Stem Cell

Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression – Signal Transduction and Targeted Therapy

Time Stamp: July 2, 2024 8:00 PM
Source Node: 627921

Highly expressed Sema3C is correlated with stemness features in HCC

To explore the mechanism of interaction between CSCs and tumor stroma, we screened out genes that were highly expressed in cirrhosis and HCC tissues compared to normal liver tissues in GSE14323 and GSE6764 databases, and intersected with genes that were elevated in sorafenib-resistant HCC xenografts based on GSE121153 dataset to obtain 13 common genes (Fig. 1a). Among the 13 genes, we looked for secretory proteins that may mediate intercellular communication. Previous studies have reported that Sema3C, as a secreted glycoprotein, was elevated in HCC tissues and correlated with tumor size, portal vein embolization, and metastasis.^15,16 Consistently, we found that Sema3C expression was higher in patients with stage III&IV than in patients with stage I&II (Fig. 1b). To detect the differential expression of Sema3C in circulatory levels between HCC patients and healthy individuals, we collected peripheral blood of 5 HCC patients and healthy people respectively and found that the concentration of Sema3C in HCC patients was higher than that of healthy individuals (Fig. 1c). By analyzing the data of TCGA-LIHC, HCC patients with elevated Sema3C expression have a worse prognosis compared to those with low Sema3C expression (Fig. 1d). Furthermore, we demonstrated that Sema3C expression was greatly enhanced at the fibrotic and advanced stages of DEN+CCl₄-induced HCC models (Fig. 1e). Meanwhile, based on this HCC model, we also preliminarily evaluated the association of Sema3C with stemness. Tissue immunofluorescence (IF) showed that Sema3C and EpCAM expression were highly expressed in areas with high infiltration of CAFs, while low expression in areas with low abundance of CAFs (Fig. 1f). Previously, we constructed two sorafenib-resistant cell lines (Huh7 and HepG2),¹⁷ the results showed that Sema3C expression was significantly increased in sorafenib-resistant HCC cells (Fig. 1g).

To further investigate the relationship between Sema3C and stemness, we analyzed its expression in both differentiated and non-differentiated HCC cell lines using the GSE36133 dataset, revealing higher expression in the latter. (Fig. 1h). In addition, by analyzing the correlations between the Sema3C and multiple stemness-related genes using the HCCDB databases, we discovered Sema3C was positively associated with CSCs markers, such as CD24, CD47, EPCAM, ICAM1, c-Myc, PROM1 (encoding CD133), SOX9, and THY1 (encoding CD90), while negatively correlated with hepatic lineage genes, such as ALB and HNF4A (Fig. 1i). Then, we compared Sema3C expression in EpCAM⁺ cells and EpCAM^– cells in the GSE5975 database and found that Sema3C was up-regulated in EpCAM⁺ cells (liver CSCs) (Fig. 1j). Given that CSCs contribute to tumor recurrence, the impact of Sema3C on HCC relapse was examined using the GSE14520 dataset. The results showed that patients with highly expressed Sema3C had more HCC recurrence events than patients with low Sema3C expression (Fig. 1k). Besides, the survival analysis, stratified by the expression of Sema3C and stemness-related genes (OCT4 and SOX9), revealed that HCC patients expressing both Sema3C and OCT4/SOX9 had the worst prognosis (Fig. 1l, m). Altogether, these results suggested that Sema3C was up-regulated during HCC progression and was associated with HCC stemness.

