CPSF6 promotes the Warburg effect and angiogenesis leading to tumor progression via c-Myc/ FBW7/ VEGF/ PD-L1 signaling axis with its siRNA synergistic potential with Sorafenib as a therapeutic target for liver cancer therapy.

doi:10.21203/rs.3.rs-3143964/v1

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Research Article

CPSF6 promotes the Warburg effect and angiogenesis leading to tumor progression via c-Myc/ FBW7/ VEGF/ PD-L1 signaling axis with its siRNA synergistic potential with Sorafenib as a therapeutic target for liver cancer therapy.

https://doi.org/10.21203/rs.3.rs-3143964/v1

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Background

Though CPSF6 was known to act as an oncoprotein via glycolysis in several cancers, the oncogenic mechanism of CPSF6 was not explored in association with the Warburg effect and angiogenesis in hepatocellular carcinoma (HCC) to date.

Methods

MTT assay, colony formation assay, cell cycle analysis and microarray were conducted in CPSF6 depleted HCC cells. Expression of CPSF6 was evaluated in HCC tissues, cell lines and by The Cancer Genome Atlas (TCGA) analysis, while molecular mechanism of CPSF6 was assessed by Western blotting, Immunofluorescence and Immunoprecipitation. The growth of Hep3B cells was monitored in BALB/c orthotopic and xenograft tumor models with Immunohistochemistry analysis. Also, tube formation assay, CAM assay and ELISA were performed for angiogenesis by CPSF6.

Results

CPSF6 was overexpressed in HCC tissues with poor survival rates compared to normal tissues. Hence, CPSF6 depletion suppressed the viability and colonies, induced apoptosis via PARP cleavages and increased sub-G1 population in Hep3B and Huh7 cells. Also, CPSF6 enhanced the stability of c-Myc via their binding through nuclear colocalization as an upstream of c-Myc, mainly in nucleoplasm. Furthermore, CPSF6 depletion activated phosphorylation of c-Myc (T58) for c-Myc degradation and inactivated c-Myc (S62), which was disturbed by FBW7 depletion or proteosomal inhibitor MG132. Additionally, CPSF6 depletion suppressed the Warburg effect by inhibition of glucose, HK2, PKM2, LDH and lactate, showed synergistic effect with Sorafenib in Hep3B cells and also inhibited angiogenesis by tube formation and CAM assays along with decreased expression and production of VEGF. Notably, CPSF6 depletion attenuated PD-L1 along with the increase of CD4/CD8 cell percentage in the splenocytes of BALB/c nude mice bearing Hep3B cells. Consistently, CPSF6 depletion reduced the growth of Hep3B cells in BALB/c mice in orthotopic and xenograft tumor models with inhibition of tumor microenvironment associated proteins by immunohistochemistry.

Conclusions

These findings suggest that CPSF6 enhances the Warburg effect for immune escape and angiogenesis, leading to cancer progression via c-Myc/ FBW-7/ VEGF/PD-L1 signaling axis with its siRNA synergistic effect with Sorafenib as a molecular target of liver cancer therapy.

Trial registration: retrospectively registered

CPSF6

Warburg effect

immune escape

angiogenesis

hepatocellular carcinoma

c-Myc stability

HK2

VEGF/PD-L1

Hepatocellular carcinoma (HCC) accounts for approximately 90% of liver cancer [1] as the third leading cause of cancer-associated mortality worldwide [2]. It is well documented that HCC progression is critically associated with metabolism [3], angiogenesis [4] and immune escape [5] related signaling pathways, since HCC is regarded a genetically and phenotypically heterogeneous cancer within tumor microenvironment (TME) [6].

Under the TME, dysregulated metabolism is regarded as a driving force for tumor initiation and progression [7, 8], since cancer cells prefer aerobic cancer metabolism for oxidative phosphorylation, which is known as the Warburg effect [9]. Thus, lactate production in cancer metabolism promotes malignant cancer progression through metastasis, angiogenesis and immunosuppression with poor prognosis [10, 11]. Also, it is well-known that extracellular acidification by the Warburg effect enhances immune evasion capacity of cancer cells via an increased ratio of M1-/M2-like macrophage populations [12, 13]. Emerging evidence indicates that HCCs can evade the immune surveillance, without being recognized by antigen-presenting cells (APCs). Thus, PD-L1 + Kupffer cells interact with PD-1 + CD8 + T cells to suppress T cell immunity [14] and elevated PD-L1 expression is critically associated with poor prognosis in HCC patients [15].

Also, angiogenesis critically contributes to the progression of HCC via the VEGF/VEGF receptor (VEGFR) signaling pathway [4, 16], which is targeted by tyrosine kinase inhibitor (TKI) such as Sorafenib [17].

Among Myc family consisting of c-Myc, N-Myc, L-Myc and S-Myc, c-Myc oncogene [18, 19] modulates genes of the biogenesis and glucose and glutamine metabolism [20, 21], while it is regulated by a variety of E3 ligases and ubiquitin proteasome system (UPS) including two F-box proteins such as FBW7, s-phase kinase associated protein 2 (SKP-2 or FBXL1) [22].

