A SMYD3/ITGB6/TGFβ1 Positive Feedback Loop Promotes the Invasion and Adhesion of Ovarian Cancer Spheroids

Background Implantation metastasis is the main means of dissemination in ovarian cancer. Our previous studies showed that SET and MYND domain-containing protein 3 (SMYD3) expression was higher in ovarian cancer spheroids than in monolayers. SMYD3 enhancement of spheroid invasion and adhesion is mediated by the downstream effectors ITGB6 and ITGAM. However, the potential mechanisms underlying the SMYD3/integrin-mediated invasion and adhesion of spheroids still need to be explored. Methods Western blotting was used to examine the expression of SMYD3, ITGB6 and downstream molecules under different treatments. Immunofluorescence was used to detect the expression of F-actin, E-cadherin and N-cadherin. Anti-ITGB6 antibody-based inhibition and dual-luciferase reporter assays were used to confirm the binding between ITGB6 and latent TGFβ1. Transwell invasion, adherence and 3D tumor spheroid invasion assays were employed to test the effects of TGFβ1 on the invasion and adhesion of ovarian cancer spheroids. ELISA was performed to assess the release of latent TGFβ1 from ovarian cancer spheroids. Results SMYD3 and ITGB6 activated the TGFβ1/Smad3 pathway and then induced the upregulation of Snail, Vimentin and N-cadherin and the downregulation of E-cadherin in 3D-cultured ovarian cancer spheroids. In this process, latent TGFβ1 could bind to ITGB6 and become activated to stimulate the Smad3 pathway. Moreover, SMYD3 and ITGB6 could facilitate the release of latent TGFβ1 from 3D-cultured ovarian cancer spheroids. Interestingly, TGFβ1 could promote the expression of SMYD3 and ITGB6 via feedback. This positive feedback loop could further amplify the biological effect and promote the invasion and adhesion of ovarian cancer spheroids. Conclusion Our results demonstrated that the SMYD3/ITGB6/TGFβ1-Smad3 positive feedback loop could promote the invasion and adhesion of ovarian cancer spheroids by upregulating the expression of N-cadherin, Snail, and Vimentin and downregulating the expression of E-cadherin. Thus, our study unmasked the mechanism of SMYD3- and ITGB6-induced ovarian cancer metastasis and provides new ideas for targeted ovarian cancer treatment.


INTRODUCTION
Ovarian cancer is the most lethal gynecological malignancy worldwide, and the lifetime risks of developing and dying from ovarian cancer are 0.72% and 0.45%, respectively (1,2). Characteristically, over 70% of epithelial ovarian cancer (EOC) patients have intra-abdominal dissemination involving local invasion of pelvic and abdominal organs but rarely have vascular metastasis at diagnosis (3). This is mainly because of the unique characteristics of implantation metastasis in EOC, including detachment from the original lesion, formation of spheroids in ascites, adherence to peritoneal mesothelial cells, and anchoring to the stroma. In addition, biological features such as the adhesion and invasion potential of spheroid cells also account for EOC recurrence (4). Therefore, more research has cast light on the mechanism of EOC implantation metastasis in recent years. The phenotype and gene expression profile of EOC cells constantly change during the processes of shedding, suspension, re-adhesion and proliferation to adapt to the microenvironment.
SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase that acts as a gene transcriptional regulator, participating in the methylation of various histone and nonhistone targets (5). Aberrant SMYD3 expression is found during carcinogenesis in multiple cancers, such as prostate cancer, breast cancer and colorectal cancer, suggesting its essential roles in tumor initiation and progression (6).
In our previous studies, we found that compared to ovarian cancer cells grown in monolayers, ovarian cancer spheroids exhibited increased SMYD3 expression associated with higher levels of H3K4me3 (5). The SMYD3-H3K4me3-integrin pathway plays an important role in the pathogenesis of implantation metastasis in ovarian cancer. SMYD3-induced enhancement of spheroid invasion and adhesion is mediated by the downstream effectors ITGB6 and ITGAM (7). However, the potential mechanisms underlying SMYD3/integrin-mediated invasion and adhesion still need to be explored.
It is widely accepted that TGFb plays a critical role in tumor progression and can mediate tumor immune escape, promote epithelial-mesenchymal transition (EMT) and help cells differentiate into more aggressive phenotypes for metastasis (8,9). TGFb1 is upregulated in ovarian cancer tissues and is associated with tumor progression, chemotherapy resistance and a poor prognosis (10). Nevertheless, more studies on the role of TGFb1 in ovarian cancer metastasis are required.
TGFb1 is secreted by cancer cells or fibroblasts in an inactive form, which consists of the 12.5-kDa carboxy-terminal region of TGFb1 and a 25-kDa amino-terminal latency-associated peptide (LAP) joined by noncovalent bonding. This configuration prevents TGFb1 from binding to its receptor (11). An acidic pH, reactive oxygen species, various kinases, thrombospondin-1 (THBS-1) and shear stress can activate latent TGFb1 (12,13). In a study on pulmonary fibrosis, ITGB6 was found to be the key activator of TGFb1 in lung epithelial cells (14). According to reports and our research base, we speculated that SMYD3/ITGB6 promotes the invasion and adhesion of ovarian cancer spheroids by changing the configuration of TGFb1-LAP and then activating TGFb1.
In this report, we found that SMYD3 and ITGB6 could promote the release and activation of latent TGFb1 and increase the phosphorylation of Smad3 activated by TGFb1 in ovarian cancer spheroids. As downstream targets of the TGFb1/ SMAD3 pathway and essential participants in EMT promotion, N-cadherin, Vimentin, E-cadherin and Snail were found to be regulated by SMYD3 and ITGB6 and could contribute to enhanced invasion and adhesion in ovarian cancer spheroids. In addition, TGFb1 could also upregulate the expression of SMYD3 and ITGB6 in return to form a positive feedback loop with the SMYD3/ITGB6/TGFb1 pathway to enhance the metastasis of ovarian cancer spheroids.

