Application of CD317 siRNA combined with AMPK inhibitor or AMPK siRNA in tumor treatment

Abstract

The invention discloses application of CD317 siRNA combined with an AMPK inhibitor or AMPKsiRNA in tumor treatment, and belongs to the technical field of biological medicines. The invention provides application of CD317 siRNA (small interfering Ribonucleic Acid) or combination of a CD317 inhibitor and an AMPK (adenosine monophosphate kinase) inhibitor or AMPKsiRNA in preparation of a medicine for treating tumor diseases. The CD317 siRNA or the CD317 inhibitor is used for inhibiting the expression of CD317 through gene silencing or gene editing, or is used for degrading CD317 protein in a targeted manner through PROTAC and LYTAC technologies. According to the invention, the expression of cancer cells CD317 is reduced through the CD317 siRNA or the CD317 inhibitor, so that AMPK signals are inhibited, and the sensitivity of tumor cells to the AMPK inhibitor is enhanced. The composition of the CD317 inhibitor and the AMPK inhibitor can remarkably promote death of tumor cells, and a novel anti-tumor method is provided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of CD317 siRNA combined with AMPK inhibitor or AMPK siRNA in tumor treatment. Background Art

[0002] Cancer seriously affects human health and life safety. Tumor is a non-hereditary genetic disease. Under the action of carcinogenic factors, the genes of normal cells change and lose the normal regulation of their growth, leading to abnormal proliferation. Tumor cells have three significant basic characteristics: immortality, migration and loss of contact inhibition. The core idea of ​​tumor treatment is to kill and eliminate tumor cells. At present, there are three main means of medical treatment for tumors: radiotherapy and chemotherapy, molecular targeted therapy, and immunotherapy. However, for almost all drugs, as the patient uses them for a long time, cancer cells will develop resistance to the drugs, causing the drugs to stop acting on cancer cells. This acquired resistance of tumor cells to drug treatment not only severely limits the effect of clinical treatment, but also is the molecular basis of tumor recurrence. Therefore, we need to develop new treatment methods or optimize existing methods. On the one hand, we can break drug resistance and enhance anti-tumor effects. On the other hand, we can enrich our "arsenal" against tumors and broaden the range of choices for tumor treatment strategies.

[0003] Adenosine monophosphate-activated protein kinase (AMPK) is a cell energy and nutrient sensor that plays a key role in cell energy homeostasis and metabolic regulation. Under normal circumstances, AMPK is a tumor suppressor. In the early stages of cancer development, it can inhibit tumor growth by regulating metabolism or phosphorylating tumor suppressors such as TET2, p53, and TSC2. However, in recent years, more and more studies have found that under some special genetic states or metabolic stress conditions (such as hypoxia, nutrient deprivation, etc.), AMPK is upregulated or activated in tumor cells. For these tumors, AMPK helps tumor cells cope with survival stress through a series of protective mechanisms, such as reducing energy consumption, delaying growth, increasing angiogenesis and inhibiting inflammatory response, which may be one of the important reasons why tumors continue to survive under harsh conditions (reference: Faubert B, Vincent EE, Poffenberger MC, Jones RG. The AMP-activated protein kinase (AMPK) and cancer: many faces of ametabolic regulator. Cancer Lett. 2015 Jan 28; 356 (2Pt A): 165-70.), that is, AMPK kinase "addiction" occurs. Studies have found that AMPK has been shown to prolong cell survival by regulating the redox state of tumor cells in orthotopic transplanted breast tumors (reference SM Jeon, NS Chandel, N. Hay. AMPK regulates NADPH homeostasis to promote tumor cell survival during energy stress. Nature, 485 (2012), pp. 661-665). In addition, in three different mouse leukemia models: acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and acute T-lymphocytic leukemia (T-ALL), AMPK gene deletion led to tumor cell death and significantly improved the survival rate of mouse leukemia (reference Eichner LJ, Brun SN. Genetic analysis reveals AMPK is required to support tumor growth in Murine Kras-Dependent Lung Cancer Models. Cell Metab. 2019 Feb 5; 29(2): 285-302.e7.doi:10.1016 / j.cmet.2018.10.005.).Other studies have found that in a model, the loss of AMPKα1 and α2 subunits makes leukemia cells sensitive to cell death caused by redox imbalance induced by nutrient restriction (reference: Saito, Y, Chapple, RH, Lin, A, Kitano, A, and Nakada, D. AMPK protects leukemia-initiating cells in myeloid leukemias from metabolic stress in the bonemarrow. Cell Stem Cell. (2015) 17, 585–596.). These results indicate that AMPK is a very potential therapeutic target for this part of tumors that rely on AMPK kinase activation. Our study also confirmed that targeted inhibition of AMPK activity or knockdown of AMPK expression using siRNA can significantly induce tumor cell apoptosis and inhibit the progression of mouse transplanted tumors, confirming the feasibility of targeting AMPK for anti-tumor. For AMPK-addicted tumors, AMPK inhibitors can disrupt tumor cell protein homeostasis and promote tumor cell death, and are potential anti-tumor drugs.

