PCDH9 target RAC1/ROS-NADPH-oxidase complex with the assistance of Pyk2 interaction through Cyclin D1 tracking

Background: Melanoma has dramatically increased during last 30 years with low ve-year survival and prognosis rate. Methods: Melanoma cells (A375 and G361) were chosen as in vitro model. The interference and overexpression of PCDH9 were infected by lentivirus. The effects of PCDH9 on melanoma cells were assessed in terms of alteration of PCDH9 such as cell viability, apoptosis, cell cycle and wound-healing assay. Moreover, expressions of PCDH9 with other genes (MMP2, MMP9, Pyk2 Cyclin D1, RAC1 and FAK) were also assessed by PCR and western blotting, immunohistochemical analysis. The protein complex immunoprecipitation was operated for protein-protein interaction (PPI) of PCDH9. Originlab 2020 was used for statistical analysis. Results: Melanoma cells treated with PCDH9 showed inuence melanoma as similar function as Hace1 that regulate RAC1/ROS-dependent NADPH oxidase complexes through targeting complex-bound RAC1 with the help of Pyk2, while RAC1 directly inuence RAC1/ROS-NADPH-oxidase complex to induce metalloproteinases (MMPs), that are involved in cell migration and proliferation. Moreover, PCDH9 could inuence the expressions of MMP2, MMP9, Pyk2, Cyclin D1 and RAC1, except FAK, decreased cell viability, cell migration and increased apoptosis, but did not exert signicant effects on cell cycle. Besides, the immunohistochemical results exhibited lower PCDH9 expression in malignant melanoma samples than benign naevus tissue or/and normal skin, and PCDH9 was expressed in the cytoplasm rather than nuclei. The results of immunoprecipitation demonstrated that PCDH9 bind with RAC1 and Pyk2, suggesting the regulation of PCDH9 to melanoma progression. Conclusion: Our results suggest that PCDH9 and RAC1 could predict for potential biomarkers prognosis of malignant interestingly, with Pyk2’s and


