Lung cancer, i.e., bronchogenic malignant neoplasms arising from airway epithelioma, is one of the most common malignancies, and 5-year survival rates range from 4% to 17%, depending on the stage of the disease at diagnosis ^[1][2]. Worldwide, over 1.5 million people annually develop non-small cell lung cancer (NSCLC), which accounts for 80–85% of all lung cancer cases ^[3][4]. Improvements in the knowledge of molecular alterations and their functional significance have the prospective to influence lung cancer diagnosis, prognostication, and treatment ^[5]. Tumor invasion and metastasis are one of the major causes of lung cancer-associated mortality. Therefore, to improve lung cancer treatment, the mechanism underlying lung cancer metastasis should be completely understood so that the establishment of methods that suppress tumor metastasis can be facilitated ^[6].

The molecular mechanisms underlying cancer cell invasion and migration are complex. The initial events are related to the proteolytic degradation of the extracellular matrix (ECM), which provides biochemical and mechanical barriers to cancer cell migration ^[7]. ECM degradation requires the expression and activity of matrix metalloproteinases (MMPs), which are known to play a major role in lung cancer by favoring the invasion of cancer cells ^[7][8]. Among the MMPs, matrix metalloproteinase -2 (MMP-2) activation is related to tumor progression and invasion ^[7][8]. Therefore, the inhibition of the MMP-2 expression regulatory pathway is an important therapeutic strategy for preventing lung cancer metastasis.

Periostin (POSTN), a secreted matrix N-glycoprotein that lacks a transmembrane domain, is a multimodular protein with a signal peptide (SP) that is crucial for secretion, a cysteine-rich domain in the EMILIN family (EMI domain) involved in the formation of multimers through cysteine disulfide bonds ^[9], a tandem of four homologous FAS1 domains (FAS1) that interact with integrins (αvβ3, αvβ5, α6β4) ^[10], and a C-terminal region (CTRL) regulating the cell–matrix organization and interactions by binding the ECM proteins such as collagens, fibronectin, tenascin C, or heparin ^[9][11]. These properties make POSTN a key player in the regulation of cell behavior and the organization of the ECM. POSTN is a protein expressed in various normal human tissues and plays a role in many normal physiological processes. It has been demonstrated that POSTN is involved in the physiological process of epithelial–mesenchymal transition (EMT). This glycoprotein plays a significant role in the fibrillogenesis of collagen ^[12] and the wound healing process ^[13]. Additionally, POSTN is also associated with pathological processes, including the development of cardiovascular diseases, inflammation, asthma, and tumorigenesis ^[14]. The mechanism by which POSTN interacts with tumors has not been fully understood. Most analyses have indicated that POSTN can be involved in regulating the ECM network ^[14]. It interacts with other ECM proteins to form an extracellular environment where cells can interact with each other to promote both growth and survival ^[15]. POSTN has also been confirmed to regulate the development of several types of human cancers by binding to the integrins to activate the Akt/PKB and FAK-mediated signaling pathways ^[16][17]. POSTN-activated signaling pathways promote cellular survival, motility, and adhesion, which are crucial in tumor growth, angiogenesis, invasion, and metastasis. ^[17]. Additionally, it induces neovascularization and supports tumor growth by inducing vascular endothelial growth factor receptor expression in vascular endothelial cells, also stimulating the survival of vascular endothelial cells through the Akt pathway ^[18][19][20]. Currently, many studies have indicated that the overexpression of POSTN is connected with tumor progression. However, others have reported that POSTN might inhibit the invasion and metastasis of bladder cancer cells ^[21]. Furthermore, Kanno et al. ^[22] demonstrated that POSTN had dual effects: the promotion and inhibition of pancreatic cancer. These results indicate the variable biological effects of POSTN in different tissues, which suggest the need for further studies on its complex and multi-aspect functions ^[23].

