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Biomedicine & Pharmacotherapy
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9
biomedicine AND PHARMACOTHERAPY
FGF/FGFR inhibitors downmodulates c-Myc oncoprotein and hampers the growth of adrenocortical carcinoma
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Sara Taranto a,b,1 D, Jessica Faletti ª,1, Mariangela Tamburello aD, Roberto Ronca a,CD, , Giulia Garattini a, Giorgia Gazzaroli a, Marta Turati ª, Edoardo Rocca , Riccardo Castelli , Andrea Abate ªD, Luca Mignani ª, Marta Laganà e, Constanze Hantel1,8, Sandra Sigala ª, Marco Presta ª, Arianna Giacomini ª İD
a Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
b Clinical Trial Center, Translational Research and Phase I Unit, ASST Spedali Civili di Brescia, Brescia, Italy
” Consorzio Interuniversitario per le Biotecnologie (CIB), Italy
d Department of Food and Drug, University of Parma, Parma, Italy
e Medical Oncology Department, ASST Spedali Civili of Brescia, Brescia, Italy
‘ Department of Endocrinology, Diabetology and Clinical Nutrition, University Hospital Zurich (USZ) and University of Zurich (UZH), Zürich, Switzerland % Medizinische Klinik und Poliklinik III, University Hospital Carl Gustav Carus Dresden, Dresden, Germany
ARTICLE INFO
Keywords:
Adrenocortical carcinoma Fibroblast growth factors (FGF) FGF/FGFR inhibitors c-Myc oncoprotein
ABSTRACT
Adrenocortical carcinoma (ACC) is a rare endocrine neoplastic disease that originates from the cortical cortex of adrenal gland. Unfortunately, after complete resection of the tumor, ACC relapses either locally or with distant metastasis in about 74 % of patients and for these patients the therapeutic options are still severely limited. In this study we demonstrate that the system composed by the fibroblast growth factors (FGFs) and their receptors (FGFRs) might represent a promising therapeutic target for ACC. Indeed, human ACC specimens and cell lines express FGF ligands and FGF receptors and show FGFR activation, suggesting the presence of an autocrine FGF/ FGFR loop of stimulation able to sustain ACC growth. Accordingly, inhibition of FGFR activation by TK inhibitors (erdafitinib and infigratinib) or FGF trapping (by NSC12) significantly hampered ACC growth and survival in vitro. Importantly, oral administration of erdafitinib strongly affected tumor growth in vivo by reducing tumor cell proliferation/survival and tumor angiogenesis. Mechanistically, FGF/FGFR inhibition in ACC cells strongly decreased the levels of the oncoprotein c-Myc and induced oxidative stress and DNA damage, leading to reduced tumor cell proliferation and increased tumor cell apoptosis. Altogether these results demonstrate for the first time the impact of FGF/FGFR blockade on ACC cell growth and survival both in vitro and in vivo. This study may set the rationale to start clinical trials investigating the therapeutic potential of FDA approved FGFR-TK inhibitors for the treatment of aggressive ACC.
1. Introduction
Adrenocortical carcinoma (ACC) is a rare endocrine neoplastic dis- ease with an incidence in adults ranging from 0.7 to 2 million cases per year [1,2]. The prognosis for ACC patients attests around a median overall survival of 3-4 years, heterogeneously distributed according to stage based on ENS@T classification and other prognostic factors [1,2]. ACC originates from the cortical cortex of adrenal gland, physiologically deputed to the production of a variety of hormones [3]. Indeed, in
addition to non-specific symptoms related to the presence of an abdominal mass, ACC also manifests symptoms due to hormonal excess in 50-60 % of patients. Hypercortisolism is the most frequent condition, followed by pure hyperandrogenism and, rarely, oestrogen or mineral- ocorticoid excess [4-6]. The metastasis process of ACC involves the peritoneum, liver, and lung more frequently [1,2,7]. The disease can also affect the bones and, occasionally, the brain [8]. Surgery performed in highly specialized center is the gold standard treatment for localized resectable disease, possibly followed by adjuvant treatment with the
* Corrosponding author.
E-mail address: arianna.giacomini@unibs.it (A. Giacomini).
1 Co-first authors.
https://doi.org/10.1016/j.biopha.2025.118677
Available online 21 October 2025
0753-3322/ 2025 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
adrenolytic drug mitotane (M) in case of high risk of recurrence [9]. In case of non-resectable/metastatic disease, the first-line systemic therapy is based on mitotane (M), either administered alone or in combination with the chemotherapy agents etoposide (E), doxorubicin (D), and cisplatin (P) (EDP-M scheme) [10,11]. Even though EDP-M can achieve complete pathological remissions in almost 7 % of patients, it is common for those receiving this regimen to experience disease relapse and resistance [12]. New therapeutic targets and strategies are therefore urgently needed [13], even though the scarcity of preclinical models so far available to study ACC make the development of new therapies extremely difficult [14].