Among potent molecular target oncogenes, cleavage and polyadenylation specific factor 6 (CPSF6) is known a component of the cellular cleavage and polyadenylation complex [23]. As a fusion partner with FGFR1 (fibroblast growth factor receptor [24], CPSF6 is involved in the progression of acute myeloid leukemia [25], HIV-1 infection [26], breast cancer [27], gastric cancer [28] and liver cancer [29]. Nonetheless, the underlying molecular mechanism of CPSF6 still remains unclear to date. Thus, in the present study, oncogenic mechanism of CPSF6 was elucidated in HCC tissues, cell lines and orthotopic & xenograft tumor models in association with the Warburg effect closely associated with angiogenesis and immune escape in TME.

Cell lines and cell culture

Human hepatocellular carcinoma cell lines such as Hep3B and Huh7 cells (HepG2, Hep3B, Huh7 and Sk-hep-1) and human embryonic kidney Hek-293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HepG2, Hep3B, Sk-hep-1 and Hek-293T cells were cultured in Dubbeco Modified Eagle’s medium (DMEM, catalog NO. LM 001–05, WelGENE, Republic of Korea). Huh7 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI, catalog NO. LM 011 − 01, WelGENE, Republic of Korea). All cells were cultured at 37°C in the aforementioned medium supplemented with 10% fatal bovine serum (FBS) and 1% antibiotic solution (100 units/ml penicillin and 100 µg/ml streptomycin) in a 5% CO₂ atmosphere. Also, all cell lines were authenticated by short-tandem-repeat profiling and without mycoplasma contamination.

cDNA microarray measurement and data analysis

Total RNA was isolated from Hep3B cells transfected with control vector and CPSF6 siRNA by using Trizol reagent (Invitrogen). RNA quality was assessed by Agilent 2100 bioanalyzer including the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was conducted by using ND-2000 Spectrophotometer (Thermo Inc., DE, USA). Based on library preparation and sequencing shown in Kim et al’s paper [30], data analysis was performed. Differentially expressed genes were determined based on counts from unique and multiple alignments using coverage in Bedtools (Quinlan AR, 2010). The RC (Read Count) data were processed based on TMM + CPM normalization method using EdgeR within R (R development Core Team, 2020) using Bioconductor (Gentleman et al.,2004). Also, gene classification was based on the searches conducted by DAVID (http://david.abcc.ncifcrf.gov/) and Medline databases (http://www.ncbi.nlm.nih.gov/). The RNA mRNA-sequencing data used in the study (GSE229281) are available in a public repository from NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229281).

RNA interference and plasmid transfection

HCC cells were seeded onto culture plate overnight and transfected with the mixtures of CPSF6 siRNA or FBW7 siRNA or SKP2 siRNA or negative control siRNA purchased from Bioneer (Korea) adjusted at 40 nM by using transfection reagent (INTERFERin®, Polyplus, France) according to manufacturer’s protocols. The transfected cells were incubated for 48h for next experiment. Also, CPSF6, V5-c-Myc, HA-Ubiquitination, HA-c-Myc WT, HA-c-Myc S62, HA-c-Myc T58 and pcDNA 3.0 plasmids purchased from Addgene (Watertown, MA, USA) were transfected into the cells by using transfection reagent (Turbofect, Thermo Fisher Scientific, Radnor, PA, USA) and then were incubated for 48h for further study.

Cytotoxicity Assay

Hep3B and Huh7 cells were transfected by CPSF6 siRNA or negative control siRNA for 48 h. The cells were seeded into 96 well plate at a density of 7×10 ³ cells/well and incubated overnight at 37 ℃ for 24h, 48h and 72h. Then 20µl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 1mg/ml, 4 Merck KGaA, Germany) was added to each well of the plate and incubated for 2 h at 37 ℃ in dark. After the supernatant was carefully aspirated, 100 µl of DMSO (Ducksan, Korea) was added and the optical density was measured by Biorad microplate reader model 680 (Biorad, USA) at 570nm.

Colony formation assay

Hep3B and Huh7 cells transfected by CPSF6 siRNA or negative control siRNA were seeded onto 6-well cell culture plate at 1×10 ³ cells/well to examine the proliferation of CPSF6 depleted cells. Medium of the cells was changed every three days and incubated for 2 weeks to form colonies under at 37 ℃ 5% CO₂ incubator. The colonies were stained with Diff Quick solution 2 (Cat No.38721, Sysmex corporation, Japan), dried overnight and counted.

Wound healing assay

Hep3B cells were transfected with siRNA-Control and siRNA-CPSF6 plasmids for 48 h. Then wound healing assay was conducted. Scratch was created with a plastic tip in control or CPSF6 siRNA transfected Hep3B cells. After 24h or 48 h incubation, the migratory activity of the cells was measured.

Cell cycle analysis

Hep3B and Huh7 cells (1 × 10⁵cells/ml) transfected by CPSF6 siRNA or negative control siRNA for 48 h were washed twice with cold PBS and fixed in 75% ethanol at − 20°C. The cells were incubated with RNase A (10 mg/ml) for 1 h at 37°C and stained with propidium iodide (50 µg/ml) for 30 min at room temperature in dark. The stained cells were analyzed for the DNA content by FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) using CellQuest Software

