To detect the expression of Arpin, and determine its correlation with clinicopathological characteristics and the prognosis of gastric cancer (GC) patients.

A total of 176 GC patients were enrolled as study subjects and classified into groups according to different clinicopathological variables. GC mucosal tissues were obtained via surgery. Another 43 paraffin-embedded tissue blocks of normal gastric epithelium (> 5 cm away from the edge of the tumor) were included in the control group. Immunohistochemistry (IHC) for the Arpin and Arp3 proteins was performed on the formalin-fixed, paraffin-embedded GC tissues. Additionally, expression of the Arpin protein in 43 normal gastric tissues was also determined using IHC.

Expression of the Arpin protein in GC was lower than that in normal gastric mucosa (30.68% vs 60.47%, P < 0.001). A χ² test of the 176 GC samples used for IHC showed that decreased Arpin expression was associated with advanced TNM stage (P < 0.01) and the presence or absence of lymph node metastasis (80.92% vs 35.56%, P < 0.001). Additionally, a significant correlation was observed between the expression of Arpin and the presence of the Arp2/3 complex in GC tissues (χ² = 30.535, P < 0.001). Moreover, a multivariate Cox regression analysis revealed that Arpin expression [hazard ratio (HR) = 0.551, P = 0.029] and TNM stage (HR = 5.344, P = 0.001) were independent prognostic markers for overall survival of GC patients. Regarding the 3-year disease-free survival (DFS), the recurrence rate of GC patients with low Arpin expression levels (median DFS 19 mo) was higher than that in the high-Arpin-expression group (median DFS 34 mo, P = 0.022).

Low Arpin levels are associated with clinicopathological variables and a poor prognosis in GC patients. Arpin may be regarded as a potential prognostic indicator in GC.

Gastric cancer (GC) remains the fourth most common malignancy and the third leading cause of cancer-related death worldwide, despite its steadily decreasing incidence and mortality since 1930s[1,2]. Although the early detection rate of GC has increased, many patients still suffer from distant metastasis, resulting in a median survival of only 3-5 mo[3-6]. At present, understanding of the multidisciplinary treatment of cancer is a concern of doctors and researchers. However, surgery is still the treatment of choice for most early solid tumors and even some advanced malignant tumors. With the progress made in molecular biology research, accumulating evidence has shown that the carcinogenesis of the gastric mucosa is a multi-factor, multi-step, and multi-stage development process that involves a variety of related genes. Moreover, stages of GC development are affected by different genes. Therefore, it is necessary to identify novel markers that can accurately reflect the biological characteristics of GC tumors, provide new therapeutic strategies, and predict clinical outcomes.

Invasion and metastasis are the two main characteristics of malignant tumors. In general, invasion and metastasis accompany the movement of cancer cells from the primary focus in cancer tissues to other normal tissues or organs, and actin polymerization is necessary for this movement. Actin, a structural protein composed of actin filaments, exists in two forms, monomers and polymers. The actin polymerization process can be divided into two distinct steps: actin monomer polymerization in the nucleus, followed by the addition of actin monomers to the formed nuclei or fibers. Cell movement results from the mutual cooperation of polymerization and depolymerization of the cytoskeleton itself and adhesion and desorption between different parts of the cell and extracellular matrix. Moreover, the formation of lamellipodia is closely associated with actin polymerization. Lamellipodia play an important role in the process of exploration of the external environment by cancer cells and the formation of new adhesion contacts with the extracellular matrix, which allow motility and spreading. The process of lamellipodiaformation is often driven by spatially and temporally regulated actin polymerization at the leading edge[7]. The molecular mechanisms of cancer cell motility and migration are more complicated than that expected, the movement and migration of cancer cells is a result of multi-step process initiated by the formation of membrane protrusions in response to migratory and chemotactic stimuli. It is generally believed that lamellipodia in driving cancer cell migration plays a main role, and it is caused by adhesion force to pull the cell body forward in the basement membrane. However, many cytokines can form new actin fibers, and each factor can form a specific network. The actin-related protein 2/3 (Arp2/3) complex, the most investigated molecule by far, is the sole machine that generates branched actin networks, and is also considered to be a key regulator of cell motility[8]. It has been reported to be involved in the development and migration of some cancers, such as pancreatic, gastric, colorectal, and breast cancer[9-12]. By binding actin filaments to the side of an existing filament and initiating branch formation, the Arp2/3 complex accomplishes its role of nucleating actin filaments[13]. Therefore, the Arp2/3 complex is thought to be involved in cancer cell invasion and metastasis and is controlled by the tumor-stromal interaction, but the specific guiding mechanism of cell invasion in tumors has not been extensively explored. Relevant research results show that the Arp2/3 complex and its activators, such as the WAVE complex, are deregulated in diverse cancers. The Arp2/3 complex plays an important role in the invasion and metastasis of tumor cells. However, it is worth noting that the Arp2/3 complex is also vital for the maintenance of normal cell function.