Sema3C promotes stemness maintenance and initiation in HCC

To further explore the effect of Sema3C on the stemness phenotype of HCC, we compared the protein levels of Sema3C across multiple HCC cell lines. Our findings revealed elevated Sema3C expression in HCC cell lines compared to normal liver cell lines, and Sema3C was also expressed in hepatic stellate cells (LX-2) to a certain extent (Fig. 2a). In addition, we analyzed Sema3C expression variations across cell types in HCC using published single-cell datasets (GSE146115, GSE146409, and GSE166635). The results indicated that Sema3C was predominantly expressed in tumor cells and, to a lesser extent, in stromal cells, while it was rarely expressed in normal hepatocytes or endothelial cells, consistent with our in vitro cell line results (Supplementary Fig. 1a). Then, Hep3B and MHCC-97L were selected for Sema3C overexpression, while HepG2 and Huh7 were chosen for Sema3C knockdown (Fig. 2b). To determine Sema3C expression in CSC populations, we compared the protein levels of Sema3C between spheroids and non-spheroids. The results showed that Sema3C expression was significantly higher in spheroids than in non-spheroids (Fig. 2c). In line with bioinformatics analysis, Sema3C overexpression up-regulated the expression of stemness-related genes (Sox2, Oct4, Nanog, EpCAM, CD133, CD90, and CD24) in HCC cells when compared to empty vector controls (Fig. 2d). To further investigate whether Sema3C functionally contributes to drug resistance, self-renewal, and tumorigenesis, we conducted MTT and sphere formation assays. Sema3C overexpression in HCC cells significantly increased resistance to sorafenib, while Sema3C knockdown led to a marked decrease in resistance. (Fig. 2e, f). Meanwhile, HCC cells overexpressing Sema3C showed increased spheroid formation, while Sema3C knockdown cells had reduced spheroid formation (Fig. 2g, h). Additionally, colony formation experiments revealed that Sema3C overexpression promoted HCC proliferation, whereas Sema3C knockdown inhibited cell proliferation (Supplementary Fig. 1b, c). We also found that apoptosis was significantly inhibited in Sema3C-overexpressing HCC cells and enhanced in Sema3C-knockdown HCC cells (Supplementary Fig. 1d, e). Transwell assays further demonstrated that Sema3C overexpression promoted cell invasion and migration, whereas Sema3C knockdown attenuated cell invasion and migration (Supplementary Fig. 1f, g).

To evaluate the impact of Sema3C on tumor-initiating ability in vivo, a limiting dilution assay was conducted by subcutaneously injecting 5 × 10⁵, 5 × 10⁴, and 5 × 10³ HCC cells into nude mice. Hep3B cells overexpressing Sema3C showed a significantly higher tumor-initiating capacity compared to control cells. In contrast, Sema3C knockdown HCC cells resulted in a significantly reduced number of tumors compared to controls (Fig. 2i). Next, to verify the stemness maintenance role of secreted Sema3C in the TME, we conducted a series of rescue experiments. On the basis of endogenous Sema3C knockdown, recombinant human Sema3C (rhSema3C) was used to stimulate HCC cells. Transwell assay found that addition of rhSema3C could reverse the impact of Sema3C knockdown on invasion and migration of HCC cells (Supplementary Fig. 2a). MTT assay showed that the rhSema3C could reverse the sensitivity to sorafenib in HCC cells with Sema3C knockdown (Supplementary Fig. 2b). In addition, in vitro sphere formation also confirmed that secreted Sema3C could restore the spheroid formation of HCC cells upon Sema3C knockdown (Supplementary Fig. 2c). In summary, these findings demonstrate that Sema3C plays a critical role in enhancing HCC stemness, chemoresistance, and tumor initiation.

