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Fascin-1 is a Novel Prognostic Biomarker Associated with Tumor Invasiveness in Adrenocortical Carcinoma
Giada Poli, Carmen Ruggiero, Giulia Cantini, Letizia Canu, Gianna Baroni, Roberta Armignacco, Anne Jouinot, Raffaella Santi, Tonino Ercolino, Bruno Ragazzon, Guillaume Assie, Massimo Mannelli, Gabriella Nesi, Enzo Lalli, Michaela Luconi
The Journal of Clinical Endocrinology & Metabolism Endocrine Society
Submitted: August 10, 2018 Accepted: November 20, 2018 First Online: November 23, 2018
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Fascin-1 is a marker of ACC invasiveness
FASCIN-1 IS A NOVEL PROGNOSTIC BIOMARKER ASSOCIATED WITH TUMOR INVASIVENESS IN ADRENOCORTICAL CARCINOMA
Giada Poli1*, Carmen Ruggiero2-5*, Giulia Cantini1, Letizia Canu1,6, Gianna Baroni6,7, Roberta Armignacco1,8, Anne Jouinot’8,9, Raffaella Santi6,7, Tonino Ercolino1,6, Bruno Ragazzon’8, Guillaume Assie,8-10, Massimo Mannelli1,6, Gabriella Nesi6,7, Enzo Lalli2-5, Michaela Luconi1,6
‘Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy
2 Université Côte d’Azur, Sophia Antipolis, 06560 Valbonne, France;
3CNRS UMR7275, Sophia Antipolis, 06560 Valbonne, France;
4NEOGENEX CNRS International Associated Laboratory, Sophia Antipolis, 06560 Valbonne, France;
5 Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, 06560 Valbonne, France;
‘Careggi University Hospital (AOUC), Florence, Italy;
Department of Surgery and Translational Medicine, University of Florence, Florence, Italy;
8 Institut Cochin, INSERM U1016, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
9 Department of Endocrinology Cochin Hospital, Assistance Publique - Hôpitaux de Paris, Paris, France.1
10 Reference Center for Rare Adrenal Diseases Reference Center for Rare Adrenal Cancer Network COMETE, Hôpital Cochin, Assistance Publique - Hôpitaux de Paris, Paris, France
ORCID numbers:
0000-0001-5186-064X
Luconi
Michaela
Received 10 August 2018. Accepted 20 November 2018.
*These authors equally contributed to the work
CONTEXT: Novel tumor markers are urgently needed to better stratify adrenocortical cancer (ACC) patients and improve therapies for this aggressive neoplasm.
OBJECTIVE: To assess the diagnostic and prognostic value of the actin-bundling protein fascin- 1 (FSCN1) in adrenocortical tumors.
DESIGN, SETTING AND PARTICIPANTS: A local series of 37 malignant/37 benign adrenocortical tumors at Careggi University Hospital and two independent validation ACC cohorts (Cochin, TCGA) from the European Network for the Study of Adrenal Tumors were studied.
MAIN OUTCOME MEASURES: FSCN1 expression was quantified by immunohistochemistry, Western Blot and quantitative RT-PCR analyses in ACC specimens; overall and disease-free survival associated with FSCN1 expression were assessed by Kaplan-Meier analysis and compared with that of Ki67 labelling index and tumor stage.
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RESULTS: In spite of the low diagnostic power, in the Florence ACC series, FSCN1 immunohistochemical detection appeared as an independent prognostic factor, also refining results obtained with staging and Ki67 labelling index. The robust prognostic power of FSCN1 levels was further confirmed in two independent ACC cohorts. A positive correlation was found between FSCN1 and Steroidogenic Factor-1 (SF-1), with a significant higher expression of both factors in ACCs at advanced stages and with at least one of the three Weiss score parameters associated with invasiveness.
Moreover, we demonstrated FSCN1 role in promoting cell invasion in a human ACC cell line only in the case of increased SF-1 dosage.
CONCLUSIONS: These findings show that FSCN1 is a novel independent prognostic marker in ACC and may serve as a potential therapeutic target to block tumor spread.
Tumor invasion markers are needed to better stratify adrenocortical cancer. We demonstrate the robust independent prognostic power of Fascin-1 in three independent adrenocortical cancer cohorts. .
INTRODUCTION
Adrenocortical cancer (ACC) is a rare endocrine tumor with poor prognosis, particularly when metastatic at diagnosis. It lacks selective and efficacious therapies, which currently consist of surgical resection (RO) and administration of the adrenolytic drug mitotane [1] in association with cytotoxic agents in advanced stages [2]. Recently, significant advances in the molecular diagnosis and prognosis of the tumor have been achieved by integrated genetic profile analysis of large patient cohorts [3-4]. Nevertheless, the mechanisms inducing an aggressive and metastatic phenotype in ACC remain elusive [5]. So far, few studies have searched for protein markers capable of not only discriminating between benign forms of adrenocortical tumors (ACA) and ACC, but also predicting tumor progression [5]. A recent proteomic study [6] identified Fascin-1 (FSCN1) as a potential malignancy marker by a two-dimensional-differential-in-gel- electrophoretic (2D-DIGE) approach performed in ACC versus normal adrenals. FSCN1 is an actin-bundling protein involved in formation of filopodia and invadopodia [7-8]. It is almost absent in most normal epithelial tissues and highly expressed in many human carcinomas [9]. Its upregulation has been associated with a poor prognostic value and metastatic spread in several carcinomas, as revealed by recent meta-analyses and systemic reviews [10-11].