RT-qPCR analysis

To evaluate the expression of CPSF6(forward primer sequence: 5’-GCTGAATATGGTGGGCATGAT-3’, reverse primer sequence: 5’-ATTTGGTGCTGCTCCTTTACC-3’), IL-6 (forward primer sequence: 5’-CCACCGGGAACGAAAGAGAA-3’, reverse primer sequence: 5’-GAGAAGGCAACTGGACCGAA-3’), TGF-β (forward primer sequence: 5’-TTGCTGAGGTATCGCCAGGAA-3’, reverse primer sequence: 5’-CTACTACGCCAAGGAGGTCA-3’) and GAPDH (forward primer sequence: 5’-AGCCACATCGCTCAGACAC-3’, reverse primer sequence: GCCCAATACGACCAAATCC-3’), RT-qPCR analysis was conducted. Total RNAs were isolated from Hep3B cells transfected with siRNA-Control and siRNA-CPSF6 plasmids for 48h by using QIAzol (Invitrogen, Carlsbad, CA, USA). Then cDNA was synthesized with oligo dT (Bioneer, Daejeon, Korea) and M-MLV reverse transcriptase (Enzynomics, Daejeon, Korea). Then RT-qPCR was performed with Light cyclerTM (Roche, Basel, Swiss) according to manufacturer’s protocol.

Tissue microarray and immunohistochemistry

HCC patient tissue microarray (80 cases) was purchased from Biomax (HLivH160CS01, Derwood, MD, USA) and immunohistochemistry (IHC) staining was performed with Discovery XT (Roche, Mannheim, Germany). Hepatocellular carcinoma and matched normal adjacent tumor tissues in the microarray plate were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned at 4µm. Sections were deparaffinized, rehydrated and incubated with 3% H₂O₂. After antigen repair and being blocked, the slides were incubated with mouse monoclonal antibody against CPSF6 (1:200) (sc-376228, Santacruz, Dallas, TX, USA) at 4°C overnight. Subsequently, the slides were incubated with secondary antibody at room temperature for 30 minutes and then with streptavidin peroxidase complex. Staining was performed using 3, 3-diaminobenzidine (DAB) substrate kit for peroxidase reaction and counterstained with hematoxylin. Finally, the slides were analyzed under a light microscope.

Western blotting.

Hep3B and Huh7 cells transfected by transfected by CPSF6 siRNA or negative control siRNA were lysed in NP40 buffer containing 50mM Tris/HCL (pH7.5), 0.5% NonidetP-40, 1mM EDTA, 120 mM NaCl, 1 mM dithiothreitol, 0.2 mM phenylmethylsufonylfluoride with protease inhibitors cocktails (Roche, Mannheim, Germany) and phosphatase inhibitors (Merck KGaA, Darmstadt, Germany). The lysates were quantified by DC Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). The protein samples were electrophoresed on 8 to 15% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked with TBST diluted 5% skim milk for 1h at room temperature or TBST diluted 5% BSA for 4 h at 4 ℃. Then they were incubated with primary antibodies of CPSF6 (Cat No. sc-376228.Santacruz, USA), PARP (Cat.No 9542, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (Cat No 9664, Cell Signaling Technology, USA), c-Myc (Cat No ab32072, Abcam, USA), FBW7 (Cat No ab109617, Abcam, Waltham, MA, USA), SKP2 (Cat.No sc-7164, Santacruz Biotechnology, Dallas, TX,USA), HK2 (Cat No 2106, Cell Signaling Technology, USA), PKM2 (Cat.No 4053, Cell Signaling Technology, USA), LDH (Cat No sc-133123, Santacruz, USA) and β-actin (Cat No A2228, Merck KGaA, USA) diluted in 5% BSA in TBST overnight at 4 ℃, washed three times for 10 min with TBST, and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, USA) for 2h. Expression was visualized by using ECL Immunoblotting detection reagent (GE Healthcare, Giles, UK).

Co-Immunoprecipitation

Hep3B cells transfected by CPSF6 siRNA or negative control siRNA were lyzed in lysis buffer (50 mM Tris–HCl, pH 7.4, 0.1% SDS, 150 mM NaCl, 1% Triton X-100, 1mM NaF, 1mM EDTA, 1mM Na₃VO₄, and 1× protease inhibitor cocktail), and then were immunoprecipitated with antibodies of CPSF6 and c-Myc. Thereafter, protein A/G sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were applied. The final precipitated proteins were subjected to immunoblotting with the indicated antibodies.

Immunofluorescence

Hep3B cells transfected by CPSF6 siRNA or negative control siRNA were fixed on poly-L-lysine-coated slide in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. The permeabilized cells were incubated with 3% bovine serum albumin (BSA) in PBS for 1 h, followed by immunostaining with mouse polyclonal CPSF6 (Cat No. sc-376228.Santacruz, USA) and rabbit polyclonal c-Myc (Cat No ab32072, Abcam, USA). Mouse and Rabbit IgG FITC antibody H & L (Abcam, Cambridge, MA, USA) was used as the secondary antibody. The immunostained cells were mounted in medium containing DAPI (Vectashield, Vector Labs, Burlingame, CA) and visualized under an FLUOVIEW FV10i confocal microscopy (Olympus Corporation, Tokyo, Japan).

Ubiquitination assay

293T cells transfected with siRNA-Control and siRNA-CPSF6 plasmids (V5-c-Myc and HA-Ubiquitin), followed by addition of 20 µM proteasome inhibitor MG132 for 2 h, and immunoprecipitated with anti-HA antibody and protein G-agarose beads, and immunoblotted with anti-HA antibody.