Cell Lines and Culture Conditions
The human EOC cell line HEY was a gift from Dr. Robert Bast's laboratory at the University of Texas Anderson Cancer Center, Houston, TX. The human EOC cell line A2780 were obtained from the Shanghai Key Laboratory of Female Reproduction Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University. HEY and A2780 cells were cultured in complete RPMI 1640 medium at 37°C in a 5% CO 2 environment. All complete media were supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ ml streptomycin.

Cell Treatments Using an Inhibitor of and an Antibody Against TGFb1
Cells were seeded in ultralow-attachment 6-well plates. For active TGFb1 and SB431542 inhibitor treatment, 10 ng/ml recombinant human TGFb1 (rhTGFb1) protein (Abcam, ab50036) was added to the culture medium for 72 h, and the medium was replaced at 48 h. Then, 10 mM SB431542 (MedChemExpress, HY-10341) was added to the medium and incubated for 6 h (equal doses of PBS and DMSO were used as the negative controls for rhTGFb1 and SB431542, respectively). The cells were collected for further western blotting and cell function assays. For the anti-ITGB6 inhibition assay, 10 mg/ml anti-ITGB6 antibody (Millipore, MAB2076Z) was added to the culture medium as a pretreatment to block ITGB6 for 12 h. Then, 10 ng/ml latent TGFb1 (CST, 5154) was added to the culture medium for 72 h (equal doses of normal mouse IgG1 (Santa Cruz Biotechnology, sc-3877) and PBS were used as the negative controls for the anti-ITGB6 antibody and latent TGFb1 experiments, respectively). The cells were collected for further western blotting and cell function assays.

Transwell Invasion Assay
A Transwell system (24-well insert, pore size: 8 mm, Corning, 3422) was used to measure the invasive ability of 3D-cultured HEY cells. The inserts were coated with 50 ml of Matrigel (BD Bioscience Pharmingen, 356234) at a 1:8 dilution and incubated at 37°C overnight. The following day, cells were trypsinized, counted and resuspended in serum-free medium. A total of 1*10 4 cells/200 ml of serum-free medium were added to the upper well of the chamber. In addition, 600 ml of complete medium was added to the lower well. After incubation for 16 h, the upper surface of the membrane was wiped with cotton swabs to remove any remaining cells, and the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 2% crystal violet. Five representative fields of each insert were imaged and counted using an Olympus light microscope at 100× magnification.