The fibroblast growth factor (FGF) family includes 18 secreted members and 4 intracellular FGF homologous factors. The secreted members are grouped into six subfamilies based on phylogenetic anal- ysis and sequence homology and include canonical (FGF1/2/5, FGF3/4/ 6, FGF7/10/22, FGF8/17/18, FGF9/16/20) and endocrine (FGF19/21/ 23) FGFs. Secreted FGFs interact with tyrosine kinase (TK) FGFRs (FGFR1-4) that trigger intracellular signaling cascades [15]. An aber- rant activation of the FGF/FGFR system has been found in different types of cancer where it plays a crucial role by affecting tumor cell proliferation and survival, angiogenesis, and drug resistance [15,16]. Indeed, several FGFR TK inhibitors as well as anti-FGFR/FGF mono- clonal antibodies and FGF ligand traps have been developed and tested in clinical trials involving mostly patients with solid tumors [17,18]. At present, four drugs belonging to the FGFR TK inhibitor (FGFR TKi) class (i.e., erdafitinib, pemigatinib, futibatinib and infigratinib) have received FDA approval for the treatment of urothelial cancer and chol- angiocarcinoma [19-21].
In the context of adrenal gland, FGF/FGFR axis acts both in physi- ological conditions (in the development and maintenance of tissue ho- meostasis) and in the pathogenesis of adrenocortical carcinoma [22]. Indeed, FGFR1 appears to be one of the most differentially expressed genes in ACC. Also, FGFR4 overexpression is observed in 88 % and 47 % of respectively pediatric and adult adrenocortical tumors, related in some cases to gene amplification [22]. Moreover, FGFR4 is higher expressed in late stages of ACC compared to lower grade tumors and associates with worst prognosis [22,23].
On these bases, this work is aimed at investigating the therapeutic potential of FGF/FGFR blockade by using two inhibitory approaches, one targeting FGF receptors with the FDA approved TK inhibitors erdafitinib and infigratinib, and the other one based on the trapping of FGF ligands at the extracellular level with the small molecule NSC12 previously identified in our laboratory [24,25].
2. Materials and methods
2.1. Cell cultures and reagents
H295R (CVCL_0458) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). H295R cells, derived from a primitive ACC [26], were cultured in DMEM/F-12, HEPES media (Gibco, Life Technologies, Carlsband, CA, USA), supplemented with 1 % ITS + Premix (Corning, NY, USA), 2.5 % Nu-Serum (Corning, NY, USA), and 1 % penicillin-streptomycin (Lonza Group AG, Basel, CH). MUC-1 cell line was kindly provided by Dr. Hantel. MUC-1 cells, established from an ACC neck metastasis of an EDP-M treated male patient [14,27], were cultured with Advanced DMEM/F12 media (Gibco, Life Technol- ogies, Carlsband, CA, USA), supplemented with 10 % FBS and 1 % penicillin-streptomycin. Both human ACC cell lines were tested regu- larly for Mycoplasma negativity and authenticated using STR profiling.
Erdafitinib (JNJ-42756493) and infigratinib (BGJ-398) were ob- tained from MedChemExpress (Monmouth Junction, NJ, USA) and NSC12 was synthetized as previously reported [25].
