Lung cancer remains the leading cause of cancer death and a growing public health burden around the world. For stage I or II NSCLS, despite the best surgical treatment, about 40% of patients die due to recurrence after tumor resection [11]. About 75% of newly diagnosed patients with NSCLS are usually in advanced stage (III or IV stage). For these patients, drug therapy is crucial to improve quality of life and prolong survival [12]. In the past few decades, the concept of modern oncology has paid more and more attention to precision medicine based on genomic variations. With the progress of next-generation sequencing and other mapping techniques in tumor analysis and the acceleration of drug development, more molecular targeted therapeutic drugs have been used in the treatment of malignant tumors, improving the survival of patients with advanced cancer [13]. Although effective targeted therapy can benefit patients to some extent in the field of NSCLS, the existing targeted drugs still do not achieve satisfactory results, and tumors almost always show inherent or acquired drug resistance. This is often the most common potential mechanism of cancer recurrence and metastasis [14–15]. Therefore, the treatment of lung cancer needs to screen more diagnostic and predictive biomarkers, which is very important for more effective diagnosis and treatment of cancer.
ASAP1 is a phospholipid-dependent GAP located on the long arm of chromosome 8, at 24.1–24.2, also known as AMAP1, DDEF1, DEF1 and Centaurin-β4. It consists of N-bar, PH, ArfGAP, ankyrin and eight E/DLPPKP repeats, a Pro-rich domain and a SH3 domain [16]. ASAP1 can catalyze the hydrolysis of GTP bound by ARF (ADP ribosylation factor) to GDP, regulate actin recombination and cytoskeleton kinetics to control cell movement, and participate in the regulation of adhesion plaque, pseudopodia invasion and plasma membrane fold formation [17–18].
Recent studies have described the relationship between ASAP1 and malignant biological behavior of tumors. Its expression is significantly increased in many kinds of tumors, such as head and neck cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, prostate cancer and so on [5]. It can significantly enhance the ability of tumor proliferation, growth, invasion and metastasis, and is expected to become a new target for tumor diagnosis and treatment. In 2005, Onodera et al. [19] found that ASAP1 was expressed at a high level in highly invasive breast cancer cells, but at a low level in noninvasive breast cancer cells and normal breast epithelial cells, and silencing ASAP1 could effectively block the invasive activity of breast cancer, indicating that ASAP1 is a component involved in different invasive activities of breast cancer. In 2008, Lin et al. [20] compared the differentially expressed genes in transplantable human metastatic prostate cancer sublines and nonmetastatic prostate cancer sublines by Long-SAGE analysis. It was found that ASAP1 gene was expressed in metastatic sublines, and the expression of ASAP1 in xenografted tumor sections and clinical samples was positively correlated with prostate cancer metastasis. In 2010, Muller et al. [5] found that the expression of ASAP1 was upregulated in gastric cancer, colon cancer, gallbladder cancer, breast cancer, bladder cancer, ovarian cancer, thyroid papillary carcinoma, esophageal cancer and head and neck squamous cell carcinoma. ASAP1 can promote the movement and invasion of colorectal cancer cells, stimulate the formation of metastasis in vivo, and is related to the poor survival of patients with colorectal cancer. In 2014, Hou et al. [10] found that the expression of ASAP1 mRNA in epithelial ovarian cancer was higher than that in the normal ovarian tissues. Further studies found that the OS time and nonrecurrence survival time of patients with high expression of ASAP1 were shorter. Those with low expression of ASAP1 showed longer survival time, and the expression of ASAP1 was significantly correlated with pelvic metastasis, which indicated that overexpression of ASAP1 was associated with poor prognosis of epithelial ovarian cancer and played an important role in the progression and metastasis of ovarian cancer. In the same year, Li et al. [21] analyzed the expression of ASAP1 in 64 cases of primary laryngeal squamous cell carcinoma (LSCC) by RT-qPCR and immunohistochemical methods. The results showed that the expression of ASAP1 was upregulated in primary LSCC and was related to lymph node metastasis and clinical stage. Further studies have shown that