Potential therapy with the inhibitor of TGF-β receptors LY2109761 for oral squamous cell carcinoma

One way of trying to control oral squamous cell carcinoma is to invest in new therapies focused on the molecular biology of receptors and their intracellular signaling pathways. This study aimed to evaluate the effect of LY2109761 (an inhibitor of TGF-β receptors) on cell migration in oral squamous cell carcinoma in vitro. Actin cytoskeleton of SCC-4 cells control and LY2109761 (1, 5 and 10 μM) treated on three-dimensional Matrigel were analysed by using confocal laser microscopy. Control and LY2109761 (1, 5 and 10 μM) treated cells that migrated through the membrane of three-dimensional cell migration assays were counted, significance was p<0.05. Control cells were seen with voluminous cytoplasm, cell cortex preserved and actin cytoskeleton well developed with well distributed actin filaments. Regardless of concentration, cells treated showed: rounded morphology and small size, scanty cytoplasm, cortical F-actin less clear that the control cells, and disruption of actin filaments. The migratory cells were inhibited by treatment with LY2109761 [F (3, 11) = 3742, p<0.0001], in a dose-dependent manner. These results suggest that LY2109761 exerts an inhibitory effect on the actin cytoskeleton and cell migration on SCC-4 cells, therefore, it is a promising therapeutic option for oral squamous cell carcinoma.


Introduction
Among head and neck tumors, oral squamous cell carcinomas account for 90% of cases, whose primary lesions may present as symptoms like difficulty in swallowing and hoarseness, and the prognosis for early cases is promising, unlike advanced cases, where regional and/or distance metastasis are common (Sanderson & Ironside, 2002). During the development of metastasis, the displacement of cells within primary site tissues, cell migration, occurs to target distant organs at secondary sites (Krakhmal et al., 2015); (Albini & Noonan, 2010;Kramer et al., 2013), when cell migration occurs (Albini & Noonan, 2010). It is important the development of new therapeutics (Capece et al., 2017), the blockade of the TGF-β signaling pathway is a promising therapeutic option (Neuzillet et al., 2015). The LY2109761 is a dihydropyrrolopyrazole inhibitor which has oral bioactivity and a potential of inhibition in vivo greater than 50% (Li et al., 2008) and acts on the intracellular domain of the TGF-β type I receptor (TβRI), through competition for the catalytic site of ATP blocking phosphorylation of the receptor and activation of the intracellular signaling pathway (Connolly et al., 2012).
The TGF-β signaling is one of the pathways involved in tumor progression (Loomans & Andl, 2014), acting in the processes of invasion and cellular migration (Drabsch & ten Dijke, 2012), regulation of growth, differentiation, proliferation and apoptosis (Zhang et al., 2014), therefore, the blockade of the TGF-β signaling pathway is a promising therapeutic option (Neuzillet et al., 2015). The LY2109761 is a dihydropyrrolopyrazole inhibitor which has oral bioactivity and a potential of inhibition in vivo greater than 50% (Li et al., 2008) and acts on the intracellular domain of the TGF-β type I receptor (TβRI), through competition for the catalytic site of ATP blocking phosphorylation of the receptor and activation of the intracellular signaling pathway (Connolly et al., 2012).
This study aimed to evaluate the effect of LY2109761 treatment, by using three-dimensional assays, on the regulation of the actin cytoskeleton and cell migration in oral squamous cell carcinoma in vitro.

Methodology
This is a prospective, experimental, quantitative and analytical study (Pereira et al, 2018). This research was dismissed from ethical evaluation by the Research Ethics Committee of the Federal University of Triângulo Mineiro for using commercially available cell lines. This study the SCC-4 cell line, originating from moderately differentiated human oral squamous cell carcinoma, was used (American Type Culture Collection -ATCC®). Cell culture was done in a humid incubator with 5% CO2/95% atmosphere at 37 °C in Dulbecco's Modified Eagle's Medium F12 (Sigma-Aldrich, St. Louis, MO) containing fetal bovine serum (FBS) 10% (Cultilab, Campinas, SP, Brasil) 100 μg/ml penicillin, 100 U/ml streptomycin (Sigma-Aldrich, St. Louis, MO), 400 ng/ml hydrocortisone (Ariston, São Paulo). Analysis of the effect of LY2109761 on three-dimensional actin cytoskeleton and cell migration in oral squamous cell carcinoma was performed in biological triplicates and experimental duplicates.