Sema3C maintains HCC stemness via a dysregulated AKT/Gli1/c-Myc signaling axis

To investigate the downstream mechanisms by which Sema3C maintains HCC stemness, pathway enrichment analysis based on the TCGA dataset was conducted. The result revealed a strong association between Sema3C and signaling pathways regulating stem cell pluripotency, consistent with our above findings. Additionally, significant KEGG enrichment was observed in the PI3K-AKT, Wnt, and Hippo signaling pathways (Fig. 3a). GSEA analysis also revealed that the PI3K-AKT and Hedgehog signaling pathways were highly enriched in HCC samples with elevated Sema3C expression (Fig. 3b). Given the role of PI3K/AKT, Wnt, Hippo, and Hedgehog pathways in stemness regulation, we detected key molecules within these pathways. Results suggested that Sema3C overexpression did not alter β-catenin or phosphorylated-YAP (p-YAP) protein levels (Fig. 3c). However, the mRNA levels of Gli1, Gli2, c-Myc, and CCND1 were increased in overexpressed-Sema3C HCC cells, and on the contrary, the levels of these target genes were decreased upon Sema3C knockdown (Fig. 3d). Consistently, western blot analysis confirmed that key mediators of the AKT and Hedgehog pathways—p-AKT, Gli1, and c-Myc—were highly expressed in Sema3C-overexpressing MHCC-97L HCC cells. Conversely, knockdown Sema3C in HepG2 cells attenuated the activated signaling. Furthermore, stimulation of MHCC-97L cells with varying doses of rhSema3C or at different time points revealed a dose- and time-dependent activation of the Gli1/AKT pathway (Fig. 3e). To further validate the significance of AKT/Gli1 signaling in Sema3C-driven HCC stemness, functional rescue experiments were performed in which a specific AKT inhibitor MK2206 was introduced into the Sema3C overexpressing HCC cells. MK2206 attenuated the stemness features induced by Sema3C overexpression, as evidenced by reduced chemoresistance in HCC cells (Fig. 3f), sphere-formation (Fig. 3g), migration, and invasion (Supplementary Fig. 2d). Previous study has demonstrated that the PI3K/AKT pathway could modulate the Hedgehog signaling pathway in renal cell carcinoma.¹⁸ Our findings indicated that MK2206 treatment blocked Sema3C-induced Gli1 protein expression in HCC cells, suggesting Sema3C could act as a mediator in potentiating AKT signaling activation of the Hedgehog pathway in HCC (Fig. 3h). This was further validated in an in vivo HCC model, where HepG2 cells with or without Sema3C knockdown were subcutaneously injected into nude mice. Upon Sema3C knockdown, the immunohistochemistry (IHC) staining revealed a decreased Ki67 expression as well as down-regulation of activated p-AKT, c-Myc, and Gli1 versus control tumors (Fig. 3I). Collectively, Sema3C-mediated activation of AKT and Gli1 signaling pathways promoted stemness maintenance of HCC.

Sema3C maintain HCC stemness via NRP1 and ITGB1

Previous studies have shown that Sema3C could bind to the neuropilins (Nrp1 or Nrp2), to mediate downstream signal transduction.¹⁹ Moreover, NRP1 was up-regulated in HCC and promoted the expansion of CSCs and tumor growth.^20,21 To this end, we examined whether NRP1 mediated the stemness maintenance for Sema3C in HCC. MHCC-97L and Hep3B cells were treated with NRP1-targeting siRNA (Supplementary Fig. 3a, b), and results showed that knockdown of NRP1 attenuated Sema3C-induced chemoresistance, self-renewal, migration, and invasion of HCC cells compared to control cells (Supplementary Fig. 3c–e). Furthermore, we examined whether Sema3C-activated AKT and Gli1 signaling depended on NRP1 binding. Our results revealed that Sema3C-induced phosphorylation of AKT, and an increase of Gli1 and c-Myc expression were reversed by NRP1 knockdown (Supplementary Fig. 3f). NRP1 binds to a variety of downstream receptors, including the plexins and the integrins family.¹⁶ To identify co-receptors mediating stemness maintenance after Sema3C binds to NRP1, Sema3C and NRP1 were overexpressed in Huh7 cells, co-immunoprecipitation combined with mass spectrometry (IP/MS) revealed that only ITGB1, which belonged to the integrin family, could bind to both Sema3C and NRP1 (Fig. 4a, b, Supplementary Fig. 4a). Analysis of the TCGA-LIHC database showed a positive correlation between Sema3C and ITGB1 expression in HCC (Supplementary Fig. 4b). Co-immunoprecipitation (Co-IP) assays in Huh7 cells also confirmed the binding of Sema3C, NRP1, and ITGB1 (Fig. 4b). Simulated molecular docking results showed that VWFA domain (amino acids 140-378) of ITGB1 bound to Sema3C (Supplementary Fig. 4c). Moreover, NRP1 knockdown in Huh7 cells inhibited ITGB1 from binding to Sema3C, while NRP1 still bound Sema3C after ITGB1 knockdown, indicating that NRP1 acted as a ligand-binding receptor, facilitating the formation of a complex with Sema3C and ITGB1 in HCC cells (Fig. 4c). Furthermore, ITGB1 expression was significantly elevated in Sema3C-overexpressing Huh7 cells but decreased in Sema3C-knockdown HepG2 cells (Fig. 4d).

