CAFs-derived rho-associated kinase1 mediated EMT to promote laryngeal squamous cell carcinoma metastasis

Background Cancer-associated fibroblasts (CAFs) play an essential role in tumorigenesis and development of cancers. Nevertheless, the specific molecular mechanism of tumorigenesis and development in Laryngeal squamous cell carcinoma (LSCC) still unknown. Methods CAFs, CPFs and NFs were isolated and identified from laryngeal cancer, para-laryngeal cancer and normal tissues. Immunofluorescent staining, Rt-PCR and Western Blot were used to detect the expression of related proteins. Wound healing, migration, invasion and animal experiments were used to examine the ability of movement, migration, invasion and metastasis of LSCC. Results ROCK1, was highly expressed in CAFs and CAFs enhanced LSCC metastasis in vivo and vitro, and downregulation of ROCK1 in CAFs inhibited the migration and invasion of LSCC cells. While increasing ROCK1 expression in NFs promoted the migration and invasion of LSCC cells. Further studies revealed that epithelial-mesenchymal transition (EMT) and JAK2/STAT3/ERK1/2 pathway might play an essential role in promoting metastasis of LSCC. In addition, inhibition activity of ROCK1 or JAK2/STAT3/ERK1/2 signal molecules significantly reduced EMT and metastasis. Conclusions CAFs-derived ROCK1 via JAK2/STAT3/ERK1/2 axis mediated EMT to promote LSCC metastasis and targeting ROCK1 might provide a potential treatment strategy for LSCC. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-023-02911-z.


Background
Laryngeal squamous cell carcinoma (LSCC) is a type of head and neck cancers [1]. Standard radical resection is now the main treatment for LSCC patients, however, the 5-year survival rate after surgery is not high, mainly because of tumors metastasis [2]. Consequently, it is of great significance to explore the specific molecular mechanism of metastasis for the treatment of LSCC patients.
As a basic process in embryonic development, Epithelial-mesenchymal transition (EMT) is the basis of normal development including wound healing and malignant epithelial neoplasms [3]. EMT is a well characterized embryological process which epithelial cells undergo a phenotypic switch by losing their cell polarity and the epithelial marker (E-catenin, β-catenin) translate into mesenchymal phenotypic cells through acquiring the mesenchymal marker (N-catenin, Vimentin). Losing the polarity of epithelial phenotypes, such as synthesis of basement membranes, can enhance migration and progression and anti-apoptosis [4]. Researches also have found that EMT is involved in tumor metastasis, during the process, tumor cells acquire mesenchymal phenotype and invasiveness through epithelial mesenchymal transformation [5].
Recently, accumulating evidences have manifested interactions between tumor and tumor microenvironment is essential for tumor metastasis. Tumor microenvironment includes tumor and stromal cells [5]. Hence, the interactions between epithelial cells and stromal cells usually act as the regulators of EMT, and the factors creating EMT are originating from the tumor and stromal cells [6]. Normally, tumor stromal cells contain fibroblasts, endothelial, macrophages and lymphocytes cells, which secrete inflammatory factors (TNF-α, TGF-β, IGF), chemokines (IL-8, IL-6, MCP-1), matrix degrading enzyme (MMP-9, MMP-2, MMP-1) and growth factor (EGF) [7]. These factors formed the microenvironment, cancer-associated fibroblasts (CAFs) are the major cells in it which plays the key role in forming the tumor microenvironment and regulating progression and metastasis [8].
Moreover, studies have found that rho-associated kinase1/2 (ROCK1, ROCK2) activation promotes proliferation, however, ROCK1 or ROCK2 inactivation reduces migration [9]. In addition, Stadler S has demonstrated that ROCK1 and ROCK2 paly an essential role in the metastasis of colorectal cancer [10]. Nevertheless, the specific molecular mechanism of tumorigenesis and development in LSCC still unknown.
In our study, we first isolated CAFs from LSCC, and found ROCK1 was highly expressed in CAFs and CAFs enhanced LSCC metastasis in vivo and vitro, and downregulation of ROCK1 in CAFs inhibited migration and invasion of LSCC cells. While increasing ROCK1 expression in normal fibroblasts (NFs) promoted migration and invasion of LSCC cells. Further study indicated that EMT and JAK2/STAT3/ERK1/2 pathway might play an essential role in promoting LSCC metastasis. In addition, inhibition of ROCK1 or JAK2/STAT3/ERK1/2 signal pathway via inhibitors significantly reduced EMT and metastasis. Our study suggested that deprivation of ROCK1 or JAK2/STAT3/ERK1/2 molecules would act as an effective treatment strategy against LSCC.