2.2. RT-PCR analysis
Total RNA was extracted using TRIzol Reagent (Invitrogen) accord- ing to manufacturer’s instructions. Two ug of total RNA were retro- transcribed with MMLV reverse transcriptase (Invitrogen) using random hexaprimers. Then, cDNA was analyzed by semiquantitative PCR using the following primers:
| FGFR1 | For: GGGCTGGAATACTGCTACAA Rev: GCCAAAGTCTGCTATCTTCATC |
| FGFR2 | For: GGATAACAACACGCCTCTCTT Rev: GCCCAAAGCAACCTTCTC |
| FGFR3 | For: TGGTGTCCTGTGCCTACC Rev: CCGTTGGTCGTCTTCTTGT |
| FGFR4 | For: AACCGCATTGGAGGCATT Rev: TCTACCAGGCAGGTGTATGT |
| FGF1 | For: TGAATGGGAACTCCCTTCC Rev: GCTTTCAAGACTCTTTGCCT |
| FGF2 | For: TGTGTCTATCAAAGGAGTGTG Rev: CCGTAACACATTTAGAAGCCA |
| FGF3 | For: ACTCTATGCTTCGGAGCAC Rev: GAGGCATACGTATTATAGCCCA |
| FGF4 | For: CCAACAACTACAACGCCTACGA Rev: CCCTTCTTGGTCTTCCCATTCT |
| FGF5 | For: ATGCAAGTGCCAAGTTCAC Rev: TGTATTGCTGAGGCATAGGT |
| FGF6 | For: GAAAGTGGCTATTTGGTGGG Rev: ATTTCCAGCAGGCTGTAGG |
| FGF7 | For: TGACTTTGCCTCGTTTATCA Rev: TGGCTACAAATGTGAACTGT |
| FGF8 | For: TCATCCGGACCTACCAACTC Rev: AATCTCCGTGAAGACGCAGT |
| FGF9 | For: TGGATTTCACTTAGAAATCTTCCC Rev: ATTCCAGAATGCCAAATCGG |
| FGF10 | For: GGAGAAAGCTATTCTCTTTCACC Rev: ATCTCCAGGATGCTGTACG |
| FGF16 | For: ACTCTATGGGTCGAAGAAACTC Rev: GAGGCATAGGTGTTGTACCA |
| FGF17 | For: CAACAAGTTTGCCAAGCTC Rev: TACTTCTCACTCTCAGCCC |
| FGF18 | For: GGACATGTGCAGGCTGGGCTA Rev: GTAGAATTCCGTCTCCTTGCCCTT |
| FGF19 | For: TTTGCTGGAGATCAAGGCA Rev: CCTCCGAGTACTGAAGCAG |
| FGF20 | For: CATTTCCTGTTGCCTCCTG Rev: CCAAGATACCGAAGAGGCT |
| FGF21 | For: ACCTGGAGATCAGGGAGGAT Rev: GCACAGGAACCTGGATGTCT |
| FGF22 | For: ATCAGACCATCTACAGTGCC Rev: ATCTTCTGCTCATCACACCT |
| FGF23 | For: ATCAGACCATCTACAGTGCC Rev: ATCTTCTGCTCATCACACCT |
2.3. Western blot analysis
Cells were washed in cold PBS and homogenized in NP-40 lysis buffer (1 % NP-40, 20 mM Tris-HCl pH 8, 137 mM NaCl, 10 % glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin). Protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad Laboratories). Expression of specific proteins were detected using specific antibodies indicated in the table below. ß-actin or GAPDH were used as loading controls. Chemi- luminescent signal was acquired by ChemiDoc™M Imaging System (Bio- Rad) and analyzed using the ImageJ software (http://rsb.info.nih.gov/i j/).
| ANTIBODY | SOURCE |
|---|---|
| Phospho-FGFR (Tyr 653/654) | Cell Signaling Technology |
| c-Myc | Cell Signaling Technology |
| Cleaved Caspase 3 | Cell Signaling Technology |
| Phospho-Histone H3 (Ser 10) | Merck Millipore |
| Nitrotyrosine | Merck Millipore |
| Phospho-H2AX (Ser 139) | Cell Signaling Technology |
| (continued on next page) |
(continued)
| ANTIBODY | SOURCE |
|---|---|
| CD31 | Dianova |
| B-Actin | Sigma-Aldrich |
| GAPDH | Santa Cruz Biotechnology |
2.4. Viable cell counting
Cells were cultured under appropriate conditions for 96 or 120 h. Propidium iodide staining (Immunostep, Salamanca, SP, EU) was used to detect PI negative viable cells by flow cytometry. Absolute cell counts were obtained by the counting function of the MACSQuant® Analyzer (Miltenyi Biotec).
2.5. Subcutaneous human xenografts
Experiments were performed according to the Italian laws (D.L. 116/ 92 and following additions) that enforce the EU 86/109 Directive and were approved by the local animal ethics committee (OPBA, Organismo Preposto al Benessere degli Animali, Università degli Studi di Brescia, Italy). Six- to eight-week-old female NOD/SCID mice were injected subcutaneously (s.c.) with H295R cells (5 x 10° cells/implant) in 200 ul of PBS. When tumors were palpable, mice were randomly assigned to receive everyday treatment with erdafitinib (30 mg/kg) or control/ vehicle DMSO in drinking water. Tumor volumes were measured with caliper and calculated according to the formula V = (D × d2)/2, where D and d are the major and minor perpendicular tumor diameters, respec- tively. At the end of the experimental procedure, tumor nodules were excised, weighed and processed for histological analysis.
