CN110151999A - Targeted drugs for inhibiting the progression of malignant melanoma - Google Patents

Abstract

The invention belongs to the field of biotechnology and medicine, and relates to a targeted drug for inhibiting the progression of malignant melanoma. The targeted drug for inhibiting the progression of malignant melanoma can regulate TNFAIP3, 14‑3‑3ζ, Prohibitin or P‑stat3 The protein expression level and transcription level of pathway molecules and the protein expression level of downstream PD‑L1 can inhibit the progression of melanoma from proliferation, invasion, metastasis and immune escape. The invention provides a targeted drug for inhibiting the progression of malignant melanoma, which can provide a theoretical basis and a key target for melanoma targeted drug therapy and combined biological therapy.

Description

Targeted drugs for inhibiting the progression of malignant melanoma

technical field

The invention belongs to the field of biotechnology and medicine, and relates to a targeted drug, in particular to a targeted drug for inhibiting the progress of malignant melanoma.

Background technique

Melanoma is a malignant tumor derived from melanocytes, and its morbidity and mortality have gradually increased in recent decades. In light-skinned people, the incidence of melanoma increases at an annual rate of 3-7%, and more than 1/5 of melanoma patients develop tumor metastasis, which is related to death. Compared with other solid tumors, melanoma has a higher proportion of invasion and metastasis, and the average age of death is lower. Therefore, the research on the molecular mechanism of the pathogenesis and progression of melanoma has also received great attention.

Previous studies on the pathogenesis of melanoma mainly focused on the genetic background and the interaction between sun exposure and melanoma. These studies suggest that there are distinct biological subtypes of melanoma with varying pathogenesis, genetic patterns, and etiological links to sun exposure. In familial melanoma, the susceptibility locus of high-penetrance melanoma is CDKN2A, which encodes P16 and P14ARF, which regulate the cell cycle through the retinoblastoma protein (Rb) and P53 pathways, respectively. In the sporadic melanoma patient population, 55-60% of melanomas were found to have BRAF mutations, while 20-30% had NRAS mutations. In one study, it was confirmed that 59% of melanomas derived from intermittent sunlight exposure had BRAF mutations, significantly higher than other types of melanomas. At the same time, the study found that among the pigmentation genes, the melanocortin-1 receptor (MC1-R) gene was associated with melanoma, and its correlation exceeded the clinical skin phenotype.

In recent years, research on the mechanism of melanoma progression has mainly focused on the role of stem cells, cell cycle regulation, aging, autophagy, and apoptosis in the development of melanoma. Studies on somatic gene mutations in melanoma and nevus have identified several key cellular pathways for nevus formation and malignant transformation. The important pathways that have been confirmed include Rb pathway, P53 pathway, RAS/MAPK pathway and PI3K/AKT pathway. In the AKT pathway, BRAF/NRAS mutations can increase the PI3K/AKT cascade amplification effect, and high expression of AKT can promote the survival of tumor cells by activating downstream mTOR to inhibit autophagy and other pathways. In addition, in terms of immune mechanism, many melanoma antigens that can be recognized by autologous T cells and antibodies have also been found, among which important antigens include: (1) mutated tumor antigens (such as mutated P16); (2) cancer/testis family Common tumor-specific antigens (such as MAGE-1, MAGE-3, NY-ESO-1, etc.); (3) Cell typing-specific differentiation antigens (such as tyrosine kinase, gp100, Melan-A/MART-1 Wait). Since melanoma is an immunogenic tumor, several immune escape mechanisms have also been found in advanced tumors, including loss of specific antigens and MHC class I molecules, and loss of secretion of immunosuppressive cytokines.