Previous studies have shown that ITGB1 facilitates sorafenib resistance and tumor formation of HCC.^22,23 Our study investigated the role of ITGB1 in HCC stemness regulation and found a positive correlation between ITGB1 expression and multiple stemness-related genes in the HCCDB database (Fig. 4e). siRNA was used to knock down ITGB1 in Huh7 cells (Fig. 4f), and results revealed that ITGB1 knockdown significantly inhibited chemoresistance, sphere-forming ability, migration, and invasion (Fig. 4g, h, Supplementary Fig. 4d). Concordantly, a subcutaneous tumor model of HCC showed that ITGB1 knockdown led to a significant reduction in tumor incidence rate (Fig. 4i). To characterize the role of ITGB1 in Sema3C-directed stemness, we transfected Huh7 cells with OE-Sema3C, with or without si-ITGB1. ITGB1 knockdown abrogated Sema3C-induced chemoresistance, self-renewal, migration, and invasion (Fig. 4j, k, Supplementary Fig. 4e). Moreover, silencing ITGB1 also antagonized Sema3C-induced upregulation of AKT phosphorylation, GlI1, and c-Myc expression (Fig. 4l). Additionally, NRP1 or ITGB1 knockdown in HCC cells weakened the stemness effect of rhSema3C, suggesting that rhSema3C relied on NRP1 and ITGB1 to exert its function (Supplementary Fig. 4f-h). Analysis of TCGA-LIHC data showed that HCC patients with high Sema3C, NRP1, and ITGB1 expression signatures had a worse prognosis (Fig. 4m). These results suggest that Sema3C regulates HCC stemness through the NRP1/ITGB1 axis.

HCC cells-derived Sema3C promotes ECM remodeling and HSCs activation

As axon guidance molecules, semaphorins establish and maintain effective communication links between axons and target cells, resulting in the formation of functional synapses.²⁴ Inspired by this, we explored whether Sema3C secreted by HCC cells interacted with other TME components to reshape the tumor niche, promoting HCC stemness. Gene Ontology (GO) pathway enrichment analysis of proteins bound to Sema3C and NRP1 revealed significant enrichment in extracellular matrix organization, integrin-mediated signaling pathway, and collagen fibril organization. These findings suggested that Sema3C may regulate the ECM in the HCC microenvironment (Fig. 5a). To assess the effect of Sema3C on the ECM remodeling, a collagen contraction assay was conducted. We found that after being treated with the supernatants of OE-Sema3C-MHCC-97L cells, the gel contraction of collagen I was significantly enhanced (Fig. 5b). The MMP and lysine oxidase (LOX) family proteins are responsible for collagen remodeling in cancer.^25,26 Overexpression of Sema3C markedly increased MMP2, MMP9, LOX, and LOXL2 levels, and treatment of MHCC-97L cells with rhSema3C elevated MMP2, LOX, and LOXL2 but not MMP9 (Supplementary Fig. 5a, b). To investigate the role of Sema3C in ECM remodeling in vivo, we performed orthotopic implantation of OE-Sema3C-MHCC-97L cells or control cells in nude mice. Tumors in mice injected with OE-Sema3C-MHCC-97L cells were significantly larger than those in control mice. Masson’s trichrome and PicroSirius red staining showed greater collagen fiber infiltration in OE-Sema3C tumors. IHC and quantitative analysis revealed increased α-SMA and collagen I expression in OE-Sema3C tumors compared to controls (Fig. 5c, d). These findings above revealed that Sema3C in HCC cells could exacerbate ECM deposition in the TME.

In addition to directly regulating ECM, considering the secretory properties of Sema3C, we explored whether HCC-derived Sema3C could transform HSCs into CAFs in a paracrine way, thereby promoting ECM production. First, we treated LX-2 cells with rhSema3C and observed morphological changes using IF. The results showed that rhSema3C treatment markedly increased the cytoplasmic volume of LX-2 cells and induced the formation of more cellular projections (Supplementary Fig. 5c). In addition, rhSema3C could also enhance the proliferation ability of LX-2 cells (Supplementary Fig. 5d).

