Application of CD317 siRNA in combination with AMPK inhibitors or AMPK siRNA in tumor treatment

Abstract

The application of CD317siRNA in combination with AMPK inhibitors or AMPKsiRNA in tumor treatment belongs to the field of biomedical technology. This invention provides the application of CD317siRNA or CD317 inhibitors in combination with AMPK inhibitors or AMPKsiRNA in the preparation of drugs for treating tumor diseases. The CD317siRNA or CD317 inhibitors inhibit CD317 expression through gene silencing or gene editing, or target and degrade CD317 protein through PROTAC or LYTAC technologies. This invention reduces CD317 expression in cancer cells by CD317siRNA or CD317 inhibitors, thereby inhibiting AMPK signaling and enhancing the sensitivity of tumor cells to AMPK inhibitors. The combination of CD317 inhibitors and AMPK inhibitors can significantly promote tumor cell death, providing a new method for anti-tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of CD317 siRNA in combination with AMPK inhibitors or AMPK siRNA in tumor treatment. Background Technology

[0002] Cancer seriously impacts human health and life. Tumors are a non-hereditary genetic disease. Under the influence of carcinogenic factors, normal cells undergo genetic changes, losing their normal regulation of growth and 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. Currently, there are three main methods of medical treatment for tumors: radiotherapy and chemotherapy, molecular targeted therapy, and immunotherapy. However, almost all drugs, as patients use them for a long time, lead to cancer cells developing resistance, causing the drugs to cease to have an effect on cancer cells. This acquired resistance of tumor cells to drug treatment not only severely limits the effectiveness of clinical treatment but is also the molecular basis for tumor recurrence. Therefore, we need to develop new treatment methods or optimize existing methods, on the one hand, to overcome drug resistance and enhance anti-tumor effects, and on the other hand, to enrich our "arsenal" against tumors and broaden the range of tumor treatment strategies.

[0003] Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy and nutrient sensor that plays a crucial role in cellular 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 through metabolic regulation or phosphorylation-mediated mechanisms targeting tumor suppressors such as TET2, p53, and TSC2. However, in recent years, increasing research has revealed that under certain specific gene states or metabolic stress conditions (such as hypoxia and nutrient deprivation), AMPK expression is upregulated or activation is enhanced in tumor cells. For this type of tumor, AMPK helps tumor cells cope with survival stress through a series of protective mechanisms, such as reducing energy consumption, slowing growth, increasing angiogenesis, and inhibiting inflammatory responses. This 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 in orthotopically transplanted breast tumors by regulating the redox state of tumor cells (Reference: SM Jeon, NSChandel, N. Hay. AMPK regulates NADPH homeostasis to promote tumor cell survival during energy stress. Nature, 485(2012), pp.661-665. Furthermore, in three different mouse leukemia models—acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and acute T-lymphoblastic leukemia (T-ALL)—AMPK gene deletion led to tumor cell death and significantly improved mouse leukemia survival (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.).Studies have also found that in one model, the loss of AMPK α1 and α2 subunits sensitized leukemia cells to cell death caused by redox imbalance induced by nutritional 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 highly promising therapeutic target for tumors that are partially dependent on AMPK kinase activation. Our study also confirmed that targeting and inhibiting AMPK activity or knocking down AMPK expression using siRNA can significantly induce tumor cell apoptosis and inhibit the progression of xenografts in mice, demonstrating the feasibility of targeting AMPK for anti-tumor purposes. For tumors addicted to AMPK, AMPK inhibitors can disrupt tumor cell protein homeostasis and promote tumor cell death, making them a potential anti-tumor drug.

[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 29-33 kDa. It is a surface molecule that has been 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 metastases, chronic B lymphocytic leukemia, and colorectal cancer tissue and plasma samples. It also specifically upregulates cancer cells in metastatic tumor cells and chemotherapy-resistant cells (see: Induction of HM1.24peptide–specific cytotoxic T lymphocytes by using peripheral-blood stem-cell harvests in patients with multiple myeloma. BLOOD, 15 NOVEMBER 2005 VOLUME 106, NUMBER 10). CD317 overexpression is not only positively correlated with clinical indicators such as poor tumor prognosis, but has also been shown to directly promote tumor development and progression in various 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 overexpression in hepatocellular carcinoma enhances EGFR activation through a lipid valve-dependent mechanism, thereby promoting hepatocellular carcinoma cell proliferation. CD317 is also highly expressed in hematologic malignancies, and CD317 knockdown can disrupt calcium balance and protein homeostasis in cancer cells, 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.).Furthermore, CD317 knockdown significantly inhibited the survival of cancer cells under nutrient deprivation (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 cancer therapy. Nevertheless, whether CD317 expression affects the AMPK signaling pathway and whether targeting CD317 can enhance the anti-tumor effect of AMPK inhibitors have not been previously reported. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to design and provide the application of CD317 siRNA combined with AMPK inhibitors or AMPK siRNA in tumor treatment. The present invention provides a novel method to enhance the anti-tumor effect of AMPK inhibitors. It mainly involves a combination strategy of small interfering RNA targeting CD317 or CD317 inhibitors combined with AMPK inhibitors and its application in anti-tumor treatment. The mechanism of action is that CD317 siRNA or CD317 inhibitors reduce the expression of CD317 in cancer cells, thereby inhibiting AMPK signaling and enhancing the sensitivity of tumor cells to AMPK inhibitors.

