Abstract
Background/Aim: Fascin, an actin-bundling protein, plays an essential role in cancer metastasis. The Hippo pathway is critical for carcinogenesis and cancer stem cell self-renewal. Mammalian STE20-like kinase (MST) is a core component of the Hippo pathway. However, whether fascin and MST2 affect melanoma remain largely unknown. This study aimed to investigate the role of fascin and MST2 in melanoma development. Materials and Methods: Surgically excised skin melanomas and the adjacent non-tumorous skin tissue from 30 cases were analyzed using immunohistochemistry for fascin and MST2. The melanoma cell line WM793 was employed for fascin and MST2 knock-down followed by western blotting, and melanoma xenografting in BALB/c mice. Results: Immunohistochemistry revealed increased expression of fascin and decreased expression of MST2 in melanoma. The reverse correlation of fascin and MST2 was statistically significant. Fascin siRNA upregulated MST2 expression; however, MST2 siRNA did not significantly affect fascin expression in the WM793. WM793 xenografting followed by fascin knock-down inhibited tumor growth significantly in the animal study. Conclusion: Fascin is a regulator of the Hippo pathway and plays an important role in melanoma development. Therefore, fascin could be a potential therapeutic target for melanoma.
Fascin (actin-bundling protein fascin-1) plays an important role in cell migration and invasion, as well as in tumor metastasis, by crosslinking the actin forming the cytoskeleton (1, 2), which stabilizes the invasive filopodia and is assumed to confer metastatic potential (3-5). Although there have been many studies on the use of fascin as a prognostic marker in epithelial cancers including pancreatic (6), colorectal (7, 8), and breast (9, 10) cancer, the association between fascin and tumorigenesis has not been well established in malignant melanoma due to the lack of relevant studies.
Mammalian STE20-like protein kinase 1 (MST1) and Mammalian STE20-like protein kinase 2 (MST2) are the closest mammalian homologs of the Drosophila Hippo kinase. The core components of the Hippo pathway are serine–threonine kinases, MST1/serine-threonine kinase 4 and MST2/serine-threonine kinase 3 (STK3), large tumor suppressor kinase 1 (LATS1) and 2 (LATS2), and scaffold protein Salvador and cofactor Mob1.
The functional loss of Hippo protein leads to a massive overgrowth of tumor cells due to the acceleration of cell cycle progression and the failure of developmental apoptosis (11-13). MST2 is involved in the functional loss of Hippo protein (13). However, the regulatory mechanism of Hippo kinase activation remains unclear.
Graves et al. (14) reported that MST2 over-expression induces apoptosis in various transformed cell lines and Zhou et al. (15) showed that MST1 and MST2 inhibit the progression of hepatocellular carcinoma by inactivating Yap1. In addition, it has been shown that the Hippo pathway is regulated by actin cytoskeleton and Hippo signaling is blocked by actin polymerization (16). MST2 phosphorylates LATS1, and MST2 homodimerization is required for its activation (17).
In mammals, TAZ (transcriptional coactivator with PDZ-binding motif) plays a key role in the development of lung cancer, breast cancer, and hepatocellular carcinoma (18-21). TAZ is highly expressed in malignant melanoma, and its knock-down inhibits tumor growth which indicates that TAZ plays a role in the carcinogenesis of malignant melanoma (22).
Malignant melanoma is well known for its invasive growth and metastatic potential and shows extremely poor prognosis due to the lack of effective therapies. Although it occurs rarely in Asian populations, recently an increasing number of people have been affected, which may be attributed to aging, increased outdoor activities, and the depletion of the ozone layer by environmental pollution (23-25). However, a limited number of studies have covered the pathophysiology of malignant melanoma, and there has been only a single study on the association between fascin and the Hippo pathway, in which fascin-MST2-TAZ signaling was identified using two melanoma cell lines (26).
Hence, this study aimed to discover factors involved in the development and progression of malignant melanoma and provide fundamental data to establish a new therapeutic strategy. The role of fascin, a novel regulator of MST2-LATS-TAZ signaling, was investigated study by immunohistochemical analysis, in vitro assays using knock-down of fascin and MST2, and in vivo studies examining tumor development and progression using fascin knock-down after xenografting of melanoma cell line WM793 into BALB/c mice.