Three-dimensional actin cytoskeleton assays
Was prepared Matrigel TM (BD Biosciences, Bedford, MA, USA) 1:2 medium and applied in glass coverslipes put in six well plates. The plates were placed in humidified incubator for 30 minutes to form a gel. It was seeded 1x10 4 SCC-4 cells/well and incubated for 30 minutes, then medium/well were added. After 24 hours of incubation, it was performed the treatment with LY2109761 (Cayman Chemical, Ann Arbor, MI, USA), at concentrations of 1, 5 and 10 μM, and for control cells medium DMSOvv was added. After 24 hours of LY treatment, cells were washed with Dulbecco's Phosphate Buffered Saline (D-PBS) with calcium and magnesium for 5 minutes. The fixation was performed with 4% paraformoaldehyde for 1 hour. After two washes with D-PBS for 5 minutes permeabilization was performed with triton X-100 (Sigma-Aldrich, St Louis, Missouri, USA) 0,2 % for 5 minutes. The blocking of nonspecific sites was done with albumin solution from bovine serum (BSA) (Sigma-Aldrich, St Louis, Missouri, USA) 1% for 5 minutes.
During 30 minutes the f-actin was stained with rhodamine-conjugated phalloidin (Molecular Probes, Eugene, Oregon, USA) 1:100 DPBS. The evidenciation of the nuclei was made for 5 minutes with 4'6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO)1: 100 D-PBS. The assembly of the slides was performed with Vectashield® (Vector Laboratories, Burlingame, California, USA). The images of actin citoskeleton experiment were obtained in confocal laser scanning microscope LSM 510 Meta (Zeiss®, Goettingen, Germany) and the entire length of the blades were analyzed by two independent observers.

Three-dimensional cell migration assays
In the upper chambers of the cell migration plate (BD BioCoat™ 24-well plate, 8.0 μm, BD Bioscience, Bedford, MA), 1x105 SCC-4 cells were seeded in medium with/without addition of treatment. To the middle of the control cells was added DMSOvv; and to the medium of the treated cells, LY2109761 at concentrations of 1, 5 and 10 μM. 500 μl of medium containing 20% FBS was added in the lower chambers of the plate. The migration plates were kept in a humid incubator with 5% CO2/95% atmosphere for 24 hours After the treatment period, cells that did not migrate on the upper surface were carefully removed with swab. The fixation of the cells that migrated through the membrane was performed with 30% methanol for 30 seconds and, staining as per manufacturer's instructions, with Instant Prov Newprov® (Pinhais, PR, Brazil). The control and treated cells that migrated through the membrane were counted in ten random fields, using a 20x objective inverted microscope (Axio Vert.A1, Zeiss ®) by one observer.

Statistical analysis
Results were analyzed using the IBM SPSS® 20.0 (Chicago, IL, USA) and graphs with Graphpad Prism (San Diego, CA, USA).The Levene variance test, the analysis of variance (ANOVA) and Tukey's post-test were used, considering p<0.05.

Results
LY2109761 treatment affected the actin cytoskeleton of OSCC in vitro. Control cells were seen with morphology: polarized, voluminous cytoplasm, eccentric spherical nuclei, cell cortex preserved and actin cytoskeleton well developed with well distributed actin filaments. Filopodia and lamellipodia were seen only in control cells ( Figure 1A). After treatment with LY2109761, regardless of concentration, the cells showed: rounded morphology and small size, scanty cytoplasm, spherical nuclei and central, cortical F-actin less clear that the control cells, and disruption of actin filaments ( Figure 1B-D). Fonte: Autores.