Cycloheximide chase assay

Hep3B cells transfected with siRNA-Control and siRNA-CPSF6 plasmids for 48h were treated with 50 µg/mL cycloheximide (CHX, Merck KGaA, Darmstadt, Germany) for 30, 60 and 120 min or 20 µmol/L MG132 for 4 h before harvest for Western blotting.

Lactate, Glucose and ATP assay

Hep3B cells were transfected with siRNA-Control and siRNA-CPSF6 plasmids for 48 h and then cell culture medium was collected. The levels of lactate (K-607), glucose (K-606) and ATP (K354) (BioVision, Milpitas, CA, USA) were measured in the collected medium by ELISA.

Glucose uptake assay

Glucose uptake was measured by using a fluorescent glucose analog, 2-NBDG (Cayman Chemicals, Ann Arbor, MI, USA). Cells seeded in glass-bottom dishes were incubated with 100 µM2-NBDG for 2 h. Fluorescent images were taken by confocal microscope (Zeiss LSM 700 microscope) with 488 nm laser as excitation source. Images at ~ 10 fields of view were acquired for each sample.

Tube formation assay

Matrigel (BD Biosciences, Bedford, MA, USA) was thawed at 4 ℃ overnight. The 48 well plates coated with 150 ml Matrigel were incubated at 37 ℃ for 30 min. After gel formation, HUVEC cells (1 x10⁵ cells/well) were seeded on the layer of polymerized Matrigel with the cultured media from CPSF6 depleted or untreated Hep3B cells. After 6 h incubation, the cells were fixed with 4% formaldehyde and randomly chosen fields were photographed under an Axiovert S 100 light microscope (Carl Zeiss, USA) at 100X magnification. Tube networks were quantified using NIH Scion image program

Luciferase reporter assay

VEGF promoter construct was cotransfected into CPSF6 depleted Hep3B cells with Renilla luciferase reporter plasmid. Two days after transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

CAM (Chick chorioallantoic membrane) assay

To examine the angiogenic activity of CPSF6, CAM assay was undertaken in vivo. Briefly, cultured media from CPSF6 depleted or untreated Hep3B cells were loaded onto a 1/4 piece of thermonox disc (Nunc, Naperville, IL, USA). The discs were applied onto the CAM of a 9-day-old embryo and incubated for 48 h. Then fat emulsion was injected under the CAM and the number of newly formed blood vessels was photographed and counted. The experiment was repeated in two groups (5 eggs per group

Synergy validation by combination of CPSF6 depletion and Sorafenib.

To determine the synergy between CPSF6 depletion and Sorafenib, cytotoxicity, cell cycle analysis and Western blotting were performed in Hep3B cells exposed to si-CPSF6 and/or Sorafenib. Combination index between CPSF6 depletion and Sorafenib

was analyzed by the Compusyn software (ComboSyn, Inc., New York, NY, USA) and SynergyFinder [31, 32]..

Establishment of CPSF6 shRNA Hep3B cell lines

To establish Hep3B cell lines stably expressing CPSF6 shRNA, control shRNA, CPSF6 shRNA lentiviral vectors and transfection mixtures (Turbofect, Thermo Fisher Scientific) were transfected into Hep3B cells. The transfected cells were grown in the medium supplemented with puromycin at 2 µg/mL for approximately 14 days to eliminate the untransfected cells. Then, the macroscopic clones were picked out and continuously passaged in the medium supplemented with puromycin (0.5 ng/mL–1.5 ng/mL). CPSF6 protein expression was evaluated in Hep3B cells transfected with control shRNA and CPSF6 shRNA by Western blotting for the following animal study

Liver tumor orthotopic & xenograft models and immunohistochemistry

All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHUASP-21-205). For the xenograft model, 1x10⁶ of LV-shControl or LV-shCPSF6 Hep3B cells were suspended in a serum-free medium and mixed with a Matrigel (Corning, Bedford, MA, USA) at a 1:1 ratio. The cell mixture was subcutaneously injected into the right flank of BALB/c athymic nude mice. All mice were sacrificed on Day 42 after implantation and isolated tumors were photographed and weighed. Also, flow cytometry analysis was conducted for CD8/CD4 percentage in the splenocytes isolated from the mice.

For orthotopic tumor model, 2x10⁶ of LV-shControl or LV-shCPSF6 Hep3B cells were suspended in a serum-free medium and mixed with a Matrigel matrix (Corning, Bedford, MA, USA) at a 1:1 ratio. Using a 31G insulin stringe, a small scratch was made in the left-lateral lobe (s) of the liver of BALB/c nude mice under anesthesia with 2% isoflurane, and the cell mixture was slowly injected. The incision site was closed with sutures as described previously [33]. All mice were sacrificed on Day 42 after implantation. In addition, immunohistochemistry was conducted in tumor sections with antibodies of for CPSF6 (Cat No. sc-376228.Santacruz, USA), c-Myc (Cat No. sc-40. Santacruz, USA), PCNA (Cat No. sc-56, Santacruz, USA), Caspase3 (Cat.No 9662, Cell Signaling Technology, USA), HK2 (Cat.No 2106, Cell Signaling Technology, USA), PKM2 (Cat.No 4053, Cell Signaling Technology, USA), LDH (Cat No. sc-133123.Santacruz, USA), VEGF (Cat No. sc-152.Santacruz, USA) and PD-L1 (Cat No.17952-1-AP, Proteintech, USA).

