More than 90 % of human cancer-related deaths are caused by metastasis, a complex process that involves several distinct steps, including the loss of cell adhesion, increased motility and invasiveness, the ability to enter and survive in circulation, egress into organs, and successful colonization at a remote site. To achieve this, cells bearing metastatic tumors must accumulate the expression of several genes that are necessary for the tumor metastatic cascade [1], [2], [3], [4]. As a result, one of the most pressing challenges is to identify the genes that regulate the pathways that promote metastasis. Lung cancer is the leading cause of cancer-related deaths [5]. Approximately 85 % of lung cancer cases are non-small-cell lung cancers (NSCLCs). While there have been advancements in the development of molecular-targeted agents and immune checkpoint inhibitors in recent years, overall survival rates for advanced NSCLC have not improved as expected, which remains a critical problem in cancer therapy. Currently, there are no well-understood mechanisms underlying the progression [6], [7]. To improve treatment outcomes and increase patient survival rates, it is crucial to better understand the molecular mechanisms involved in the metastasis-promoting processes of NSCLC.

Calponin is an actin-activated myosin ATPase inhibitor originally discovered in chicken gizzard smooth muscle [8]. The human genome contains three different isoforms of calponin: basic calponin (h1-calponin, CNN1), neutral calponin (h2-calponin, CNN2) and acidic calponin (h3-calponin, CNN3) [9], [10], [11]. These isoforms contain a conserved N-terminal calponin homology (CH) domain, two actin-binding sites in the middle region, and two actin binding sites in the C-terminal region. A growing number of studies have demonstrated that calponins interact with cytoskeletal proteins other than actin, including tropomyosin, myosin and tubulin [12], [13], [14], [15]. Several cellular activities related to cytoskeletal function are also regulated by calponins, including proliferation, migration, adhesion, and differentiation. Specifically, h2-calponin is expressed in a wide range of smooth muscle and non-muscle cells. Apart from regulating the cytoskeleton in many types of cells, h2-calp serves a range of other functions. Liu and Jin suggested that h2-calp is expressed in three distinct types of cells, including cells under high mechanical tension, cells with high rates of proliferation, and cells actively migrating. Research on epidermal keratinocytes and fibroblasts shows h2-calp expression is controlled by mechanical tension in the cytoskeleton [16]. A number of recent studies have investigated the expression of h2-calp in breast cancer, prostate cancer, and other cancers, providing evidence that h2-calp is involved in tumor growth [17], [18]. In other cancer types, however, h2-calp's expression pattern and biological function remain unclear.

Chromosome Xp22.2 encodes the ribosomal S6 kinase 2, or RPS6KA3 (p90RSK2 or RSK2). The RSK2, one of four members of the RSK protein family, relays upstream signals of the mitogen-activated protein kinases (MAPK) pathway [19]. This protein kinase is composed of two kinase domains separated by a linker region containing multiple phosphorylation sites. N-terminal kinase domains are responsible for RSK2 substrate phosphorylation, whereas C-terminal kinases play auto regulatory roles [20]. MAPK binds to the C terminus of RSK2 and phosphorylates it, initiating a series of highly orchestrated phosphorylation events through MAPK-mediated C-terminus kinase phosphorylation, ultimately activating RSK2 [21], [22], [23]. The cAMP-responsive element-binding protein (CREB) and heat shock protein 27 (HSP27) are phosphorylated and activated by RSK2 and are important for the RSK2-mediated invasive ability in head and neck squamous cell carcinoma (HNSCC) [24].

Here, we provide evidence that h2-calp knockdown(KD) promotes NSCLC cells metastasis in vitro and in vivo through activation of RSK2/HSP27/CREB signaling. These results have implications for the understanding of potential function of h2-calponin in NSCLC progression.