CAFs with high ROCK1 expression enhanced LSCC metastasis
In our study, CAFs, Cancer Para-laryngeal Fibroblasts (CPFs) and NFs were isolated and identified from laryngeal cancer, para-laryngeal cancer and normal tissues. It is well known that in comparison with NFs, the phenotype of CAFs was significantly different. CAFs express specific molecules, including α-SMA, FSP1, NG2 and PDGF-β receptor, et al. [11,12]. The specific markers were determined by Western Blot, the expressions of FAP, α-SMA, FSP1, NG2 and PDGF-β were significantly elevated in CAFs (**P < 0.01, Fig. 1A and B). Here, we established a co-culture system in vitro (Fig. 1C), in which fibroblasts were indirectly co-cultured with laryngeal cancer Hep2 cells line, which separated by a semi-permeable membrane (pore size of 0.6 μm). At the appropriate time point, Hep2 cells were either assayed by way of the wound healing, migration, invasion. As showed in Fig. 1D and E, Hep2 co-cultured with CAFs cells (Hep2/ CAFs cells) enhanced movement than Hep2 co-cultured with NFs (Hep2/NFs cells, **P < 0.01). Migration assay showed that Hep2/NFs cells (105 ± 8.6) migrated less than Hep2/CAFs cells (286 ± 15.2). Invasion assay also showed similar results, Hep2/NFs cells (51 ± 6.5) invaded less than Hep2/CAFs cells (101 ± 4.0, *P < 0.05, **P < 0.01, Fig. 1F and G). To further illustrate CAFs played a determinant role in metastasis. Hep2/CAFs cells and Hep2/ NFs cells respectively inoculated via tail vein into 4-weekold male immunodeficient mice. Six weeks after inoculation, Hep2/CAFs cells (5.8 ± 0.7) demonstrated larger and more frequently lung metastases as compared to Hep2/ NFs cells (2 ± 1.5, *P < 0.05, Fig. 1H and I). In our study, the mRNA and protein expressions of ROCK1 considerably high expressed in CAFs in comparison with CPFs and NFs (*P < 0.05, **P < 0.01, Fig. 1J L). Data above indicated that Hep2 cells co-cultured with CAFs compared to Hep2 cells co-cultured with NFs enhanced LSCC metastasis in vivo and vitro and ROCK1 was highly expressed in CAFs.

CAFs-derived ROCK1 promoted EMT of LSCC
Accumulating evidences suggested that EMT, a wellcharacterized embryological process, had been identified to play a critical role in cancer metastasis [13]. In order to examine the role of CAFs-derived ROCK1 in mediating EMT, Hep2 cells were co-cultured with NFs/ROCK1 (Hep2/NFs/ROCK1 cells), NFs/vector (Hep2/NFs/vector cells), CAFs/si-ROCK1 (Hep2/CAFs/si-ROCK1 cells) or CAFs/parental (Hep2/CAFs/parental cells) in a previously described co-culture system respectively. At the appropriate time point, Hep2 cells were either assayed by way of the IF and Western Blots. As Fig. 3A C and 3D showed that the expression of E-cadherin was markedly decreased, while the expressions of N-cadherin, Slug and Vimentin were observably increased in Hep2/ NFs/ROCK1 cells when compared to Hep2/NFs/vector cells (*P < 0.05). In contrast, Fig. 3B and E F showed that expression of E-cadherin was markedly increased, while the expressions of N-cadherin, Slug and Vimentin were markedly decreased in Hep2/CAFs/si-ROCK1 cells when compared to Hep2/CAFs/si-NC cells (*P < 0.05). All data indicated CAFs with high expression of ROCK1 promoted EMT in LSCC.

