KCTD1 Upregulation in HCC Tissues
To measure the clinical significance of KCTD1 in malignant tumors, GEPIA based on The Cancer Genome Atlas (TCGA) data is analyzed to examine the expression of KCTD1 across various tumor samples and paired normal tissues. KCTD1 expression is upregulated in most tumor tissues (Fig. 1a) and the HCC database shows that KCTD1 expression increases in HCC tissues when compared with non-tumor liver tissues (Fig. 1b). The expression level of KCTD1 in 10 normal livers and 70 HCCs is detected by IHC analysis. KCTD1 is highly expressed in 50% of adjacent normal liver tissues and detected in 10 (14%) of the 70 HCCs as indicated by intense staining (3+). 23 (33%) of the 70 HCCs are moderately positive (2+) and 37 (53%) are weakly positive or negative for KCTD1 expression (1+/0) (Fig. 1c-d). Clinicopathologic association analysis of the 70 HCCs reveals that KCTD1 expression is associated with the advanced clinical stage of HCC (Additional file 1: Table S1). Low KCTD1 expression is observed from low-grade hepatocellular cancers (I/II), whereas high KCTD1 expression is detected from high-grade HCC (III/IV) (P<0.05) (Fig. 1d). In 30 paired HCC and adjacent non-tumor samples, KCTD1 expression shows a high proportion of upregulation in the HCC samples compared with non-tumor samples as analyzed by quantitative RT-PCR (Fig. 1e, Additional file 2: Table S2). Furthermore, high-level KCTD1 expression is correlated negatively with the overall survival of patients according to Kaplan-Meier Plotter database (Fig. 1f) and therefore, KCTD1 expression increases significantly in high-grade HCC tissues (III/IV) compared to adjacent normal liver tissues.
KCTD1 Overexpression Enhances both in vitro and in vivo Proliferation, Migration and Metastasis of HCC Cells
The effects of KCTD1 overexpression on the malignancy of human HCC are analyzed by examining the expression profile of the KCTD1 protein in the HCC cell lines. The relatively low expression of KCTD1 proteins is detected in human hepatoma cell lines HepG2, Hep3B, and Huh7, while high expression of KCTD1 is found from MHCC97H and SMMC7721 cells (Additional file 3: Figure S1a). The HCC cell lines with low expression and high expression of KCTD1 are selected for KCTD1 overexpression and knockdown, respectively. To evaluate the role of KCTD1 in HCC, full-length KCTD1 is ligated into the pGC-FU lentiviral vector and stable expression HCC cell lines (pFLAG-KCTD1 and pFLAG-NC) are generated with HepG2 and Huh7 cells (Figure S1b). Western blotting demonstrates overexpression of KCTD1 in both HCC cell lines (Fig. 2a). KCTD1 overexpression results in more viable HCC cells (Fig. 2b), increased colony number, and larger HCC cell size than the control cells (Fig. 2c and Figure S1c), indicating the strong tumorigenicity of KCTD1. Transwell cell migration assays indicate that overexpression of KCTD1 results in a significant increase in the cell motility (Fig. 2d and Figure S1d). Moreover, transwell Matrigel invasion assays reveal that more KCTD1 cells than control cells are invasive (Fig. 2e and Figure S1e). These results suggest that KCTD1 increases the in vitro proliferation, migration, and invasion of HCC cells.
To corroborate the similar effect of KCTD1 overexpression on the tumorigenic ability of HCC cells in vivo, KCTD1 LV-infected Huh7 cells are injected subcutaneously into the two points of the back of female nude mice, individually. Within 4 weeks, the mean tumor weight and volume in the KCTD1 group increase significantly in comparison with the control group mice (Fig. 2f-g, and Figure S1f-g). HE staining confirms that the HCC cells are arranged tightly in the KCTD1 group (Fig. 2h) and IHC staining shows that KCTD1 overexpression increases the expression of the proliferation marker Ki67, endothelial marker CD31, and M2 macrophage marker Arg1 in the subcutaneous tissues derived from Huh7 cells (Fig. 2i and Figure S1h). These findings indicate that KCTD1 overexpression enhances the tumorigenic ability of HCC cells in vivo.