down-regulating the expression of ASAP1 in LSCC can significantly inhibit the invasive ability of LSCC, and a higher level of ASAP1 gene is associated with a shorter trend of progression-free survival, indicating that ASAP1 is also an important prognostic marker for LSCC. In 2020, Tsutaho et al. [22] found that the high expression of ASAP1 in pancreatic cancer was associated with increased PD-L1 and fibrosis, and with poor clinical prognosis. However, so far, there is no indepth study on the effect of ASAP1 on the malignant biological behavior of NSCLS and its possible mechanism. In the present study, it was found that the high expression rate of ASAP1 in NSCLS was significantly higher than that in paracancerous tissues (P < 0.001). The expression of ASAP1 in NSCLS tissues with a T2 + T3 + T4 infiltration depth was higher than that with a T1 infiltration depth (P = 0.003). Furthermore, the expression in patients with lymph node metastasis was higher than that in patients without lymph node metastasis (P = 0.004), and the expression of ASAP1 at stage Ⅱ, stage Ⅲ and Ⅳ was higher than that at stage I (P = 0.003). The recurrence rates of patients in the group with high expression of ASAP1 and low expression of ASAP1 were 39.39% (26/66) and 5.57 (1/18), respectively (P = 0.006). Among recurrent and nonrecurrent patients, the high expression rate of ASAP1 was 96.30% (26/27) and 70.18% (40/57), respectively (P = 0.006). The OS rate of patients with high and low expression of ASAP1 was 65.15% (43 /66) and 94.44% (17/18), respectively, and the difference was statistically significant (P = 0.023). Therefore, the present results suggested that ASAP1 may serve important roles in the malignant proliferation, invasion and metastasis of NSCLC, and may be used as a molecular index to evaluate the malignant biological behavior of NSCLC as well as predict the recurrence and prognosis of NSCLC.
FAK is a nonreceptor tyrosine protein kinase, which was originally detected in chicken embryo fibroblasts transformed by Rous sarcoma virus (v-src) [23]. FAK is encoded by a protein tyrosine kinase 2 (PTK2) gene located in the 8q24.3 region of the chromosome, which consists of an amino terminal (FERM) domain, an activation domain and a carboxyl terminal adhesion spot targeting (FAT) domain [15]. Previous studies have shown that FAK is involved in the regulation of a variety of cell processes, including growth factor signaling, cell cycle progression, cell survival, cell movement, angiogenesis, epithelial to mesenchymal transformation, the establishment of cancer stem cells and an immunosuppressive tumor microenvironment (TME), it is overexpressed in lung, ovarian, breast, and other cancers. In patients with hepatocellular carcinoma and acute myeloid leukemia, the degree of mRNA expression is related to the progression of invasive and metastatic phenotypes and poor prognosis of epithelial and mesenchymal tumors [15, 24]. In recent years, with the continuous progress of research, the exploration of FAK in NSCLC has already made some progress. A study by Ceccarelli et al. [24] published in 2006 analyzed the expression of FAK in NSCLS by immunohistochemical staining. The results showed that the level of FAK in tumor tissues was higher than that in non-tumor tissues. Western blotting and Quantitative Real-time-PCR showed that there was a statistically significant correlation between the up-regulation of FAK and higher stages of the disease, indicating that FAK was involved in tumorigenesis. In 2008, Liu et al. [25] found that down-regulation of FAK promoted apoptosis of NSCLC A549 cells and down-regulated the expression of p-ERK, p-PI3K, p-Src and p-p38. In 2013, Ji et al. [26] measured the expression of FAK protein in NSCLC tissues by immunohistochemistry and the expression of FAK gene in frozen and normal control NSCLC tissues by Quantitative Real-time-PCR. The results showed that the overexpression of FAK was significantly correlated with NSCLC positive lymph node metastasis, stage of cancer and adenocarcinoma subtype, and the increased expression of FAK gene was significantly correlated with lymph node metastasis. In addition, the overexpression of FAK was significantly correlated with poor OS in the survival analysis. It is confirmed that the overexpression of FAK is a promising pathological factor for predicting invasive behavior and prognosis in patients with NSCLC. In 2016, Tang et al. [27] found that FAK inhibition can lead to persistent DNA damage and sensitivity to radiotherapy in mutant KRAS NSCLC cells, indicating