The aim of the present study was to assess the diagnostic and prognostic value of FSCN1 compared with the current histopathological parameters in a local monocentric series of 74 adrenocortical tumours (37 ACCs and 37 ACAs). Results were further confirmed in two independent ACC validation cohorts. Finally, we investigated the role of this protein in promoting tumor cell invasion in a human ACC cell line that overexpresses the transcription factor Steroidogenic Factor-1 (SF-1) resulting in a more aggressive and invasive phenotype [12].
MATERIALS AND METHODS
Patients and ethical approval
All patients, or their parents in the case of pediatric patients, gave their written informed consent to the study. The study consists of a local cohort of n=37 patients affected by malignant (ACC) and n=37 by benign (ACA) adrenal tumors, whose clinical characteristics are detailed in Tables 1 and 2, respectively. All patients underwent surgical removal of the tumor at Careggi University Hospital in Florence (Florence series). Tumor samples were snap frozen and stored at -80℃ until protein/mRNA extraction, or were formalin-fixed/ paraffin embedded for
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immunohistochemistry. The study was approved by the Local Ethical Committee (Prot.2017-277 BIO 59/11, 27/09/2017).
Histology and immunohistochemistry
Histological diagnosis of ACC and ACA was recorded by a reference pathologist (G.N.) on tumor tissue removed at surgery. Tumor specimens were evaluated according to the Weiss score system in which the presence of three or more criteria highly correlates with malignant behavior [13].
The Ki67 labelling index (Ki67 LI) was evaluated as a proliferation marker to assess ACC prognosis using the anti-human Ki67 antibody (1:40 dilution, MIB-1, Dako, Carpenteria, CA, USA). Ki67 positive nuclei were counted in 1000 tumor cells and Ki67 LI was expressed as the percentage of labelled cells.
Tumor stage was evaluated according to the revised TNM classification of ACC proposed by the European Network for the Study of Adrenal Tumours [14].
Immunohistochemical analysis was performed as previously described [6]. Briefly, 3 um sections were de-paraffinized, hydrated with graded ethanol concentrations until distilled water. Sections were stained with anti FSCN1 antibody (1:100 dilution, sc-46675, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti synaptophysin antibody (prediluted, MRQ-40, Cell Marque, Rocklin, CA, USA) after treatment with 3.0% hydrogen peroxide in PBS. Immunohistochemical analysis was carried out using DAKO EnVision™M FLEX (Dako, Carpenteria, CA, US). Negative controls were incubated without the primary antibody.
Immunostaining results were analyzed using a light microscope at high magnification. FSCN1 staining intensity was evaluated independently by two investigators blind to the clinical data (G.N. and R.S.). The inter-observer agreement for the scoring system was evaluated by using the Cohen k coefficient (0.98) and confirmed by using Pearson’s correlation coefficient (0.95). In the case of discrepancy, a score was agreed on by a joint re-evaluation of the slides. Cytoplasmic staining intensity was estimated using a score of 0, 1, 2, or 3, which corresponded to negative, weak, moderate, and strong intensity. The proportion of positive tumor cells was calculated for each specimen and scored 0 if 0%, 0.1 if 1 to 9%, 0.5 if 10 to 49%, and 1 if >50% of tumor cells were positive for FSCN1. A semiquantitative H-score was then calculated by multiplying the staining intensity grade by the proportion score [15]. The cut-off point for separating samples with high or low FSCN1 expression was between the H-scores < 2 or ≥ 2.
SDS-PAGE and Western blot analysis
Tissue samples were homogenized by mechanical disruption with Ultraturrax T10 basic IKA (Werke Gmbh & Co, Staufen, Germany) in RIPA lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton-100, 1 mM Na3 VO4, 1 mM PMSF). Cell samples were prepared as previously described [16].
After protein measurement using the Bradford method, equal amounts of proteins for each cell and tissue sample (30 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Immobilon, Merck Millipore, Milan, Italy). Membranes were incubated overnight at 4℃ with the following primary antibodies: anti FSCN1 (sc-46675, Santa Cruz Biotechnology), anti VAV2 (ab52640, Abcam, Cambridge, UK), anti SF-1 (07-618, Millipore), anti beta-tubulin (T4026, Sigma-Aldrich), anti beta-actin (sc-1615, Santa Cruz Biotechnology), and anti GFP (ab6556, Abcam) followed by species-specific peroxidase-conjugated secondary IgG (1:2000) incubation at room temperature for 1 hour. Image acquisition was performed with a ChemiDoc XRS instrument (BIO-RAD Labs, CA, USA). Semiquantitative densitometric analysis of FSCN1 band intensity was performed by Quantity One software of analysis (BIO-RAD Labs),
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with a grading score of low or high intensity, corresponding to absence/band intensity below or above the intensity of the FSCN1 band of the internal standards for ACC (ISACc) or ACA (ISACA). ISACC and ISACA consist of the same total protein amount derived from equal amounts of all ACC or ACA samples, respectively, run in each gel. All Western blots were repeated in at least three independent experiments. Beta-actin or beta-tubulin was used as internal loading standards to normalize protein expression.