Adhesion Assay
A 24-well plate was coated with 200 ml of Matrigel at a 1:50 dilution and air-dried in a biosafety cabinet for 6 h. Then, for blocking non-specific binding, 200 ml of serum-free medium containing 0.1% BSA (bovine serum albumin, Mpbio, 02FC007710) for 1 h. 3D-cultured cells were trypsinized, counted and resuspended in serum-free medium. A total of 3*10 4 cells/200 ml of serum-free medium were added to each Matrigel-coated well and incubated in the incubator for 1-2 h. Nonadherent cells were removed by washing with PBS. Adherent cells were fixed with 4% paraformaldehyde and stained with 2% crystal violet. Five representative fields of each insert were imaged and counted using an Olympus light microscope at 40× magnification.

3D Tumor Spheroid Invasion Assay
This assay was performed as previously described (15). HEY cells (2×10 4 cells/ml) were plated in ultralow-attachment 96-well round-bottomed plates in 200 ml of medium/well. rhTGFb1 protein (10 ng/ml) was added to treat the cells in the TGFb1+DMSO and TGFb1+ SB431542 groups. The same volume of PBS was added to treat cells in the PBS+DMSO and PBS+SB431542 groups. After 3 days of incubation, 100 ml of culture medium was gently removed from each well, and 100 ml of Matrigel matrix was gently dispensed into each bottom well. The plate was transferred into a 37°C incubator, and the Matrigel matrix, which contained 20 ng/ml rhTGFb1 or 20 mM SB431542 according to the experimental design, was allowed to solidify. One hour later, 100 ml of serum-free growth medium containing 10 ng/ ml rhTGFb1 or 10 mM SB431542 was gently added to each well. An image was recorded for each tumor spheroid at 0, 24, and 48 h to dynamically observe the 3D tumor spheroid invasion ability. Representative fields of spheroids were randomly counted using an Olympus light microscope at 100× magnification. The diameters of the spheroids were measured by ImageJ software.

Dual-Luciferase Reporter Assay
A fragment of the PAI-1 promoter (-799~71 bp) was amplified and cloned into the pGL3 vector, which contains firefly luciferase (pGL3-PAI-1). Luciferase activity was measured using a dualluciferase reporter assay system at 24h posttransfection according to the manufacturer's instructions (Promega, E1910). Normalized data were calculated as the ratio of firefly/ renilla luciferase activities.

ELISA
The TGFb1 concentration in the supernatant was measured with a Quantikine ELISA Human TGFb1 kit (R&D Systems). A2780 cells were first subjected to siRNA-mediated gene silencing treatment for 48 h. A total of 6×10 5 cells were suspended in 1.5 ml of culture medium, added to a single well of a six-well ultralow-attachment plate, and then cultured for 72 h. The culture medium was not changed during the culture period. Then, the culture medium was collected in a 1.5 ml conical tube and centrifuged at 3,000 rpm for 15 minutes. The concentration of TGFb1 in the culture medium was determined using a human TGFb1 Quantikine ELISA kit (R&D Systems). HCl and NaOH/ HEPES were used to convert latent TGFb1 into the activated form.

Statistical Analysis
Statistical analysis and graphing were conducted using GraphPad Prism 8. ImageJ was used for protein quantification. Statistically significant differences were determined by Student`s t-test, and p values <0.05 were considered significant.