KCTD1 overexpressing cells and control Huh7 cells are injected into the tail veins of nude mice (6-week old, female) to check for the lung metastasis, the metastatic nodules on the surfaces of lungs and livers are examined after six weeks (Figure S1i). Tumor foci in the mouse lungs are revealed by HE staining. The mice from the KCTD1 group have larger lung metastatic nodules, while a few smaller nodules are detected from mice with control cells (Fig. 2j). Metastatic nodules also appear from mouse liver tissues obtained from the KCTD1 group. All in all, these findings demonstrate that KCTD1 overexpression promotes the in vivo invasion and metastasis of HCC cells.
To further confirm whether KCTD1 overexpression leads to malignant transformation, we infect NIH3T3 mouse embryonic fibroblast cells with KCTD1 LV and NC LV, examine the infection efficiency by GFP fluorescence (Additional file 4: Figure S2a), and confirm KCTD1 overexpression in the NIH3T3 cells by Western blot analysis (Figure S2b). KCTD1 overexpression enhances the malignant phenotype (Figure S2c) and cell growth (Figure S2d) and induces increased colony formation of the NIH3T3 cells (Figure S2e). The oncogenesis-promoting effect of KCTD1 on tumor formation in vivo is examined. 2×105 NIH3T3 cells are subcutaneously injected into healthy BALb/c nude female mice and KCTD1-overexpressing cells form larger tumors in mice, but the control cells form significantly smaller tumors (Figure S2f-g), suggesting that the oncogenic capabilities of KCTD1 enhance malignant transformation of NIH3T3 cells.
KCTD1 Knockdown suppresses the in vitro and in vivo Proliferation, Migration and Metastasis of HCC Cells
To confirm the role of KCTD1 in human HCC, KCTD1 shRNA is ligated into the GV248 lentiviral vector (GeneCopoeia) and the stable cell lines (GFP-KCTD1 shRNA and GFP-NC shRNA) are screened and established in MHCC97H and SMMC7721cells (Additional file 5: Figure S3a). Western blotting demonstrates knockdown of KCTD1 expression in both MHCC97H and SMMC7721 cell lines (Fig. 3a). KCTD1 knockdown decreases the HCC viable cell number (Fig. 3b) and suppresses the number and size of HCC cell colonies (Fig. 3c and Figure S3b). Moreover, the wound-healing assays show that KCTD1 knockdown suppresses HCC cell migration by about 50% (Fig. 3d and Figure S3c). Transwell migration assays indicate that knocking down KCTD1 results in significant decrease in the cell motility (Fig. 3e and Figure S3d). Matrigel invasion assays disclose that a significantly smaller proportion of HCC cells with KCTD1 knock down invaded through the Matrigel-coated chamber compared to the number of control HCC cells (Fig. 3f and Figure S3e). The data show a strong antitumorigenic role for KCTD1 downregulation.
The influence of KCTD1 knockdown on the tumorigenicity of HCC cells is examined in vivo. MHCC97H cells stably infected with KCTD1 shRNA-LV or control NC-LV are s.c. injected into the two points of the back of five nude mice, individually and after 2 weeks, the mean tumor weight and volume of the KCTD1-knockdown group decrease compared with the NC group (Fig. 3g-h and Figure S3f-g). HE staining confirms that the MHCC97H cells are arranged loosely in the KCTD1 shRNA group (Figure S3h) and IHC analysis shows that KCTD10 knockdown inhibits the expression of Ki67, CD31, and Arg1 (Fig. 3i and Figure S3i). KCTD1 shRNA cells and control MHCC97H cells are intravenously injected via the tail vein into nude mice (6-week old, female) and after six weeks, the metastatic nodules are calculated on the surfaces of mouse lungs and livers (Figure S3j). The mice in the KCTD1 shRNA group have increasingly fewer and smaller lung metastatic nodules than those in the NC shRNA group (Fig. 3j-k). Tumor foci in mouse lungs are detected by HE staining, while the liver morphology does not change obviously in the two groups of mice (Fig. 3l). Altogether, these results demonstrate that KCTD1 downregulation suppresses HCC cell proliferation, migration, and metastasis in vivo, suggesting that KCTD1 knockdown markedly suppresses the tumorigenic ability of HCC cells.