Ubiquitination is a highly conserved post-translational modification that can lead to changes in intracellular protein interactions, functions, and stability. It plays an important role in a variety of cellular processes, such as cell cycle, transcriptional regulation, and signal transduction, regulating almost every aspect of cellular physiological functions. At the same time, recent studies have found that ubiquitination is also closely related to autophagy: in muscle atrophy cells, forkhead proteins O (FOXO) 1, 3, 4, which are necessary for AKT activation, can simultaneously regulate autophagy and ubiquitination. system; and the ubiquitin kinase PINK1 can also phosphorylate ubiquitin, activate the ubiquitin ligase parkin and recruit autophagy receptors on the mitochondrial outer membrane to promote autophagy. The ubiquitination process is regulated by three types of enzymes: the ubiquitination activator (E1) can activate ubiquitination through an ATP-dependent reaction, which connects the C-terminus of the ubiquitin molecule and the E1 enzyme through a thioester bond; the ubiquitin conjugation The enzyme (E2) receives ubiquitin from the E1 enzyme by thiolation; the ubiquitin ligase (E3) facilitates the transfer of ubiquitin to the lysine residues of the matrix molecule, forming isomeric peptides. Ubiquitin can be further ubiquitinated through receptor lysine residues or polyubiquitinated N-termini. Deubiquitination is the reverse process of ubiquitination regulated by deubiquitinating enzymes (DUBs), which hydrolyze ubiquitin-subunit peptide bonds to reverse ubiquitination. More than one hundred genes encoding deubiquitinating enzymes have been found in humans, including carboxy-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Josephins, and JAB1/MPN/MOV34 metalloenzymes (JAMMs) and others. Except JAMM family, all are zinc metalloproteases. DUBs have multiple roles in regulating the ubiquitination pathway, including removing mono/polyubiquitin from protein subunits, activating ubiquitin by cleaving ubiquitin from precursor molecules, etc. At present, many studies have found that deubiquitinating enzymes are closely related to the occurrence and development of tumors. In breast cancer cells, deubiquitinase OTUD3 can inhibit tumorigenesis by regulating PTEN stability; deubiquitinase USP4 can also maintain the stability of PRL-3 and promote colon cancer cell proliferation.

TNFAIP3, also known as tumor necrosis factor-alpha-inducible protein 3 (TNFAIP3), is a ubiquitin-editing enzyme with negative immunoregulatory effects. Studies have found that the ovarian tumor (OTU) domain of TNFAIP3 can function as a deubiquitinating enzyme. Protein structure studies found that there is a cysteine protease fold at the TNFAIP3-OTU residue, which plays a central role in its catalytic hydrolysis. At the same time, the study of the fourth zinc finger structure (ZnF4) of TNFAIP3 found that this structure enables TNFAIP3 to have ubiquitin ligase activity and bind to the ubiquitin chain linked by Lys48. TNFAIP3 is restrictedly expressed in thymus, spleen and other lymphoid tissues and organs, and plays a core regulatory role in inflammatory response. In immune cells, overexpression of TNFAIP3 can deubiquitate through receptor-associated interaction protein silk/threonine kinase (RIPK1), TNF receptor-associated factor 2 (TRAF2), TRAF6 and NF-κB essential regulator (NEMO). Inhibition of NF-κB pathway, which plays a role in inflammatory response. In damaged hepatocytes, TNFAIP3 can regulate the level of phosphorylated STAT3 by inhibiting suppressor of cell signaling 3 (SOCS3), affect the IL-6/STAT3 pathway and promote liver regeneration, and SOCS3 has also been clearly confirmed to have an inhibitory effect on melanoma. cancer effect. In addition, studies have found that TNFAIP3 plays a regulatory role in various tumor tissues and cells. In various lymphoma tissues such as marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma and Burkitt lymphoma, overexpression of TNFAIP3 can inhibit the activity of NF-κB, and can Inhibit tumor cell proliferation and promote its apoptosis. In solid tumors, TNFAIP3 can inhibit the proliferation and metastasis of liver cancer cells by inhibiting the expression of TNF-α and Twist1, and overexpressing TNFAIP3 can reduce the resistance of pancreatic cancer to the antineoplastic drug gemcitabine. It is suggested that TNFAIP3 may play an important role in several tumors through multiple pathways.

Recent studies have shown that programmed cell death protein 1 (PD-1) carried by immune cells such as CD8 ⁺ T cells and tumor cell membrane surface ligand (PD-L1) play an important role in the mechanism of tumor immune escape. It has been reported in the literature that Stat3 is one of the main transcription factors of PD-L1 in tumor cells, and its expression and function play an important role in regulating the expression of PD-L1. As the main regulator of stat3 expression and function, the role of TNFAIP3 in the mechanism of tumor immune escape cannot be ignored. In the research of our research group, it was found that TNFAIP3 can regulate the expression and function of stat3 through 14-3-3ζ phosphorylation and Prohibitin protein expression, and then regulate tumor progression.