Key EMT Factors Are Promoted Along With SMYD3 and ITGB6 Upregulation During Ovarian Cancer Cell Spheroid Formation
According to our previous study, SMYD3 is overexpressed in ovarian cancer ascites spheroids compared with primary ovarian cancer tissues. Using a 3D culture model to mimic the suspended growth conditions in ascites, we found that SMYD3 could enhance the adhesion and invasion of ovarian cancer spheroids and promote metastasis of ovarian cancer in vivo by upregulating the expression of ITGB6 and ITGAM (7). Compared with 2Dcultured HEY and A2780 cells, the corresponding 3D-cultured cells showed higher expression of SMYD3 and ITGB6, which indicated that the ovarian cancer spheroids had a more invasive phenotype. In addition, we found that the expression of Ncadherin, Snail, Vimentin and E-cadherin, which are essential molecules in the EMT process, was also increased in 3D-cultured HEY and A2780 cells ( Figure 1A). Tumor cells can be more invasive during the EMT process. With this in mind, what is the mechanism of the upregulation of N-cadherin, Snail, and Vimentin and downregulation of E-cadherin during 2Dcultured ovarian cancer cell transformation into 3D spheroids? Do these EMT-related genes contribute to SMYD3/ITGB6mediated spheroid metastasis? Both these questions need to be answered. The phosphorylation level of Smad3 in 2D-and 3D-cultured HEY and A2780 cells was evaluated by western blot analysis. In the histogram of the western blot quantification, "*" refers to p < 0.05 and "**" refers to p < 0.01. Smad3, as a well-known downstream signal of TGFb1, can be activated by phosphorylated ALK5 after binding of TGFb1 to its receptor (16). Since the TGFb1/Smad3 signal transduction pathway is involved in inducing EMT in ovarian cancer (17), we aimed to identify whether the TGFb1/Smad3 pathway is more activated in 3D-cultured ovarian cancer spheroids than in 2D-cultured ovarian cancer cells. As shown in Figure 1B, higher phosphorylation levels of Smad3 were found in 3D-cultured HEY and A2780 cells than in 2D-cultured cells, which indicated that TGFb1/Smad3 might play a role in promoting the EMT process in ovarian cancer spheroids.
Activation of the TGFb1/Smad3 Pathway Is Conducive to the Regulation of EMT-Related Genes in 3D-Cultured Ovarian Cancer Spheroids A review of the previous literature about the induction of EMT by TGFb1 showed that the epithelial markers E-cadherin was repressed while the mesenchymal markers Vimentin and Ncadherin were induced during the induction of EMT by TGFb1 (18). Since phosphorylated Smad3, N-cadherin, Snail and Vimentin were all increased in 3D-cultured ovarian cancer spheroids and E-cadherin was decreased in 3D-cultured ovarian cancer spheroids, we aimed to determine whether Ncadherin, Snail, Vimentin and E-cadherin are target molecules of the activated Smad3 pathway in 3D-cultured ovarian cancer spheroids. First, we used rhTGFb1 to treat 3D-cultured HEY and A2780 cells for 24, 48 and 72 hours. Over time, the amount of phosphorylated Smad3 increased under the condition of constant expression of total Smad3, which demonstrated that TGFb1 could stimulate the Smad3 pathway. In addition, the expression of N-cadherin, Snail and Vimentin was also increased and the expression of E-cadherin was decreased after treatment with rhTGFb1 ( Figure 2A). Subsequently, SB431542, an inhibitor of ALK5, was added to the supernatant of 3Dcultured HEY and A2780 cells and incubated for 6 hours. As the phosphorylation of Smad3 was inhibited by SB431542, the expression of N-cadherin, Snail and Vimentin also declined, and the expression of E-cadherin was enhanced ( Figure 2B). Furthermore, we found that the regulation of EMT-related genes induced by rhTGFb1 could be restrained by SB431542 ( Figure 2C). These findings showed that TGFb1 could stimulate the Smad3 pathway and contribute to the increases in the expression of Snail and N-cadherin. Finally, we used Transwell invasion, adherence and 3D tumor spheroid invasion assays to validate the functional effects of rhTGFb1 and SB431542 on the EMT process. RhTGFb1 enhanced the invasion and adhesion of 3D-cultured HEY cells, and these effects were inhibited when SB431542 was added simultaneously (Figures 2D, E).

