Targeting the transcriptional activity of STAT3 by a novel fusion protein

Background The continuous activation of transcription factors drives many diseases, including tumors, autoimmune disease, neurodegenerative disease, and male infertility. Thus, Blocking the transcriptional activity of these proteins may inhibit disease progression. In this study, we developed a new method to specifically inhibit the activity of the transcription factor STAT3. Methods Fusing the transcriptional inhibitory domain KRAB with STAT3 successfully blocked the transcription activity of STAT3 in cancer cells without affecting its function in the mitochondria and lysosomes. Results the expression of KRAB-STAT3 fusion protein inhibited the growth of tumor cells. Conclusions The KRAB-STAT3 fusion protein provides a novel approach for drug development for the treatment of cancer or autoimmune diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09837-1.


Background
The abnormal activation of numerous signaling pathways lead to diseases such as tumors, autoimmune diseases, neurodegenerative diseases, and male infertility. Attempts have been made to block the continuous transcriptional activation, such as inducing protein degradation through (Proteolysis-targeting chimeras, PROTAC ) [1], blocking protein-protein interaction through antibodies [2], and silencing or knocking out gene expression using (RNA interference, RNAi ) [3] or Clustered regularly interspaced palindromic repeats (CRISPR)- [4]. The transcription factor signal transducer and activator of transcription 3 (STAT3) is a critical promoter during tumorigenesis [5]. In the inactive state, STAT3 is a monomer and localized in the cytoplasm. When phosphorylated on tyrosine 705 (Y705), STAT3 forms a homodimer with another STAT3 or a heterodimer with other STAT family members. Then, STAT3 is transpositioned into the nucleus by importin-β1 [6] or importin-a3 [7]. The nucleus STAT3 binds to the promoter of the target genes, including c-Myc, cyclinD1, and MCL-1 [8,9], and initiates transcription.
If STAT3 is ov2ctivatedated, it may cause cancer or autoimmune diseases. Many small molecules targeting the phosphorylation site of STAT3 have been developed [10][11][12]. However, due to the structural similarity between STAT proteins and the existence of a heterodimer, it is difficult to specifically inhibit the transcription activity of STAT3 by small molecules. STAT3 also plays a critical role in mitochondria [13] and lysosomes [14]. Moreover, STAT3 diminished markedly in the cell by PROTAC, RNAi, or CRISPR may cause side effects. Therefore, developing a novel strategy to block the abnormal STAT3 activity is an urgent requirement.
Kruppel-associated box (KRAB) is a transcriptional repressor domain commonly found in eukaryotic zinc finger proteins [15]. It is fused with Cas9 to silence the gene expression by inducing the transcriptional inhibition [16,17]. In the present study, we fused the KRAB domain to the N terminal of STAT3 to make a Trojan horse and found that this KRAB-STAT3 (K-S) fusion protein specifically inhibits STAT3 transcription and does not affect the function of the mitochondria and lysosomes.

Mice
Six-eight-week-old nude mice were purchased from Charles River (Beijing, China) and maintained at the Laboratory Animal Center of Sichuan University. An equivalent of 1 × 10 5 cells was injected subcutaneously, and tumor volume was measured with a caliper (length and width). At the end of the experiment, all animals were anesthetized with 10% pentobarbital sodium and killed by cervical dislocation. The present study was approved by the Animal Ethics Committee of Sichuan University (20211478A). All the experimental protocols were approved and carried out in accordance with the relevant guidelines and regulations of the Animal Ethics Committee of Sichuan University and (Animal Research: Reporting of In Vivo Experiments, ARRIVE) guidelines.

Cell proliferation analysis in vitro
Cell proliferation was monitored using BeyoClick ™ EdU-555 (Beyotime Biotechnology, C0075L) according to the manufacturer's instructions. Briefly, 1 × 105 cells were cultured in six-well plates with 10 mM EdU containing complete medium for 2 h and fixed with Immunol Staining Fix Solution (Beyotime Biotechnology, P0098) at room temperature for 15 minutes. Then, the cells were permeabilized with Immunostaining Permeabilization Buffer with Triton X-100 (Beyotime Biotechnology, C0075L) at room temperature for 15 minutes. Subsequently, 500 μL Click Additive Solution (Click Reaction Buffer: 430 μL, CuSO 4 : 20 μL, azide 555: 1 μL, and Click Additive Solution 50 μL) and Hoechst 33342 were added to the cell and incubated in the dark at room temperature for 30 minutes. Finally, cell proliferation was analyzed using FACSAria SORP (BD Biosciences, USA) and FlowJo program.