POSTN is an extracellular matrix N-glycoprotein that is a major constituent of the desmoplastic stroma around solid tumors ^[18]. POSTN can directly exert its functions on tumor cells in paracrine or autocrine mode. Additionally, POSTN supports oncogenesis not only by activating intracellular pathways but also through its impact on ECM desmoplasia. The desmoplastic stroma of a malignant neoplasm constitutes a tumor microenvironment that supports tumor growth and invasion. Nevertheless, specific molecular mechanisms defining how POSTN remodels distinct tumor microenvironments have not been fully accounted for ^[18][24].

POSTN upregulation has been demonstrated for many cancer types, such as non-small cell lung cancer (NSCLC) ^[25][26], invasive ductal breast cancer (IDC) ^[27], pancreatic ^[28], and ovarian cancer ^[29], and is consequently defined as a tumor-enhancing factor ^[17]. Only a few reports in bladder cancer and osteosarcoma have shown POSTN as a tumor-inhibiting factor ^[21][30]. To confirm this, analysis of POSTN expression is required in a larger cohort of various cancer cases.

Consistent with studies related to other cancers ^[18][31], POSTN was involved in the motility and in vitro invasive potential of lung cancer cells. The data suggest that POSTN plays a crucial role in the multistep cascade process of cancer metastasis. Invasion of the basement membrane and ECM is critical for metastasis of NSCLC, which depends on degradation of these components, particularly by MMP, proteolytic enzymes widely associated with increasing cancer-cell growth, tumor invasion, and metastasis ^{[7][32][33][34]}.

It has been recently shown that POSTN may support adhesion and migration of ovarian epithelial cancer cells by interacting with αvβ3 and αvβ5 integrins ^[11][29]. Orecchia et al. ^[35] also showed that the proliferating activity of melanoma cells was inhibited by the addition of antibodies directed against POSTN elements involved in the interaction with both αvβ3 and αvβ5 integrins, which showed that such an interaction was crucial for tumor growth ^[11]. Therefore, it is believed that the identification of molecules that mediate the association of cancer cells with CAFs is the most crucial challenge. It is also significant to identify signaling pathways and molecules in cancer cells that can be activated upon direct interaction with CAFs. Therefore, the analysis of interactions between these cells will be the subject of the future research.

These findings are in accordance with the observations of Soltermann et al. ^[36], who showed that in NSCLC, epithelial POSTN was also significantly associated with several clinicopathological parameters such as squamous cell carcinoma histotype, higher stage, and higher pT as well as larger tumor size. However, it should be emphasized that compared to the studies, those authors used a different patient pool size. A similar study was obtained by Zhu et al. ^[37], who indicated that high POSTN levels in cancer cells of ovarian cancers were correlated with advanced late stages (III/IV) and cancer recurrence. The above observations were also confirmed in the case of renal cell carcinoma by Morra et al. ^[38]. In their study, they showed that higher levels of POSTN in cancer epithelial cells correlated with higher tumor stage, lymph node metastases, and poor overall survival. Interestingly, in line with the researchers' previous studies ^[25][39], the intensity of immunoreactivity of POSTN in epithelial cancer cells increased with the malignancy grade of the tumors and had an impact on patient overall survival. The survival analysis demonstrated that a high epithelial expression of POSTN in NSCLC and in AC and SCC subtypes was associated with poor patient outcomes. This indicates the influence of POSTN in cancer development. Moreover, a multivariate analysis showed that POSTN expression in epithelial cancer cells can be an independent positive prognostic factor in the whole NSCLC patient cohort as well as in AC and SCC groups. Similarly, Ben et al. ^[40] demonstrated that in pancreatic ductal adenocarcinoma (PDAC), high POSTN expression in cancer epithelial cells was indicative of poor prognosis compared to the adjacent tissue. Furthermore, studies by Riener et al. ^[41], related to liver tumors, indicated that POSTN expression in cancer epithelial cells was associated with reduced overall survival and correlated with tumor grade. A similar trend was found. Similar results were also reported in oesophageal squamous cell carcinoma by Wang et al. ^[42], who found that high POSTN expression correlated with poor prognosis and shorter overall survival, which is in line with the tendency demonstrated.