RNA isolation and quantitative real-time RT-PCR
mRNA isolated from tissues of the local ACC cohort was subjected to quantitative real-time RT- PCR (qRT-PCR) for the following gene transcripts: SF-1/NR5A1, FSCN1 and GAPDH (Taqman gene expression assay, Applied Biosystems: Hs000610436_m1, Hs0060251_m1, 4352934). The amount of target, normalized to the endogenous reference gene (GAPDH) and relative to a calibrator (Stratagene), was calculated by 2-44Ct
Cell culture
The H295R/TR SF-1 cell line has been developed and fully characterized in our laboratory [12, 16]. Cells were cultured in Dulbecco’s modified Eagle’s medium-F12 supplemented with penicillin-streptomycin, 2% NuSerum (BD Biosciences), 1% ITS+ (BD Biosciences) and blasticidin (5 µg/ml) - zeocin (100 µg/ml) (both from Cayla InvivoGen). Doxycycline (Sigma- Aldrich) was used at the concentration of 1 ug/ml in experiments involving increased SF-1 expression.
Immunofluorescence and filopodia detection and quantification
This was performed as described [16] using Alexa Fluor-594 phalloidin (Invitrogen) to visualise F-actin. For each condition, between 120 and 360 cells were scored.
Transwell invasion assay through Matrigel
This was performed as described [16] using the CytoSelect 24-well cell invasion assay kit according to the manufacturer’s instructions (CBA-100, Cell Biolabs). In invasion experiments, the FSCN1 inhibitor G2 (Xcessbio Biosciences) was used at the concentration of 10 uM.
Knock-down and rescue experiments
The following siRNAs were used in knockdown experiments:
- siFascin #1: GAGCAUGGCUUCAUCGGCU [17]
- siFascin #2: CACGGGCACCCUGGACGCCAA [17]
- VAV2: AGUCCGGUCCAUAGUCAAC [16]
- control siC (medium GC; Invitrogen)
Cells were transfected by Amaxa nucleofection, as described [16]. For rescue experiments, plasmids encoding GFP (pEGFP-C2; Clontech), GFP-X. tropicalis fascin [18] and RNAi insensitive GFP-VAV2 [16] were co-transfected together with the siRNAs.
Statistical analysis
Data was expressed as mean±SD or in cell experiments as mean±SEM. Statistical analysis was performed by SPSS 24.0 (Statistical Package for the Social Sciences, Chicago, US) for Windows. Correlation analyses were carried out using a x2 test for categorical and Pearson’s/Spearman’s test for parametric/ nonparametric continuous variables, respectively. The inter-observer agreement for the scoring system was evaluated by using the Cohen k coefficient and confirmed by using Pearson’s correlation coefficient. The cut-off for strong agreement chosen for the k coefficient was 0.81, and 0.75 for the Pearson coefficient [19]. Differences in
continuous variables were analyzed by means of the Student’s t test for independent data to compare two classes of data, or 1-way ANOVA with Bonferroni’s correction for multiple testing.
For FSCN1 mRNA analysis three independent ACC cohorts were studied (local, Cochin and TCGA). The local cohort consisted of n=21 ACC samples where the amount of FSCN1 normalized on GAPDH expression was measured by qRT-PCR Taqman analysis (Taqman gene expression assay, Applied Biosystems: Hs0060251_m1, 4352934). The Cochin cohort included 48 ACCs (Gene Expression Omnibus data set GSE49280 and ArrayExpress data set E-TABM- 311) [3], and the TCGA cohort included 78 ACCs (https://gdc-portal.nci.nih.gov/
projects/TCGA-ACC) [4]. Patients’ data is listed in Tab.3. For the Cochin cohort, all samples were normalized using the Robust Multiarray Average algorithm (Bioconductor affy package), and probe set intensities were then averaged per gene symbol. For the TCGA cohort, mRNA sequencing data was extracted from Broad Institute GDAC Firehose (TCGA data version 2015_08_21), and all calculations were performed on log2 values of RSEM-normalized read counts. Differential abundance was measured with moderated t test (limma R package). To avoid introducing bias by identification of the best cut-off, the median value was used in each cohort.
Š
RESULTS
Diagnostic power of FSCN1 expression measurement
FSCN1 expression was evaluated by immunohistochemistry and Western blotting in samples from a series of n=37 ACCs and n=37 ACAs diagnosed and operated at Careggi University Hospital. Patients’ clinico-pathological characteristics are described in Table 1 (ACCs) and Table 2 (ACAs).
FSCN1 immunohistochemical expression was high (H score ≥ 2) in 79% (26/33) of ACCs, whereas an intense FSCN1 band was detected in 71% (15/21) of ACC by Western blot analysis. Conversely, only 55% of ACAs (20/36) showed a strong immunoreactivity for FSCN1 (H score ≥ 2) and the corresponding band was observed with high intensity in 17% (6/36) of ACAs by Western blotting, with x2=4.2, p=0.04 (in IH), and x2=17.1, p<0.001 (in Western blotting), between ACCs and ACAs for the two techniques, respectively. Correlation between the two techniques was more stringent and significant in ACCs than ACAs (x=11, p=0.001, and x2=6, p=0.014, respectively).