Immunofluorescence
Cells grown on coverslips were fixed with Immunol Staining Fix Solution at room temperature for 5 minutes and blocked in QuickBlock ™ Blocking Buffer for Immunol Staining (Beyotime P0260) at 37 °C for 30 minutes. Then, the cells were washed with phosphate-buffered saline (PBS, pH 7.2) for 30 min and incubated with primary antibodies at a 1:100 dilution in 5% bovine serum albumin (BSA) at 4 °C overnight, followed by incubation with secondary antibodies at a 1:200 dilution at 37 °C for 1 h and Hoechst 33342 staining at room temperature for 5 minutes. Finally, the cells were mounted with Antifade Mounting Medium (Beyotime Biotechnology, P0128) and examined under a fluorescence microscope (Olympus, IX73, Japan). The primary antibodies used in this study were mouse anti-Stat3 monoclonal antibody (Cell Signaling Technology, 9139, USA), rabbit anti-Flag monoclonal antibody (Cell Signaling Technology, 14,793), and rabbit anti-c-Myc monoclonal antibody (Cell Signaling Technology, 18,583) and the secondary antibodies were donkey anti-rabbit IgG (H + L) Alexa Fluor Plus 488 (Invitrogen, A32766, USA) and donkey anti-mouse IgG Alexa Fluor Plus 555 (Invitrogen, A32773).

Transwell assay
For the migration assay, 1 × 10 5 cells were cultured in the upper chamber with 200 μL FBS-free DMEM medium, while 600 μL DMEM medium with 10% FBS was added to the lower chamber. Cells were incubated at 37 °C in a 5% CO 2 for 48 h. Subsequently, the cells on the top side of the upper chamber were removed gently using a wet cotton swab, and the remaining cells on the bottom side were stained with 0.1% crystal violet and analyzed under a microscope (Olympus, CKX41).

Lysosomal pH estimation by flow cytometry
Lysosomal pH was measured using the lysosomal pH detection kit (Beijing Biolab Technology, HR8268) according to the manufacturer's instructions. Briefly, 1 × 10 5 A375 cells were cultured in the cell culture medium containing P02 for 5 min. Then, lysosomal pH was analyzed on FACSAria SORP (BD Biosciences) using FlowJo software.

Flow cytometric sorting
Flow cytometric sorting was performed as described previously [18]. A375 and HeLa cells were infected with Plvx-KRAB-STAT3-3XFlag lentivirus for 7 days. The cells were digested with trypsin (0.25 mg/mL, Gibco 25,300,054), and the supernatant was removed by centrifugation (500×g, room temperature, 3 minutes). The cells were filtered through a 70-μm membrane, sorted with FACSAria SORP (BD Biosciences), and analyzed with FlowJo software.

Examination of cellular ATP content
The ATP level was detected using ATP Assay Kit (Beyotime Biotechnology, S0026), according to the manufacturer's instructions. Briefly, the cells were lysed using 200 μL lysis buffer and clarified by centrifugation at 4 °C, 12000×g for 5 minutes. Then, 200 μL working solution was added to 20 μL supernatant, and the luminescence was measured on a microplate reader (Molecular Devices, SpectraMax i3x).

Cell component separation
Mitochondria were isolated using the Cell Mitochondria Isolation Kit (Beyotime, C3601), according to the manufacturer's instructions. Briefly, cells were lysed using lysis buffer (250 mM sucrose; 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA) on ice for 10 minutes, and the cell debris was removed by centrifugation at 4 °C, 1000×g for 5 min. The supernatant was clarified by centrifugation at 12000×g for 30 minutes; the mitochondria were in the sediment, and the cytoplasmic component was in the supernatant. Subsequently, the nuclear protein was separated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027). Briefly, cells were lysed using Buffer A, vortexed for 5 s, and placed on ice for 15 minutes. Then, Buffer B was added, and the mixture was vortexed for 5 s and incubated on ice for 1 minute, followed by centrifugation at 4 °C, 16000×g for 5 minutes. The cytoplasmic component was in the supernatant, and nuclear proteins were in the precipitate.