Background
Cutaneous melanoma, a type of skin cancer, develops in melanocytes. According to data from the National Cancer Institute, the incidence of melanoma has rocketed over the last 30 years, even the Cancer Research UK estimated that melanoma has an incidence rate of 3.1/10000 and a mortality rate of 0.8/10000 worldwide. With lower ve-year survival rate of metastatic melanoma (MM), the prediction appeared essential. Albeit numbers of validated biomarkers have been con rmed in melanoma, Chinese population has been demonstrated to gain different subtypes of melanoma from the Caucasian population [1], therefore the already-being biomarkers haven't predicted well in prognosis of Chinese melanoma patients. Hence, a novel biomarker, feasible is urgently needed.
In order to gure out which genes could be relevant to Chinese population, bioinformatic technologies have been applied that PCDH9 have been chosen, while RAC1 always play opposite role was found in our natural bioproduct against melanoma study [2,3]. PCDH9 belongs to protocadherins, one of the largest subfamilies of calcium-dependent adhesion proteins [4]. Previous studies have demonstrated that the overexpression of PCDH9 could suppress different cancers [5,6] and tumor cells by arresting cell cycle at G0/G1 phase [7,8], it also can predict prognosis of MM patients [5,9]. Oppositely, RAC1 has been believed to affect actin cytoskeleton and regulate cell adhesion, motility, cycle, apoptosis, membrane tra cking and even transcription [10]. Both of them play important roles in tumor biology by modulating cell processes [11,12]. Furtherly, activities of RAC1 have been reported of involving different stages of oncogenesis such as initiation, progression, invasion and metastasis [13], even it was ranked as the third most frequently occurring mutation in melanoma induced by UV [14,15]. Additionally, some reported ROS's involving in tumor cell migration and invasion [16,17] and a key component of NAPDH-oxidasecomplex formed by RAC1, one of the major enzymatic sources of ROS in various tissues [18]. While it's reported that ROS/RAC1-NADPH-oxidase complex are involved in endothelial migration by mediation of angiotensin-1 (Ang-1) and vascular endothelial growth factor (VEGF) [19,20]. As known, the endothelial migration is essential for tumor cell invasion, where ROS/RAC1-NADPH-oxidase complex induce expression of matrix metalloproteinases (MMPs) after growth factor and tumor promoter stimulation [21,22]. Tumor-associated MMPs family has been studied for over half a century and are known as mediators in some cell processes [23]. Among them MMP2 has been believed to act as a pro-tumorigenic and pro-metastatic factor in different cancers besides melanoma [24], while higher MMP9 levels have been found in melanoma patients than healthy population that made it to be considered as a promising marker for melanoma [25]. Additionally, previous studies have found that MMP9 is activated during invasion of melanoma cells [25,26]. Therefore, we have chosen MMP2 and MMP9 as tumor metastasis indicators to explore the mechanism of how PCDH9 and RAC1 act on melanoma cells.
Proline-rich tyrosine kinase 2 (Pyk2) is a paralogue to focal adhesion kinase (FAK) in vertebrates [27] and, in absence of FAK, can execute the same therapeutic effects [28][29][30]. Increasing Pyk2 and FAK expression could trigger neoplastic activation and promote invasiveness with metastasis of different cancers, highlighting that Pyk2 is a potential therapeutic target for cancer [31,32]. Some target molecules can in uence, either increasing or decreasing, the function of Pyk2 by interaction [30]. Another key molecule of melanoma is the cell cycle regulator Cyclin D1 that has been demonstrated as an oncogene in cutaneous melanoma [33], and it could affect tumorigenesis via nuclear tra cking by multiple mechanisms [34]. Therefore, the main objective of this investigation was to clarify the role of PCDH9 and RAC1 in melanoma. After proceeding overexpression and interference of PCDH9 by GV358-PCDH9 lentivirus and GV358-SiRNA lentivirus in melanoma cell lines (A375 and G361), certain assays (cell viability, apoptosis and cell cycle assays, PCR, western blot analysis) and evaluation of PCDH9 expression by immunohistochemical (IHC) [35] in patients' skin biopsy were performed to explore the alterationin uence of PCDH9 to melanoma cells.
The expression of MMP2 is a prognostic marker for MM patients that could independently predict patient survival [36], its expression has been related to the poorer survival of melanoma patients [36], and the varieties of PCDH9 expression in melanoma cells were in accordance with this result. MMP9 could block the EGFR-ERK/AKT signaling pathway to suppress MM's angiogenesis and it has been considered as a key angiogenic factor [37]. The results of MMP9 expression in this study exhibited a negative correlation with PCDH9, as well as a positive correlation with RAC1. According to previous studies the role of Cyclin D1 in melanoma cells is debatable. Some studies suggested that the upregulated expression of Cyclin D1 enhances metastases [38], while others found a downregulation of Cyclin D1 in metastatic tumor and upregulation in tumor progression [39]. Even some histopathological studies showed that there is no signi cant difference in the expression of Cyclin D1 [40]. In the present study, we found that the expression of Cyclin D1 increased along with the overexpression of PCDH9, which is negatively correlated with MMP2 and MMP9.