Representative images of FSCN1 staining scores are illustrated in Fig.1A. FSCN1 expression was assessed by semiquantitative Western blot analysis of representative ACC and ACA samples, demonstrating the relatively lower expression of FSCN1 in benign vs. malignant neoplasms (Fig.1B).
Notably, FSCN1 distribution was not homogenous in all ACC samples, tending to concentrate at the periphery of the tumor mass in advanced ACCs (Fig.2B). Non-tumoral tissue areas were negative for FSCN1 (Fig.2B, asterisks). Any artefact due to non-homogenous fixation (rim effect) could be excluded, since immunostaining of tumor serial sections showed strong and diffuse reactivity for neuroendocrine markers, i.e.synaptophysin (Fig.2C).
Prognostic power of FSCN1 expression
In ACC cases, no significant correlation was found between clinico-pathological parameters (age, sex, tumor diameter, functional activity, stage, Weiss score and Ki67 LI) and FSCN1 detected by immunohistochemistry, Western blot or qRT-PCR.
To assess the value of FSCN1 in predicting disease-free (DFS) and overall (OS) survival, we constructed Kaplan-Meier curves stratifying ACC patients according to high and low FSCN1
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immunohistochemical expression: FSCN1 proved to be significantly associated with DFS (Fig.3A) and OS (Fig.3B).
When estimating the prognostic value of tumor stage (Fig.3C,D) and Ki67 LI (Fig.3E,F) in the Florence ACC series, the addition of FSCN1 immunohistochemical reactivity as a second parameter for stratification in Kaplan-Meier analysis significantly improved prediction of DFS (Fig.3G,I) and OS (Fig.3H,J).
Expression levels of the adrenocortical lineage marker SF-1 have previously been proposed as an independent immunohistochemical prognostic marker in ACC associated with tumor aggressiveness [15,20]. We therefore measured both SF-1 and FSCN1 gene expression by qRT- PCR in the Florence ACC cases, and found a significant correlation between SF-1 and FSCN1 transcript levels (r=0.773, p<0.001), n=21, Fig.4A).
When stratifying patients in two groups based on their clinical parameters (Stage: 3-4 vs. 1-2; Ki67 LI: ≥10 vs. < 10), FSCNI expression detected by qRT-PCR was significantly higher in tumor high-risk groups (Fig.4B). Interestingly, FSCN1 levels were significantly higher in tumors displaying at least one of the three Weiss score parameters associated with invasion (i.e. sinusoidal, venous and capsular invasion), “invasive” Weiss score (Fig.4B). A significant positive correlation was found between FSCN1 expression classes (low and high, with cut-off value defined as the median of FSCN1 gene expression distribution) and Weiss score, stratified according to at least one of the three parameters associated with invasion (x == 4.11, p=0.043). Stratification of the Weiss score by invasion characteristics significantly correlated (x=11.5, p=0.001) with stage stratification by tumor aggressiveness (stage 1-2 vs. 3-4). These findings suggest that FSCN1 is associated with invasive ACC capabilities. Similar results were obtained for quantitative SF-1 expression (Fig.4C), when cases were stratified according to tumor stage, further confirming that SF-1 is overexpressed in aggressive ACCs [15-20].
Similar to what was found for FSCN1 protein expression, Kaplan-Meier analysis obtained by stratifying patients in low and high expression levels of FSCNI transcript showed that it correlated with DFS (Fig.5A) and OS (Fig.5B). Notably, prognostic analysis performed on two independent validation cohorts of ACC patients, the Cochin [3] and TCGA [4] series, confirmed the significant role of FSCNI expression in both tumor recurrence (Fig.5C,E) and OS (Fig.5D,F). The calculated hazard risk (HR) values were comparable for DFS and OS, respectively, in all three cohorts (Fig.5).
FSCN1 is involved in supporting the invasive phenotype of ACC cells overexpressing SF-1 Since FSCN1 has been implicated in the migration, invasion and metastasis of several cancer cell types [18,21-22], we investigated its role in regulating the invasive phenotype of ACC H295R cells. We used the H295R cell model overexpressing SF-1 in a doxycycline (Dox)-inducible fashion (H295R/TR SF-1), having previously demonstrated that increased SF-1 dosage in H295R cells affects cytoskeleton remodeling and invasive properties through upregulation of the guanine nucleotide exchange factor VAV2 [16]. Two different siRNAs were able to efficiently downregulate FSCNI expression in H295R/TR SF-1 cells (Fig.6A). Importantly, FSCNI knock- down could significantly inhibit filopodia formation associated with the increased SF-1 dosage in H295R cells (Fig.6B), although it had no effect on the number of cells showing filopodia under conditions of basal SF-1 dosage. Similarly, FSCN1 knock-down or pharmacological inhibition with G2, a small molecule that hinders FSCN1 actin-bundling activity, blocked cell invasion through Matrigel only when SF-1 dosage was increased, with no effect on their invasive properties in the presence of SF-1 basal levels (Fig.6C-E). This phenotype is strikingly similar to the effects of VAV2 knock-down, which also selectively inhibits H295R cell invasion only
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under conditions of increased SF-1 dosage [16]. For this reason, we tested whether VAV2 and FSCN1 could compensate for the effects of each other’s knock-down in invasion assays. As shown in Fig. 6F, transfection of expression vectors encoding GFP-Fascin-1 and GFP-VAV2 could both rescue the effect of FSCN1 knock-down in H295R/TR SF-1 cells treated with Dox. GFP-VAV2 but not GFP-Fascin-1 completely rescued the effect of VAV2 knock-down under the same conditions. Knock-down specificity by siFSCN1- siVAV2 and expression of the GFP- fusion proteins is shown in Fig.7.