Contents of the invention

In order to solve the above technical problems in the background technology, the present invention provides a targeted drug for inhibiting the progression of malignant melanoma that can provide a theoretical basis and a key target for melanoma targeted drug therapy and combined biological therapy.

In order to achieve the above object, the present invention adopts the following technical solutions:

A targeted drug for inhibiting the progression of malignant melanoma, characterized in that: the targeted drug for inhibiting the progression of malignant melanoma can regulate the protein expression of TNFAIP3, 14-3-3ζ, Prohibitin or P-stat3 pathway Level and transcription level and inhibit the protein expression level of downstream PD-L1, and then inhibit the progression of melanoma from proliferation, invasion, metastasis and immune escape.

The above-mentioned targeted drugs for inhibiting the progression of malignant melanoma can inhibit the expression of TNFAIP3 or P-stat3 protein.

The aforementioned targeted drugs for inhibiting the progression of malignant melanoma can promote the expression of 14-3-3ζ or Prohibitin protein.

The above-mentioned targeted drugs for inhibiting the progression of malignant melanoma are TNFAIP3 small interfering RNA, short hairpin interfering RNA, and plasmids or lentiviruses carrying small interfering RNA or short hairpin interfering RNA; or the above-mentioned drugs for inhibiting malignant melanoma Progressive targeted drugs are agonists of 14-3-3ζ or prohibitin.

The aforementioned targeted drug for inhibiting the progression of malignant melanoma also includes targeted drug stabilizers and/or excipients capable of enhancing the effect of the targeted drug for inhibiting the progression of malignant melanoma.

The above adjuvants are one or more pharmaceutically acceptable carriers and/or excipients.

The application of a targeted drug for inhibiting the progression of malignant melanoma in the prevention and treatment of melanoma.

The application of a targeted drug for inhibiting the progression of malignant melanoma in the prevention and treatment of malignant melanoma.

The advantages of the present invention are:

The present invention provides a targeted drug for inhibiting the progression of malignant melanoma, which can inhibit the proliferation, invasion and metastasis of malignant melanoma, and the signaling pathway target of immune escape, that is, TNFAIP3 can negatively regulate 14-3-3ζ and Prohibitin protein expression, thereby promoting Stat3 phosphorylation, thereby promoting melanoma proliferation, invasion and metastasis, and immune escape. Inhibition of TNFAIP3 can inhibit the above biological behaviors of tumors through this signaling pathway, thereby inhibiting tumor progression. In this study, the expression levels of TNFAIP3 in melanoma and nevus cells were analyzed in cells and tissues, and the effects of TNFAIP3 on the proliferation, invasion, metastasis and immune escape of melanoma were studied using cell and animal models, and the relationship between TNFAIP3 and melanoma was explored. The relationship between tumor growth and development, and through cell function experiments, it was clarified that TNFAIP3 regulates 14-3-3ζ and Prohibitin, and then regulates the mechanism of the Stat3 phosphorylation signaling pathway in melanoma, and established a stable transformation through RNA interference technology Cell line, to explore the connection between TNFAIP3 and 14-3-3ζ/Prohibitin/Stat3 pathway in melanoma, and observe its effect on melanoma progression. The invention provides a new signaling pathway for targeted therapy and biological combination therapy of melanoma, and has broad application prospects in the field of medicine. The present invention takes the role and mechanism of TNFAIP3 in melanoma as the key entry point, and uses molecular cell biology techniques to systematically study the targeting of TNFAIP3/14-3-3ζ/ The specific molecular mechanism of the Prohibitin/Stat3 signaling pathway, and the obtained results provide a theoretical basis and key targets for the update of melanoma targeted drugs and combined biological treatment programs.

Description of drawings

Figure 1 shows that silencing TNFAIP3 can inhibit the proliferation of melanoma cells A2058 and A375 through CCK-8 experiments;

Figure 2 Definitively knocking out TNFAIP3 can significantly reduce the volume of tumor mass (a), growth rate (b) and average mass (c) through tumor-bearing experiments in nude mice;

Figure 3 shows that knocking down TNFAIP3 can significantly inhibit the lung metastasis of tumor mass through animal experiments;

Figure 4 shows that TNFAIP3 can inhibit 14-3-3ζ phosphorylation (b) and Prohibitin protein (a) expression through Western Blot experiments;

Figure 5 is a chromatin immunoprecipitation experiment to verify that Stat3 can bind to the PD-L1 promoter region and transcriptionally regulate PD-L1 expression.

