FSCN1 as a new druggable target in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a high risk of relapse and metastatic spread. The actin‐bundling protein fascin (FSCN1) is overexpressed in aggressive ACC and represents a reliable prognostic indicator. FSCN1 has been shown to synergize with VAV2, a guanine nucleotide exchange factor for the Rho/Rac GTPase family, to enhance the invasion properties of ACC cancer cells. Based on those results, we investigated the effects of FSCN1 inactivation by CRISPR/Cas9 or pharmacological blockade on the invasive properties of ACC cells, both in vitro and in an in vivo metastatic ACC zebrafish model. Here, we showed that FSCN1 is a transcriptional target for β‐catenin in H295R ACC cells and that its inactivation resulted in defects in cell attachment and proliferation. FSCN1 knock‐out modulated the expression of genes involved in cytoskeleton dynamics and cell adhesion. When Steroidogenic Factor‐1 (SF‐1) dosage was upregulated in H295R cells, activating their invasive capacities, FSCN1 knock‐out reduced the number of filopodia, lamellipodia/ruffles and focal adhesions, while decreasing cell invasion in Matrigel. Similar effects were produced by the FSCN1 inhibitor G2‐044, which also diminished the invasion of other ACC cell lines expressing lower levels of FSCN1 than H295R. In the zebrafish model, metastases formation was significantly reduced in FSCN1 knock‐out cells and G2‐044 significantly reduced the number of metastases formed by ACC cells. Our results indicate that FSCN1 is a new druggable target for ACC and provide the rationale for future clinical trials with FSCN1 inhibitors in patients with ACC.


| INTRODUCTION
Metastases are produced by the dissemination of cancer cells beyond their organ of origin. Their formation is a complex, multistep and overall very inefficient process; this probably explains why a long delay may exist between primary tumor formation and the detection of metastases. Overall, metastases are responsible for the largest number of cancer-related deaths. 1,2 A number of anticancer therapies are focused on counteracting cancer cell spreading in the body and formation of metastases. Most of those therapies are rationally designed by targeting critical factors implicated in the metastatic process. 3 Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a high risk of relapse and metastatic spread. 4 Current treatments for ACC only have a limited efficacy in advanced-stage disease and rely upon the use of the adrenolytic agent mitotane 5 and combination chemotherapy in patients with progressive disease. 6 Even if immunotherapy has been shown to reduce tumor burden in some patients with ACC, its efficacy is not established at present. 7 Activation of cytoskeleton dynamics by growth factor signaling and/or genomic alterations in cancer cells is crucial for their migration, invasion and dissemination. 8 One important protein regulating migration and invasion in cancer cells is fascin (FSCN1), encoded by the FSCN1 gene in chromosome 7p22.1. FSCN1 is an actin-bundling protein involved in the formation of filopodia and invadopodia. 9,10 It is almost absent in most normal epithelial tissues, while it is expressed at high levels in many cancers. [11][12][13][14] Its upregulation has been associated with poor prognosis and metastatic spread in several carcinomas, as revealed by meta-analyses and systemic reviews. 13,14 Small molecule compounds inhibiting FSCN1 have shown their potential to inhibit metastasis formation in animal models of several types of cancer. [15][16][17] FSCN1 is overexpressed in ACC compared with normal adrenal tissue. 18 Its expression levels were significantly correlated to both shortened disease-free survival (DFS) and overall survival (OS) in three different cohorts of ACC patients 19 and high pre-operative circulating FSCN1 levels were predictors of recurrence. 20 FSCN1 has been shown to synergize with transcription factor SF-1 and the Rho/Rac guanine nucleotide exchange factor (GEF) VAV2 21 in enhancing the invasion properties of ACC cancer cells. 19 Based on those results, we investigated the effects of FSCN1 inactivation by CRISPR/Cas9 or pharmacological blockade on the invasive properties of ACC cells, both in vitro and in an in vivo metastatic ACC model in zebrafish. Our results indicate that FSCN1 is a new druggable target for ACC and provide the rationale for future clinical trials with FSCN1 inhibitors in patients with ACC.

| ACC patients
Diagnosis of ACC was done on the basis of routine histopathological analysis. Tumor stage was evaluated according to the European Network for the Study of Adrenal Tumors classification. 22 The clinical characteristics of the patients and their tumors' genetic alterations are reported in Table S1.