Materials and Methods
Reagents. Rabbit anti-fascin antibody (Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-MST2 antibody (Thermo Fisher Scientific, Waltham, MA, USA) were used as primary antibodies for immunohistochemical staining. For RNAi studies, fascin siRNA (5’-3’ sense GCCUGAAGAAGAAGCAGAU) (Dharmacon, Lafayette, CO, USA) and MST2 siRNA (5’-3’ sense CAAGAGUCAUGA AAAUUGUtt) (Invitrogen, Carlsbad, CA, USA) were used. For in vivo delivery, fascin siRNAs were encapsulated in liposomes and injected intravenously through the tail vein. Anti-β-actin (AC-15) was obtained from Sigma-Aldrich. Horseradish peroxidase-conjugated secondary antibodies (sc-2054, 2055) used for western blots were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
Clinical samples. Clinical samples diagnosed as primary invasive melanoma between 2010 and 2019 at the Chosun University Hospital were collected. From these, 30 samples which contained a substantial amount of both primary tumor and adjacent nonneoplastic skin tissue were randomly selected regardless of sex, age, and location. This study was approved by the institutional review board (Permission number: CHOSUN 2020-11-033).
Cell culture. Melanoma cell line WM793 (ESTDAB Melanoma Cell Bank, Tübingen, Germany) was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin (Calbiochem, San Diego, CA, USA) at 37°C.
Animals. Six-week-old female BALB/c mice (Samtako Bio Korea, Gyeonggi-do, Republic of Korea) were used. Feed and water were supplied ad libitum under controlled conditions: 22-26°C temperature, 50% humidity, and a 12/12 h light/dark cycle. The BALB/c mice were bred for two weeks under free-ranging conditions after the inoculation of WM793 cell line into the dorsal subcutaneous tissue. At 14 days after inoculation, 10 μl/g of 1 mg/ml fascin siRNA was injected into three mice, and the same volume of saline was injected into the other three mice to be used as controls, via the tail vein using a 29 G needle. After 72 h, the animals were sacrificed, and the tumor tissues were excised, measured, and fixed in 10% neutral buffered formalin.
Immunohistochemical staining. Paraffin sections of 4 μm thickness were obtained, stained using the BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ, USA) and incubated with antibodies at a dilution of 1:200 for fascin and 1:2,000 for MST2. Tris-buffered saline was used instead of primary antibodies for negative controls, and counter staining was performed using Mayer’s hematoxylin.
In the case of staining for both fascin and MST2, intracytoplasmic brown staining was considered positive and classified into 1+ (weakly positive), 2+ (moderately positive), and 3+ (strong positive) according to the staining intensity.
RNA interference and western blot analysis. WM793 cells were transfected with siRNA oligonucleotides using an Amaxa™ Transfection System (Lonza Bioscience, Basel, Switzerland) and grown for 72 h prior to exposure to neutral pH medium. Cell extracts were prepared for immunoblotting by homogenization in ice-cold buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 5% glycerol, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10 mM Na3VO4, complete Mini protease inhibitor cocktail tablet (Roche, Basel, Switzerland), and 2 mM PMSF. After centrifugation at 12,000 × g for 10 min, aliquots of the supernatants containing 50 μg protein were subjected to SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The blot was incubated with indicated antibodies overnight and visualized using the ECL-plus western blotting detection system from Amersham Biosciences (Little Chalfont, UK).
Histopathologic examination. The tumor tissues excised from the animals were fixed in 10% neutral buffered formalin for 24 h, and paraffin blocks were made. Glass slide specimens were sectioned from the paraffin blocks at 4 μm thickness and examined under a light microscope after conventional hematoxylin and eosin staining.
Statistical analysis. All numerical data are presented as the mean±SE of at least three independent experiments. Student’s t-test was used for simple comparisons. p<0.05 was considered to indicate a statistically significant difference.
Results
Immunohistochemical analysis of clinical samples. Immunohistochemical evaluation of nonneoplastic skin tissue adjacent to the melanoma was possible in 21 cases and revealed weak positive staining (1+) for fascin and strong positive staining (3+) for MST2 in all.
Immunohistochemical analysis of the melanomas, however, revealed weak, moderate, and strong staining for fascin in one, 15, and 14 cases, respectively (Figure 1), and negative, weak, and moderately positive staining for MST2 in two, 17, and 11 cases, respectively (Figure 2). Fascin expression showed a negative correlation with MST2 expression (p=0.0001) (Figure 3). However, fascin and MST2 staining intensities were not associated with tumor invasiveness.