DISCUSSION
Recent ACC guidelines suggest the importance of integrating molecular analysis with classical clinical parameters to improve the management of this rare and aggressive tumor [23]. Over the last decade, significant advances have been made in identifying alterations in candidate genes and their expression. Nevertheless, very few routinely screenable protein markers have been recognized as being associated with classical clinical parameters, thus allowing improvement in ACC prognostication [5]. Given that these proteins mark the metastatic signature of ACC, they could also represent potential targets for the development of novel personalized anti-cancer therapies.
In the present paper, we confirm our previous finding that FSCN1 is differentially expressed in ACCs and normal adrenals [6], with no FSCN1 reactivity in normal tissue areas, but strong positivity in neoplastic areas. We now demonstrate that FSCN1 cannot statistically be considered a valid marker for differential diagnosis between malignant and benign neoplasms; however, FSCN1 protein levels in the Florence series were generally lower in ACA than ACC samples.
Our previous paper [6] also demonstrated that FSCN1 expression is absent in normal adrenal cortex, confirming results obtained for other organs and their tumor counterparts [9]. This suggests that FSCN1 displaces other actin-bundling proteins in the transformed cells, promoting and stabilizing formation of filopodia and invadopodia, specialized and organized actin-rich cell protrusions that favor cell migration [8].
Interestingly, FSCN1 localization in ACCs is highly heterogeneous, from diffuse to focalized expression. The tendency to localize at the tumor border may be the consequence of acquiring migratory and invasive properties in certain subpopulations of the tumor cells, which become more prone to invading the surrounding tissues. FSCN1 expression in breast cancer has also been demonstrated as a key feature in supporting trans-endothelial migration [24], a pivotal step in the metastatic process.
Our data shows that FSCN1 levels are significantly higher in the more aggressive tumors, when stratified for stage and Weiss score parameters of invasion (at least for one positive parameter among sinusoidal, venous and capsular invasion), but not for Ki67 LI, implying that FSCN1 correlates with the metastatic potential rather than the proliferative properties of the tumor. We suggest that the Weiss score parameters may differ in weight in relation to the proliferative or invasive properties of the tumor, an aspect that could be considered for better stratification of ACCs. Notably, cases in the Florence series revealed a significant correlation between advanced stages (3-4) and “invasive” Weiss score. We observed a similar behavior for SF-1 immunostaining, with higher levels in aggressive than indolent ACCs, and a significantly high correlation between the expression of these two markers obtained by qRT-PCR.
As described for SF-1 [15, 20], Kaplan-Meier analysis of the Florence ACC cohort showed that FSCN1 immunohistochemical expression can also significantly predict disease recurrence and OS. Moreover, when combined with stage or Ki67 LI, FSCN1 can refine their
prognostication power, so providing a useful protein marker for a more accurate stratification of recurrence risk in patients with ACC. Until now, Ki67 LI has been considered the most powerful predictor of disease recurrence and survival in ACC patients after complete tumor resection [25]. However, due to difficulties in its standardization and reproducibility for many tumor types, including ACC [26], other histopathological parameters, such as the mitotic index [27] or VAV2 [28] have been tested. Here, we have shown that FSCN1 and Ki67 LI are independent parameters and are likely to be associated with two different tumor characteristics, i.e. invasive/metastatic potential and proliferation. Notably, when considering the risk of recurrence, FSCN1 greatly improved the predictive prognostic power of Ki67 LI and stage. In addition, the similar results obtained for the three independent ACC cohorts show that quantitative FSCNI gene expression is a robust independent prognosticator, as no significant correlation was found between FSCN1 and clinico-pathological parameters. Interestingly, three different and independent analytical techniques were employed for each of the three cohorts regarding gene expression quantitative evaluation and correlation analysis, but very similar results in terms of HR were obtained in all three cohorts. These findings not only strengthen our results, but also suggest that different methods can be routinely applied to measure FSCNI abundance.
Together with VAV2, FSCN1 is one of the few histopathological markers associated with invasion, and combined with genetic parameters, that could be used as an independent factor to further improve patient stratification.
In addition to its prognostic value, FSCN1 may also represent a novel therapeutic target for ACC, particularly in the advanced stages, where therapeutic options are rather disappointing and call for new and more effective drugs. Our results demonstrate that interfering with FSCN1 by gene silencing or chemical inhibition significantly reduced the increased tumor migration and invasiveness observed in a H295R cell line conditionally overexpressing SF-1, and consequently more aggressive than its wild type counterpart.
Repression of invadopodia and filopodia formation by inhibiting FSCN1 is not only important in deterring the migratory ability of cancer cells, but it may also be pertinent to potential immunotherapy strategies. It has been demonstrated that glioma cells silenced for FSCNI were more susceptible to cytotoxic lymphocyte attack [29].