Effects of overexpressed PCDH9 on cell viability and apoptosis
The overexpression of PCDH9 reduced the proliferation of melanoma cells. Overexpressed PCDH9 groups showed indeed a lower viability than control groups (Fig. 3A, B). As time passed, the viability of melanoma cells trended to stabilize, but PCDH9 overexpressed groups had less viable cells than control groups in different durations (Fig. 3C, D), and the differences between PCDH9 and control groups were signi cant (24, 48, 72 and 96 hours) (Fig. 3A, B).
The expression of PCDH9 signi cantly affected apoptosis of melanoma cells as well. Overexpression increased apoptosis (p < 0.05) (Fig. 3E), while interference decreased apoptosis (p < 0.05) in A375, a similar phenomenon happened in G361 with more modest effects (Fig. 3F).
Recent studies have found low PCDH9 expression in various cancer types [9,41,42]. The above phenomenon agreed with our investigation where alterations of PCDH9 could in uence the viability of melanoma cells. Meanwhile, the percentage of apoptosis cells was affected by the change in PCDH9.
2.3. Effects of overexpressed PCDH9 on cell cycle and wound healing Regarding the cell cycle arrest, although we found differences between control and interfered PCDH9 groups in G361 cells, there was no discrepancy between overexpressed PCDH9 or interfered PCDH9 groups and other groups (blank and control groups) in both cell lines (A375 and G361) ( Fig. 4A-D). Therefore, the changes in PCDH9 did not affect melanoma cell regulation and the cell cycle regulator Cyclin D1 modulation revealed no signi cant effect on cell cycle. Previous investigations of hepatocellular carcinoma (HCC) found that PCDH9 suppresses HCC cells by inducing cell cycle arrest at G0/G1 phase [7]. This result suggests that PCDH9 and Cyclin D1 could affect melanoma cell through other mechanisms.
With respect to cell migration, after quantifying the scratched boundary by Image J (Fig. 4E, F), the results revealed that the relative density decreased with the duration of cell culture in blank and control groups (p < 0.001), while the relative wound density did not change in a marked way in overexpressed PCDH9 groups (p > 0.05) (Fig. 4G, H). The relative wound density of scratched boundary was signi cantly different in overexpressed PCDH9 groups compared with blank and control groups after 24 hours and 48 hours (p < 0.001) (Fig. 4I, J). This phenomenon is similar to the correlation found between PCDH9 and other tumor cells (HCC) [42,43]. To conclude, overexpressed PCDH9 could signi cantly inhibit melanoma cell migration, by decreasing cell viability and increasing apoptosis.

Interaction of PCDH9 with RAC1 and Pyk2
The interaction between Pyk2 and other proteins, as well as targeted molecules, could affect the function of Pyk2 by interaction as previously demonstrated [2,38]. In the current study, expression of Pyk2 in melanoma cells was signi cantly affected by the alteration of PCDH9 (Fig. 1B, C) (Fig. 2B). Therefore, we explored the interaction between "PCDH9 and Pyk2", "PCDH9 and RAC1" by Co-IP. "PCDH9 and Pyk2", "PCDH9 and RAC1" were co-immunoprecipitated with PCDH9-FLAG in both cell lines (A375-PCDH9-Flag and G361-PCDH9-Flag), while there was no protein in negative control (Supplementary Figure S1). It was demonstrated that PCDH9 interacted with the protein Pyk2 and the protein RAC1. We assumed in accordance with previous investigations that PCDH9 may serve the function as Hace1 that regulate RAC1/ROS-dependent NADPH oxidase complexes through similar mechanism [44], but Hace1 target complex-bound RAC1 directly, while PCDH9 need the help of Pyk2 [44].
2.5. PCDH9 protein expressed differences in normal skin, pigmented nevus and melanoma tissue tested by IHC stains Immunohistochemical results showed that the positive percentage of PCDH9 expression was lower in human melanoma tissue than in normal skin or/and pigmented nevus tissue; additionally, PCDH9 was mainly expressed in the cytoplasm, while a small amount was expressed in the nuclei. The positive percentage of PCDH9 was expressed in normal skin or/ and pigmented nevus tissue, while only 23.3% (7/30) in melanoma tissue, which was lower than non-tumor tissue ( Table 1, Fig. 5).  [44]. High DNA damage susceptibility tend to Carcinogenesis. Meanwhile, previous exploration of how Cyclin D1 affects tumorigenesis speculated that it could play a role in nuclear tra cking rather than overexpressing itself [34,44]. PCDH9 might be tra cked from nuclei to cytoplasm by the assistance of Cyclin D1.