Immunohistochemical staining for fascin. A: Nests of early stage melanoma shows diffuse strong positive cytoplasmic immunoreactivity (arrow) in the tumor focus. However, the adjacent non-tumorous epidermis (asterisk) shows weakly positive immunoreactivity confined to the basal layer. B: Deeply invasive advanced melanoma shows diffuse strong positive immunoreactivity (arrows). Scale bars measure 200 μm (A) and 100 μm (B).
Immunohistochemical staining for MST2. A: Early stage melanoma shows weakly to moderately positive cytoplasmic immunoreactivity (arrows) in the tumor area. The adjacent non-tumorous area (asterisk) shows moderately positive immunoreactivity in the full-layer of the epidermis. B: More advanced melanoma shows weakly to moderately positive immunoreactivity (arrows) in the tumor area. However, the adjacent non-tumorous epidermis (asterisk) shows strong positive immunoreactivity in the full-layer of the epidermis. Scale bars measure 200 μm (A) and 100 μm (B).
Comparison of Fascin and MST2 immunoreactivity in each corresponding case. Histogram demonstrates statistically significant negative correlation between fascin and MST2 immunoexpression.
Fascin knock-down effect. Western immunoblotting following knock-down of fascin by transfection with fascin siRNA oligonucleotides in WM793 cells showed a significant increase in the levels of MST2 compared to the control group (Figure 4).
Immunoblotting for Fascin knock-down. Fascin siRNA oligonucleotides transfection increases MST2 expression in WM793 melanoma cell line.
MST2 knock-down effect. Western immunoblotting following knock-down of fascin by transfection with MST2 siRNA oligonucleotides in WM793 cells showed no significant effect on the levels of fascin compared to the control group (Figure 5).
Immunoblotting for MST2 knock-down. MST2 siRNA oligonucleotides transfection does not increase fascin expression in WM793 melanoma cell line.
Fascin siRNA-treated animals. Following the establishment of xenograft by inoculation of WM793 cells, fascin was knocked down by transfection with liposome-encapsulated fascin siRNA oligonucleotides. After 72 h, the BALB/c mice were sacrificed, and the tumor tissues were excised.
The mean largest diameter was 1.33 cm in the group treated with fascin siRNA and 1.93 cm in the control group, which showed a significant difference between the groups (p<0.05) (Figure 6).
BALB/c mice xenograft experiment. Excised tumor sizes of the liposome encapsulated fascin siRNA injection group are smaller than those of the control group. Histogram shows statistically significant decrement of tumor size in the fascin knock-down group (p<0.05).
Histopathologic findings of the animals. Micro-focal necrosis was observed in one case in the group treated with fascin siRNA (Figure 7A), whereas severe central tumor necrosis was observed in all three cases in the control group (Figure 7B).
Histopathologic findings of the xenograft experiment. A: Fascin siRNA treated group shows micro-foci of tumor necrosis (asterisks) in the tumor center (left). Higher magnification shows malignant melanoma with melanin pigmentation (right). B: Control group shows massive tumor necrosis (asterisks) in the tumor center (left). Higher magnification shows malignant melanoma with melanin pigmentation (right). Hematoxylineosin staining. Scale bars measure 200 μm (Lt.) and 50 μm (Rt.).
Discussion
Cutaneous malignant melanoma occurs from melanocytes in the basal layer of the epidermis. Although malignant melanoma can occur in any site where melanocytes exist, it occurs most commonly in the skin. Malignant melanoma is defined as malignant transformation of melanocytes or pre-existing nevus cells and has a very high malignant potential. Superficial spreading melanoma accounts for 70% of all melanoma cases in the West including the United States, whereas acral lentiginous melanoma is the most common type accounting for 52–80% of all melanoma cases in the East including Korea (24, 25).
Moreover, it was rare and of little interest in the East compared to the West. Recently, however, there has been a sharp increase in cases as observed in other skin cancers including squamous cell carcinoma and basal cell carcinoma (27-29). Despite this epidemiological change, a limited number of studies have covered the pathophysiology of malignant melanoma in Asians.