SMYD3 and ITGB6 Can Activate the TGFb1/Smad3 Pathway and Regulate the Expression of EMT-Related Genes in 3D-Cultured Ovarian Cancer Spheroids
To determine why SMYD3 and ITGB6 could make ovarian cancer spheroids more invasive, we downregulated the expression of SMYD3 and ITGB6 in 3D-cultured HEY and A2780 cells. As shown in Figure 3A, after SMYD3 silencing, the expression of its target gene ITGB6 was also decreased. In addition, the phosphorylation levels of Smad3 were consequently reduced, along with decreased expression of N-cadherin, Snail, and Vimentin and increased expression of E-cadherin. After ITGB6 silencing, the expression changes in p-Smad3, N-cadherin, Snail, Vimentin, Ecadherin were consistent with those observed with SMYD3 silencing ( Figure 3B). In addition, using immunofluorescence assay, we also demonstrated that when we inhibited the expression of SMYD3 and ITGB6, the expression of E-cadherin significantly increased. And as the 3D-cultured ovarian cells were treated with rhTGFb1, it significantly decreased. The expression of N-cadherin showed opposite tendency to the one of E-cadherin ( Figures 3C, D).
Considering to the expression changes of EMT-related genes, we should also pay attention to the morphological changes of 3D-cultured ovarian cancer cells. The epithelial-like structure of 3D-cultured spheroid is more solid round. And the mesenchymal-like structure of 3D-cultured is grape-like or spindle like (19). The 3D-cultured HEY and A2780 spheroid with SMYD3 or ITGB6 silencing presented the robust cell-cell adherence and more solid structure compared with 3D-cultured HEY and A2780-NC spheroid ( Figure 3C). The 3D-cultured HEY and A2780 spheroid presented the loose cell-cell adherence and spindle-like structure when it was exposed to rhTGFb1 ( Figure 3D). Therefore, SMYD3 and ITGB6 could stimulate the TGFb1/Smad3 pathway and regulate the expression of Ncadherin, Snail, Vimentin, E-cadherin in ovarian cancer spheroids and promote the EMT process.

Latent TGFb1 Can Bind to ITGB6 and Release Active TGFb1 to Stimulate the Smad3 Pathway
How can SMYD3 and ITGB6 stimulate the TGFb1/Smad3 pathway? TGFb1 is produced as a latent precursor and functions in an active form. Therefore, the activation of latent TGFb1 is a crucial regulatory event. ITGB6 was reported to be important in stringently localized activator of latent TGFb1 at epithelial surfaces (20). Therefore, we performed a dualluciferase reporter assay to identify whether ITGB6 plays a role in the activation of TGFb1. Plasminogen activator inhibitor-1 (PAI-1) is a well-known target gene of TGFb1 (21). When we downregulated the expression of ITGB6 in 3D-cultured HEY cells, the relative luciferase activity of PAI-1 was decreased accordingly (p<0.0001) ( Figure 4A). These results suggested that ITGB6 could stimulate the TGFb1 pathway. Then, we used latent TGFb1 to treat 3D-cultured HEY and A2780 cells. As a result, the Smad3 pathway was activated. However, after adding a monoclonal antibody against ITGB6 to inhibit latent TGFb1 binding to ITGB6, latent TGFb1 failed to stimulate the Smad3 pathway ( Figure 4B). Therefore, latent TGFb1 could be transformed into the active form by binding to ITGB6 and then induce the activation of the Smad3 pathway.
As the binding partner of ITGB6, ITGAV has also been reported to be able to activate latent TGFb1 in tumors (22, 23). It is necessary to investigate whether ITGAV could regulate the activation of latent TGFb1 in 3D-cultured ovarian cancer spheroids and whether ITGAV is involved in the ITGB6regulated activation of latent TGFb1 in 3D-cultured ovarian cancer spheroids. First, we silenced the expression of ITGAV in 3D-cultured ovarian cancer spheroids using siRNA. After treatment with latent TGFb1, the phosphorylation level of Smad3 did not decrease in ITGAV knockdown cells. Therefore, ITGAV might have no effect on the activation of latent TGFb1 in 3D-cultured ovarian cancer spheroids. Then, we used 10 mg/ml anti-ITGB6 antibody to block ITGB6 for 12 h in 3D-cultured NC-ITGAV and si-ITGAV cells. After that, 10 ng/ml latent TGFb1 was added to the culture medium for 72 h. We found that anti-ITGB6 antibody was more effective in inhibiting the phosphorylation level of Smad3 in 3D-cultured NC-ITGAV cells than in 3D-cultured si-ITGAV cells, implying that ITGAV might play a role in ITGB6-regulated activation of latent TGFb1 in 3Dcultured ovarian cancer spheroids ( Figure 4C).