2.6. Histological analyses
Tumor samples were fixed in formalin and embedded in paraffin. Formalin-fixed, paraffin-embedded samples were sectioned at a thick- ness of 3 um, dewaxed, hydrated, and processed for immunohisto- chemistry with rabbit anti-human/mouse pFGFR (Cell Signaling Technology), rabbit anti-human/mouse c-Myc (Cell Signaling Technol- ogy), rabbit anti-human/mouse nitrotyrosine (Millipore), rabbit anti- human/mouse pH2AX (Cell Signaling Technology), rabbit anti-human phospho-Histone H3 (Chemicon), rabbit anti-human/mouse cleaved Caspase3 (Cell Signaling Technology), or rat anti-mouse CD31 (Dia- nova) antibodies.
Sections were finally counterstained with Carazzi’s hematoxylin before analysis by light microscopy. Images were acquired with the automatic high-resolution scanner Aperio System (Leica Biosystems, Wetzlar, Germany, EU) and image analysis was carried out using the open-source ImageJ software.
2.7. Statistical analyses
Statistical analyses were performed using Prism 9 (GraphPad Soft- ware). Student’s t test for unpaired data (2-tailed) was used to test the probability of significant differences between two groups of samples. For more than two groups of samples, data were analyzed with a 1-way analysis of variance and corrected by the Bonferroni multiple compari- son test. Tumor volume data were analyzed with a 2-way analysis of variance and corrected by the Bonferroni test. Differences were considered significant when p < 0.05.
3. Results
3.1. Adrenocortical carcinoma cells express FGFs and FGFRs
To assess the expression of secreted FGF ligands (FGFs) and their
receptors (FGFRs) in ACC, we first analyzed datasets from The Cancer Genome Atlas (TCGA) by using the cBioPortal software platform. These datasets included genomic and proteomic data from 92 ACC patients (Table S1). Notably, about 66 % of ACC cases analyzed showed alter- ations of the FGF/FGFR system with about 15 % of patients presenting alterations of both FGFs and FGFRs (Fig. 1A-B, Table S2). Particularly, about 25 % of ACC cases showed high expression of at least one FGFR, FGFR1 and FGFR4 being the most upregulated (10 % and 13 % of all cases, respectively) (Fig. 1C and Fig. S1). Also, 58 % of ACC cases showed high expression of at least one secreted FGF (Fig. 1D), being FGF18 the most upregulated (21 % of all cases) (Table S2 and Fig. S2). High levels of FGFR and FGF expression were due mainly to mRNA upregulation and in some cases to gene amplification, whereas very rare structural genetic mutations were detected (Fig. S1 and S2). In keeping with these findings, H295R and MUC-1 ACC cell lines express all the four FGFRs and several secreted members of the FGF family (Fig. 1E). Also, both cell lines show the constitutive activation of FGFR signaling as demonstrated by the expression of phosphorylated (Tyr653/654) FGFR (pFGFR) in the absence of exogenous stimuli (i.e, without addition of exogenous FGFs) in the cell medium (Fig. 1F). These data strongly indicate the presence of an autocrine FGF/FGFR loop of stimulation in ACC cells (Fig. 1G).
3.2. FGF/FGFR inhibition affects ACC cell growth and survival in vitro
In order to assess the role of the FGF/FGFR system in ACC cell growth, we utilized two inhibitory approaches, one targeting the TK activity of FGFRs and the other one trapping the FGF ligands at the extracellular level. To this aim, H295R and MUC-1 cells were treated with increasing doses of the FGFR selective TK inhibitors erdafitinib and infigratinib (BGJ398) or with increasing doses of the pan FGF trap small molecule NSC12 (Fig. 2). Both FGFR-TK blockade (by erdafitinib or BGJ398) and FGF trapping (by NSC12) significantly reduced the growth and survival of H295R and MUC-1 cells with IC50 ranging from 2 to 7 µM (Fig. 2). Of note, FGF/FGFR blockade similarly affected ACC cells sen- sitive (H295R) and resistant (MUC-1) to the first-line therapy EDP-M [14]. These data indicate that autocrine FGF/FGFR stimulation may play a pivotal role in ACC biology.
3.3. The FGFR selective TK inhibitor erdafitinib hampers ACC growth in vivo
We next assess the therapeutic potential of the FDA approved TK inhibitor erdafitinib in vivo by using an ACC xenograft model. To this aim, H295R cells were subcutaneously implanted into immunodeficient mice. When tumors were palpable (28 days after tumor implantation), mice received everyday treatment with erdafitinib (30 mg/kg) or vehicle in drinking water (Fig. 3). In keeping with in vitro data, in vivo FGFR-TK blockade by erdafitinib significantly reduced the rate of growth of H295R xenografts compared to vehicle-treated animals with a significant decrease of tumor weight at the end of the experiment (day 49 after tumor implantation) (Fig. 3A).