Conclusion
According to previous studies, MMP2 and MMP9 could represent a biomarker of malignant melanoma [24,25], while Pyk2 and FAK expression could trigger neoplastic activation and promote invasiveness with metastasis of different cancers [31,32]. We explored and performed a series of investigations, including cell viability assay, apoptosis assay, PCR, Co-IP, of interference and overexpression of PCDH9 by GV358-PCDH9 lentivirus and GV358-SiRNA lentivirus respectively in melanoma A375 and G361 cell lines.
According to our results, overexpressed PCDH9 triggered upregulated expressions of Pyk2 and Cyclin D1, while RAC1, MMP1 and MMP9 were downregulated, and apoptosis of melanoma cell lines was promoted. Interfered PCDH9 induced downregulation of Pyk2 and Cyclin D1, while RAC1, MMP1 and MMP9 were upregulated and decreased apoptosis. However, the alteration of PCDH9 expression did not affect melanoma cell regulation in a signi cant manner. It has been suggested that PCDH9 could affect melanoma cells by different mechanisms. Specimen investigations of IHC assay exhibited lower PCDH9 expression in malignant melanoma specimens than in benign naervus tissue or/and normal skin, furtherly proving the indicative function of PCDH9; our study revealed that PCDH9 was mainly expressed in the cytoplasm rather than in nuclei. The tra cking of PCDH9 might be through Cyclin D1, and Cyclin D1 could play a more important role in tra cking than in cell regulation. Co-IP demonstrated the interaction between "PCDH9 and Pyk2", "PCDH9 and RAC1".

Sample collection and Preparation
Tissues (human normal skin tissue (n = 45), human pigmented nevus (n = 30) and primary malignant melanoma tissue (n = 30)) were collected and prepared as para n specimens until use. These tissues were ethically acquired from the outpatient clinic of the A liated Hospital of Guangdong Medical University with Chinese population (Han people) with personal identi ers redacted.

Transfection
Melanoma cells (A375 and G361) were seeded in 6 well plates (1 × 10 5 cell/well) the day before transfection and were transfected by two types of lentiviruses (siRNA and PCDH9). Control group were transfected with the empty vector. Blank group were treated with transfection reagent only. Transfection was performed using Genechem Transfection Reagent (Shanghai, China), according to the manufacturer's instructions. Seventy-two hours after transfection, cells were observed by uorescent inverted microscope for being screened by puromycin. The e ciency of PCDH9 alteration in melanoma cells was detected by Real-Time PCR, Western blot analysis.

Cell viability by Cell Counting Kit-8 (CCK-8)
Cells were seeded into 96-well plates at a density of 2 × 10 5 cells/well and treated by non-transfected plasmid, transfected with empty plasmid and transfected with PCDH9 overexpressed plasmid as explained above. Apoptosis was analyzed by cytometric analysis, using FITC Annexin V Apoptosis Detection Kit (company name). Cells were seeded in 6-well plate at a density of 1×10 6 cells/well. Brie y, cells were treated with Camptohtecin stock solution (1μL in DMSO) and incubated for 5 hours at 37°C. After that, the cells were centrifuged (1000 rpm for 10 min), washed twice with cold PBS and resuspended in 1X Binding 5 μL of FITC Annexin V and 5 μL PI (Bio-Rad) were added to cell suspension and incubated, protected from light, for 15 min at room temperature. Finally, samples were analyzed using the BD FACS Canto II ow cytometer.

Cell cycle assay
Cell cycle was analyzed using the ow apoptosis. Brie y, Cells were seeded in 6-well plates at a density of 1×10 6 cells/well. Cells were then detached, centrifuged at (1000 rpm for 10 min) and then vortexed with 5 mL cold 75% ethanol. Cells were incubated at -20°C for 2 hours, washed twice with PBS to remove ethanol. Cells were resuspended in 0.5 mL PI/RNase staining Buffer (Bio-Rad) for 15 min at room temperature, samples were analyzed using the BD FACS Canto II ow cytometer.