A recent paper reports that the specific FSCN1 competitive inhibitor G2, we used in our in vitro experiments, could block breast cancer invasion and metastatic colonization in a mouse model, with no toxicity or side effects after 2-month treatment [22]. This molecule is highly specific for FSCN1 as it binds directly to one of the actin-binding sites on the protein, stabilizing FSCN1 in its inactive conformation and blocking filopodia formation. Since FSCN1 is expressed at low levels in normal tissues and overexpressed in ACC and other tumors, its inhibition seems a promising strategy, as it acts specifically on tumor cells.
VAV2 is a direct SF-1 dosage-dependent target gene that, through its GEF activity, is essential to induce increased migration in Matrigel when SF-1 expression is forced in the H295R ACC cell line [16]. Remarkably, our results show that FSCN1 and VAV2 are both required in filopodia formation and invasiveness only under conditions of increased SF-1 dosage in H295R cells. In particular, silencing or inhibition of FSCN1 suppressed the increased cell invasiveness when SF-1/VAV2 is overexpressed (Fig. 4C, D). Transfected VAV2 could rescue the effect of FSCN1 knock-down on cell invasion, but FSCN1 overexpression could not completely compensate for the effect of VAV2 silencing. These results are consistent with a model whereby VAV2 acts to enhance Cdc42 and Rac1 activation and induce actin polymerization, and finally
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FSCN1 stabilizes the actin bundles. The final result of this process is the induction of cytoskeletal remodeling and increased cell invasion (Fig.8).
Recently, FSCN1-induced expression and filopodia formation have been reported to mediate the migratory and invasive effects exerted by linoleic acid in an in vitro cell line model of breast cancer (30). This unsaturated acid can also stimulate steroidogenesis in adrenocortical cells in vitro (31). Functional ACCs, particularly cortisol-secreting forms, are associated with worse prognosis (32,33). Interestingly, in the Florence series, association analysis between FSCN1 immunoreactivity and cortisol secretion in ACCs displays a trend toward significance x2=3.5, p=0.06, which suggests that dietary unsaturated fatty acids, such as linoleic acid (LA), may trigger FSCN1 upregulation in ACC cells, eliciting tumor progression and invasiveness. Further studies investigating any correlation between LA levels and FSCN1 expression in ACC are mandatory to validate this hypothesis.
FSCN1 expression also occurs in benign tumors but not in normal tissue, thus excluding its use as a diagnostic marker in adrenocortical neoplasms. A similar distribution of FSCN1 has been reported in both benign and malignant colorectal tumors, despite the prognostic power maintained by this marker in adenocarcinomas (34). However, progression from the benign to the malignant form is still debated for adrenocortical tumors (35,36). All together, these findings indicate that FSCN1 may be an essential but not exclusive factor to trigger malignancy, suggesting that additional factors are necessary for acquiring the malignant phenotype. VAV2, which cooperates with FSCN1 to induce ACC cell invasion, may be one of these factors. Further studies are required to elucidate the exact mechanisms by which FSCN1, SF .- 1 and VAV2 may concur to support ACC invasive properties.
We recognize as a limitation of this study that, for each analysis and correlation, a variable number of ACC samples were considered, since some data were not available for all patients and some techniques could not be applied to all samples (FSCN1 immunohistochemistry and Western blot analysis, FSCN1 and SF-1 RNA expression). In particular, FSCN1 immunostaining could not be performed in the validation cohorts, due to the retrospective nature of data available and the lack of tissue samples.
a FUNDINGS
In conclusion, survival analysis conducted in three ACC independent cohorts shows that FSCN1 expression is a robust independent prognostic marker. Since FSCN1 is a key protein in promoting tumor cell functions involved in invasion, it could also serve as a potential therapeutic target to specifically interfere with ACC spread and metastasis.
Acknowledgements
We thank Dr. M. Parsons for her kind gift of the GFP-Fascin-1 expression vector and for critical reading of the manuscript, and F. Aguila for artwork: all the figures in the manuscript are original creations.
This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) Investigator Grant 2015 to M. L. (grant # IG2015-17691) and AIRC-CRF Multi-user Equipment Program 2016 to M. L. (grant # 19515). Seventh Framework Program (FP7/2007-2013) under grant agreement nº 259735 ENS@T-Cancer.
Associazione Italiana per la Ricerca sul Cancro
http://dx.doi.org/10.13039/501100005010, IG2015-17691, Michaela Luconi; Seventh Framework Programme http://dx.doi.org/10.13039/501100004963, FP7/2007-2013,
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Massimo Mannelli; Associazione Italiana per la Ricerca sul Cancro -Cassa Risparmio Firenze, 19515, Michaela Luconi
Correspondence to: Michaela Luconi, PhD, Endocrinology Unit, Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale Pieraccini 6-50139 Firenze, Italia, Tel: +39 055 2758239, e-mail:
DISCLOSURE SUMMARY:
The authors have nothing to disclose.
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8. Johnson HE, King SJ, Asokan SB, Rotty JD, Bear JE, Haugh JM. F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling. J Cell Biol. 2015;208:443-55.