SMYD3 and ITGB6 Can Facilitate the Release of Latent TGFb1 From 3D-Cultured Ovarian Cancer Spheroids
Since we found that SMYD3 and ITGB6 could upregulate the expression of Snail and N-cadherin via the activation of the TGFb1/Smad3 pathway, whether SMYD3 and ITGB6 can enhance the release of latent TGFb1 needed to be explored. We used ELISA to quantify the amounts of active and total TGFb1, and the difference was considered the amount of latent TGFb1. We found that the latent TGFb1 level was decreased after downregulating SMYD3 or ITGB6 (both p<0.0001) ( Figures 5A, B). Hence, SMYD3 and ITGB6 could not only activate latent TGFb1 but also increase the release of latent TGFb1 from 3D-cultured ovarian cancer spheroids.

TGFb1 Can Promote the Expression of SMYD3 and ITGB6 via Feedback
We verified that ITGB6 could activate latent TGFb1. Subsequently, we found that TGFb1 could also promote the expression of SMYD3 and ITGB6 in 3D-cultured HEY and A2780 cells ( Figure 6A). SB431542 reduced the expression of SMYD3 and ITGB6 ( Figure 6B). The upregulation of SMYD3 and ITGB6 due to the activation of TGFb1 could be inhibited by SB431542 ( Figure 6C). These findings demonstrated that TGFb1 promotes the expression of SMYD3 and ITGB6 via the Smad3 pathway. As shown in Figure 6D, latent TGFb1 facilitated the expression of SMYD3 and ITGB6. After the binding of latent TGFb1 to ITGB6 was blocked, the expression of SMYD3 and ITGB6 also declined. Thus, the SMYD3/ITGB6/TGFb1 positive feedback loop drove the upregulation of Snail and N-cadherin, which promoted invasion, adhesion and EMT progression during ovarian cancer metastasis.