We collected conditioned medium (CM) from HCC cells with varying Sema3C expression levels and treated LX-2 cells for 48 h. The CM from OE-Sema3C-HCC cells or rhSema3C significantly increased the mRNA levels of ACTA2, COL1A1, ELN, and TGF-B1 (Supplementary Fig. 5e, f), as well as the protein expression of α-SMA and collagen I in LX-2 cells (Fig. 5e, f). Additionally, ELISA assay confirmed that Sema3C secreted by HCC cells promoted TGF-β1 production in LX-2 cells (Fig. 5g). Then the effect of Sema3C on the biological function of HSCs was verified. We found that CM from OE-Sema3C-HCC cells or rhSema3C significantly increased the capacity of LX-2 cells to chemoresistance, migration, and invasion as compared to controls (Fig. 5h, i). To verify the communication between HSCs and HCC cells, we collected CM from OE-Sema3C-activated LX-2 cells and co-cultured it with Hep3B cells. The results revealed that activated LX-2 upregulated stemness-related genes and enhanced chemoresistance in Hep3B cells, indicating that activated LX-2 cells were sufficient to induce stemness maintenance in HCC cells (Supplementary Fig. 5g, h).

To explore the impact of Sema3C-mediated interactions between HCC cells and HSCs on tumorigenesis in vivo, we constructed a xenograft HCC model by injecting HCC cells and HSCs subcutaneously into nude mice (Fig. 5j). Animals co-injected with OE-Sema3C Hep3B cells and LX-2 cells had a greater tumor weight and volume than all other groups (Fig. 5k, l). It is therefore suggested that HCC cells-derived Sema3C is involved in both remodeling ECM and activation of HSCs to form a supportive niche in TME.

IL-6 and cholesterol biosynthesis are responsible for Sema3C-mediated HSCs activation

According to the above findings, we explored the potential mechanism by which Sema3C induces HSCs activation. Thus, an RNA sequencing assay was performed based on LX-2 cells using rhSema3C or its control treatment. We identified 1383 differentially expressed genes (DEGs), with 1019 upregulated and 364 downregulated. IL6 and IL8 were the most significantly upregulated in the rhSema3C-treated groups compared to controls (Fig. 6a). Meanwhile, analysis of TCGA databases revealed a strong positive correlation between Sema3C expression and IL6, IL8 (CXCL8) expression in HCC (Supplementary Fig. 6a, b). To determine whether Sema3C directly promoted IL6 and IL8 production, we treated LX-2 cells with rhSema3C or supernatants from OE-Sema3C HCC cells, and only IL6 mRNA level was significantly upregulated (Fig. 6b). Moreover, ELISA experiments confirmed that gradient doses of rhSema3C or supernatants of OE-Sema3C-MHCC-97L cells increased IL6 secretion in LX-2 cells (Fig. 6c). To further investigate the biological processes involved in Sema3C-regulated HSCs activation, enrichment analysis of DEGs from transcriptome sequencing was performed. We found that Sema3C not only positively regulated IL6 production but also influenced cholesterol biosynthesis and metabolic processes in HSCs (Supplementary Fig. 6c-e). Consistently, rhSema3C significantly increased the total cholesterol content in LX-2 cells in vitro (Supplementary Fig. 6f). We identified the major DEG involved in cholesterol biosynthesis and metabolism, finding that HMGCS1, INSIG1, FASN, and HMGCR expression levels were significantly increased in the rhSema3C-treated groups (Supplementary Fig. 6g). Considering that HMGCR is the rate-limiting enzyme in the mevalonate pathway for cholesterol biosynthesis and its high expression in various tumor tissues promoted tumor progression.^27,28 Therefore, we explored whether Sema3C affected cholesterol metabolism mainly by regulating HMGCR expression in HSCs. The results showed that rhSema3C treatment upregulated HMGCR mRNA and protein levels in LX-2 cells (Supplementary Fig. 6h, i). To elucidate the pathway by which Sema3C promoted IL6 secretion and cholesterol synthesis, we first investigated whether the PI3K/AKT pathway, known to be activated in HCC cells, was also involved in regulating LX-2 cells. However, the results suggested that rhSema3C could not increase AKT phosphorylation in LX-2 cells (Supplementary Fig. 6j). GSEA analysis revealed significant enrichment of the NF-κB pathway in rhSema3C-treated LX-2 cells (Fig. 6d). Treatment with rhSema3C or supernatants from OE-Sema3C HCC cells increased NF-κB (p65) phosphorylation in LX-2 cells (Fig. 6e, Supplementary Fig. 6k). To verify the involvement of NF-κB in Sema3C-induced HSCs activation, we pre-treated LX-2 cells with the NF-κB inhibitor-Bay 11-7082 and found that subsequent rhSema3C stimulation did not increase α-SMA or HMGCR expression (Fig. 6f, Supplementary Fig. 6l), and it also antagonized IL6 secretion and cholesterol synthesis (Fig. 6g, Supplementary Fig. 6m). Next, to explore whether Sema3C promotes HSCs activation via NRP1 and ITGB1, we performed Co-IP experiments showing that CM from Sema3C-expressing Hep3B cells induced NRP1-ITGB1 interaction in LX-2 cells (Fig. 6h). Silencing NRP1 in LX-2 cells using siRNA abrogated rhSema3C-induced IL6 secretion, NF-κB phosphorylation, α-SMA and HMGCR expression, and cholesterol synthesis (Fig. 6i, j, Supplementary Fig. 6n-p). To identify the involvement of ITGB1 in Sema3C-mediated HSCs activation, we found that ITGB1 protein levels were significantly increased in LX-2 cells treated with rhSema3C or supernatants of OE-Sema3C-HCC cells (Fig. 6k). Knockdown of ITGB1 similarly antagonized Sema3C-induced IL6 secretion, NF-κB phosphorylation, α-SMA and HMGCR expression, and cholesterol synthesis (Fig. 6l, m, Supplementary Fig. 6q-s). Additionally, phenotypic experiments demonstrated that knocking down NRP1 or ITGB1 reduced rhSema3C-induced TGF-β1 secretion, as well as proliferation, invasion, and migration of LX-2 cells (Fig. 6n–p). These findings suggested that Sema3C promoted HSCs activation by upregulating IL6 secretion and cholesterol synthesis through NRP1 and ITGB1.