Statistical analysis

To statistical analysis of the data, Sigmaplot version 12 software (Systat Software Inc., San Jose, CA, USA) was used. All data were expressed as means ± standard deviation (SD) from one representative of at least three independent experiments. Two-tailed t-Student’s test and one-way ANOVA analysis were performed for comparison of two groups. The statistically significant difference was considered p < 0.05 between control and CPSF6 depleted groups

Overexpression of CPSF6 in HCC patient tissues and cytotoxic and apoptotic effects of CPSF6 depletion in HCCs.

Human tissue array showed overexpression of CPSF6 in HCC patient tissues compared to adjacent normal liver tissue (Fig. 1A). Also, TCGA analysis revealed the highly mRNA expression of CPSF6 in HCC patients with poor overall survival rate compared to normal control (Supplementary Fig. 1A, 1B). Consistently, CPSF6 was overexpressed in Ⅰ-Ⅵ stages of HCC (Supplementary Fig. 1C) and also was highly expressed at mRNA level in various cancer cell lines, including breast, liver, colon and prostate cancers in order (Supplementary Fig. 1D). To assess the cytotoxic and anti-proliferative effects of CPSF6 depletion in HCCs, MTT and colony formation assays were conducted in Hep3B and Huh7 cells. As shown in Fig. 1B, CPSF6 depletion exerted significant cytotoxicity in Hep3B and Huh7 cells. Likewise, colony formation assay revealed that the numbers of colonies were significantly reduced in CPSF6 depleted HCCs compared to untreated control (Fig. 1C). To confirm the cytotoxicity by CPSF6 depletion is due to apoptosis, cell cycle analysis and Western blotting were conducted for PARP, CPSF6 and β-actin in Hep3B and Huh7 cells. Here CPSF6 depletion increased sub-G1 population and cleaved PARP in Hep3B and Huh7 cells (Fig. 1D, E). Furthermore, CSPF6 depletion inhibited the migratory activity in Hep3B cells by wound healing assay (Fig. 1F).

Differentially expressed gene profile and related signaling pathways in CPSF6 depleted Hep3B cells.

The cDNA microarray assay revealed differentially expressed gene profile and signaling pathways in CPSF6 silenced cells (Fig. 2A, 2B). In addition, the gene ontology analysis classified affected genes and signaling pathways into apoptosis, cell cycle, cell migration, cell proliferation, cell migration, cell cycle and angiogenesis (Fig. 2B). Of note, metabolism-related genes (22%), cell cycle-related genes (14%), cell death- related genes (12%) and apoptosis-related genes (10%) were critically involved in CPSF6-depleted Hep3B cells (Fig. 2C). Herein, metabolism related genes were differentially expressed with upregulation (red) or downregulation (blue) (Fig. 2D).

CPSF6 modulated c-Myc in HCCs through their binding and colocalization.

To assess the correlation between CPSF6 and c-Myc, cBioPortal database analysis, Western blotting, Immunoprecipitation and Immunofluorescence were conducted. Despite the weak correlation between CPSF6 and c-Myc with Spearman correlation coefficient (r = 0.11) by cBioPortal database (Fig. 3A), the endogenous expression of CPSF6 was in parallel with that of c-Myc in HCCs such as HepG2, Hep3B, Huh7 and Sk-Hep1 cells (Fig. 3B). The colocalization of CPSF6 and c-Myc was found in nucleus rather than in cytoplasm of Hep3B cells by Immunofluorescence (Fig. 3C). Consistently, CPSF6 depletion decreased the expression of c-Myc in Hep3B and Huh7 cells (Fig. 3D). Of note, Immunoprecipitation validated the binding between CPSF6 and c-Myc in Hep3B cells, regardless of depletion or overexpression of CPSF6 (Fig. 3E, 3F). Furthermore, CPSF6 (green) depletion decreased the expression of c-Myc (red) in Hep3B cells by Immunofluorescence. (Fig. 3G).

CPSF6 depletion induced c-Myc degradation mediated by FBW7 and

inhibited c-Myc stability in HCCs.

Since E3 ubiquitin ligase is participated in c-Myc degradation [34], c-Myc ubiquitin ligases such as FBW7 and SKP2 plasmids were transfected into Hep3B cells. Here FBW7 overexpression enhanced c-Myc degradation in CPSF6 depleted Hep3B cells (Fig. 4A) and CPSF6 overexpression reduced the ability of FBW7 to inhibit c-Myc expression in Hek-293 cells (Fig. 4B), while FBW7 depletion restored the expression of c-Myc, but not by SKP2 depletion (Supplementary Fig. 2). Furthermore, CPSF6 depletion enhanced the ubiquitination of c-Myc induced by HA-Ub by ubiquitination assay in Hek-293 cells. (Fig. 4C). Also, proteosomal inhibitor MG132 reversed the reduced expression of c-Myc by CPSF6 depletion in Hep3B cells (Fig. 4D). Of note, the half-life of c-Myc protein was remarkably reduced by CPSF6 depletion in the presence of protein synthesis inhibitor cycloheximide in Huh7 and Hep3B cells (Fig. 4E), implying c-Myc stability control by CPSF6. Also, depletion or overexprssion of CPSF6 regulated phosphorylation of c-Myc (S62) and c-Myc (T58) (Fig. 4F). In contrast, depletion or overexprssion of c-Myc did not affect the expression of CPSF6 (Figure. 4G)

CPSF6 depletion suppressed glycolysis, and its related genes in HCCs.