Detailed ways

The present invention provides a targeted drug for inhibiting the progression of malignant melanoma, which can inhibit the proliferation, invasion and metastasis of malignant melanoma, and the signaling pathway target of immune escape, that is, TNFAIP3 can negatively regulate 14-3-3ζ and Prohibitin protein expression, thereby promoting Stat3 phosphorylation, thereby promoting melanoma proliferation, invasion and metastasis, and immune escape. Inhibition of TNFAIP3 can inhibit the above biological behaviors of tumors through this signaling pathway, thereby inhibiting tumor progression.

The targeted drug used to inhibit the progression of malignant melanoma provided by the present invention is the study of the role and mechanism of the ubiquitin editing enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3) in malignant melanoma, as well as targeting TNFAIP3 and its downstream Application of phosphorylated 14-3-3ζ, prohibitin and Stat3-related drugs or biological agents in the treatment of melanoma. Molecular biology and animal experiments have proved that in malignant melanoma, TNFAIP3 can promote the growth, invasion, metastasis and PD-L1-mediated immune escape of melanoma cells and tumor masses. TNFAIP3 can inhibit the phosphorylation of 14-3-3ζ and the expression of prohibitin in tumor cells through its ubiquitin editing function, thereby promoting the phosphorylation of Stat3 and promoting tumor progression. siRNA, shRNA or gene editing related technologies targeting TNFAIP3 and its downstream phosphorylation 14-3-3ζ, prohibitin and Stat3 can effectively control the proliferation, invasion, metastasis and immune escape of melanoma, and can be applied to melanoma target to biological therapy.

The present invention will be further described in detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto.

Example 1

The CCK-8 experiment was used to verify that silencing TNFAIP3 can inhibit the proliferation of melanoma cells, and the results are shown in Figure 1. Cell culture: the control cells of the melanoma cell line A2058/A375 cells and the siTNFAIP3 cells were seeded in 96-well plates at a density of 5000 cells/well; 37 ° C, 5% CO ₂ conditions in a constant temperature incubator, respectively After culturing for 0h, 24h, 48h, and 72h, the medium was aspirated, and 100uL of CCK-8 reagent (1:10 diluted in the medium) was added. After incubation at 37°C for 2h in the dark, the OD ₄₅₀ absorbance value was measured with a microplate reader. From Figure 1 (NC: negative control; shTNFAIP3(1): TNFAIP3 shRNA interference fragment 1; shTNFAIP3(2): shRNA interference fragment 2), it can be seen that the growth rate of tumor cells was significantly slowed down after silencing TNFAIP3.

Example 2

The nude mouse tumor-bearing experiment confirmed that knocking out TNFAIP3 can significantly reduce the growth rate of the tumor mass, the results are shown in Figure 2 (Figure 2 is the subcutaneous injection of the human melanoma A375 cell line into nude mice, and the mice were sacrificed after 21 days of continuous observation , take out the growing tumor mass, observe and take pictures; shNC: negative control; shTNFAIP3(1): TNFAIP3 shRNA interference fragment 1; shTNFAIP3(2): shRNA interference fragment 2). Animal model construction: the control group and the tumor cells transfected with shTNFAIP3 short hairpin RNA by lentivirus were subcutaneously injected into immunodeficient nude mice at a quantity of 1×10 ⁷ each, and the tumor mass size was measured twice a week, and observed for 3 The nude mice were sacrificed one week later, and the volume and weight of the tumor mass were measured. It can be seen from Figure 2 that knocking out TNFAIP3 can significantly reduce the growth rate of the tumor mass (b), as well as significantly reduce the volume size (a) and weight (c) of the tumor mass.

Example 3

Animal experiments have verified that knocking down TNFAIP3 can significantly inhibit the ability of tumor mass to metastasize to the lung, and the results are shown in Figure 3. Animal model construction: the control group and mouse melanoma cells transfected with shTNFAIP3 short hairpin RNA by lentivirus were injected into the tail vein of C57 mice at ¹ ×107 cells per mouse, and the C57 mice were sacrificed after 2 weeks of observation , the lungs of the mice were taken to observe the formation of metastases. From Figure 3 (the murine melanoma cell B16F10 was injected into the C57 mouse by tail vein, 21 days later, it was executed and the lungs were taken out for observation. NC: negative control; shTNFAIP3-1: B16F10 cells were transfected with TNFAIP3 shRNA interference fragment 1; shTNFAIP3 -2: B16F10 cells were transfected with shRNA interference fragment 2.) It can be seen that knocking down TNFAIP3 can significantly inhibit the ability of mouse tumor cells to form metastases in the lungs.