| Cell lines
H295R (RRID:CVCL_0458; derived from the primary tumor of a female patient with ACC) were obtained from ATCC. H295R/TR SF-1 GFP luc (a H295R subclone with Dox-inducible overexpression of the transcription factor SF-1), 21 MUC-1 (derived from a neck metastasis of a male patient with ACC), 23 CU-ACC2 (RRID: CVCL_RQ01; derived from a liver metastasis of a female patient with ACC) 24 and JIL-2266 cells (derived from the primary tumor of a female patient with ACC) 25 were generated and cultured as described. 21,[23][24][25] All cell lines were authenticated using STR profiling within the last 3 years and all experiments were performed with mycoplasma-free cells.

| Transwell invasion assay through Matrigel
It was performed as previously described. 21

| Proliferation and cell cycle analysis
Cell proliferation was evaluated by cell counting using a Countess 3 automatic cell counter (Thermo Fisher). Doubling times were calculated using the formula: Lamellipodia were defined as sheet-like protrusive structures extending from the cell edge and consisting mostly of dynamic, criss-crossed actin filaments, with ruffles being of similar morphology but not adhered and moving centripetally toward the main cell body. Quantification of focal adhesion size and number was performed by application of a threshold to all images using ImageJ to isolate and identify focal adhesions between 1 and 5 μm in size, as previously described. 28 Focal adhesion size was normalized to the total cell area and showed as the percentage of the cell occupied by focal adhesions.
In all analyses, at least 90 cells were evaluated. All samples were processed equally and evaluated blindly regarding sample identity.
2.9 | Reverse transcription quantitative polymerase chain reaction It was performed as described 21 29 and was previously shown to efficiently inhibit beta-catenin-dependent proliferation and target gene expression in those cells. 29 Results were calculated using the ΔΔC T threshold cycle method. 30

| Cancer cell xenografts in zebrafish
The adult transgenic zebrafish (Danio rerio) line Tg(kdrl:EGFP) was maintained as described. 36 Breeding of adult male and female zebrafish was carried out by natural crosses, and embryos were collected and raised in fish water with incubation at 28.5 C until the experi- Xenografts of vehicle-treated and Dox-treated (1 μg/ml) control and FSCN1 KO were realized as previously described. 37 Briefly,

| Statistical analyses
They were performed using GraphPad Prism software version 9.4, considering a P value <.05 as the threshold for a significant difference.

| FSCN1 inactivation in ACC cells induces defects in cell attachment to substrate and proliferation
To study the role of FSCN1 in an ACC cell model, several H295R cell clones were produced where FSCN1 was inactivated by CRISPR/Cas 9 recombination. To be able to study the effect of FSCN1 inactivation on biological effects caused by overexpression of the transcription factor SF-1, the H295R/TR SF-1 GFP luc subclone was used to perform FSCN1 knockout (KO), where SF-1 dosage can be increased by Dox treatment. 19,21 Two cell clones with successfully targeted FSCN1 and two clones transfected with a control sgRNA were selected at random for further experiments (Figure 2A). We verified the absence of recombination in potential off-target sequences in the FSCN1 KO clones (Table S2).  Figure S4 and Table S4).

| Effects of FSCN1 inactivation on steroidogenesis, gene and protein expression, and metabolic profiles in ACC cells
H295R cells are a differentiated ACC cell line producing a large variety of steroids. 32,40 Since cytoskeletal dynamics are known to affect steroid production in adrenocortical cells, 42 we compared the concentrations of several steroids in the culture medium in control and FSCN1 KO cells, both in basal conditions and after stimulation with FSK, a pharmacological activator of adenylate cyclase. FSCN1 inactivation had no effect on steroid production, both in basal conditions (Table S5) or after stimulation with FSK ( Figure S5).
To assess the impact of FSCN1 inactivation on global gene and protein expression in H295R cells, we performed RNA-seq and proteomic profiling of control and FSCN1 KO clones. A distinct set of genes was differentially expressed in FSCN1 KO cells compared with control ( Figure 3A and Table S6). Pathways related to cell architecture and motility were significantly enriched among those DEG ( Figure 3B). In proteomic analysis, only seven proteins were found to be differentially expressed in FSCN1 KO cells compared with control (Table S7).
Among those, adseverin/scinderin (SCIN) and visinin-like 1 (VSNL1) were consistently upregulated in FSCN1 KO cells, both at the transcript and protein levels ( Figure 3A, C and Tables S6 and S7). SCIN and VSNL1 mRNA upregulation in FSCN1 KO cells was confirmed by RT-qPCR ( Figure 3D). FSCN1 has been recently associated to the control of oxidative phosphorylation through regulation of the mitochondrial actin cytoskeleton. 26 To evaluate the impact of FSCN1 inactivation on global metabolic profiles in H295R cells, we performed untargeted metabolomics and identified a set of metabolites which were all depleted in FSCN1 KO compared with control cells (Table S8). Those included aspartic-glutamic acid and some pyrimidines.