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Management of Adrenocortical Carcinoma in Adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol. 2018 Jul 24. pii: EJE-18-0608. doi: 10.1530/EJE-18-0608. [Epub ahead of print]
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28. Sbiera S, Sbiera I, Ruggiero C, Doghman-Bouguerra M, Korpershoek E, de Krijger RR, Ettaieb H, Haak H, Volante M, Papotti M, Reimondo G, Terzolo M, Luconi M, Nesi G, Mannelli M, Libé R, Ragazzon B, Assié G, Bertherat J, Altieri B, Fadda G, Rogowski-Lehmann N, Reincke M, Beuschlein F, Fassnacht M, Lalli E. Assessment of VAV2 Expression Refines Prognostic Prediction in Adrenocortical Carcinoma. J Clin Endocrinol Metab. 2017;102:3491- 3498.
29. Hoa NT, Ge L, Erickson KL, Kruse CA, Cornforth AN, Kuznetsov Y, McPherson A, Martini F, Jadus MR. Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity. Am J Transl Res. 2015;7:271-84.
0. Gonzalez-Reyes C, Marcial-Medina C, Cervantes-Anaya N, Cortes-Reynosa P, Salazar EP. Migration and invasion induced by linoleic acid are mediated through fascin in MDA-MB- 231 breast cancer cells. Mol Cell Biochem. 2018;443:1-10.
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Fig.1. FSCN1 protein expression in adrenal tumors. (A) Representative immunohistochemical staining of FSCN1 (right panel, 20X magnification) for each intensity score (0-3) coupled with hematoxylin-eosin section (left panel) in ACC specimens. (B) Representative Western blotting for FSCN1 performed on ACCs and ACAs with different intensity grading (high and low, evaluated vs. the respective internal standards, ISAcc and ISACA). Lanes were normalized with respect to actin.
Fig.2. FSCN1 localization in ACC. Hematoxylin-eosin staining of a representative advanced ACC (5X magnification, A). Immunohistochemistry shows positivity concentrated at the tumor border for FSCN1 (B) but not for synaptophysin (C). Asterisks indicate normal tissue areas, negative for FSCN1 immunohistochemistry.
Fig.3. FSCN1 as detected by IH predicts disease free survival and overall survival in ACC. Disease free (DFS) and overall (OS) survival Kaplan-Meier curves according to low/high classes of FSCN1 immunohistochemical expression (A and B), low (1-2)/high (3-4) stages (C and D), Ki67 LI <10/≥10 classes (E and F), or when samples were stratified in 3 classes combining FSCN1 and stage (G and H) or FSCN1 and Ki67 LI (I and J); p values determined using a Log- Rank test, and number of cases in each group are indicated.
Fig.4. FSCN1 and SF-1 quantitative gene expression in ACC stratified for clinical characteristics. (A) Positive correlation between FSCNI and SF-1 gene expression (with GAPDH as a reference gene) evaluated by Taqman quantitative real time RT-PCR in ACC samples; r=0.773, p<0.001, n=21. Bar graphs represent mean±SEM FSCNI (B) and SF-1 (C) gene expression (with GAPDH as a reference gene) evaluated by Taqman quantitative RT-PCR in ACC samples stratified for stage, Ki67 LI, and Weiss score parameters of invasiveness (“invasive” Weiss score defined as at least one positive parameter among sinusoidal invasion, venous invasion, capsular invasion); p values (*p<0.05, ** p<0.005, and *** p<0.001) were calculated by Student’s t-test. Cut-off values to define each class and number of samples (n) are indicated.
Fig.5. High FSCN1 transcript levels are a negative prognostic factor in ACC: a multicenter analysis. Disease free (DFS) and overall (OS) survival Kaplan-Meier curves according to FSCN1 transcript classification in low/high levels in three independent ACC cohorts, local (A,B), Cochin (C,D) and TCGA (E,F). Log rank and Hazard Risk (HR) are indicated, with respective p values.
Fig.6. FSCN1 selectively regulates cytoskeleton remodeling and invasion in H295R cells overexpressing SF-1. (A) Immunoblots showing FSCN1, SF-1, VAV2 and beta-tubulin expression in H295R/TR SF-1 cells transfected with a control siRNA (siC) or two different siRNAs directed against FSCN1 (siFSCN1 #1 and siFSCN1 #2), in basal culture conditions or
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after treatment with doxycycline (Dox; 1 ug/ml) to induce SF-1 overexpression, as indicated. (B) Data is reported as mean+SEM percentage of filopodia-forming cells in H295R/TR SF-1 cells transfected with siC or siFSCN1 #1 in basal culture conditions or upon Dox treatment in n=3 independent experiments; *** p<0.001, 1-way ANOVA with Bonferroni’s correction for multiple testing. (C,D) Invasion through Matrigel by H295R/TR SF-1 cells transfected with siC or siFSCN1 #1 (C), with siC or siFSCN1 #2 (D), as well as treated or untreated with the FSCN1 inhibitor G2 (10 µM) (E), under basal culture conditions or upon Dox treatment. Data is reported as meantSEM percentage of Matrigel-invading cells over ctrl in H295R/TR SF-1 cells, under basal culture conditions or upon Dox treatment; * p<0.05 and *** p<0.001, 1-way ANOVA with Bonferroni’s correction for multiple testing in n=3 (C) or n=4 (E) independent experiments; ** p<0.01 Mann-Whitney test in n=6 independent experiments (D). (F) Data is reported as meantSEM percentage of Matrigel-invading cells over ctrl in H295R/TR SF-1 cells treated with Dox transfected with siC, siFSCN1 #1 or siVAV2 and expression plasmids for GFP, GFP- FSCN1 or GFP-VAV2, as evaluated in n=3 independent experiments; * p<0.05, ** p<0.01, *** p<0.001, ns (not significant). 1-way ANOVA with Bonferroni’s correction for multiple testing.