Signal molecules of JAK2, STAT3 and ERK1/2 were of great importance in LSCC metastasis
Tumor metastasis is a multifactorial process [14], signal molecules might play a crucial role in LSCC metastasis. To interrogate the role of signal molecules in CAFsinduced ROCK1 mediating EMT to promote LSCC metastasis. As Fig. 4A and B showed that the expressions of p-JAK2, p-STAT3 and p-ERK1/2 were markedly increased in Hep2/NFs/ROCK1 cells when compared to Hep2/NFs/vector cells (*P < 0.05, **P < 0.01). Consistent with these observations, Fig. 4C and D showed that the expressions of p-JAK2, p-STAT3 and p-ERK1/2 were markedly decreased in Hep2/CAFs/si-ROCK1 cells when compared to Hep2/CAFs/parental cells (**P < 0.01). All data just indicated that signal molecules of JAK2, STAT3 and ERK1/2 were of great importance in LSCC metastasis, while the upstream and downstream relationship between them were not clear.
These observations match what the expression of E-cadherin was markedly increased, while the expressions of N-cadherin, Slug and Vimentin were markedly decreased (*P < 0.05, **P < 0.01, Fig. 7G H). These data indicated CAFs-derived ROCK1 mediated EMT to aggrandize the movement, migration and invasion ability of LSCC via activating ROCK1/JAK2/STAT3/ERK1/2 axis.

Discussion
Interactions between tumor and tumor microenvironment is essential for tumor metastasis. Microenvironment includes tumor and stromal cells [5]. CAFs, the activated fibroblasts in tumor stroma, is important modifiers of tumor progression. CAFs are known to be a heterogeneous group of cells, mainly derives from normal epithelial cells, mesenchymal fibroblasts, bone marrow mesenchymal stem cells, vascular beds, or epithelium. In comparison with NFs, the phenotype and function were significantly different. CAFs can express specific molecules, α-SMA, FAP, FSP1/S100A4, NG2 and PDGF-receptor, et al. [19]. CAFs, which act as a "stromal response", plays a prominent role in tumor microenvironment [20]. Therefore, a better understanding with the molecule mechanism of metastasis is of great significance for developing effective clinical prevention programs and new targeted therapies for LSCC.
First and foremost, we isolated CAFs, CPFs and NFs from laryngeal cancer tissues, para-laryngeal cancer tissues and normal tissues. The specific markers were determined by Western Blot, CAFs highly expressed of α-SMA, FAP, NG2 and FSP1 and enhanced LSCC metastasis in vitro and vivo. Interestingly, we found ROCK1 was highly expressed in CAFs. This might indicate that CAFs with the high expression of ROCK1 played a core effect in enhancing movement, migration and invasion of LSCC.
It was well known that ROCK1/2 played a role in regulating cell cycle, proliferation and mitosis. Activation of ROCK1 could induce cell proliferation, inversely, inhibition ROCK1 could reduce migration [9]. Stadler et al. also found that CAFs could promote colorectal cancer metastasis through ROCK1 [10]. Here, we co-cultured CAFs and NFs with laryngeal cancer Hep2 cell line respectively. We found ROCK1 was highly expressed in CAFs, and ROCK1 might promote LSCC metastasis in vivo and vitro. To further explore CAFs-derived ROCK1 promoted LSCC metastasis, we down-regulated ROCK1 expression in CAFs and up-regulated ROCK1 expression in NFs respectively, the results showed that the expressions of CAFs associative phenotypes markedly decreased, and the ability of movement, migration and invasion was also obviously reduced in Hep2 cells co-cultured with deprivation of ROCK1 in CAFs. On the contrary, the expressions of CAFs associative phenotypes markedly increased, and the ability of movement, migration and invasion was also obviously increased in Hep2/ NFs/ROCK1 cells. All that indicated that CAFs-derived ROCK1 authentically promoted LSCC metastasis.
Accumulating evidence have indicated EMT, characterized by loss of polarity of epithelial cells, is important modifiers of tumor progression. The epithelial markers (E-cadherin, β-catenin) acquired the mesenchymal markers (N-cadherin, vimentin, ZEB2) to form mesenchymal cells thus these transformed epithelial cells acquire fibroblast like properties and exhibit reduced cell-cell adhesion and increased motility [21]. In order to examine the role of CAF-derived ROCK1 in contemplating EMT in LSCC, IF and Western Blot were applied to inspect the expressions of EMT associated phenotype markers in Hep2/NFs/ROCK1, Hep2/NFs/vector, Hep2/CAFs/ parental and Hep2/CAFs/si-ROCK1 cells respectively. Results showed that epithelial phenotype marker (E-cadherin) levels were markedly decreased, and mesenchymal phenotype markers (N-cadherin, Slug and Vimentin) levels were markedly increased in Hep2/NFs/ROCK1 cells. The results were reversed in Hep2/CAFs/si-ROCK1 cells, which indicated that CAFs-derived ROCK1 mediated EMT to promote LSCC metastasis.
Furthermore, to further explore the molecular mechanism of ROCK1 mediated EMT to accelerate LSCC metastasis. Signal molecules play a crucial role in LSCC metastasis. Signal molecular pathway of JAK2 played an important role in cancer metastasis of cancer. The expression of STAT3 and phosphorylated STAT3 increased in gastric cancer in comparison with normal stomach [22]. The activation of STAT3 was positive in early gastric cancer, poorly differentiated adenocarcinoma and metastatic lymph node tissues [24]. ERK1/2 was generally located in cytoplasm, but when it was activated, it could across the nuclear membrane and transfer to nucleus [24]. ERK1/2 could induce cyclin D1 expression and accelerated cell mitosis to promote cell proliferation [26].
In summary, ROCK1 was highly expressed in laryngeal CAFs, CAFs-derived ROCK1 mediated EMT via activating JAK2/STAT3/ERK1/2 signal pathway to promote LSCC metastasis. These findings exhibited that stromal ROCK1 could enhance LSCC initiation and progression, and targeting ROCK1 might provide a potential treatment stratagem for LSCC.

