GC is currently one of the major malignancies affecting humans. The abnormal expression of tumor-related genes is considered one of the main mechanisms of tumorigenesis and tumor progression in GC. Targeted therapy for cancer driver genes is a novel path for the prevention and treatment of GC.
As a multifunctional scaffold protein, ASAP1 regulates cell movement, participating in and regulating focal adhesions, invading pseudopodia, and fold formation in the plasma membrane.6,12−14 With rapid improvements in molecular biotechnology, ASAP1 is expected to become a novel tumor diagnostic marker. Biological therapy targeting ASAP1 may have an enormous impact on the development of antitumor drugs. Hence, it is important to study the influence and mechanism of ASAP1 on malignant biological behavior of GC.
The results of this study showed that the mRNA and protein expression levels of ASAP1 in GES-1 epithelial cells derived from normal human gastric mucosa were significantly lower than that in the three GC cell lines, namely, BGC823, MGC803, and MKN45 (all P < 0.05), suggesting that ASAP1 may be closely related to gastric tumorigenesis and tumor progression, which deserves further investigation.
Activation of oncogenes or inactivation of tumor suppressor genes leads to abnormal cell proliferation, differentiation, and apoptosis, which should be key to tumorigenesis and tumor progression. In 2009, Sabe et al.15 pioneered the use of RNA interference (RNAi) and showed that siRNA-mediated ASAP1 silencing effectively inhibited the aggressive activity of breast cancer. In 2017, Yang et al.16 showed that knockdown of ASAP1-IT1 effectively inhibited bladder cancer stem cells, thereby inhibiting the recurrence, metastasis, and chemotherapy resistance of bladder cancer. In 2018, Zhang et al.17 showed that interference of ASAP1-IT1 inhibited the proliferation, migration, and metastasis of non-small cell lung cancer cells. Guo et al.18 showed that knocking out ASAP1-IT1 significantly reduced the number of cholangiocarcinoma colonies. Liu et al.19 showed that the application of RNAi technology downregulated ASAP1 expression, in turn, inhibiting REF52 cell spread and migration. In 2018, Zhang et al.20 showed that ASAP1 overexpression promoted the migration and invasion of ovarian cancer cells, enhanced cancer cell proliferation and survival, and inhibited the apoptosis of ovarian cancer cells. In this study, knockdown of ASAP1 significantly reduced proliferation and colony formation, increased the number of apoptotic cells, and decreased the wound healing ability of BGC823 and MKN45 cells, whereas ASAP1 overexpression had opposite effects, indicating that knocking down ASAP1 inhibits the development of gastric tumors.
The malignant biological behavior of tumor cells is closely related to angiogenic factors. VEGFA is one of the most important positive regulators of angiogenesis. It provides nutrition for the proliferation and growth of tumor cells and the establishment of vascular network by increasing the permeability of blood vessels. VEGFA plays an important role in the transport of nutrients for tumors before angiogenesis. In 2011, Hashimoto et al.21 showed that RNAi of ASAP1 significantly inhibited VEGFA-induced lumen formation, possibly affecting the angiogenesis of breast cancer via the GEP100-Arf6-ASAP1-cortacin pathway. This study showed that knocking down ASAP1 in BGC823 GC cells significantly reduced VEGFA protein expression, which subsequently blocked relevant signal transduction pathways, thereby suppressing tumor angiogenesis, proliferation, and growth, and even the invasion and metastasis of malignant GC.