CAFs-derived TGF-β1 up-regulates Sema3C expression via AP1 in HCC cells

Research has shown that HSCs were the main source of CAFs in the TME, and CAFs could cross-talk with tumor cells to influence tumor progression in a paracrine way.⁷ Thus, we wondered whether Sema3C-induced CAFs activation could in turn regulate Sema3C expression in HCC cells to promote stemness maintenance, thereby creating a vicious cycle between stromal cells and tumor cells. First, analysis of the HCCDB database revealed a positive correlation between Sema3C expression and markers associated with CAFs, including ACTA2, VIM, FAP, PDGFRB, S100A4, and collagen-associated molecules, including COL1A1, COL1A2, FLNA, ELN, and LOX (Fig. 7a). We also found Sema3C to be significantly associated with the stromal score as calculated by using the R package “ESTIMATE” (Supplementary Fig. 7a). Furthermore, analysis using various algorithms in the TIMER2.0 database indicated a positive correlation between Sema3C expression and CAFs infiltration in HCC (Supplementary Fig. 7b). Immunofluorescence was employed to examine the spatial relationship between Sema3C expression and α-SMA⁺ CAFs in both human HCC samples and a mouse model of HCC induced by DEN+CCl₄. We found that Sema3C expression was also increased in samples with high abundance of α-SMA⁺ CAFs infiltration, and the proximity of Sema3C to CAFs also indicated that Sema3C in HCC cells were constantly communicating with CAFs directly or indirectly (Fig. 7b, c). Furthermore, analysis of the TCGA-LIHC database revealed that HCC patients with high Sema3C expression and the presence of FAP⁺ CAFs subset showed a significant association with advanced tumor stages/T stages compared to those with low Sema3C expression and FAP⁺ CAFs. This suggested a clinical relevance of the interaction between Sema3C-expressing HCC cells and FAP⁺ CAFs in in driving malignant progression of HCC (Fig. 7d).