[0004] CD317, also known as bone marrow stromal antigen 2 (BST-2), Tetherin or HM1.24, is a type II transmembrane glycoprotein with a molecular weight of 29-33kda. It is a surface molecule. It is overexpressed in multiple myeloma, B lymphoma, glioblastoma multiforme, primary lung cancer, head and neck squamous cell carcinoma, endometrial cancer, brain cancer, breast cancer with bone metastasis, chronic B lymphocytic leukemia, colorectal cancer tissue and plasma specimens. At the same time, it is specifically upregulated in metastatic tumor cells and chemotherapy-resistant cells (see: Induction of HM1.24 peptide-specific cytotoxic T lymphocytes by using peripheral-blood stem-cell harvests in patients with multiple myeloma. BLOOD, 15 NOVEMBER 2005 VOLUME 106, NUMBER 10). Overexpression of CD317 is not only positively correlated with clinical indicators such as poor tumor prognosis, but has also been shown to directly promote tumor development in a variety of cell and animal models (see: Overexpression of Transmembrane Protein BST2 is Associated with Poor Survival of Patients with Esophageal, Gastric, or Colorectal Cancer. Ann Surg Oncol. 2017; 24(2): 594-602.). Our study found that CD317 is overexpressed in hepatocellular carcinoma, enhancing EGFR activation in a lipid valve-dependent manner, thereby promoting liver cancer cell proliferation. CD317 is also highly expressed in hematological tumors. Knockdown of CD317 can disrupt cancer cell calcium balance and protein homeostasis, promoting tumor cell death (CD317 maintains proteostasis and cell survival in response to proteasome inhibitors by targeting calnexin for RACK1-mediated autophagic degradation. Cell Death Dis. 2023 May 20; 14(5): 333.).In addition, CD317 knockdown also significantly inhibited the survival of cancer cells in a nutrient-deprived environment (CD317 Promotes the survival of cancer cells through apoptosis-inducing factor. J Exp Clin Cancer Res. 2016 Jul 22; 35 (1): 117.). These results indicate that CD317 is a potential target for tumor therapy. However, whether CD317 expression affects the AMPK signaling pathway and whether targeting CD317 can enhance the anti-tumor effect of AMPK inhibitors has not been reported before. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide the application of CD317 siRNA combined with AMPK inhibitor or AMPK siRNA in tumor treatment. The present invention provides a new method for enhancing the anti-tumor effect of AMPK inhibitor. It mainly relates to the combination strategy of small interfering RNA targeting CD317 or CD317 inhibitor combined with AMPK inhibitor and its application in anti-tumor. The mechanism of action is that CD317 siRNA or CD317 inhibitor reduces the expression of cancer cell CD317, thereby inhibiting AMPK signal and enhancing the sensitivity of tumor cells to AMPK inhibitor.