[0006] On the one hand, the present invention provides the application of CD317 siRNA or CD317 inhibitor in combination with AMPK inhibitor or AMPK siRNA in the preparation of drugs for treating tumor diseases.

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

[0008] The application described herein includes the AMPK inhibitor 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 technologies.

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

[0011] In the aforementioned application, the CD317 siRNA or CD317 inhibitor enhances the production of misfolded proteins induced by AMPK inhibitors, thereby promoting protein homeostasis collapse.

[0012] In the aforementioned application, the CD317 siRNA or CD317 inhibitor enhances the number of tumor cells that die induced by AMPK inhibitors.

[0013] The application in question includes cervical cancer as one of the tumor diseases.

[0014] A pharmaceutical composition that enhances the sensitivity of tumor cells to AMPK inhibitors, characterized in that it comprises one or more of CD317siRNA 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] siRNA knockdown of CD317 can inhibit AMPK activity, thereby enhancing AMPK inhibitor-induced collapse of tumor cell protein homeostasis and cell death. The CD317 inhibitor and AMPK inhibitor combination of this invention can significantly promote tumor cell death. Therefore, the combined application of AMPK inhibitors and CD317 inhibitors holds promise as a new and potential approach for cancer treatment. Attached Figure Description

[0017] Figure 1 A graph showing the protein levels of phosphorylated AMPK (p-AMPK), CD317, and the internal control β-Actin as detected by Western blot;

[0018] Figure 2 A map showing the K48 ubiquitination levels in the NS and SS fractions detected by Western blot;

[0019] Figure 3 This image shows the apoptosis detected by flow cytometry, where PI-positive cells are dead cells. Detailed Implementation

[0020] The present invention will be described in detail below through specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments 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 treatment

[0023] 1) The cervical cancer cell line HeLa was injected at a dose of 4×10 5 Cells were seeded at a density per well in 6-well plates;

[0024] 2) After overnight culture, CD317 siRNA mixtures were transfected separately (si317 mix ) and control siRNAs, of which si317 mix This is a mixture of two siRNA sequences, 1 and 2, targeting CD317 (see Table 1 below), in equal volume ratios. Sequence information is shown in Table 1 below, where 1 and 2 are two siRNA sequences targeting CD317, each containing both a sense strand and an antisense strand. Their sequences are shown below:

[0025] Table 1. Transfection with Si317 mix Sequence information of control siRNAs

[0026]

[0027] 3) After 48 hours, change the medium, add 5 mM metformin (Met) for 2 hours, collect the cells, and extract the total protein.

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

[0029] 3. Test Results

[0030] The results are as follows Figure 1 As shown, CD317 knockdown significantly inhibits AMPK activation (p-AMPK reduction), and this inhibition cannot be reversed after treatment with the AMPK agonist Met, indicating that reducing CD317 expression can significantly inhibit AMPK activation, or even antagonize the activation effect of agonists on AMPK.

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

[0032] 1. Cell treatment

[0033] 1) The cervical cancer cell line HeLa was injected at a dose of 4×10 5 Cells were seeded at a density per well in 6-well plates;

[0034] 2) After overnight culture, CD317 siRNA mixtures were transfected separately (si317 mix ) and control siRNAs;

[0035] 3) After 36 hours, change the medium, add CC to a final concentration of 5 μM and treat for 12 hours. Collect the cells and extract the NS and SS components of the protein. NS: NP-40 soluble, which is the protein that is soluble in NP-40 lysis buffer. SS: SDS soluble, which is the protein that is insoluble in NP-40 but soluble in 2% SDS.

[0036] 2. Western blot analysis of K48 ubiquitination levels (polyUb) in NS and SS fractions. 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 the production of CC-induced misfolded proteins to promote protein homeostasis collapse.

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

[0040] 1. Cell treatment

[0041] 1) The cervical cancer cell line HeLa was injected at a dose of 2×10⁻⁶. 5 Cells were seeded at a density per well in 12-well plates;

[0042] 2) After overnight culture, CD317 siRNA mixtures were transfected separately (si317 mix ) and control siRNAs;

[0043] 3) Change the medium after 36 hours, add CC to a final concentration of 5 μM and treat for 18-24 hours, then collect the cells;

[0044] 2. Flow cytometry analysis of cell death

[0045] 1) Collect the cell suspension. Centrifuge the cell suspension at 3200 rpm and 4℃ 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 in 400 μL of PI staining solution (0.05 mg / mL);

[0047] 3) Stain at 4℃ in the dark for 15 minutes;

[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, CD317 knockdown significantly enhanced cell death levels in the CC-treated group (%PI). + The increase indicates that CD317 knockdown can significantly enhance CC-induced tumor cell death.

Claims

1. Application of CD317 siRNA combined with an AMPK inhibitor in the preparation of drugs for treating tumor diseases; wherein the AMPK inhibitor is Compound C, and the tumor disease is cervical cancer; The CD317 siRNA is a mixture of sequence 1 and siRNA 2 in equal proportions. Each siRNA sequence contains a sense strand and an antisense strand. The sense strand of sequence 1 is SEQ ID NO.1, the antisense strand of sequence 1 is SEQ ID NO.2, the sense strand of sequence 2 is SEQ ID NO.3, and the antisense strand of sequence 2 is SEQ ID NO.4.

Images