KCTD1 Enhances HUVEC Tube Formation and Matrigel Plug Angiogenesis with Increased M2 Macrophage Infiltration
The single cell RNA sequencing data showed KCTD1 was expressed in endothelial cells of HCC (Additional file 6: Figure S4a). RNA sequencing analysis reveal that KCTD1 expression is positively correlated with vascular related gene expression (Fig. 4a). Moreover, KCTD1 influences the expression of the vascular marker CD31 and so whether KCTD1 modulated angiogenesis in HCC is studied. A stable HUVEC line that overexpressed KCTD1 protein is generated (Fig. 4b) and it is found that KCTD1 enhances proliferation and migration of these HUVECs in vitro (Fig. 4c-f, Figure S4b-d). KCTD1 overexpression induces HUVEC tube formation (Fig. 4g) and increases the branch counts of formed tubes and the number of HUVEC-formed tubes (Figure S4e). The supernatant from KCTD1-overexpressing Huh7 and HepG2 cells increases the number of HUVEC-formed tubes (Fig. 4h and Figure S4f-g), while the supernatant from KCTD1-knockdown MHCC97H cells decreases the number of HUVEC-formed tubes (Figure S4h). The effects of KCTD1 overexpression on new vessel formation in Matrigel plugs are investigated by subcutaneously injecting growth factor-reduced Matrigel with 800 ng/ml bFGF, HUVECs and Huh7 cells into the lower backs of 5-week-old female nude mice. The Matrigel plugs containing control cells are transparent indicating no new blood vessel formation, whereas the plugs containing KCTD1 cells are red and blood and blood vessels appear in two weeks (Fig. 4i). Correspondingly, KCTD1 overexpression induces phosphorylation of AKT, and VEGFR2 and promotes the expression of VEGF, HIF-1α and ARNT in HUVECs (Fig. 4j). These finding indicate that KCTD1 overexpression promotes the angiogenesis through activating the VEGF signaling cascade in vitro and in vivo.
Tumor-associated macrophages (TAMs) as a vital population in the tumor microenvironment (TME) are closely associated with tumor survival, angiogenesis, chemoresistance and immunosuppression, [20] and the correlation between the expression of CD31 and the M2 macrophage marker Arg1 in the HCC samples is analyzed. CD31 expression is upregulated in HCC tissues compared with healthy, adjacent liver tissues (Fig. 5a-b). The expression of CD31 is positively associated with KCTD1 expression in the HCC cases and high levels of CD31 are primarily observed from HCCs with high KCTD1 expression (Additional file 7: Table S3). The TCGA database indicates that KCTD1 is positively related with another vascular marker CD34 (Fig. 5c) and KCTD1 expression is positively associated with Arg1 expression in HCCs (Fig. 5d-e and Additional file 8: Table S4). The TIMER software shows that KCTD1 expression is positively correlated with the abundance of macrophage in HCCs (Fig. 5f) and the TCGA database reveals that KCTD1 expression is closely associated with the M2 macrophage in HCCs (Additional file 9: Figure S5A). By using qRT-PCR, lower levels of the M2 markersTGF-β, IL-10 and Arg1 are detected but higher levels of the M1 markersIL-1β/IL-12/TNFα/iNOS are observed from the KCTD1-knockdown HCC cells (Fig. 5g). The U937-differentiated M0, M1, M2 cells are cultured (Figure S5b) and M0 cells are cocultured with the supernatant from MHCC97H cells. The levels of M1-macrophage marker genes IL-12, IL-1β, TNFα and iNOS are increased, while the level of M2-macrophage marker genes TGF-β, IL-10 and Arg1 is downregulated in the M0 cells cocultured with the supernatant from KCTD1-knockdown MHCC97H cells (Figure S5c). KCTD1 knockdown could induce M0 macrophage expressing M1 characteristic genes, and polarize M0 macrophages into M1 macrophages, indicating that KCTD1 expression is associated with the tumor microenvironment.