These experimental results, obtained using clinical material, were further supported by in vitro studies. POSTN silencing using shRNA (short-hairpin RNA) significantly inhibited the migratory and invasive capabilities of lung cancer cells (A549.shRNA) compared to control A549.CTRL cells. The researchers conducted additional experiments to consolidate the mechanistic study of the effect of POSTN on migration and invasion.Protein expressions of integrin-αvβ3 and PI3K/pAKT were reduced in the cells representing the loss-of-function phenotype (A549.shRNA) compared to control cells, indicating the silencing of POSTN in the integrin-signaling pathway during the course of NSCLC. POSTN is capable of binding to integrins, including -αvβ3, -αvβ5, and -α6β4, thereby promoting activation of specific integrin-mediated signaling pathways such as Akt/PI3K signaling pathways, which leads to increased cell survival, angiogenesis, invasion, and metastasis. Consequently, the researchers may assume that POSTN silencing may inhibit NSCLC progression by blocking the αvβ3 integrin/ PI3K/AKT signaling pathway. POSTN silencing statistically significantly decreased the protein expression level of MMP-2 as well as the enzyme activity of MMP-2 in A549.shRNA cells, which likely contributes to decreasing the migratory and invasive ability of lung cancer cells. POSTN silencing might modulate the tumor microenvironment by affecting MMP-2, a protein acting as a metastasis-associated factor of the tumor microenvironment, which serves an important role in the degradation of the basement membrane and the invasion of cancer cells ^[6][7][8]. Therefore, POSTN promotes the invasive ability of lung cancer cells, at least partly via tumor microenvironment factor MMP-2, highlighting MMP-2 as an effector of POSTN signaling in lung cancer cells ^[43]. Furthermore, to the best of the knowledge, this is the first study to show a possible relationship between the knockdown of POSTN expression in lung cancer cells in vitro and the downregulation of MMP-2. The αvβ integrin/ERK signaling pathway is one of the mechanisms by which MMP-2 expression is upregulated in tumor cells ^[44]. As a result, POSTN could also induce the upregulation of MMP-2 expression via the αvβ3 integrin/ERK pathway. Such a mechanism was demonstrated by Watanabe et al. ^[45]. The functional analyses in human periodontal ligament cells revealed that POSTN regulated MMP-2 expression via the αvβ3 integrin/ERK signaling pathway. Moreover, Yan et al. ^[46] found that POSTN overexpressing 293T cells showed increased MMP-9 activation. In turn, overexpression of POSTN in the bronchial epithelial cell line BEAS-2B promoted the epithelial expression of MMP-2 and MMP-9 in a TGF-β-dependent manner ^[47]. Similar results were also reported in glioma by Wang et al. ^[43]. In their study, they showed that POSTN promoted glioma cell invasiveness in vitro, accompanied by MMP-9 expression. A recent study of renal cell carcinoma cells ^[33] also demonstrated that POSTN overexpression increased the activity of MMP-2 and MMP-9. Furthermore, they noticed that FAK knockdown attenuated MMP levels, cell migration, and invasion, which were all enhanced by POSTN, suggesting that POSTN plays a critical role in the multistep cascade process of cancer metastasis ^[33]. Similar conclusions were drawn by Ouanouki et al. ^[48], who indicated that silencing of POSTN inhibited U-87 glioblastoma cell migration and invasive potential, which is in line with the tendency demonstrated.

POSTN increases the invasive ability of lung cancer cells by increasing their migratory properties, thus affecting the expression of matrix metalloproteinases such as MMP-2, an endopeptidase playing a crucial role in the carcinogenesis of lung cancer, with functions in cell proliferation, tumor invasion, and metastasis.Exogenous POSTN increased MMP-2 expression, correspond to previous data from Burgess et al. ^[47], who reported that POSTN induced the expression of MMP-9 in differentiated primary epithelial cells. Similarly, Kanno et al. ^[22] revealed that a high concentration of recombinant POSTN promoted cell migration in pancreatic cancer cells, which is also in line with the tendency observed.

In summary, the important functional and molecular mechanisms of POSTN was shown in tumor invasion. Silencing of POSTN could inhibit the migration and in vitro invasive potential of lung cancer cells, most probably via the downregulation of MMP-2 expression and activity as well as integrin-signaling related proteins.