Example 4

After knocking out TNFAIP3 in tumor cells with small interfering RNA for 48 hours, the cells were collected to extract proteins, and Western Blot was used to analyze the effects of knockout/overexpression of TNFAIP3 on the expression levels of 14-3-3ζ, prohibitin, and phosphorylated Stat3 proteins in tumor cells. The results See Figure 4. Cellular proteins were collected, and after SDS electrophoresis, the proteins were transferred to PVDF membranes, blocked with 5% BSA at room temperature for 1 h, and coated with primary antibodies corresponding to the proteins to be detected (among them, Anti-P-STAT3, Anti-P- 14-3-3ζ, Anti-prohibitin, Anti-TNFAIP3, Anti-GAPDH were purchased from Abcam Company), and incubated overnight at 4°C. The next day, the primary antibody was recovered, and the PVDF membrane was washed 3 times with TBST, 10 min each time, and then the secondary antibody (Goat anti-rabbit IgG-HRPG, Goatanti-mouse IgG-HRP purchased from SANTA Company) was applied, and incubated at room temperature for 1 h. Then wash with TBST 3 times, 10min each time. Afterwards, luminescent liquid was evenly applied and observed on a gel imaging system. The results are shown in Figure 4, interference/overexpression of TNFAIP3 can significantly negatively regulate the phosphorylation of 14-3-3ζ (b) and the expression of Prohibitin protein (a), while positively regulate the phosphorylation of Stat3.

Example 5

Chromatin immunoprecipitation experiments verified that Stat3 can bind to the PD-L1 promoter region and transcriptionally regulate PD-L1 expression. See Figure 5 for the results. Experimental data show that Stat3 can clearly bind to the PD-L1 promoter region sequence, suggesting that Stat3 is a PD-L1 transcription factor that can transcriptionally regulate PD-L1 expression.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (8)

1. A targeted drug for inhibiting the progression of malignant melanoma, characterized in that: the targeted drug for inhibiting the progression of malignant melanoma can regulate TNFAIP3, 14-3-3ζ, Prohibitin or P-stat3 pathway molecules Protein expression level and transcription level and inhibit the protein expression level of downstream PD-L1, and then inhibit the progression of melanoma from proliferation, invasion, metastasis and immune escape. 2. The targeted drug for inhibiting the progression of malignant melanoma according to claim 1, characterized in that: the targeted drug for inhibiting the progression of malignant melanoma can inhibit the expression of TNFAIP3 or P-stat3 protein. 3. The targeted drug for inhibiting the progression of malignant melanoma according to claim 1, characterized in that: the targeted drug for inhibiting the progression of malignant melanoma can promote the expression of 14-3-3ζ or Prohibitin protein. 4. The targeted drug for inhibiting the progression of malignant melanoma according to claim 1, 2 or 3, characterized in that: the targeted drug for inhibiting the progression of malignant melanoma is small interfering RNA of TNFAIP3, short Hairpin interfering RNA and a plasmid or lentivirus carrying small interfering RNA or short hairpin interfering RNA; or the targeted drug for inhibiting the progression of malignant melanoma is an agonist of 14-3-3ζ or prohibitin. 5. The targeted drug for inhibiting the progression of malignant melanoma according to claim 4, characterized in that: the targeted drug for inhibiting the progression of malignant melanoma also includes a targeted drug stabilizer and/or can enhance The adjuvant for the effect of the targeted drug used to inhibit the progression of malignant melanoma. 6. The targeted drug for inhibiting the progression of malignant melanoma according to claim 5, characterized in that: the adjuvant is one or more pharmaceutically acceptable carriers and/or excipients. 7. The application of a targeted drug for inhibiting the progression of malignant melanoma in the prevention and treatment of melanoma. 8. The application of a targeted drug for inhibiting the progression of malignant melanoma in the prevention and treatment of malignant melanoma.