Accordingly, immunohistochemical analysis of H295R tumor xeno- grafts showed high levels of FGFR phosphorylation that were drastically reduced in erdafitinib-treated animals compared to vehicle-treated mice (Fig. 3B). This was accompanied by a significant reduction of tumor cell proliferation as revealed by the staining for the proliferation marker phospho-Histone H3 (pHH3) (Fig. 3B). Various members of the FGF family expressed by ACC cells (e.g. FGF1, FGF2, FGF4, FGF5, and FGF8) have been shown to be endowed with a potent pro-angiogenic activity [28], thus suggesting that FGFs secreted by ACC cells in vivo may also exert a paracrine pro-angiogenic function in ACC. This hypothesis was supported by the presence of elevated levels of FGFR phosphorylation in endothelial cells of H295R xenografts (Fig. 3C). On this basis, we investigated the effect of FGFR-TK blockade on ACC neovascularization in vivo. As shown in Fig. 3C, treatment with erdafitinib strongly reduced
E
H295R
A
B
FGF altered
39
14
8
FGFR altered
31
92
61
31
☒ FGF/FGFR alterations (66.3%)
Not FGF/FGFR altered
☐ no FGF/FGFR alterations (33.7%)
C
3
D
5
2
7
5
6
3
1
92
1
19
92
40
2
69
21
FGFR1 (5.4%)
☐ FGFR2 (3.3%)
☒ FGFR1+FGFR4 (3.3%)
☐ FGFR2+FGFR3 (1.1%)
☒ one FGF altered (22.8%)
☒ FGFR3 (2.2%)
two FGFs altered (20.7%) ☒
☒ FGFR4 (6.4%)
☒ FGFR2+FGFR4 (1.1%)
☐ FGFR3+FGFR4 (2.2%)
☐ three FGFs altered (7.6%)
no FGFR alterations (75.0%)
☒ more then three FGFs altered (5.4%)
☐ no FGF alterations (43.5%)
F
RT-PCR
WB
MUC-1
H295R
MUC-1
H295R
MUC-1
H295R
MUC-1
KDa
FGFR1
FGF1
FGF17
120/145
pFGFR
FGF2
FGF18
FGFR2
42
ß-Actin
FGF4
FGF19
FGFR3
FGF5
FGF20
G
FGFR4
FGF6
FGF21
Autocrine stimulation
GAPDH
FGF8
FGF22
FGF
FGF9
FGF23
FGF10
GAPDH
FGFR
vascular FGFR phosphorylation in H295R xenografts. This was paral- leled by a significant reduction of tumor vascularization, as revealed by immunohistochemical analysis of CD31+ vessels that appear also less elongated and anastomized in tumors treated with erdafitinib when compared to controls (Fig. 3D).
3.4. Blockade of FGFR activation strongly reduces c-Myc protein levels in ACC cells
The activation of FGFR signaling is involved in c-Myc stabilization in different tumor types, FGF trapping or FGFR-TK inhibition leading to the
A
H295R
Erdafitinib
BGJ398
NSC12
Viable Cell Counts (% Ctrl)
Viable Cell Counts (% Ctrl)
100-
100
Viable Cell Counts (% Ctrl)
100
75
75
75
IC50 ~ 7.4 µM
50
IC50 - 1.93 µM
50
50
IC50 ~2.9 µM
25
25
25
0-
0-
0-
0.1
0.3
1.0
3.2
10.0
30.0
0.1
0.3
1.0
3.2
10.0
0.1
0.3
1.0
3.2
10.0
μΜ
μΜ
μΜ
100-
100-
100
PI+ Cells (%)
75
PI+ Cells (%)
75
PI+ Cells (%)
75
50
50
50
25
25
25
0
0
0
DMSO
0.1
0.3
1
3
6
10
30
DMSO
0.3
1
3
6
DMSO
0.1
0.3
0.1
10
1
3
6
10
μΜ
μΜ
μΜ
B
MUC-1
Erdafitinib
BGJ398
NSC12
Viable Cell Counts (% Ctrl)
100-
Viable Cell Counts (% Ctrl)
100-
Viable Cell Counts (% Ctrl)
100-
75
75
75
IC50 ~ 5.5 UM
IC 50~ 3.8 μΜ
IC50~5.3 μΜ
50
50
50
25
25
25
0-
0-
0-
0.1
0.3
1.0
3.2
10.0
30.0
0.1
0.3
1.0
3.2
10.0
30.0
0.1
0.3
1.0
3.2
10.0
30.0
μΜ
μΜ
μΜ
100
100-
100-
PI+ Cells (%)
75
PI+ Cells (%)
75
PI+ Cells (%)
75
50
50
50
25
25
25
0.
