Modern cell biology and bioinformatics have revealed that multiple cell signaling pathways play an important role in the initiation, survival, growth, invasion, migration, metastasis, and drug resistance of hepatocellular carcinoma. A deeper understanding of these pathways is fundamental to the design of novel molecular and gene therapies. Accurately identifying differentially expressed genes associated with hepatocellular carcinoma progression in each patient can help personalize drug selection. In addition, characteristic molecular markers of some pathways are useful for early detection of hepatocellular carcinoma and prediction of efficacy, while evaluating treatment outcomes in clinical practice and trials.
1. Tyrosine kinase-dependent pathway
Tyrosine kinases are a class of enzymes that catalyze ATP-dependent phosphorylation at a variety of receptors, including EGFR, VEGFR, PDGFR, FGFR, HGF/c-MET receptors, and c-KIT receptors. These enzymes play a key role in the growth, survival, and invasion of cancer cells. Tyrosine kinase overexpression has been found in many cancers, including hepatocellular carcinoma, and is therefore targeted by small molecule inhibitors such as sorafenib, lenvatinib, cabozantinib and regorafenib.
1.1 Vascular endothelial growth factor (VEGF) pathway
The rapid growth of tumors requires a large blood supply, and most tumors exhibit uncontrolled neovascularization, known as angiogenesis, which plays a key role in tumor metastasis. TACE treats hepatocellular carcinoma by blocking the blood supply to tumors, but it faces challenges such as heterogeneity, poor drug permeability, and complex tumor microenvironment. VEGF family peptides (such as VEGF, VEGF-B, VEGF-C, etc.) regulate neovascularization, among which VEGFR-2 is the most important in tumor angiogenesis. Clinical samples showed that VEGF gene is highly expressed in hepatocellular carcinoma and is a potential target for gene therapy, and VEGF levels can be used as a biomarker for diagnosis and prediction of treatment response.
1.2 Epidermal growth factor (EGF) pathway
EGF regulates cell growth, survival, proliferation, and differentiation, and its receptor EGFR has been extensively studied in a variety of cancers, such as liver, breast, and lung cancer. In hepatocellular carcinoma, the EGF pathway regulates the inflammatory environment by inducing CXCL5 and CXCL8. Phase II clinical trials of small molecule inhibitors such as erlotinib and vatanib, as well as cetuximab, an anti-EGFR monoclonal antibody. These inhibitors can block the EGF pathway and induce apoptosis in HCC cells, but EGFR mutations may lead to drug resistance. The study found that there were seven EGFR mutations in HCC cells, all of which were resistant to erlotinib. EGF was highly expressed in HCC, and transgenic mice overexpressing EGF had a higher incidence of liver cancer. Clinical data suggest that overexpression of EGF and EGFR is associated with a high incidence and poor prognosis of HCC in patients with HBV or HCV infection. EGF/EGFR knockout leads to HCC cell cycle arrest and apoptosis, therefore, the EGF pathway is a good candidate for HCC gene therapy.
1.3 Fibroblast growth factor (FGF) pathway
The FGF family contains more than twenty proteins that are responsible for cell proliferation, metabolism, differentiation, and survival. Among them, FGF19 is highly expressed in patients with hepatocellular carcinoma and is associated with poor prognosis. When healthy, FGF19 promotes liver regeneration. Studies in transgenic mice have shown that high expression of FGF19 in mice leads to primary liver cancer.
FGF19 induces multiple signaling pathways in hepatocellular carcinoma and regulates endoplasmic reticulum stress and promotes immune escape. The multi-targeted kinase inhibitors lenvatinib and BLU9931 show positive effects on the FGF19 pathway.
1.4 血小板衍生生长因子（PDGF）
PDGF is a tyrosine kinase-dependent growth factor that has been implicated in cell growth and tissue regeneration. High levels of PDGF are associated with chronic liver diseases such as hepatocellular carcinoma, liver fibrosis, and cirrhosis. PDGF plays a key role in hepatocellular carcinoma-associated fibrosis and is an important therapeutic target. Pharmacological strategies include neutralizing antibodies, aptamers, soluble receptors, and small molecule inhibitors.
1.5 干细胞生长因子受体（c-KIT）
c-KIT is a tyrosine kinase-dependent receptor that binds to stem cell growth factor (SCF). In hepatocellular carcinoma, c-KIT is overexpressed and has multiple mutations that promote cell survival, migration, and angiogenesis. HBV and HCV promote hepatocellular carcinoma stem cell production by activating c-KIT. Drugs such as imatinib mesylate and sorafenib have an effect on c-KIT, but should be used with caution to avoid negative effects.
1.6 肝细胞生长因子（HGF/c-MET）
HGF regulates hepatocyte regeneration and growth. In hepatocellular carcinoma, HGF overexpression promotes tumor cell proliferation, survival, and metastasis by activating c-MET receptors. c-MET can be used as a prognostic marker and an indicator of chemoresistance in hepatocellular carcinoma. Small molecule inhibitors such as PHA665752 and AMG 337 inhibit the HGF/c-MET pathway. Knockdown of c-MET has also shown efficacy in inhibiting hepatocellular carcinoma growth and reversing drug resistance.