Based on mRNA-Seqencing data in CPSF6 depleted Hep3B cells (Fig. 2C, 2D), the effect of CPSF6 depletion on glycose metabolism was explored in HCCs by Western blotting, ELISA, Flow cytometry and Immunofluorescence. Regarding protein–protein interactions (PPI). The binding scores between c-Myc and HK2 or PKM2 or LDH were 0.642, 0.728 and 0.769, respectively, by STRING database (Fig. 5A). Also, Spearman correlation coefficients between CPSF6 and HK2 or PKM2 were 0.36 and 0.52, respectively (Fig. 5B). Actually, Western blotting revealed that CPSF6 deletion suppressed protein expression of HK2, PKM2 and LDH in Hep3B cells (Fig. 5C). Furthermore, CPSF6 depletion decreased the levels of glucose, lactate and ATP in the culture supernatants of Hep3B cells by ELISA (Fig. 5D, 5E, 5F). Consistently, CPSF6 depletion reduced glucose uptake in Hep3B cells by Immunofluorescence and flow cytometry analysis (Fig. 5G, 5H). However, it is noteworthy that depletion of HK2, LDH, PD-L1 and VEGF did not affect the expression of CPSF6 in Hep3B cells (Supplementary Fig. 4).

CPSF6 depletion suppressed immune escape and its related genes in HCCs.

Given that metabolism product lactate is involved in immune escape of tumor cells under tumor microenvironment [35], we can expect that CPSF6 may regulate the genes related to immune escape. Indeed, the close correlation of PD-L1 with VEGF or CD4 or CD8 were predicted by STRING database (Fig. 6A). Likewise, Spearman's correlation coefficient data reveal that CPSF6 has positive correlation with PD-L1 (r = 0.12), while it has inverse correlation with CD8(r= -0.48) or CD4(r= -0.12) (Fig. 6B). Also, CPSF6 depletion attenuated the expression of PD-L1 in Hep3B cells (Fig. 6C). Indeed, Immunopreciptation verified the direct binding between CPSF6 and PD-L1 (Fig. 6D). Furthermore, CPSF6 deletion abrogated mRNA expression of IL-6 and TGF-β as proinflammatory and immune evasion related proteins (Fig. 6E) and also suppressed protein expression of TGF-β and SMAD1/2/3 along with activation of a tumor suppressor FOXP3 in Hep3B cells (Fig. 6F). Notably, the culture supernatant from CPSF6 depleted Hep3B cells significantly enhanced the proliferation of splenocytes from BALB/C mice in absence or presence of Con A (2µg/mL) compared to untreated control (Fig. 6G). Consistently, the percentage of CD4/CD8 was significantly increased in the splenocytes of BALB/c mice bearing CPSF6 depleted Hep3B cells compared to untreated control (Fig. 6H).

CPSF6 depletion abrogated angiogenesis in HCCs.

cBioPortal database showed the close correlation between CPSF6 and VEGF by Spearman's correlation coefficient (r = 0.35) (Fig. 7A). Here CPSF6 depletion attenuated VEFG expression (Fig. 7B) and also significantly decreased mRNA level of VEGF in CPSF6 depleted Hep3B cells compared to untreated control (Fig. 7C). Likewise, CPSF6 depletion significantly reduced VEGF production in the culture supernatants from CPSF6 depleted Hep3B cells compared to untreated control by ELISA (Fig. 7D). Consistently, CPSF6 depletion significantly reduced VEGF promoter luciferase activity compared to untreated control in Hep3B cells transfected with VEGF-Luc reporter (Fig. 7E). Furthermore, to validate the angiogenic activity of CPSF6, tube formation assay and CAM assay were conducted with the culture supernatants from Hep3B cells transfected by CPSF6 siRNA or negative control siRNA. Here tube formation assay showed that CPSF6 depletion significantly reduced the number of tube formed networks in HUVECs compared to untreated control (Fig. 7F). Similarly, CAM assay revealed that CPSF6 depletion significantly reduced the number of neovascularization networks in CAMs compared to untreated control (Fig. 7G).

CPSF6 depletion showed synergistic antitumor effect with Sorafenib in Hep3B cells.

To check synergistic potential of CPSF6 depletion with Sorafenib, MTT assay, Western blotting and cell cycle analysis were conducted in Hep3B cells. Herein CPSF6 depletion significantly enhanced cytotoxicity with Sorafenib in Hep3B cells (Fig. 8A), which was confirmed by CompuSyn analysis that combination index(CI) was below 1, and by SynergyFinder showing red color above 10 between CPSF6 depletion and Sorafenib (Fig. 8B). Consistently, CPSF6 depletion enhanced inhibitory effect of Sorafenib on c-Myc, pro-PARP and pro-caspase 3 compared to Sorafenib alone in Hep3B cells (Fig. 8C). Likewise, CPSF6 depletion significantly increased sub G1 population to 21.88% and 51.71% with Sorafenib compared to Sorafenib alone (10.82% and 16.55%) at 1.25 and 2.5 µΜ, respectively, in Hep3B cells (Fig. 8D).

CPSF6 depletion suppressed the growth of Hep3B cells in orthotopic and xenograft tumor models.