0-
0-
DMSO
0.1
0.3
A
3
6
10
30
DMSO
0.1
0.3
M
3
6
10
30
DMSO
0.1
0.3
1
3
6
10
30
μΜ
μΜ
μΜ
B
pFGFR
pHH3
C
DMSO
Erdafitinib
A
350-
Vehicle
+ Erdafitinib 30 mg/kg
0.4
V
Erdaf
Tumor volume (mm3)
300
Tumor weight (g)
250
0.3
200
*
150
0.2
100
**
**
50
0.1
0-
30 35 40 45 5
50
0.0
Days post implantation
V
Erdaf
30mg/kg
0
1
DMSO
Erdafitinib
pFGFR+ Area (%)
60
pHH3+ Cells (nº/field)
50
40
40
30
20
20
10
0
0
DMSO
Erdafitinib
DMSO
Erdafitinib
pFGFR
D
CD31
DMSO
20
CD31+ Area (%)
15
10
5
Erdafitinib
0
DMSO
Erdafitinib
-
proteasomal degradation of the c-Myc protein [29-33]. So far, little is known about the role of c-Myc in ACC growth and progression. Analysis of the same datasets from The Cancer Genome Atlas (TCGA) used for FGF and FGFR expression revealed that about 9 % of ACC patients show altered c-Myc expression due to gene amplification (1.1 %), high mRNA levels (5.4 %), and high protein levels (2.2 %) (Fig. 4A and S3). Inter- estingly, 100 % of patients with c-Myc alterations also showed altered expression of FGF and/or FGFR (Table S2). Also, a recent work by Pennannen et al. demonstrated that c-Myc protein expression in ACC
samples is associated to malignancy and shorter survival [34]. Accord- ingly, TCGA data analysis revealed that higher c-Myc mRNA expression was observed in recurred/progressed (R/P) patients compared to dis- ease free (DF) patients (Fig. 4B) and correlated with worse overall sur- vival (Fig. 4C).
In keeping with these findings, inhibition of the FGF/FGFR axis by erdafitinib, BGJ398, or NSC12 significantly reduced c-Myc protein levels in ACC cells (Fig. 4D). Accordingly, immunohistochemical anal- ysis of H295R tumor xenografts showed high levels of c-Myc protein that
A
B
C
Tumor Adrenocortical Carcinoma (v32)
Disease free status
tcga - 79 - tpm - gencode36 MYC (ENSG00000136997.21) Expression cutoff: 7.55 (min.grp=8) WITH_SURV (n=49)
8
MYC mRNA expression
15
1.0
high (n=23) low (n=26)
0.9
**
92
12
OS probability
0.8
0.7
0.6
9
-
0.5
0.4
84
6
0.3
0.2
0.1-
☒ c-Myc alterations (8.7%)
3
raw p 0.026
bonf p 0.874
0.0
☐ no c-Myc alterations (91.3%)
DF R/P
0
20
40
60
80
100
120
140
160
Follow up in months
D
H295R cells
MUC-1 cells
E
Erdafitinib
BGJ398
NSC12
c-Myc
DMSO
DMSO
Erdafitinib
BGJ398
NSC12
KDa
pFGFR
120/145
PFGFR
120/145
DMSO
60
c-Myc
57/65
c-Myc
57/65
GAPDH
39
GAPDH
39
40
20
☐ pFGFR
c-Myc
☐ PFGFR
c-Myc
Densitometry (fold change vs Ctrl)
1.2
Densitometry (fold change vs Ctrl)
c-Myc+ Area (%)
1.2.
1.0
1.0
**
0.8
0.8
Erdafitinib
0
**
*
**
**
*
*
0.6
*
*
0.6
*
DMSO
Erdafitinib
**
0.4
0.4
0.2
0.2
0.0
0.0
Erdafitinib
BJG398
NSC12
Erdafitinib
BJG398
NSC12
Erdafitinib
BJG398
NSC12
Erdafitinib
BJG398
NSC12
were drastically reduced in erdafitinib-treated animals compared to vehicle-treated mice (Fig. 4E).
3.5. FGFR inhibition triggers oxidative stress, DNA damage, and ACC cell death
In previous works we have demonstrated that the decrease of c-Myc protein levels induced by FGF/FGFR blockade triggers oxidative stress and DNA damage, leading to tumor cell death [29,31,32]. On this basis, we investigated whether the same molecular mechanism is recapitulated also in ACC cells. To this aim, H295R cells were treated for 24 h with 10 uM erdafitinib and assessed for oxidative stress and DNA damage. As shown in Fig. 5A, the decrease of FGFR phosphorylation and c-Myc protein levels was paralleled by a significant increase of the oxidative stress and DNA damage markers nitrotyrosine and yH2AX, respectively. These effects eventually led to impaired H295R cell proliferation and induction of cell death after 96 h of treatment with 10 µM erdafitinib (see Fig. 2A).