In 2013, Dang et al[14] reported a new protein, Arpin, which contains 220 amino acid residues that are localized to the cell membrane. It is encoded by the C15ORF38gene, which is present in multicellular animals and unicellular amoebas. Interestingly, this protein contains a carboxyl structure region but no C-terminal end, helix, or single actin binding domain. However, it does inhibit the Arp2/3 complex, resulting in the inhibition of actin polymerization. Arpin contains the putative binding site of the Arp2/3 complex[14], which has been reported to be closely related to the development and migration of cancer cells due to its key role in filopodia initiation[15,16]. Arpin can guide the direction of cell movement, and it is also known as “the steering factor”. However, this process is not dependent on the formation of lamellipodia and is not based on actin dynamics. Arpin acts in a dose-dependent manner and induces the cell to move. Arpin and its acidic motif can compete with nucleation promoting factors for Arp2/3 binding, thereby inhibiting the activation of the Arp2/3 complex[14,17], resulting in the inhibition of actin filament polymerization. Related research has shown the significant relationship between decreased Arpin, the clinical features of breast cancer, and the poor prognosis of breast cancer patients with low Arpin expression relative to the 5-year replase-free survival[18]. However, little is known about the expression of Arpin in GC tissue and its relationship with the clinicopathological characteristics and prognosis of GC patients. In this study, we investigated the issues mentioned above, preliminarily determined the correlation between Arpin and GC as well as between Arpin and the Arp2/3 complex, and provided a theoretical and experimental basis for further research of the specific signaling transduction mechanism of Arpin and the Arp2/3 complex in advanced gastric carcinoma.

To our knowledge, the present study is the first to report the clinical significance of Arpin expression in GC patients, although reduced expression of Arpin and its influence on poor prognosis need to be confirmed in further follow-up studies with larger samples.

In this study, all participants with GC were treated surgically. The sections were obtained from postoperative gastric specimens. In addition, the Arpin and Arp3 IHC analysis was conducted using postoperative gastric slides. The results showed that the Arpin protein was significantly decreased in tumor samples relative to the normal tissues in the control group. Therefore, we speculate that due to the decreased expression of Arpin, tumor cells acquire the ability to invade and metastasize in GC, and the degree of malignancy of tumors is closely related to the clinical stage. We then assessed the correlation between Arpin expression and tumor staging. We found that low Arpin expression was significantly associated with advanced TNM stage, and we concluded that Arpin could play a significant role in tumor biology. In the later stage, lower Arpin expression levels indicate greater malignancy. Whether patients in TNM stage IV, with liver metastasis and peritoneal metastasis are correlated with the expression of Arpin deserves intensive study. Further research may accompany with the expression of Arpin with sensitivity of anti-cancer drug and the correlation of the expression of Arpin with cadherin. Because tumor invasion depth, lymph node metastasis, and distant metastasis are the main pathological basis of malignant tumor staging, we further analyzed the correlation between Arpin expression and local invasion of regional lymph nodes. The IHC results showed that low Arpin expression was significantly associated with the depth of invasion and local lymph node metastases. The results of this research may provide new approaches for determining whether loss of Arpin function is associated with increased cell motility in the epithelial-mesenchymal transition during the progression of cancer. Moreover, the inhibitory effect of Arpin on cell migration could potentially be used to control metastasis. However, the tumor size had no significant effect on the test results. Due to the characteristics of the samples and other factors, our results may be different from previous experimental results.

The expression of the Arp2/3 complex was detected in gastric carcinoma tissue in our experiments. Some observations suggest that stromal cells that express Arp2/3 move and grow in advance of the cancer cells to prepare an environment that facilitates cancer cell invasion. Myofibroblasts, which are considered cancer-induced stromal cells, have been shown to affect the adhesion and movement of cancer cells[19]. Additionally, Arpin inhibits the Arp2/3 complex at the lamellipodium tip. Our research results showed that the expression of Arpin and Arp2/3 complex expression in gastric carcinoma were significantly correlated. Determination of whether a negative correlation exists between these two proteins and the specific mechanism concerning how these proteins affect cell migration will require further study with larger samples. Our experiments may have also indirectly shown a decrease in Arpin expression in GC tissues.

More significantly, in order to investigate the effect of Arpin on the prognosis of GC patients, we compared the 3-year DFS in the Arpin low expression group and the high expression group. Consistently, our study demonstrated that Arpin expression and the TNM stage were independent prognostic indicators of DFS. We propose that Arpin likely plays a role in GC metastasis and the prognosis. However, the exact molecular events leading to cancer metastasis and a poor prognosis have not yet been well elucidated, and further research is required.

The present study had some limitations. We could not visualize the specific signal transduction and regulation mechanism of Arpin and the Arp2/3 complex in GC tissue due to technical difficulties. Arpin is a newly discovered protein, its impact on GC patients and the specific mechanism that it guides cell migration may help us develop new therapeutic target. The study of mechanism about signal path is the direction of our future research. Thus, more work is required in the future.

In summary, the present study showed that Arpin was decreased in GC tissues. Additionally, this low expression pattern was found to be significantly correlated with aggressive clinicopathological features. In addition, the specific regulatory mechanisms of Rac, WAVE, Arpin and the Arp2/3 complex in the development of GC are still unclear. In recent years, targeted therapeutics for key molecular drivers of cancer progression have been developed[20]. Arpin may be used as a potential biomarker that could provide important information about tumor progress and could even be a possible target for GC therapy.