Fig.7. Specificity of knock-down and rescue experiments in H295R cells. (A) Specificity of FSCN1 and VAV2 knock-down by the respective siRNAs. (B) Expression of GFP, GFP-FSCN1 and GFP-VAV2 proteins in the experiments shown in Fig. 5F.
Fig.8. A schematic hypothesis of the role of VAV2 and FSCN1 in promoting cytoskeletal remodeling and invasion of cancer cells. By its GEF activity, VAV2, a gene transcriptionally upregulated following SF-1 overexpression in ACC cells, favors activation of Cdc42 and Rac1, which promote cytoskeleton remodeling and filopodia - lamellipodia/ruffles formation. These structures are stabilized by FSCN1, leading to increased cell migration and invasion.
| Mean (SD) | N Patients | % | |
|---|---|---|---|
| Age at surgery (years) | 45 (17) | 37 | 100 |
| Sex | |||
| Male | 12 | 32 | |
| Female | 25 | 68 | |
| Secretion | |||
| Non secreting | 10 | 27 | |
| Glucocorticoids | 14 | 38 | |
| Sex Steroids | 9 | 24 | |
| Mineralocorticoids | 1 | 3 | |
| NA | 3 | 8 | |
| Tumor diameter (cm) | 8.8 (5.7) | 37 | 100 |
| Ki67 LI (%) | 19.3 (20.7) | 37 | 100 |
| Weiss score | 5.8 (1.8) | 37 | 100 |
| ENSAT Stage | |||
| I | 7 | 19 | |
| II | 10 | 27 | |
| III | 14 | 38 | |
| IV | 4 | 11 | |
| NA | 2 | 5 | |
| Metastases | Lung, liver, bone, pancreas, contralateral adrenal | 9 | 24 |
| Surgery | 37 | 100 |
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| MTT therapy | 18 | 49 | |
| Other Chemotherapies (EDP, taxol) | 10 | 27 | |
| Radiotherapy | 3 | 8 | |
| Follow-up from surgery (months) | 59.2 (49.5) | 37 | 100 |
Etoposide-Doxorubicin-Cisplatin combined chemotherapy (EDP), Mitotane (MTT), Not available (NA).
| Mean (SD) | N Patients | % | |
|---|---|---|---|
| Age at diagnosis (years) | 53 (13) | 37 | 100 |
| Sex | |||
| Male | 15 | 40 | |
| Female | 22 | 60 | |
| Secretion | |||
| Non secreting | 7 | 19 | |
| Sex Steroids | 15 | 40 | |
| Mineralocorticoids | 7 | 19 | |
| NA | 8 | 22 | |
| Tumor diameter (cm) | 3.0 (2.1) | 37 | 100 |
| Follow-up surgery/diagnosis (months) | 30.7(16.8) | 37 | 100 |
| Surgery | 37 | 100 |
LE
Table 3. General characteristics of the Cochin and TCGA validation ACC cohorts.
Characteristics
| Cochin cohort (n=48) | TCGA cohort (n=78) | |
|---|---|---|
| Age (Median [range]) | 44 [18-82] | 50 [14-77] |
| Sex | ||
| Female | 37 | 47 |
| Male | 11 | 31 |
| ENSAT stage | ||
| I | 4 | 9 |
| II | 26 | 36 |
| III | 2 | 16 |
| IV | 16 | 15 |
| NA | - | 2 |
Detailed characteristics of the Cochin and TCGA cohorts are available in references [3] and [4], respectively. Not available (NA)
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200-
400-
300
150-
200-
200-
100-
100-
50-
0
0
0
sic
+
+
sic
+
+
+
siFSCN1
+
+ +
siFSCN1
+ +
G2 Dox
+
+
Dox
+
OX
+
F
ns
200
*
**
ADV
invading cells (% of ctrl)
**
1
150
100
50
0
sic
+
siFSCN1 siVAV2 GFP
+
+
+
+
+ +
+
+
+
GFP - Fascin GFP - VAV2
+
+
+
+
ADVANCE ARTICLE: JCEM
A
siVAV2
siFSCN1 #1
SİVAV2
sic
sic
kDa
kDa
VAV2
100
FSCN1
55
ß-tubulin
55
ß-tubulin
55
ADVANCE ARA
B
kDa
130
100
70
GFP
55
25
ß-tubulin
55
siC
+
siFSCN1
+ + + +
siVAV2
GFP GFP - Fascin GFP - VAV2
+ + + T + + +
+
+ +
filopodia ARTICLE
VAV2
CDC42
actin polymerization
FSCN1
DIRECTIONAL MIGRATION
ADVAN’G
THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
ADVANCE ARTICLE: JCEM
ENDOCRINE SOCIETY
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