DISCUSSION
Ovarian cancer is the leading cause of death among gynecological malignancies. Worldwide, the incidence rate of ovarian cancer is seventh among those of female malignant cancers, while the mortality rate is eighth, and the 5-year survival rate is below 45% (24). The main metastatic mode of ovarian cancer is the transcoelomic pathway. Briefly, cells disseminate from the primary epithelial ovarian tumor and float as spheroids in the ascites. Metastatic cells then attach to peritoneal organs and form macrometastatic colonies (25). Both genetic changes and epigenetic changes contribute to the initiation and transcoelomic process of ovarian cancer. This disease is characterized by "genomic chaos" caused by high chromosomal instability due to a massive number of copy number abnormalities and chromosomal alterations (26,27). Therefore, epigenetic enzymes may play a great role in regulating the transcoelomic spread of ovarian cancer. According to the previous work of our team, SMYD3, a histone methylation transferase, was found to be expressed at higher levels in ascites-derived spheroids than in primary ovarian tumor cells from EOC patients. In ovarian cancer spheroids, SMYD3 was found bind to H3K4me3 at the ITGB6 and ITGAM promoter regions to upregulate the expression of ITGB6 and ITGAM, which was verified to enhance the invasion and adhesion of ovarian cancer spheroids (7). ITGB6 is an essential member of the integrin family. The integrin family is documented to be involved in various cancer processes (such as tumor initiation, metastasis and drug resistance), and ITGB6 is also reported to be associated with the progression and metastasis of oral squamous cell carcinoma, bladder cancer and colorectal carcinoma (28)(29)(30)(31). Previously, we found that FIGURE 2 | Activation of the TGFb1/Smad3 pathway is conducive to the upregulation of EMT-related genes in 3D-cultured ovarian cancer spheroids. (A) Changes in the expression of N-cadherin, Snail, Vimentin and E-cadherin and the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with 10 ng/ml rhTGFb1 protein for 24, 48 and 72 h (B) Changes in the expression of N-cadherin, Snail, Vimentin and E-cadherin and the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with 10 mM SB431542 for 6h. (C) Changes in the expression of N-cadherin, Snail, Vimentin and E-cadherin and the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with or without rhTGFb1 and SB431542. RhTGFb1 protein (10 ng/ml) was added to the culture medium for 72 h Then, 10 mM SB431542 was added to the medium and incubated for 6 h (equal doses of PBS and DMSO were used as the negative controls for rhTGFb1 and SB431542, respectively). (D) Transwell invasion and adhesion assays revealed changes in the invasion and adhesion of 3D-cultured HEY cells after treatment with or without rhTGFb1 and SB431542. (E) 3D tumor spheroid invasion assays revealed changes in the invasion and adhesion of 3D-cultured HEY cells after treatment with or without rhTGFb1 and SB431542. RhTGFb1 protein (10 ng/ml) was added to treat cells in the rhTGFb1+DMSO and rhTGFb1+ SB431542 groups. The same volume of PBS was added to treat cells in the PBS+DMSO and PBS+SB431542 groups. After 3 days of incubation, 100 ml of culture medium was gently removed from each well, and 100 ml of Matrigel matrix was gently dispensed into each bottom well. Matrigel matrix containing 20 ng/ml rhTGFb1 was used in the rhTGFb1+DMSO and rhTGFb1+ SB431542 groups, and 20 mM SB431542 was used in the rhTGFb1+ SB431542 and PBS+SB431542 groups. One hour later, 100 ml of serum-free growth medium containing 10 ng/ml rhTGFb1 was gently added into each well of the rhTGFb1+DMSO and rhTGFb1+ SB431542 groups. SB431542 (10 mM) was gently added to the rhTGFb1+SB431542 and PBS+SB431542 groups. "*" refers to p < 0.05, "**" refers to p < 0.01, "***" refers to p < 0.001, and "****" refers to p <0 .0001. (D) Immunofluorescence assay exhibited the expression changes of F-actin, E-cadherin and N-cadherin in both 3D-cultured HEY and A2780 cells after rhTGFb1 treatment. In the histogram of the western blot quantification, "*" refers to p < 0.05, "**" refers to p < 0.01, and "***" refers to p < 0.001. Changes in the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after treatment with or without latent TGFb1 and anti-ITGB6 antibody. Anti-ITGB6 antibody (10 mg/ml) was added to the culture medium as a pretreatment to block ITGB6 for 12 h. Then, 10 ng/ml latent TGFb1 was added to the culture medium for 72 h (equal doses of normal mouse IgG1 and PBS were used as the negative controls for the anti-ITGB6 antibody and latent TGFb1, respectively). (C) Changes in the phosphorylation level of Smad3 in 3D-cultured HEY and A2780 cells after silencing of ITGAV. NC-ITGAV and si-ITGAV were transfected into HEY and A2780 cells. After 6 h, the cells were resuspended in ultralow-attachment plates for 3D culture. Then, 10 mg/ml anti-ITGB6 antibody was used to block ITGB6 for 12 h in 3D-cultured NC-ITGAV and si-ITGAV cells, and equal doses of normal mouse IgG1 were used as a negative control. Subsequently, 10 ng/ml latent TGFb1 was added to the culture medium and incubated for 72 h in each group. In the histogram of the quantification, "*" refers to p < 0.05, "**" refers to p < 0.01, and "***" refers to p < 0.001.
ITGB6 could promote the invasion and adhesion of ovarian cancer spheroids. However, the underlying molecular mechanisms should be further explored.