Wound-healing assay
Cells were seeded into 6-well plates at a density of 1.5×105 cells/well until reach on con uency (80-100 %) and scratched by a sterile 10 μL pipette tip. Cells were washed twice with PBS; then complete medium was added to allow cells moving into the gap, photographed by using an inverted microscope DMI3000B (Leica, Germany) at 0h, 24h and 48h. The image J (Maryland, USA) was used to measure the wound space. Migration rate was calculated as the proportion of initial scratch distant of each sample and the mean distance between both borderline remaining cell free after migration.

Quantitative Real-Time PCR analysis
Each frozen pellet of melanoma cells (A375 and G361), treated in different experimental conditions were homogenized in a lysis buffer. The para n specimens were depara nizated included two 100% xylene changes (xylene I-10 min, xylen II-10 min) followed by rehydration with a graded series of ethanol (anhydrous ethanol I-5min, anhydrous ethanol II-5 min, 95% ethanol-5 min, 85%-5 min, 75%-5 min) and then rinsed under distilled running water for 3-5 min. Antigen retrieval consisted of a two-minute incubation of slides in citric acid retrieval solution heated to 98°C with a commercial steamer following a cool down step to room temperature (cold water and ice pack were added), slides were transferred into a wet box and were then rinsed three times by PBS.
After protein blocking, primary antibodies (1:200) were incubated at 4°C overnight. After being in room temperature for 30 min, the slides were washed 3 times for 3 min each by PBS. After removing PBS and protein blocking, secondary antibodies (1:1000) were added at room temperature for 1 h. The slides were then washed 3 times for 3 min each by PBS. After removing PBS, 1 drop of prepared DAB solution (1 ml A:1 drop B:1 drop C) for DAB staining was added and the slides were observed under microscope. After being rinsed in running water for 10 min, hematoxylin was added for 1 min and then the slides were washed by water for 5 min. The slides were then dehydrated in a series of ethanol (75%, 85%, 95%, 100%) and 100% xylene changes, and mounted with a coverslip with dry neutral resin.

Protein complex immunoprecipitation (Co-IP) assay
PCDH9-Flag was overexpressed in A375 and G361 cell lines by Lentiviruses Infection and cultured by DMEM with 10% FBS in 10 cm petri dishes, as mentioned above. After discarding the cell media and washing once by iced PBS, the cells were lysed in a buffer containing TBS with 1% Triton X-100 and PMSF, 1 mL in each dish. The solution was collected in 1.5 mL Eppendorf tubes and after centrifugation (1000 rpm for 15 min), the supernatant was collected. For the input sample preparation, 80 μL of protein solution with 20 μL (5×loading buffer) were boiled. The leftover solution was incubated with 2 μL of anti-Flag antibody at 4°C overnight. Protein G Agarose beads (40 μL each tube) were added and the solution was shaken for 3 h at 4 °C. Then after being centrifuged (2500 rpm for 5 min), the supernatant was removed. The sediments were washed ve times by lysis buffer with PMSF (1mL each time) and after the addition of loading buffer (40 μL) were boiled for 10 min at 100°C. Control group was incubated with mouse IgG.

Statistical Analysis
Data are presented as the mean ± SD from at least 3 independent experiments. Statistical comparisons between two groups were made using Student's t tests. Differences among three or more groups were compared by one-way ANOVA followed by least signi cant difference post hoc tests (Originlab 2020, Northampton, MA, USA), p<0.05 was considered as statistically signi cant, while p<0.01 was considered as highly statistically signi cant.

Declarations
Ethics approval and consent to participate: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of the A liated Hospital of Guangdong Medical University. Written informed consent was obtained from individual or guardian participants.
Consent for publication: Availability of data and material.
Availability of data and materials: All data generated or analyzed during this study are included in this published article [and its supplementary information les.