2. PI3K/AKT/mTOR 通路
The PI3K/AKT/mTOR pathway plays a key role in hepatocellular carcinoma (HCC) progression, with approximately 50% of HCC patients showing hyperactivity. This signaling pathway regulates cell cycle, growth, metabolism, and survival, and provides downstream signals for other cell growth pathways such as EGF and IGF. Transcriptome analysis showed that AKT was highly expressed in HCC tissues in 23% of HCC patients, which was closely related to recurrence and poor prognosis of HCC after surgery.
Bioinformatics analysis found that the mTOR signal was abnormal in half of HCC cases. PTEN is a tumor suppressor that inhibits PI3K/AKT/mTOR signaling, and the PTEN gene is mutated or downregulated in about half of HCC cases, resulting in activation of this pathway.
Sirolimus and everolimus are selective mTOR inhibitors investigated for the treatment of HCC after SOR therapy failure. Recent meta-analyses have shown that these two drugs improve survival in HCC patients after liver transplantation. Studies have shown that everolimus in combination with the all-AKT inhibitor MK-2206 can synergistically inhibit the proliferation of three HCC cell lines, Hep3B, HepG2 and Huh7. However, a phase III clinical trial failed to show an increase in survival in patients with HCC who were intolerant of advanced SER. In another phase II clinical trial, the combination of SOR and the mTOR inhibitor tessirolimus did not achieve the desired effect in patients with advanced HCC. Clinical results of PI3K/AKT/mTOR inhibitors have been mixed, suggesting the need to develop more innovative therapies and gene therapy as a promising option.
3. PLK1 pathway
PLK1 is a serine/threonine kinase that plays multiple roles in cell cycle progression, primarily by regulating spindle checkpoints in M phase. PLK1 is overexpressed in a variety of tumors, resulting in uncontrolled cell proliferation beyond mitotic checkpoints. Studies have shown that PLK1 expression in hepatocellular carcinoma samples is 12 times higher than in normal tissues. Knockdown of PLK1 can significantly reduce the biological activity of hepatoma cells and significantly reduce the growth of hepatocellular carcinoma xenograft in nude mice.
Arbutus Biopharma has developed TKM-080301, a gene silencing therapy targeting PLK1, which has been evaluated in Phase I/II clinical trials to evaluate its safety, pharmacokinetics and preliminary anti-tumor activity. Phase I results showed a favorable biosafety profile, but phase II results did not show an improvement in overall survival.
4. RAS/RAF/MEK/ERK通路
RAS/RAF/MEK/ERK is a complex signaling pathway that affects cell proliferation, differentiation, angiogenesis, and survival. Its building blocks and downstream proteins are called mitogen-activated protein kinases (MAPKs). The level of MEK1/2 phosphorylation in HCC tissues is 7-fold higher than in neighboring tissues. Studies have found that RAS/RAF/MAP abnormalities contribute to the malignant transformation of hepatocytes. RAF1 protein is upregulated in approximately 60% of hepatocellular carcinoma patient samples. HBV integrates into the host genome and induces hepatocellular carcinoma by activating the RAS/RAF/MEK/ERK pathway.
RAS/RAF/MEK/ERK signaling is a therapeutic target for a variety of kinase inhibitors, such as sorafenib and lenvatinib. Despite progress, clinical outcomes remain suboptimal. CI-1040 is a small molecule inhibitor that selectively targets MEK/ERK, and the Phase I clinical trial results are encouraging, but the Phase II results are disappointing. Second-generation MEK/ERK inhibitors PD0325901 exhibited higher pharmacological properties and efficiency in vitro and in vivo, but also failed in phase II clinical trials. These challenges make gene therapy a potential solution. For example, knockdown of ERK2 can significantly inhibit the proliferation of hepatocellular carcinoma cells by silencing the BRAF gene to inhibit the growth of a mouse model of hepatocellular carcinoma.
5. Hedgehog (Hh) 通路
The Hedgehog (Hh) pathway plays a key role in liver disease and includes SHH, IHH, and DHH ligands. These ligands inactivate PTCH1, which in turn activates SMO, simultaneously activates GLI transcription factors, and increases cell proliferation, differentiation, and invasion. The HH pathway is overactive in 50% of hepatocellular carcinoma cases, is associated with tumor progression, and leads to liver fibrosis. HHIP, an antagonist of Hh signaling, is downregulated in hepatocellular carcinoma and liver fibrosis and becomes a target for gene therapy. Vismodegib is an inhibitor of the Hh pathway that has shown efficacy in preclinical studies, but clinical data are insufficient. RNAi-mediated knockdown of SMO inhibits hepatocellular carcinoma cell growth.