| Inactivation of FSCN1 expression or pharmacological antagonism in ACC cells induces defects in Matrigel invasion in vitro
Overexpression of transcription factor SF-1 is a marker of malignancy in ACC. 43 An increased SF-1 dosage in the H295R cell line considerably increases their invasive properties in vitro and in vivo. 19,21 We have previously shown that FSCN1 knockdown or treatment with the FSCN1 small molecule inhibitor G2 impaired Matrigel invasion of H295R cells triggered by SF-1 overexpression. 19 We then investi-   Figure S1. n (independent experiments) = 6. Mean ± SD is shown. ****P < 0.0001, t test. Bottom: Representative micrographs of control and FSCN1 KO cells taken 48 h after plating. Scale bar, 20 μm. (D) FSCN1 KO cells proliferation is slower compared with control cells. Data are derived from the combined analysis of control #1 and #2 (orange line) and FSCN1 KO #1 and #2 H295R clones (violet line). Cells were cultured without adding Dox in the culture medium. Data for individual clones are shown in Figure S3. n (independent experiments) = 6. Mean ± SD is shown. ***P < .001, t test.

| DISCUSSION
We have shown in this preclinical study that FSCN1 has an important role in regulating ACC cell invasion in vitro and in increasing their metastatic potential in an in vivo zebrafish model.  links between FSCN1 and regulation of cell proliferation in ACC cells.
A limitation of this study is the absence of FSCN1 "put-back" experiments to confirm its function in FSCN1 KO cells. However, we believe that the data presented here are robust, since they were obtained by analysis of two different knock-out clones, where the presence of mutations in off-target genes produced by our CRISPR geneinactivating strategy was thoroughly excluded, as described in section 2.3 and shown in Table S2. Moreover, FSCN1 "put-back" data were shown in our previous study, 19 which fully validated the results obtained after FSCN1 knock-down by siRNA. Another limitation is the lack of validation of most transcriptomic and proteomic results obtained. However, we focused on the two genes that were commonly found to be upregulated in FSCN1 KO cells both in transcriptomic and proteomic studies (SCIN and VSNL1) and validated the results obtained by RT-qPCR ( Figure 3D). Furthermore, it has to be underlined that metastasis is a multistep process including migration of the tumor cells from their primary site, dissemination in the circulatory system, colonization and survival in secondary sites. 8 The zebrafish model we employed to study the effects of FSCN1 gene inactivation or pharmacological antagonism on metastasis formation in vivo does not allow to discriminate among those different steps.
We have shown here that either FSCN1 gene inactivation or treatment with the specific FSCN1 inhibitor G2-044 inhibited H295R cell invasion in Matrigel and in an in vivo metastatic ACC model in zebrafish. Remarkably, the FSCN1 inhibitor also antagonized the invasion of other ACC cell lines which express lower levels of FSCN1 compared with H295R ( Figure 4F). These data suggest that FSCN1 has an important function in the invasion process of ACC cell lines independently from its relative expression and that its inhibition may then be of therapeutic relevance even in cancers expressing low amounts of this protein. G2-044 is an orally available FSCN1 inhibitor which has an improved potency compared with previous inhibitors. 17 A phase I clinical trial that employed this molecule showed efficacy in some patients with advanced, treatment-refractory solid cancers. 46 Another trial is presently ongoing to study the effect of G2-044 monotherapy or in combination with an anti-PD-(L)1-immune checkpoint inhibitor on metastatic cancers (NCT05023486). In addition to its direct involvement in the metastatic process, recent studies have highlighted the effects of FSCN1 on the antitumoural immune response. In the TCGA ACC cohort, FSCN1 expression is negatively correlated with intratumoural effector and memory T cell signatures 47 and with PDL1 expression. 48 In our study, FSCN1 KO in H295R cells strongly upregulated expression of NLRP1, a gene encoding a key protein in the inflammasome 49 (Table S5). In syngeneic mouse models of various cancers FSCN1 inhibition by G2-044 synergized with immune checkpoint inhibitors and increased the numbers of intratumoural activated dendritic cells and activated CD8 + T cells, leading to increased animal survival. 50 It is likely that the success of innovative therapies for ACC will be dependent on the combination of immunotherapies with treatments that counteract the immunosuppressive tumor microenvironment typical of those cancers. 7 Our study provides the rationale and preclinical data in support of testing the efficacy of FSCN1 inhibitors in clinical trials including patients with ACC.

AUTHOR CONTRIBUTIONS
The work reported in the paper has been performed by the authors, unless clearly specified in the text.