LY2109761 treatment inhited cell migration of oscc in vitro.
There was a significant difference between the number of migratory cells inhibited by treatment with LY2109761 [F

Discussion
This study demonstrated through three-dimensional assays that treatment with LY2109761, a TβRI inhibitor, affected the actin cytoskeleton and inhibited cell migration in OSCC. Among the steps involved in the formation of metastasis, the reconfiguration of the actin cytoskeleton, the main responsible for cell migration, plays a crucial role in cellular motility (Sun et al., 2015), which makes the LY2109761 a promising target for antimetastatic drugs.
Metastatic formation begins in the primary tumor with loss of cell-cell adhesion, motility, and interactions of tumor cells with extracellular matrix and basement membrane, invading the stroma, entering blood and/or lymphatic vessels, reaching the target organ, extravasate and expand the tumor at a distant site (Howell & Grandis, 2005). In this study treatment with the specific inhibitor of TβRI, LY2109761, has affected biological processes that occur in the formation of metastasis.
The cell surface initiates cell movement by means of structural rearrangements in the actin cytoskeleton, such as the formation of filopodia and lamelipodia (Yamaguchi & Condeelis, 2007). The filopodia has the ability to probe the environment and direct the migration (Lehtimäki et al., 2016), and the lamelipodia are responsible for the attachment of the cell to the migration site and generation of the necessary force for the movement (Yamaguchi & Condeelis, 2007). LY2109761 reduced the amount of filopodia and lamelipodia in SCC-4 cells.
Tyrosine kinase receptors (RTKs) are transmembrane proteins located on the cell surface and are important regulators of several key processes such as proliferation, differentiation, migration, survival (Lemmon & Schlessinger, 2010) and TGF-β receptors are RTKs (Chen et al., 2020). The increase of RTK's activation is an important cancer characteristic (Lemmon & Schlessinger, 2010) leading to higher oncogenic mutation (Blume-Jensen & Hunter, 2001).
This excessive activation and numerous mutations of tumors made RTKs an important target of new cancer treatments because of the direct effects on tumor cells (Hunter, 2014). In tumors TGF-β signaling pathway plays a progression role, stimulating cell motility and consequently metastasis (Drabsch & ten Dijke, 2012), promoting changes in the cytoskeletal architecture of tumor cells (Yang, 2010). LY2109761 exerted an inhibitory effect on the cytoskeleton, demonstrating that of this pathway may play a meaningful role in the cellular motility in OSCC.
In cells from the Tca8113 line of OSCC, treated with TGF-β for 24 hours, there was a phenotypic changes from epithelial cells to elongated fibroblast-like cells (Bu & Chen, 2017). This study demonstrated that control cells exhibited elongated morphology and reorganization of the actin cytoskeleton, while the LY2109761, TGFβ inhibitor, treatment leads cells to became smaller, rounded and with fewer organized actin filaments.
There are different forms of cell migration, the main ones being ameboid and mesenchymal. Elongated cells are associated with mesenchymal migration, while rounded cells are related to ameboid migration. It is known that cells have the ability to modify the morphology and mode of migration according to external conditions and intracellular regulation, and such adaptations facilitate the formation of metastasis (Clark & Vignjevic, 2015). LY2109761 seems to have an anti-metastatic potential as demonstrated in cell studies with glioblastoma (Joseph et al., 2013) and in colorectal cancer cells applied in mice (Zhang et al., 2010). In this study, the treatment with LY2109761 lead to a reduction of cell migration rate, and it could be indicated a relation with the anti-metastatic potential of this inhibitor.

Conclusion
The results of this study showed that control SCC-4 cells were elongated, and cells treated with LY2109761 were rounded, and the rate of cell migration reduced. Therefore, the inhibitor LY2109761 is a promising therapeutic option for OSCC since it exerts an inhibitory effect on the actin cytoskeleton and cell migration in SCC-4 cells of OSCC by blocking the TGF-β signaling pathway. Further studies such as evaluation of processes such as apoptosis and cell proliferation should be performed to analyze the effects of LY2109761, as well as preclinical and possibly clinical studies for this inhibitor to be used in the treatment of OSCC.