[0006] In one aspect, the present invention provides the use of CD317 siRNA or CD317 inhibitor combined with AMPK inhibitor or AMPK siRNA in the preparation of a drug for treating tumor diseases.

[0007] The application of four combinations includes: 1. Application of CD317 siRNA combined with AMPK inhibitor in the preparation of drugs for treating tumor diseases. 2. Application of CD317 inhibitor combined with AMPK inhibitor in the preparation of drugs for treating tumor diseases. 3. Application of CD317 siRNA combined with AMPK siRNA in the preparation of drugs for treating tumor diseases. 4. Application of CD317 inhibitor combined with AMPK siRNA in the preparation of drugs for treating tumor diseases.

[0008] In the application, the AMPK inhibitor includes Compound C.

[0009] The application is characterized in that the CD317 siRNA or CD317 inhibitor inhibits CD317 expression through gene silencing or gene editing, or targets and degrades CD317 protein through PROTAC or LYTAC technology.

[0010] In the application, the CD317 siRNA or CD317 inhibitor significantly inhibits the activation of AMPK by reducing CD317 expression.

[0011] In the application, the CD317 siRNA or CD317 inhibitor enhances the production of misfolded proteins induced by AMPK inhibitor to promote the collapse of protein homeostasis.

[0012] In the application, the CD317 siRNA or CD317 inhibitor enhances the number of tumor cell death induced by AMPK inhibitor.

[0013] In the application, the tumor disease includes cervical cancer.

[0014] A pharmaceutical composition capable of enhancing the sensitivity of tumor cells to AMPK inhibitors, characterized in that it comprises one or more of CD317 siRNA or CD317 inhibitors, and one or more of AMPK inhibitors or AMPK siRNAs.

[0015] Compared with the prior art, this application has the following beneficial effects:

[0016] Knocking down CD317 using siRNA can inhibit AMPK activity and enhance the collapse of tumor cell protein homeostasis and cell death induced by AMPK inhibitors. The CD317 inhibitor and AMPK inhibitor composition of the present invention can significantly promote tumor cell death. Therefore, the combined use of AMPK inhibitors and CD317 inhibitors is expected to provide a new potential solution for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a Western blot to detect the protein levels of phosphorylated AMPK (p-AMPK), CD317, and internal reference β-Actin;

[0018] Figure 2 Western blot was used to detect the K48 ubiquitination level in NS and SS fractions;

[0019] Figure 3 This is a diagram of apoptosis detected by flow cytometry, in which PI-positive cells are dead cells. DETAILED DESCRIPTION

[0020] The present invention is described in detail below by specific examples. The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention. The test methods in the following examples, unless otherwise specified, are conventional methods, and the experimental materials used are all commercially available products.

[0021] Example 1: Western blot detection of the effect of CD317 knockdown on AMPK activation

[0022] 1. Cell processing

[0023] 1) Cervical cancer cell line HeLa was cultured at 4×10 5 Cells / well were seeded in 6-well plates at a density of;

[0024] 2) After overnight culture, CD317 siRNAs mixture (si317 mix ) and control siRNAs, among which si317 mix It is a mixture of two siRNA sequences 1 and 2 targeting CD317 (see Table 1 below) in equal volume ratio. The sequence information is shown in Table 1 below, where 1 and 2 are two siRNA sequences targeting CD317, and each siRNA sequence contains a sense strand and an antisense strand. Their sequences are shown below:

[0025] Table 1 Transfection of si317 mix Sequence information of siRNAs and control siRNAs

[0026]

[0027] 3) After 48 hours, the medium was changed and 5 mM metformin (Met) was added for 2 hours. The cells were collected and the total protein was extracted.

[0028] 2. Western blot was used to detect the protein levels of phosphorylated AMPK (p-AMPK), CD317 and internal reference β-Actin.

[0029] 3. Test results

[0030] The results are as follows Figure 1 As shown in the results, CD317 knockdown significantly inhibited the activation of AMPK (reduced p-AMPK), and this inhibitory effect could not be restored after treatment with the AMPK agonist Met, indicating that reducing CD317 expression can significantly inhibit the activation of AMPK and even antagonize the activation effect of agonists on AMPK.