To verify the dynamic communication between HCC cells and CAFs, primary CAFs were isolated from HCC clinical samples (Supplementary Fig. 7c). Then, HCC cells were cocultured with the CM of CAFs in vitro to confirm this hypothesis. The results indicated that treatment with CAFs-derived CM increased Sema3C expression in HCC cells compared to the control (Fig. 7e, Supplementary Fig. 7d). Previous studies have shown that TGF-β1 secreted by activated CAFs was involved in the regulation of tumor cells,²⁹ and our above KEGG enrichment analysis also showed that Sema3C might be related to the TGF-β1 signaling pathway (Fig. 3A). We investigated whether TGF-β1 in CAF supernatants could enhance Sema3C expression in HCC cells. Analysis of the TCGA database showed a positive correlation between Sema3C and TGF-β1 expression in HCC (Supplementary Fig. 7e). Moreover, treatment of HCC cells with gradients of TGF-β1 in vitro significantly upregulated Sema3C mRNA levels (Supplementary Fig. 7f).

We explored the mechanism of TGF-β1-mediated Sema3C upregulation in HCC cells by analyzing the transcription factor binding sites in the Sema3C gene promoter region. Three AP1 binding sites were identified upstream (−1846 to −776 nucleotides) of the transcription start site (TSS) (Fig. 7f). Comparative analysis of AP1 family gene expression in MHCC-97L and Huh7 cells revealed that c-Jun and c-Fos exhibited the highest expression levels among the family genes (Supplementary Fig. 7g). As expected, silencing c-Jun/c-Fos with siRNA significantly inhibited Sema3C expression in HCC cells (Fig. 7g, h). Through ChIP-qPCR analysis, it was observed that stimulation with TGF-β1 led to a notable increase in the binding of phosphorylated c-Jun (p-c-Jun) and phosphorylated c-Fos (p-c-Fos) to the Sema3C promoter (Fig. 7i). Furthermore, the levels of p-c-Jun and p-c-Fos were increased with a dosage TGF-β1 treatment (Fig. 7j). To verify the regulatory effect of the TGF-β1-AP1 signaling axis on Sema3C, we performed rescue experiments using galunisertib (an inhibitor of TGF-β receptor 1), or a TGF-β1–neutralizing antibody. The results showed that galunisertib could block TGF-β1-induced Sema3C expression and phosphorylation of c-Jun and c-Fos (Fig. 7k). Besides, both galunisertib and TGF-β1–neutralizing antibody could antagonize CAFs-CM-mediated Sema3C expression (Fig. 7l, m). Collectively, AP1 signaling was accountable for the TGF-β1–mediated Sema3C upregulation in HCC cells.

Sema3C inhibition enhances sorafenib efficacy in HCC mouse model

The above studies have identified that Sema3C was up-regulated in sorafenib-resistant HCC cells and this elevated expression of Sema3C led to sorafenib resistance in vitro. However, targeted inhibitors for Sema3C are not commercially available nowadays. Therefore, we examined the therapeutic targeting of Sema3C by intravenous injection of rAAV8-shSema3C alone or in combination with sorafenib in a DEN+CCl₄-induced HCC mouse model. At 20 weeks, the mice were injected with the rAAV8-shSema3C or its control rAAV8-shNTC via the tail vein. Three weeks later, the mice were segregated into 4 groups and then administrated with sorafenib at 30 mg/kg or DMSO control for 3 weeks (Fig. 8a). We randomly selected 3 mice from each group to verify the knockdown efficiency of Sema3C after removing liver tumors (Fig. 8b). We found that the combination of rAAV8-shSema3C and sorafenib significantly inhibited liver-to-body weight, tumor numbers, and the maximum size of tumors (Fig. 8c–f). Notably, The combination treatment of rAAV8-shSema3C and sorafenib significantly improved survival compared to mice treated with DMSO control, sorafenib alone, or rAAV8-shSema3C alone (Fig. 8g). IHC analysis of proliferative marker Ki67, Collagen I, and EPCAM in the excised livers from the four treatment groups revealed a marked decrease in the rAAV8-shSema3C/sorafenib combination group, suggesting knocked down of Sema3C did effectively impair proliferative capacity, stromal deposition, and stemness niche of the tumor (Fig. 8h). Collectively, these results suggested that inhibition of Sema3C sensitized HCC cells to sorafenib and synergically reduced tumor growth.