Statistical analysis
Each set of experiments was repeated at least three times. All statistical analyses were performed using Graph-Pad Prism 9. The quantitative data were presented as mean ± standard deviation (SD).

Construction of the KRAB-STAT3 fusion protein
STAT3 has four isoforms (α, β, γ, and δ) [19,20]. Alternative splicing generates STAT3α and STAT3β; STAT3α is the full-length form of STAT3 and acts as a tumor promoter, while STAT3β lacks a transcriptional activation domain. Therefore, STAT3α was used in this study. Moreover, STAT3 was activated to form a homodimer or heterodimer to bind to DNA. Owing to its proximity to the DNA binding domain, the C-terminal of STAT3 is not suitable for modification. Therefore, we fused the KRAB domain to the N-terminal of STAT3 and the flag sequence to the C-terminal. Also, a linker was inserted between KRAB and STAT3. The total length of KRAB-Linker-STAT3-Flag was 868 amino acids, and the total molecular weight was 111 kDa (Fig. 1A). Then, we packaged the virus containing the KRAB-STAT3 coding sequences in 293 T cells to obtain lentiviral particles and infected the A375 or HeLa cells. Subsequently, the cells were screened under puromycin selection, and the KRAB-STAT3-expressing cells were obtained by flow cytometry (Fig. 1B). To further confirm the KRAB-STAT3 fusion protein-expressing cell line, we detected the Flag tag using immunofluorescence and identified the cells positive for KRAB-STAT3 fusion protein by flag tag (Fig. 1C). In addition, Western blot analysis of STAT3 showed that STAT3α was the main form of endogenous STAT3. Also, a 110-kDa fusion protein was detected above the band of STAT3α, which was KRAB-STAT3 fusion protein. The results further confirmed the expression of KRAB-STAT3 fusion protein in monoclonal cell lines (Fig. 1D, E). KRAB-STAT3 forms dimers in HEK293 cells after interleukin (IL)-6 treatment (Fig. 1F). The fusion protein also interacts with endogenous STAT3 (Fig. 1G). Then, the cellular components were isolated, and KRAB-STAT3 was located in the cytoplasm and nucleus (Fig. 1H) but not in mitochondria (Fig. 1I).

KRAB-STAT3 inhibits STAT3 target gene expression
STAT3 is activated in tumor cells, resulting in overexpression of the downstream target genes. To determine whether KRAB-STAT3 negatively regulates the expression of downstream target genes, we used RT-PCR to detect the mRNA level of the target genes. Also, the expression of KRAB-STAT3 reduced the transcription of c-Myc, CyclinD1, and Mcl-1 ( Fig. 2A-D). However, the decrease in the mRNA level was not equal to the decrease in protein level because many events, such as mRNA longevity and ribosome translation efficiency, could cause inconsistency between the mRNA level and protein level  [21]. Therefore, we detected the protein level of these target genes by Western blot and found that the levels of c-Myc, CyclinD1, and Mcl-1 proteins were decreased significantly (Fig. 2E-H). In addition, we used immunofluorescence and found that the expression of c-Myc was decreased in KRAB-STAT3-expressing cells. We also found maximal STAT3 accumulation in the nucleus in most KRAB-STAT3-expressing cells (Fig. 2I, J). However, the percentage of nuclear STAT3 fluorescence [22] is less in KRAB-STAT3-expressing cells (Fig. 2K).

KRAB-STAT3 promotes apoptosis and inhibits proliferation
STAT3 is a widely recognized oncogene, and the continuous activation of STAT3 causes malignant transformation of normal cells [23]. MCL1, the target gene of STAT3, inhibits apoptosis [24]. The inhibition of phosphorylation of STAT3 induces apoptosis in cultured pancreatic cancer cells [25] and solid and hematological tumors [26]. To further confirm the role of KRAB-STAT3 on apoptosis, we detected the cell death rate in the fusion proteintransfected HeLa and A375 cells by flow cytometry. Also, the expression of KRAB-STAT3 slightly increases the apoptosis of HeLa (Fig. 3A,B) and A375 cells (Fig. 3C,D). Moreover, the proliferation-promoting protein cyclinD1 is the target gene of STAT3 [27]. STAT3 promotes cell growth in neurocytes [28], hepatocellular carcinoma cells [29], and colon cancer cells [30]. Next, the overexpression of KRAB-STAT3 decreased the cell proliferation in HeLa (Fig. 3E,F) and A375 cells (Fig. 3G,H). Cell cycle analysis showed that KRAB-STAT3-expressing arrested the A375 cells in G0 and G1 phases (Fig. 3I-K).