To confirm the above-mentioned in vitro results, animal studies were conducted in orthotopic and xenograft tumor models of Balb/c male athymic nude mice bearing CPSF6 LV-shRNA or LV-shControl transfected Hep3B cells for 6 weeks (Fig. 9A and 9C). Before animal study, CPSF6 LV-shRNA activity was confirmed by protein expression of c-Myc, HK2, PKM2 and LDH and cell viability compared to intact Hep3B cells (Supplementary Fig. 3A, 3B). In a xenograft tumor model, the growth of Hep3B cells implanted into the right flank of to Balb/c nude mice was significantly suppressed in CPSF6 LV-shRNA Hep3B group compared to LV-shControl group (Fig. 9B). Similarly, in an orthotopic tumor model by direct injection of Hep3B cells into livers of Balb/c nude mice, CPSF6 depletion significantly reduced the volumes of Hep3B cells and liver weights compared to sham control group (Fig. 9C, D, E, F). Furthermore, Immunohistochemistry revealed CPSF6 depletion decreased the expression of CPSF6, c-Myc, PCNA, HK2, PKM2, LDH, VEGF and PD-L1 and increased the expression of caspase 3 compared to untreated control, while histopathological changes were found in LV-shControl group more than in CPSF6 LV-shRNA Hep3B group by H&E staining (Fig. 9G).

In our current work, the oncogenic mechanism of CPSF6 was explored in HCC tissues, cell lines and xenograft & orthotopic tumor models. TCGA database reveals that the mRNA overexpression of CPSF6 in HCC patients predicts poor overall survival rate compared to normal control and also CPSF6 was overexpressed in Ⅰ-Ⅵ stages of HCC. Consistently, CPSF6C was overexpressed in HCC patient tissues compared to adjacent noncancerous tissues by human tissue array, implying oncogenic potential of CPSF6.

Herein CPSF6 depletion suppressed the viability and the number of colonies, induced apoptosis via cleaved-PARP and increased sub-G1 population in Hep3B and Huh7 cells, indicating the cytotoxic and apoptotic effect of CPSF6 depletion in HCCs, which was supported by Tan et al’s paper [29] that CPSF6 is critically involved in HCC progression by upregulation of NAD(P)H quinone dehydrogenase 1(NQO1) through alternative polyadenylation.

Recent evidence reveals that NQO1 stabilizes the tumor suppressor p53 [36] and is negatively associated with a well-known oncogene c-Myc by decreasing Nrf2 stability [37]. Thus, to explore the oncogenic mechanism of CPSF6, the association of CPSF6 with c-Myc was elucidated in HCCs. Indeed, CPSF6 was endogenously expressed in parallel with that of c-Myc in HepG2, Hep3B, Huh7 and Sk-Hep1 cells. Consistently, CPSF6 was found to enhance the stability of c-Myc through nuclear colocalization by cycloheximide assay and Immunofluorescence, which was also confirmed by their binding mainly in nucleoplasm by Immunoprecipitation. It is well documented that Myc levels are regulated in normal cells via targeted degradation by the ubiquitin-proteasome system (UPS) including FBW7 or SKP2 [38]. Actually, CPSF6 depletion promoted c-Myc degradation in Hep3B cells, which was disturbed by FBW7 depletion or proteosomal inhibitor MG132, but not SKP2, strongly demonstrating close interaction between CPSF6 and c-Myc or FBW7. Furthermore, CPSF6 depletion attenuated the expression of c-Myc, while c-Myc depletion did not affect c-Myc and depletion of HK2, LDH, PD-L1 and VEGF did not affect the expression of CPSF6 in Hep3B cells, indicating CPSF6 as an upstream of c-Myc or HK2, LDH, PD-L1 and VEGF.

Emerging evidence denotes that tumor microenvironment (TME) can be a therapeutic target for effective cancer therapy, since TME is modulated by components related to tumor initiation, metastasis, immune response, metabolism and angiogenesis [39–41]. Since c-Myc is involved in glycolysis[42] and cancer metabolism [43], the effect of CPSF6 was examined on the metabolism in Hep3B cells. Based on microarray data on effect of CPSF6 on cancer metabolism and angiogenesis in CPSF6 depleted Hep3B cells, CPSF6 depletion suppressed glucose uptake, lactate and ATP and lactate by ELISA and also attenuated the protein expression of HK2, PKM2 and LDH in Hep3B cells, indicating the potent role of CPSF6 in cancer metabolism and glycolysis that is associated with the Warburg effect [44]. It is interesting that the Warburg effect confers immune evasion ability on cancer cells against macrophage-mediated phagocytosis [12] and also induces angiogenesis in endothelial cells in the TME [45].

Accumulating evidence reveals that tumor cells secrete pro-angiogenic factors including VEGF and bFGF to form an abnormal vascular networks leading to aggressive tumor progression [46, 47] through the crosstalk between angiogenesis and immune regulation in the TME [47]. As expected, CPSF6 depletion significantly reduced VEGF production as well as VEGF promoter luciferase activity compared to untreated control in Hep3B cells. Furthermore, CPSF6 depletion significantly reduced the number of tube formed networks in HUVECs compared to untreated control by tube formation and CAM assays, implying angiogenic property of CPSF6 as a fusion partner with FGFR1.

It is well documented that the cancer cells can often escape from immune surveillance in the TME to inhibit the cytotoxic function of tumor-antagonizing immune cells including CD8⁺ cytotoxic T cells and effector CD4⁺ T cells via EGFR/PD-L1 signaling [48, 49]. Notably, CPSF6 depletion attenuated the expression of PD-L1 along with the increase of CD4/CD8 cell percentage in the splenocytes of BALB/c nude mice bearing Hep3B cells, implying an important role of CPSF6 in immune evasion.