that FAK is a new regulator of DNA damage repair in mutant KRAS NSCLC, and its pharmacological inhibition can lead to radio-sensitization, which may be beneficial for tumor treatment. These results suggest that FAK can also be used as a molecular index to evaluate the biological behavior and prognosis of patients with NSCLC. The present study found that the high expression rate of FAK in NSCLC was significantly higher than that in paracancerous tissues (P < 0.001). The expression of FAK in NSCLC tissues with a T2 + T3 + T4 infiltration depth was higher than that with a T1 infiltration depth (P = 0.006). Furthermore, the expression of FAK at stage Ⅱ, stage Ⅲ and Ⅳ was higher than that at stage I (P = 0.014). The recurrence rates of patients in the group with high expression of FAK and low expression of FAK were 37.68% (26/69) and 6.67% (1/15), respectively (P = 0.030). Among recurrent and nonrecurrent patients, the high expression rate of FAK was 96.30% (26/27) and 75.44% (43/57), respectively (P = 0.030). The OS rate of patients with high and low expression of FAK was 66.7% (46/69) and 93.33% (14/15), respectively, and the difference was statistically significant (P = 0.003). The results suggested that FAK plays a promoting role in the occurrence and development of NSCLC, and its abnormal expression might lead to the biological behavior of malignant proliferation, invasion and metastasis of NSCLC, which is highly related to lung cancer recurrence. Therefore, FAK may also be used as a molecular index to evaluate the biological behavior, predict the recurrence and prognosis of NSCLC.
So far, researches have confirmed that ASAP1 is a multidomain protein, which is rich in proline structure and can bind to the corresponding protein to exert its biological function. As a member of human ArfGAP family containing BAR domain, it has been identified as a regulator of actin cytoskeleton and focus adhesion (FA) complex [16]. FA is a large and mature form of integrin adhesion complex, which contains activated integrins and large cytoplasmic plaques composed of proteins that mediate the binding of actin filaments to integrins and initiate very important signals for proliferation, survival and differentiation. FAK is a key protein, which mediates the signal transduction of FAs [28]. In 2002, Liu et al. [29] confirmed that ASAP1 regulates the assembly of FAs through the combination of its CH3 domain and FAK, and there is an interaction between them. The present study showed that there were 65 cases of consistent high expression of ASAP1 and FAK in NSCLC, and the Kappa value was 0.812 (P < 0.001), indicating that there was a high correlation between the expression of ASAP1 and the expression of FAK. The consistent high expression of ASAP1 and FAK was closely related to the age, TNM classification, depth of tumor invasion and lymph node metastasis (P < 0.05). The recurrence rate of patients in the group with consistent high expression of ASAP1 and FAK and consistent low expression of ASAP1 and FAK were 40.00%(26/65) and 7.14%(1/14), respectively (P = 0.027). This result suggested that ASAP1 and FAK can jointly promote the malignant biological behavior of NSCLC.
At present, FAK has been considered as a promising target for the development of small molecular anticancer drugs. However, most of the drugs developed against FAK have not achieved satisfactory results. Among them, FAK inhibitor (Defactinib) only showed moderate antitumor activity in phase II clinical trials of KRAS mutant NSCLC [30–31]. Solid tumors are composed of a complex heterogeneous mixture of cancer cells, immune cell populations, and stromal cells; therefore, combined therapy is needed in most cases for the persistence of the treatment [32]. The present study found that ASAP1 and FAK play a synergistic role in promoting the occurrence, development, invasion and metastasis of NSCLC, and are closely related to the survival status, indicating that ASAP1 is expected to become a potential target for FAK combined therapy.
In short, further experiments on ASAP1 and FAK in vivo and in vitro are needed to explore the molecular mechanism of the role of ASAP1 and FAK in the occurrence and development of NSCLC to further deepen the understanding of the malignant biological behavior of NSCLC. It will provide important insights for early diagnosis, early treatment and prognosis evaluation of NSCLC. Additionally, new targets and therapeutic strategies for basic research and clinical new drug development of NSCLC are required for a more comprehensive approach.