The malignant biological behavior of tumor cells is closely related to programmed cell death. Cysteinyl aspartate specific proteinases (caspases) are a group of proteases related to apoptosis and cytokine maturation. Caspase 3 is an important protease and an execution factor of apoptosis. It often exists in the form of inactive zymogen. The activation of caspase 3 marks an irreversible process of apoptosis.22 Cleaved-caspase 3 is the active fragment of activated caspase 3. Song et al.23 showed that the activation of caspase 3 induces apoptosis in GC cells. Related studies have shown that the rate of positive expression of cleaved-caspase 3 in cervical cancer tissues is low and is negatively correlated with the differentiation of cervical cancer tissues. Patients with high cleaved-caspase 3 expression levels have a better prognosis.24–26 PARP is a deoxyribonucleic acid repair enzyme that maintains the stability of genetic materials and is activated during DNA damage and repair, thereby regulating apoptosis. After cleavage by activated caspase 3, PARP is transformed into cleaved-PARP that in turn trigger apoptosis.27 Wang et al.28 showed that compared with healthy gastric tissues, PARP expression is upregulated in GC tissues. This study shows that cleaved-caspase 3 and cleaved-PARP protein expression were downregulated in BGC823 ASAP1 knockdown GC cells, suggesting that knocking down ASAP1 promotes the expression of cleaved-caspase 3 and cleaved-PARP proteins, further confirming that ASAP1 regulates apoptosis of GC cells.
The invasion and metastasis of tumor cells are continuous processes that involve the interaction between cancer cells and host cells. MMPs are a class of proteolytic enzymes that are closely related to the invasion and metastasis of malignant tumors. Among these, MMP2 and MMP9 both induce and enhance the formation of blood vessels and invade blood vessels and lymphatic vessels through degradation, destruction, and infiltration, eventually leading to tumor invasion and metastasis.29 Downregulation of MMP2 and MMP9 inhibits the invasion and metastasis of tumor cells.30–31 In this study, knockdown of ASAP1 significantly downregulated the expression of MMP2 and MMP9 proteins and inhibited the invasion and migration of GC cells.
Invasion and metastasis of tumor cells often require a specific cell phenotype. The EMT is a rapid, reversible, temporary process that occurs under certain specific physiological and pathological conditions when epithelial cells lose their normal polarity and tight connection between cells and convert from epithelial phenotype to mesenchymal phenotype. It mainly manifests as downregulation of the epithelial marker E-cadherin, upregulation of the mesenchymal marker N-cadherin, and the transformation of keratin into Vimentin as the core of cytoskeleton.32 Among these, E-cadherin and N-cadherin are key components of the epithelial monolayer cell connection.33 Vimentin is the main component of the intermediate silk protein family that maintains cell integrity and provides stress resistance and promotes tumor growth and invasion.34–35 In 2017, Li et al.36 blocked ASAP1 expression by RNAi, which reversed the EMT process of breast cancer cells. In 2018, Zhang et al.20 showed that ASAP1 overexpression enhanced tumor cell motility and invasiveness of ovarian cancer cells. In this study, knockdown of ASAP1 inhibited the phenotype loss of GC epithelial cells and reduced the appearance of mesenchymal cell phenotypes, which was manifested by the upregulation of E-cadherin and downregulation of N-cadherin and Vimentin, thereby confirming that knockdown of ASAP1 effectively reverses the EMT process and inhibits invasion and metastasis of malignant tumors.
This study also used nude mouse xenograft models to evaluate the effects of knocking down ASAP1 in BGC823 gastric tumorigenesis in vivo, and to verify the relationship between ASAP1 and GC. The animal experiments showed that the growth rate of the subcutaneously transplanted tumors with ASAP1 knockdown was significantly slower and tumor weight was also significantly reduced, confirming that the proliferation and growth of ASAP1-knockdown BGC823 GC cells were significantly inhibited.
In conclusion, we show that ASAP1 is highly expressed in GC cells. ASAP1 overexpression significantly enhances the proliferation, reduces apoptosis, and improves the invasion and migration ability of GC cells. Knocking down ASAP1 significantly reduces the proliferation, promotes the apoptosis, and weakens the invasion and migration of GC cells. Downregulation of VEGFA expression reduces angiogenesis, increases the expression of cleaved-caspase 3 and cleaved-PARP, and decreases the activity of MMPs and EMT in GC cells, thereby effectively suppressing the malignant biological behavior of GC cells. Future studies may present novel opportunities for the prevention and treatment of GC.