Furthermore, CPSF6 depletion significantly enhanced cytotoxicity and sub G1 population and also suppressed the expression of c-Myc, pro-PARP and pro-caspase 3 compared to Sorafenib alone in Hep3B cells, strongly demonstrating the synergistic potential of CPSF6 depletion with Sorafenib for liver cancer therapy. Nonetheless, further studies are still required on animal study by combination therapy of Sorafenib and CPSF6 selective target compound or agent in the near future

In animal studies to validate the in vitro effect of CPSF6, CPSF6 depletion suppressed the volumes of Hep3B cells and liver weight in BALB/c mice in orthotopic and xenograft tumor models and also attenuated the expression of c-Myc, PCNA, VEGF, PD-L1, HK2, PKM2 and LDH and activated the expression of caspase 3 by immunohistochemistry, strongly demonstrating that CPSF6 can be a therapeutic target in liver cancer therapy.

In this study to explore the oncogenic mechanism of CPSF6, CPSF6 was overexpressed in HCC tissues with poor prognosis. Consistently, CPSF6 enhanced the viability and the colony formation via c-Myc stability targeted by FBW7, promoted the Warburg effect by the increase of glucose, HK2, PKM2, LDH and lactate, increased angiogenesis by tube formation and CAM assays along with increased VEGF promoter activity and also upregulated PD-L1 by reducing the percentage of CD4/CD8 cells. Also, CPSF6 depletion enhanced synergistic antitumor effect with Sorafenib in Hep3B cells and also reduced the growth of Hep3B cells in BALB/c mice in orthotopic and xenograft tumor models with inhibition of c-Myc, PCNA, VEGF, PD-L1, HK2, PKM2 and LDH and activation of caspase 3 (Fig. 10). These findings highlight evidence that CPSF6 enhances the Warburg effect for angiogenesis and immune escape, leading to cancer progression via VEGF/PD-L1 and c-Myc/ FBW-7 signaling axes as a synergistic therapeutic potential with Sorafenib for liver cancer therapy.

CPSF6: Cleavage and Polyadenylation Specific Factor 6; MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP: Poly (ADP-ribose) polymerase; ELISA: enzyme-linked immunosorbent assay ;FBS: fatal bovine serum; FBW7: F-box and WD repeat domain-containing 7 ; HCC: hepatocellular carcinoma; HUVEC: Human Umbilical Vein Endothelial Cell; IHC: immunohistochemistry; TCGA: The Cancer Genome Atlas; TME : tumor microenvironment; PD-L1: Programmed death-ligand 1; PPI: protein–protein interactions; SKP2: S-phase kinase-associated protein 2 ; UPS: ubiquitin proteasome system; VEGF: vascular endothelial growth factor; bFGF: basic endothelial growth factor; CAM: Chick chorioallantoic membrane; HK2: hexokinase-2;, PKM2: pyruvate kinase muscle isozyme 2; LDH: lactate dehydrogenase; PCNA: Proliferating cell nuclear antigen

Ethics statement

This study was approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHSASP-21-205). All animal experiments were performed in accordance with animal protocols approved by the Kyung Hee University.

Consent for publication

All authors revised and approved the manuscript.

Availability of data and materials

The RNA mRNA-sequencing data used in the study (GSE229281) are available in a public repository from NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229281). Other data supporting the findings of this study are available from the corresponding authors.

Competing interests

The authors declare no competing interests.

Authors' contributions

DYS performed in vitro experiments; DYS, HJL, and CHA performed animal study together; HJL, JEP, CHA and BSS helped to organize and analyze the data; DYS drafted this manuscript and BK and DYS corrected it; SHK supervised this study and provided funding.

Funding

This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (No.2021R1A2C2003277).

Acknowledgments

We acknowledge that patient’s raw data were used for bioinformatic analysis in our study, since TCGA database provides relevant data for free for research.

Conflict of interest

The authors declare that they have no competing interests.

Availability of data and materials

All data and materials presented in our study can be found in this published article and supplementary data.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (No. 2020R1A2C1004816) and (No.2021R1A2C2003277).

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CPSF6 promotes the Warburg effect and angiogenesis leading to tumor progression via c-Myc/ FBW7/ VEGF/ PD-L1 signaling axis with its siRNA synergistic potential with Sorafenib as a therapeutic target for liver cancer therapy.

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Abstract

Figures

Background

Materials and methods

Cell lines and cell culture

cDNA microarray measurement and data analysis

RNA interference and plasmid transfection

Cytotoxicity Assay

Colony formation assay

Wound healing assay

Cell cycle analysis

RT-qPCR analysis

Tissue microarray and immunohistochemistry

Co-Immunoprecipitation

Immunofluorescence

Ubiquitination assay

Cycloheximide chase assay

Lactate, Glucose and ATP assay

Glucose uptake assay

Tube formation assay

Luciferase reporter assay

CAM (Chick chorioallantoic membrane) assay

Establishment of CPSF6 shRNA Hep3B cell lines

Liver tumor orthotopic & xenograft models and immunohistochemistry

Statistical analysis

Results

CPSF6 depletion induced c-Myc degradation mediated by FBW7 and

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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