EMT is one of the phenotypic plasticity processes relevant to metastasis. During EMT, epithelial cells lose polarity and develop a stromal phenotype, increasing invasiveness. Cells with features of EMT are able to migrate, invade and undergo metastatic dissemination. TGFb is a common inducer of the EMT process (32). TGFb signaling promotes EMT by inducing the expression of several pleiotropic transcription factors, also known as "master regulators" of EMT (such as Snail, N-cadherin, Ecadherin and Vimentin). Proteins of the Smad family are the major effectors that control the transduction of intracellular signaling initiated by the TGFb superfamily of cytokines. SMAD-dependent signaling can be activated by an active TGFb1 ligand initially binding to TGFbRII, followed by recruitment of ALK5 (TGFbRI) to the plasma membrane (33). Here, we observed that in ovarian cancer spheroids, the Smad3 Then, 10 mM/ml SB431542 was added to the medium and incubated for 6 h (equal doses of PBS and DMSO were used as the negative controls for rhTGFb1 and SB431542, respectively). (D) Changes in the protein expression of SMYD3 and ITGB6 in 3D-cultured HEY and A2780 cells after treatment with or without latent TGFb1 and anti-ITGB6. In the histogram of the western blot quantification, "*" refers to p < 0.05 and "**" refers to p < 0.01. pathway was activated by TGFb1 and upregulated the expression of Snail and N-cadherin, which are crucial genes in the EMT process. The three isoforms of TGFb (TGFb1, TGFb-2 and TGFb-3) are always produced as inactive cytokines that cannot bind to their receptor and are not functional unless they are activated. An integrin-mediated mechanism makes an essential contribution to TGFb activation in vivo. TGFb activation has been demonstrated to be caused by the RGD tripeptide motif in the LAP region of latent TGFb binding to specific integrin receptors (20). Khalid Puthawala et al. reported that integrin alpha(v)beta6-mediated TGFb activation is required for radiation-induced lung fibrosis (34). Laura L Koth et al. found that integrin alpha(v)beta6-mediated TGFb activation regulates the homeostasis of phospholipids and collectins in the lungs (35).
To date, few articles have stated the role of ITGB6-mediated TGFb activation in tumor progression. In our study, we found that downregulated expression of ITGB6 could reduce the activation level of the Smad3 pathway when ovarian cancer spheroids were treated with rhTGFb1. When ovarian cancer spheroids were treated with latent TGFb1, the activation of the Smad3 pathway could also be inhibited if ITGB6 was blocked by a specific antibody. After the expression of SMYD3 or ITGB6 was altered, the expression of N-cadherin, Snail, Vimentin and E-cadherin and the morphological structure of the spheroids were consequently changed. Hence, we determined that ITGB6mediated TGFb activation is involved in regulating the expression of N-cadherin, Snail, Vimentin and E-cadherin and promoting EMT progression in ovarian cancer spheroids. In addition, as the binding partner of ITGB6, ITGAV, had no independent effect on activating latent TGFb1 in 3D-cultured ovarian cancer spheroids. However, ITGAV might be play a role in the ITGB6-regulated activation of latent TGFb1 in 3Dcultured ovarian cancer spheroids. Therefore, in future, more studies should be done to explore the role of ITGAV in TGFb1/ SMAD signaling activation. Interestingly, attenuating the expression of SMYD3 and ITGB6 also decreased the release of latent TGFb1 from ovarian cancer spheroids, which further demonstrated the regulatory roles of SMYD3 and ITGB6 in TGFb1 pathway activation.
Surprisingly, we also found that the expression of SMYD3 and ITGB6 could be upregulated after TGFb1 treatment, which formed a positive feedback loop among SMYD3/ITGB6/TGFb1 to enhance the invasion and adhesion of ovarian cancer spheroids by regulating the expression of E-cadherin, Ncadherin, Snail and Vimentin (Figure 7). Several previous studies also showed that TGFb could induce the expression of SMYD3 and ITGB6. Denise et al. stated that SMYD3 was specifically regulated by TGFb in iTreg cells (36). Mingyan Xu et al. reported that TGFb1 could induce ITGB6 transcription via JunB-and CBP-mediated histone hyperacetylation in oral squamous cell carcinoma (37). Chao Jing et al. reported that miR-17/20a could reduce the expression of ITGB6 by attenuating the activation of TGFb and phosphorylation of SMAD2/3 in esophageal squamous cell carcinoma (38).
In conclusion, we detailed one essential pathway that contributed to the SMYD3-and ITGB6-mediated enhancement of invasion and adherence in ovarian cancer spheroids. Our study also emphasized the significance of choosing SMYD3 and ITGB6 as potential targets for the treatment of ovarian cancer transcoelomic metastasis. In ovarian cancer spheroids, SMYD3 can increase the expression of ITGB6. When latent TGFb1 binds to ITGB6 on the cell surface, TGFb1 is activated and upregulates the expression of N-cadherin, Snail, Vimentin as well as downregulates E-cadherin to enhance the invasion and adhesion of ovarian cancer spheroids by stimulating the Smad3 pathway. Ovarian cancer spheroids can release more latent TGFb1 at the same time. In addition, TGFb1 can also facilitate the expression of SMYD3 and ITGB6 in a feedback manner.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
All experiments were approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine. The informed consents were obtained from all patients included in the study.