Cell cultures
Human laryngeal cancer Hep2 cell lines were preserved in our laboratory. Cells were repeatedly cultured in DMEM with 10% FBS containing 100 IU/ml penicillin and 100 IU/ml streptomycin in a humidified cell incubator with an atmosphere of 5% CO 2 at 37 °C. Laryngeal cancer tissue was cut from the edge of the tumor, para-cancer tissue was taken from 4 to 5 cm away from the edge of the tumor and adjacent non-tumor tissues was derived from the normal tissue of the larynx. Tumor tissues, adjacent tissues and adjacent non-tumor tissues were mechanically minced into small pieces (1-1.5 mm 3 ) and seeded onto 10 cm petri dishes in DMEM with 10% FBS containing 100 IU/ml penicillin and 100 IU/ml streptomycin. A homogeneous group of fibroblasts, produced after 7-14 days of culture, would be used in the experiments.

Immunofluorescent staining
5 × 10 4 cells were seeded into slides (Millipore, MA, USA) and fixed with 4% paraformaldehyde (PFA) for 30 min. Slides were rinsed with PBS for 3 times, blocking slides with 5% BSA for 1 h at room temperature and incubating slides with primary antibodies at 4 °C overnight. Next day, rinsing slides with PBS for 3 times and incubating slides with secondary antibodies in the dark at room temperature for 1 h. Antibodies included anti-ROCK1, anti-E-cad and anti-Vimentin, Alexa Fluor® 488 goat and Alexa Fluor® 555 which were purchased from Abcam, Cambridge, MA, USA were used to IF staining. Visualizing nuclei with DAPI in the dark for 5 min. Analyzing slides by fluorescent microscopy (10x).

Wound healing assay
Inoculating 1 × 10 6 cells into 6-well plates and scratching cells by using 20 µl pipette tips after overnight