KRAB-STAT3 inhibits the migration and invasion in vitro
Invasion and metastasis are major characteristics of tumor cells and also the leading causes of cancer-related deaths [31]. The over-activation of STAT3 promotes cell invasion and metastasis [32,33]. STAT3 inhibitors, piperine [11], pyrimethamine [10], and salidroside [34], inhibit cancer cell metastasis. Transwell assay demonstrated that KRAB-STAT3 expression reduced the invasiveness of HeLa and A375 cells (Fig. 4A-C). Moreover, lysosomal pH, mitochondria reactive oxygen species (ROS), mitochondrial membrane potential, and ATP production were detected using flow cytometry, and no difference was observed between KRAB-STAT3 fusion proteinexpressing cells and control cells (Fig. 4D-K).

KRAB-STAT3 inhibits tumor growth in vivo
To further elucidate the inhibitory effect of KRAB-STAT3 on the in vivo growth of cancer cells, the KRAB-STAT3expressing A375 cells were implanted subcutaneously in nude mice. Next, we found that the expression of KRAB-STAT3 slows tumor growth (Fig. 5A). In addition, tumor weight and volume were lower in KRAB-STAT3positive cells (Fig. 5B,C). To analyze the effect of fusion protein on the metastatic ability of A375 cells, A375 cells were implanted into nude mice by tail vein injection. We observed that the mice xenografted with KRAB-STAT3expressing tumor cells had a prolonged survival time (Fig. 5D).

Discussion
After dimerization, STAT3 formed a pincer-like structure. The head of the pincer was the DNA-binding and SH2 domains, located in the middle and C-terminal of STAT3. If the KRAB domain was fused to the C-terminal of STAT3, the protein might not bind to DNA due to the severe steric hindrance. Therefore, KRAB domain was fused to the N-terminal of STAT3 in this study. Moreover, a linker was inserted between the KRAB domain and STAT3 to weaken the protein rigidity and maintain the transcriptional inhibitory activity of KRAB. Furthermore, STAT3 has two common isoforms: STAT3α and STAT3β. STAT3α but not STAT3β contains the transcriptional activation domain. Some studies suggested that the β-form STAT3 inhibits the transcription function of STAT3α competitively because it binds to the same DNA sequence. Moreover, STAT3α is widely known as an oncogene [19,35]. Therefore, in this study, STAT3α was used to modulate the transcriptional function of endogenous STAT3. The percentage of nuclear STAT3 fluorescence was decreased in KRAB-STAT3 expressing cells, this may be caused by the disruption of the positive feedback loop of STAT3 signaling. IL6 is known as a target gene of STAT3 [9,[36][37][38], which further act as an upstream activator of STAT3. KRAB-STAT3 reduces the transcription of IL6 and thus the content of STAT3 in the nucleus. Further studies are needed to confirm this hypothesis.
Furthermore, STAT3 is localized in the mitochondria [39]. However, KRAB-STAT3 does not translocate into the mitochondria, which could be attributed to the low level of phosphorylation on serine 727 of STAT3. The expression KRAB-STAT3 inhibits cancer cell proliferation both in vitro and in vivo, caused by inhibition of STAT3 signaling through KRAB-STAT3. The over-activated STAT3 signaling in cancer cells induces target gene expression; these genes include cyclinD1 (promotes cell growth) and the BCL2 family member MCL-1 (reduces apoptosis). Taken together, the persistent transcript of these genes fuels tumor growth. However, when these are blocked by KRAB-STAT3, the tumor growth was slowed down. Therefore, KRAB-STAT3 provided a novel method to block tumor growth.
AAV is a widely used gene delivery vector [40]. Using AAV to deliver the target genes to tumor cells is a critical application. KRAB-STAT3 is only 2.6 kbp and can be packaged into AAV and delivered to cancer cells using tumor-targeting nanoparticles. Therefore, the fusion of the inhibitory domain to a transcript factor is a promising tool in disease treatment.

Conclusion
The present study provides a new approach to treating cancer, which could be used in drug development to treat the diseases caused by out-of-control transcription factors.