A recent study by Kang et al. (26) using the melanoma cell lines WM793 and WM39, revealed that fascin is involved in the development of malignant melanoma through the Hippo pathway. However, this was an in vitro study, and further studies are required.
We examined the expression pattern of fascin using immunohistochemical analysis of excised samples of malignant melanoma to identify its role in clinical cases. The result showed that fascin was weakly expressed in peritumoral nonneoplastic skin tissue mainly confined to basal cells but, on the contrary, it was generally over-expressed (moderately to strongly positive) in melanoma tissue. In addition, fascin staining intensity was not associated with tumor invasiveness. Ma et al. (30) reported that fascin expression is correlated with metastasis but not with the progression and survival rate in malignant melanoma, which is similar to our results. However, no metastatic melanoma samples were included in this study and therefore, additional studies are required. According to immunohistochemical analysis of tissue samples of oral squamous cell carcinoma, fascin expression has been associated with clinicopathological parameters of patients and tumor progression through activation of the AKT and MAPK pathways (31). Thus, to clarify the association between the expression of fascin and the progression, metastasis, and patient survival, further studies evaluating more clinical cases and various clinicopathological parameters are needed.
Immunohistochemistry was performed to determine the correlation with MST2, which plays a key role in the Hippo pathway. The results showed a strong positivity in all layers of peritumoral nonneoplastic skin tissue except the keratin layer but no or decreased expression (weakly to moderately positive) in melanoma tissue. Within the tumor, MST2 was not expressed homogeneously but showed a variegated pattern of staining. However, MST2 staining intensity was not associated with tumor invasiveness as seen in fascin immunohistochemical analysis. Although there have been few studies on MST2 in malignant melanoma, it has been reported that Hippo kinases (MST1/2) inhibit the proliferation of hepatocellular carcinoma by suppressing YAP/TAZ transcription activity (32), and that the elimination of MST1 and MST2 in the liver rapidly induce the development of hepatocellular carcinoma by activating YAP/TAZ, STAT3, Wnt/β-catenin, and Notch signaling (33). Similarly, decreased MST2 expression was associated with the oncogenesis of malignant melanoma in this study, which indicates a negative correlation between fascin and MST2 in melanoma development.
Moreover, treatment of WM793 melanoma cells with fascin siRNA oligonucleotides or MST2 siRNA oligonucleotides showed that fascin knock-down increased MST2 expression, whereas MST2 knock-down had no significant effect on fascin expression, which suggests that MST2 is a downstream target of fascin.
Our animal study where fascin siRNA oligonucleotides were inoculated via the tail vein 2 weeks after melanoma xenografting by subcutaneous injection of WM793 melanoma cells in BALB/c mice showed a significant decrease in tumor size. This is consistent with the results of immunohistochemical analysis using clinical samples as described above. Based on these results, fascin knock-down may lead to a decrease in tumor size by increasing the expression of MST2 involved in the Hippo pathway. Histopathological evaluation of animal specimens showed prominent tumor central necrosis in the control group, contrary to mild necrosis observed in tumor cells in the fascin knock-down group. Further studies are needed to clarify whether this difference in tumor necrosis can be attributed to the role of fascin in the Hippo pathway or it is merely ischemic central necrosis according to tumor size.
Conclusion
Fascin, an actin-bundling protein, is known to be involved in the invasion and metastasis of various cancers, and the Hippo pathway is known to participate in cancer development. However, the role of these components are not well studied in malignant melanoma.
Immunohistochemically, melanoma carcinogenesis was significantly associated with over-expression of fascin and decreased expression of MST2, and fascin and MST2 showed a negative correlation. In addition, fascin knockdown induced MST2 over-expression, leading to a decrease in tumor size.
Taken together, the activation of the Hippo pathway by regulating fascin can be considered as a novel therapeutic strategy for malignant melanoma.
Acknowledgements
The present study was supported by grants from the Chosun University, Gwangju, Republic of Korea (2019).
Footnotes
Authors’ Contributions
Data curation: Byung-Soo Kang. Funding acquisition: Sung-Chul Lim. Methodology: Byung-Soo Kang, Sung-Chul Lim. Supervision: Sung-Chul Lim. Validation: Sung-Chul Lim. Writing – original draft: Byung-Soo Kang. Writing – review & editing: Sung-Chul Lim.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received April 2, 2021.
- Revision received April 13, 2021.
- Accepted April 14, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