KCTD1 Activates the HIF-1α/VEGF Pathway by Interacting with HIF-1α
Hypoxia-inducible factor 1 alpha (HIF-1α) mediates the hypoxia-induced expression of VEGF, which plays a central role in angiogenesis and tumor progression [21]. Since KCTD1 has a key role in angiogenesis, whether KCTD1 regulates the HIF-1α/VEGF pathway in HCC cells is investigated. KCTD1 overexpression promotes the expression of the p-ARK/HIF-1α-ARNT/VEGF cascade in Huh7 cells (Fig. 6a), while KCTD1 knockdown decreases the expression of these proteins in MHCC97H cells (Fig. 6b). Notably, KCTD1 affects the expression of GPC3 and AFP, which are HCC clinical biomarkers. Decreased levels of ARNT and HIF-1α are observed from the KCTD1 shRNA group according to the mouse subcutaneous tumor tissues (Additional file 10: Figure S6a). The KCTD1 protein stability assays are performed and as shown in Fig. 6c-d, more significant decrease in HIF-1α/ARNT proteins is induced in the KCTD1-knockdown MHCC97H cells than in the control cells treated with different concentrations of cycloheximide (CHX) at different time points. In contrast, an increasing amount of HIF-1α proteins is measured from the KCTD1 overexpressing Huh7 cells treated with CHX (Figure S6b). CoCl2 induces the expression of HIF-1α in the HCC cells [22], KCTD1 overexpression leads to increased HIF-1α proteins, but KCTD1 knockdown results in decreased HIF-1α proteins (Figure S6c). These data strongly suggest that KCTD1 regulates the stability of HIF-1α/ARNT heterodimer proteins.
To further disclose the regulatory relationship of KCTD1 and HIF-1α/ARNT genes, co-immunoprecipitation assays are carried out. Endogenous HIF-1α is detected from immune complexes of overexpressed KCTD1 (Fig. 6e) and endogenous ARNT cannot appear from the immunoprecipitates of overexpressed KCTD1 (Fig. 6f), suggesting an interaction between KCTD1 and HIF-1α. As reported, ARNT forms a heterodimer with HIF-1α [23-25]. HIF-1α is found from immunoprecipitates of ARNT, while preimmune IgG could not precipitate any specific protein (Fig. 6g). Chromatin immunoprecipitation using anti-KCTD1 antibodies shows enrichment of VEGF promoter fragments with HIF-1α-binding sites, indicating that KCTD1 forms a complex with HIF-1α/ARNT and binds with the VEGF promoter via HIF-1α binding sites [26] (Fig. 6h). Taken together, these data show that KCTD1 is able to bind with the HIF-1α/ARNT heterodimer to regulate the VEGF signaling pathway.
Sorafenib Downregulates KCTD1 Expression to Increase the Cytotoxicity of HCC Cells both in vitro and in vivo
HCC has a poor prognosis due to its high incidence of recurrence and metastasis and postoperative chemotherapy is necessary because of high drug resistance [27]. The effects of KCTD1 knockdown on the chemosensitivity of HCC cells are studied. The MTT absorbance decreases by 27% in the KCTD1-knockdown HCC cells compared to the control cells, while 69% reduction in the MTT absorbance appears from the KCTD1-knockdown HCC cells treated with sorafenib (Fig. 7A and Additional file 11: Figure S7a), suggesting that the combination of KCTD1 knockdown with sorafenib increases the HCC cell cytotoxicity.