[0031] Example 2: Western blot detection of the effect of CD317 knockdown on AMPK inhibitor (Compound C, CC)-induced protein homeostasis collapse

[0032] 1. Cell processing

[0033] 1) Cervical cancer cell line HeLa was cultured at 4×10 5 Cells / well were seeded in 6-well plates at a density of;

[0034] 2) After overnight culture, CD317 siRNAs mixture (si317 mix ) and control siRNAs;

[0035] 3) After 36 hours, the medium was changed and CC was added at a final concentration of 5 μM for treatment for 12 hours. The cells were collected and the NS and SS component proteins were extracted respectively; NS: NP-40soluble, i.e., proteins soluble in NP-40 lysis buffer; SS: SDS soluble, i.e., proteins insoluble in NP-40 but soluble in 2% SDS.

[0036] 2. Western blot detection of K48 ubiquitination levels (polyUb K48 ).

[0037] 3. Test results

[0038] The results are as follows Figure 2 As shown, CD317 knockdown significantly enhanced the K48 ubiquitination level in the SS fraction of the CC-treated group, indicating that CD317 knockdown can significantly enhance CC-induced misfolded protein production to promote protein homeostasis collapse.

[0039] Example 3: Flow cytometry detection of the effect of CD317 knockdown on tumor cell death induced by AMPK inhibitor CC

[0040] 1. Cell processing

[0041] 1) Cervical cancer cell line HeLa was cultured at 2×10 5 Cells / well were seeded in 12-well plates at a density of;

[0042] 2) After overnight culture, CD317 siRNAs mixture (si317 mix ) and control siRNAs;

[0043] 3) After 36 hours, the medium was changed and CC with a final concentration of 5 μM was added for 18-24 hours, and the cells were collected;

[0044] 2. Analysis of cell death by flow cytometry

[0045] 1) Collect the cell suspension, centrifuge the cell suspension at 3200 rpm, 4°C for 5 min, discard the supernatant, and collect the cells in the precipitate;

[0046] 2) Wash the cells once with pre-cooled PBS, and then resuspend the cells with 400 μL PI staining solution (0.05 mg / mL);

[0047] 3) Stain at 4°C in dark for 15 min;

[0048] 4) Flow cytometry detection of apoptosis: PI-positive cells are dead cells.

[0049] 3. Test results

[0050] The results are as follows Figure 3 As shown in Figure 2, CD317 knockdown significantly enhanced the cell death level in the CC-treated group (%PI + This indicated that CD317 knockdown could significantly enhance CC-induced tumor cell death.

Claims

1. Use of CD317 siRNA or CD317 inhibitor combined with AMPK inhibitor or AMPK siRNA in the preparation of drugs for treating tumor diseases.

2. The use according to claim 1, characterized in that The AMPK inhibitor includes Compound C.

3. The use according to claim 1, characterized in that The CD317 siRNA or CD317 inhibitor inhibits CD317 expression through gene silencing or gene editing, or targets and degrades CD317 protein through PROTAC or LYTAC technology.

4. The use according to claim 1, characterized in that The CD317 siRNA or CD317 inhibitor significantly inhibits the activation of AMPK by reducing the expression of CD317.

5. The use according to claim 1, characterized in that The CD317 siRNA or CD317 inhibitor enhances the production of misfolded proteins induced by AMPK inhibitor to promote protein homeostasis collapse.

6. The use according to claim 1, characterized in that The CD317 siRNA or CD317 inhibitor enhanced the amount of tumor cell death induced by AMPK inhibitor.

7. The use according to claim 1, characterized in that The tumor disease includes cervical cancer.

8. A pharmaceutical composition capable of enhancing the sensitivity of tumor cells to AMPK inhibitors, characterized in that: It comprises one or more of CD317 siRNA or CD317 inhibitor, and one or more of AMPK inhibitor or AMPK siRNA.

Images