phenylalanine (1E) to simulate the dephosphorylation state. The results showed that after Y279/Y357/Y488 mutation, the binding of ATG14 to Beclin1 was weakened, which preliminarily suggested that the above three sites are the binding sites of cPAP and ATG14, and the results are shown in Figure 1.

The binding proteins of cPAP in 293T cells were preliminarily analyzed by mass spectrometry, and the autophagy-related protein ATG14 was screened out; the binding of cPAP and ATG14 was verified by Co-IP; the pan-tyrosine phosphorylation antibody was used to confirm that cPAP can change the tyrosine phosphorylation level of ATG14; and the point mutation experiment preliminarily screened out three target tyrosine sites: Y279/Y357/Y488.

It is planned to overexpress cPAP, and further verify the changes in the levels of phosphorylated ATG14 and important autophagy proteins through Western Blot, GFP LC3, electron microscopy and other techniques; further use phosphorylation modification mass spectrometry to determine all tyrosine phosphorylation sites on ATG14; then use point mutation of ATG14 (Y becomes D and F) to determine the effect of phosphorylation modification on the downstream autophagy function of ATG14; combine mass spectrometry and point mutation results to customize phosphorylation antibodies of related sites, and use point mutation experiments to verify whether the antibody specificity is good; further analyze the effect of related sites on the autophagy function of ATG14 to demonstrate the importance of this site.

In summary, after all 13 tyrosine (Y) sites were simulated to be dephosphorylated (F) mutated, the three sites Y279/Y357/Y488 showed that the binding of ATG14 to Beclin1 was weakened. This indicates that cPAP affects the binding of ATG14 to Beclin1 by dephosphorylating ATG14, thereby inhibiting the initiation of autophagy, and three tyrosine phosphorylation sites were found.

In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (6)

1. Use of a tyrosine phosphorylation inhibitor of prostatic isotype acid phosphatase or/and ATG14 in the preparation of a drug for preventing or treating prostate cancer or castration-resistant prostate cancer, wherein the tyrosine phosphorylation site is at least one of Y279, Y357 and Y488. 2. Use of a drug that inhibits at least one of the tyrosine phosphorylation sites Y279, Y357 and Y488 of ATG14 in the preparation of a drug for preventing or treating prostate cancer or castration-resistant prostate cancer. 3. A drug for preventing, alleviating and/or treating prostate cancer or castration-resistant prostate cancer, characterized in that the drug contains a tyrosine phosphorylation inhibitor of prostatic isotype acid phosphatase and/or ATG14, and the phosphorylation site is at least one of Y279, Y357 and Y488. 4. The drug for preventing, alleviating and/or treating prostate cancer or castration-resistant prostate cancer according to claim 3, characterized in that the drug also contains an autophagy inhibitor. 5. The drug for preventing, alleviating and/or treating prostate cancer or castration-resistant prostate cancer according to claim 4, characterized in that the drug also contains a Beclin 1 protein inhibitor. 6. The use according to any one of claims 2 to 5, characterized in that the prostate cancer or castration-resistant prostate cancer cells are 22RV1 and PC32.