After 24-h treatment with sorafenib, cell apoptosis of the KCTD1 shRNA-infected and parental MHCC97H cells is detected by flow cytometry. The rate of apoptosis increases for cells with sorafenib treatment and KCTD knockdown, as independently and synergistically compared to the proportion of the control cells (Fig. 7B and Figure S7B). Sorafenib markedly decreases the expression levels of KCTD1, HIF-1α, and ARNT proteins (Fig. 7c and Figure S7c). The effects of KCTD1 downregulation and sorafenib on tumor formation in subcutaneous and intrahepatic mouse models are probed. Both treatments synergistically decrease the size of subcutaneous tumors and 4 mice display complete tumor remission (Fig.7d and Figure S7d). Both treatments result in smaller tumor foci in the mouse livers than the control group and single treatment group (Fig. 7e and Figure S7e). Clinically, the low expression of KCTD1 leads to a prolonged median overall survival of 30.9% in 29 sorafenib-treated patients with recurrent HCC, although the sample number is so small that there is no statistical significance (Figure S7f). The results reveal that KCTD1 knockdown and sorafenib synergistically inhibit HCC progression.
miR-129-5p Targets the KCTD1 3′ UTR and Decreases the Oncogenic Effects of KCTD1 in HCC in vitro as well as in vivo
Because KCTD1 downregulation suppresses HCC progression, efficient miRNAs are predicted by multiple algorithms (TargetScan, miRWalk, RNA22 and miRanda) to identify miRNAs targeting the KCTD1 3′ UTR and three potential miR-129-5p binding sites are found (Fig. 8a). Wildtype or mutated KCTD1 3′-UTR luciferase reporter vectors are cotransfected into HEK293 cells with miR-129-5p mimics. The overexpression of miR-129-5p suppresses the luciferase activities of 3′ UTR of the KCTD1 gene (Fig. 8b) and decreases the protein expression of KCTD1 (Fig. 8c). However, miR-129-5p showed no effect on the luciferase activity of mutated miR-129-5p MRE seed sequences of KCTD1 (Additional file 12: Figure S8a), indicating that the predicted three miR-129-5P binding sites mediate binding of miR-129-5p with KCTD1. These data indicate that miR-129-5p specifically targets the 3′ UTR region of KCTD1 gene and downregulates KCTD1 expression.
To demonstrate the critical role of miR-129-5p in KCTD1-mediated HCC cell growth and angiogenesis, MHCC97H cells are infected with miR-129-5p LV (Figure S8b), miR-129-5p dramatically suppresses growth (Fig. 8d) and migration of the MHCC97H cells (Fig. 8e and Figure S8c). The supernatant from miR-129-5p-overexpressing MHCC97H cells suppresses significantly the tube-like structure formation of the HUVECs (Fig. 8f and Figure S8d). miR-129-5p is positively linked with overall survival time of patients with HCC according to TCGA database previously reported [28] (Figure S8e). The results indicate that the tumor inhibitor miR-129-5p decreases the tumor-promoting and vascularization effects of HCC.
Finally, mouse HCC models are adopted to analyze the combined effects of miR-129-5p and sorafenib. Firstly, miR-129-5p suppresses subcutaneous tumor formation in nude mice (Fig. 8g and Figure S8f) and exosomes from the supernatant of miR-129-5p-infected MHCC97H cells are injected into the tail veins of an intrahepatic tumor model in nude mice leading to a smaller size of tumor foci in the livers. The combination of both exosomes and sorafenib treatment synergistically reduces the size of the tumor foci in liver tissues compared with those in the control groups (Fig. 8h and Figure S8g). The morphology of isolated exosomes is examined by TEM (Figure S8h) and expression of exosome markers CD81 and CD63 is confirmed (Figure S8i). Expression level of miR-129-5p is markedly increased in exosomes from the supernatant of miR-129-5p-LV infected MHCC97H cells (Figure S8J) and these results suggest that miR-129-5p combined with sorafenib can suppress intrahepatic tumor development in vivo.