Similar results were observed in vivo (Fig. 5B). Indeed, immuno- histochemical analysis of H295R tumors from the experiment shown in Fig. 3A revealed a strong increase of nitrotyrosine and yH2AX levels in erdafitinib-treated mice when compared to controls. This was accom- panied by a significant increased tumor cell death, as assessed by the analysis of the apoptotic marker cleaved caspase 3 (Fig. 5B).
4. Discussion
Adrenocortical carcinoma (ACC) is a rare endocrine neoplastic dis- ease with few therapeutic options. Meagre experimental evidence re- ported in the literature suggests that the FGF/FGFR system may play a crucial role in the growth of ACC. Indeed, FGFR1 and FGFR4 and their ligands FGF8, FGF9, FGF19 and FGF21 have been found significantly upregulated in patient-derived ACC samples [22,23]. Importantly, a negative correlation between FGFR1 and FGFR4 expression and patient survival endpoints has also been shown [23].
Data reported herein strongly strengthen the studies so far present in the literature supporting a role for the FGF/FGFR system in the biology of ACC and demonstrate the therapeutic potential of targeting FGFRs or FGF ligands in this type of aggressive solid cancer. Indeed, our findings demonstrate that ACC cells express both FGF ligands and receptors and basally show FGFR activation, suggesting the presence of an autocrine FGF/FGFR loop of stimulation able to sustain the growth and survival of ACC cells. Accordingly, inhibition of FGFR activation by the selective FGFR-TK inhibitors erdafitinib and BGJ398 or trapping extracellular FGFs by the FGF trap NSC12 significantly hampered in vitro the growth and survival of ACC cells both sensitive (H295R) and resistant (MUC-1) to the first-line therapy EDP-M [14]. Importantly, oral treatments with erdafitinib strongly affected tumor growth in vivo by reducing tumor cell proliferation/survival and tumor angiogenesis.
Mechanistically, FGF/FGFR inhibition strongly reduced the protein levels of the oncoprotein c-Myc in ACC cells causing oxidative stress, DNA damage, reduced tumor cell proliferation, and eventually leading
A
Erdafitinib 10 LM
B
Nitrotyrosine
YH2AX
cCaspase3
DMSO
DMSO
KDa
pFGFR
120/145
c-Myc
57/65
Nitrotyrosine
32/66
Erdafitinib
YH2AX
15
GAPDH
39
Densitometry (fold change vs Ctrl)
3
Nitrotyrosine+ Area (%)
100
YH2AX+ Cells (nº/field)
800
cCasp 3+ Cells (nº/field)
400
2
75
600
300
*
50
400
200
1
*
** I
25
200
100
0
0
0
0
PFGFR
c-Myc
Nitrotyrosine
YH2AX
DMSO
Erdafitinib
DMSO
Erdafitinib
DMSO
Erdafitinib
to tumor cell apoptosis. These findings are in keeping with our previous work demonstrating the pivotal role of FGFR activation in stabilizing the intracellular levels of the oncoprotein c-Myc and the role of c-Myc in preventing mitochondrial oxidative stress [29]. So far little is known about the expression of the oncoprotein c-Myc in ACC cells and its role in ACC growth and progression. However, the Wnt/ß-catenin signaling, which is known to sustain the expression of MYC gene [35,36], is often found deregulated in ACC cells [37]. Indeed, H295R cells used in this work harbor an activating mutation in the gene codifying for ß-catenin (CTNNB1) thus causing the constitutive activation of the transcription activity of ß-catenin [14]. Accordingly, we observed high expression of c-Myc protein in H295R cells both in vitro and in vivo. Also, our data demonstrate that about 9 % of ACC patients show altered c-Myc expression, due to gene amplification, high mRNA levels, and/or high protein levels. Importantly, higher c-Myc mRNA expression is observed in recurred/progressed patients compared to disease free patients and correlates with worse overall survival. This is in keeping with a recent work by Pennannen et al. demonstrating that c-Myc protein expression in ACC samples associates with malignancy and shorter survival [34].