6. JAK/STAT 通路
The JAK/STAT pathway regulates cell responses to growth factors (e.g., EGF, PDGF, VEGF) and cytokines (e.g., IL-6) and inhibits cell proliferation, differentiation, and apoptosis. JAK/STAT dysregulation in hepatocellular carcinoma (HCC) leads to uncontrolled cell growth through mutations or inhibitory modulators. STAT3 inhibitors (e.g., CIMO) are highly toxic to hepatocellular carcinoma cells, inhibiting tumor growth in mice. STAT3 gene knockout inhibits hepatocellular carcinoma cell growth and angiogenesis, making it suitable for gene therapy. In addition, STAT3 is associated with tumor-promoting M2 tumor-associated macrophages and is a potential target for anti-cancer immunotherapy.
7. Survival pathways
In addition to the previously mentioned pathways, the following pathways also play an important role in maintaining the survival and anti-apoptosis of hepatocellular carcinoma cells, and are therefore associated with chemoresistance and are important targets for gene therapy.
7.1 Midkine (MK)
MK is a heparin-bound growth cytokine that is especially important in fetal life. Aberrant expression of MK in adulthood is closely related to a variety of diseases such as tumors.
Studies have shown that MK activates the PI3K/AKT pathway by binding to the anaplastic lymphoma kinase (ALK) receptor, which in turn activates MAPKs, up-regulates STAT3 and down-regulates caspase-3, thereby anti-SOR efficacy. MK gene silencing increased the sensitivity of HepG2 cells to SOR through siRNA and reversed SOR resistance in vivo, resulting in the elimination of SAR-resistant hepatocellular carcinoma in mouse models.
7.2 c-Jun-N-terminal-kinase (JNK)
JNK is a member of MAPKs that mediates the anti-apoptotic activity of hepatocellular carcinoma, promotes cell proliferation, differentiation, migration, and invasion, and mediates inflammation and fibrosis through the TGFβ/SMAD pathway. Studies have shown that JNK knockdown enhances CD95-mediated apoptosis via siRNA and induces cell cycle arrest in the G2/M phase of HepG2 cells.
7.3 Myeloid cell leukemia-1 (Mcl-1)
Mcl-1 is an anti-apoptotic protein in the Bcl-2 family that plays an important role in a variety of cancers, including hepatocellular carcinoma. Mcl-1 gene knockdown increases the sensitivity of hepatocellular carcinoma to chemotherapy and TRAIL treatments through siRNA, and eliminates drug resistance by enhancing caspase-3-mediated apoptotic activity.
7.4 Bmi-1
BMI-1 is an overexpressed oncogene in hepatocellular carcinoma that plays an important role not only in carcinogenesis, but also in stem cell pluripotency, embryogenesis, and cell proliferation. Studies have shown that siRNA silencing of the Bmi-1 gene inhibits the proliferation and aggressiveness of hepatocellular carcinoma cells and increases their sensitivity to 5-fluorouracil (5-FU) therapy.
7.5 P-糖蛋白 (P-gp)
P-gp is a membrane transporter that mediates molecular efflux. Upregulation of P-gp in cancer cells is the adaptive mechanism for their evasion of chemotherapy, and P-gp efflux is the basic mechanism of resistance to a variety of chemotherapeutic agents such as doxorubicin, paclitaxel, 5-FU, and cisplatin. Therefore, P-gp is referred to as the multidrug resistance 1 protein (MDR1). There have been many reports in HCC research on gene therapy targeting P-gp to reverse chemoresistance.
8. Inflammatory pathways
Inflammation is the body's response mechanism to stress or cell damage by recruiting cytokines such as tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) to increase blood supply and infiltrate the affected area. Unfortunately, an uncontrolled inflammatory response can have a severe impact on tissues and even contribute to the development of cancer. Hepatocellular carcinoma secretes a variety of pro-inflammatory signals, induces persistent inflammatory microenvironment in the liver, and promotes the proliferation, migration, angiogenesis, and genomic instability of the tumor microenvironment.
8.1 Lysic acid (LPA) and autophagy factor (ATX) pathways
LPA is an endogenous phospholipid whose production is largely dependent on the autophagy factor (ATX) enzyme, which is also a carrier of the LPA receptor. The LPA/ATX pathway is highly correlated with the occurrence of inflammation-related liver cancer through the TNFα/NFkB pathway. Studies have shown that knockdown of the LPA/ATX signaling pathway by RNAi-targeting LPA receptor 6 (LPAR6) inhibits hepatocellular carcinoma tumor formation in a mouse xenograft model.
8.2 转化生长因子β1 (TGFβ1)
TGFβ1 is an important pro-inflammatory signaling pathway in a variety of cancers, and its activation stimulates the metastasis of SMADs to the nucleus and activates a variety of pro-inflammatory transcription factors. In addition, hepatic Kupffer cells oversecrete TGFβ1 in response to liver injury and subsequently activate hepatic stellate cells (HSCs), which may lead to liver fibrosis and progression to cirrhosis and hepatocellular carcinoma. Recent studies have shown that targeting both TGFβ1 and SMO through gene knockdown can reprogram activated HSCs to quiescent HSCs and reverse liver fibrosis in mice. Other studies have found that combined immunotherapy, silencing TGFβ1 and cyclooxygenase-2 (COX-2) can inhibit cancer growth.
Source: International Hepatobiliary Information