Altogether these results demonstrate for the first time that FGF/FGFR blockade can represent a promising novel therapeutic strategy for ACC. Although we are aware of the limitation of this study due to the scarcity of preclinical models so far available to study ACC, data herein reported shed a light in the potential efficacy of FGF/FGFR inhibitors for the treatment of ACC and may provide important information that could be rapidly translated into the clinics. Indeed, this study may set the ratio- nale to start clinical trials investigating the therapeutic potential of FDA approved selective FGFR-TK inhibitors (i.e pemigatinib, futibatinib, erdafitinib) for the treatment of aggressive ACC with poor prognosis. Also, our findings highlight the oncoprotein c-Myc as a new target in ACC that can be indirectly inhibited by FGF/FGFR blockade. On these bases and in a translational perspective, it would be worth to assess the levels of FGFR activation and c-Myc expression in patient-derived samples as new predictive biomarkers for the use of FGF/FGFR in- hibitors in ACC.
CRediT authorship contribution statement
Luca Mignani: Methodology, Investigation. Andrea Abate: Meth- odology, Investigation. Constanze Hantel: Writing - review & editing, Investigation. Jessica Faletti: Writing - review & editing, Methodology, Investigation, Data curation. Marta Laganà: Methodology, Investiga- tion. Sara Taranto: Writing - review & editing, Methodology, Investi- gation, Data curation. Marta Turati: Methodology, Investigation. Riccardo Castelli: Methodology, Investigation. Edoardo Rocca: Methodology, Investigation. Marco Presta: Writing - review & editing, Supervision. Roberto Ronca: Writing - review & editing, Methodology, Investigation. Sandra Sigala: Writing - review & editing, Supervision. Mariangela Tamburello: Methodology, Investigation. Giorgia Gaz- zaroli: Methodology, Investigation. Arianna Giacomini: Writing - original draft, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization. Giulia Garattini: Methodology, Investigation.
Ethics approval and consent to participate
Animal Experiments were performed according to the Italian laws (D.L. 116/92 and following additions) that enforce the EU 86/109 Directive and were approved by the local animal ethics committee (OPBA, Organismo Preposto al Benessere degli Animali, Università degli Studi di Brescia, Italy).
Consent for publication
Not applicable.
Funding
AG, RR, MP, SS were supported by AIRC (IG 2023 - ID. 28939, IG 2019 - ID. 23151, IG 2019 - ID. 18493, IG 2022 - ID. 27233, respec- tively). GG is grateful to Consorzio Interuniversitario per le Bio- tecnologie (CIB) and Fondazione Guido Berlucchi for their support.
Competing interests
The authors declare no competing interests.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors thank Prof. Alfredo Berruti (University of Brescia and Medical Oncology Department, ASST Spedali Civili of Brescia, Italy) for his precious scientific support in the field of ACC, and Dr. Alessia Tribbia (University of Brescia) for her technical support.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2025.118677.
Data availability
Data will be made available on request.
References
[1] M. Fassnacht, O.M. Dekkers, T. Else, E. Baudin, A. Berruti, R. de Krijger, et al., European society of endocrinology clinical practice guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors, Eur. J. Endocrinol. 179 (4) (2018) G1-G46.
[2] M. Fassnacht, G. Assie, E. Baudin, G. Eisenhofer, C. de la Fouchardiere, H.R. Haak, et al., Adrenocortical carcinomas and malignant phaeochromocytomas: ESMO- EURACAN clinical practice guidelines for diagnosis, treatment and follow-up, Ann. Oncol. 31 (11) (2020) 1476-1490.
[3] D.C. De Silva, B. Wijesiriwardene, The adrenal glands and their functions, Ceylon Med. J. 52 (3) (2007) 95-100.
[4] A. Vaidya, M. Nehs, K. Kilbridge, Treatment of adrenocortical carcinoma, Surg. Pathol. Clin. 12 (4) (2019) 997-1006.
[5] A. Berruti, M. Fassnacht, H. Haak, T. Else, E. Baudin, P. Sperone, et al., Prognostic role of overt hypercortisolism in completely operated patients with adrenocortical cancer, Eur. Urol. 65 (4) (2014) 832-838.
[6] M. Fassnacht, R. Libe, M. Kroiss, B. Allolio, Adrenocortical carcinoma: a clinician’s update, Nat. Rev. Endocrinol. 7 (6) (2011) 323-335.
[7] D.S. Freire, S.A. Siqueira, M.C. Zerbini, B.L. Wajchenberg, M.L. Correa-Giannella, A.M. Lucon, et al., Development and internal validation of an adrenal cortical carcinoma prognostic score for predicting the risk of metastasis and local recurrence, Clin. Endocrinol. 79 (4) (2013) 468-475.
[8] A. Turla, M. Lagana, V. Cremaschi, M. Zamparini, L. De Maria, F. Consoli, et al., Outcome of brain metastases from adrenocortical carcinoma: a pooled analysis, J. Endocrinol. Invest. 47 (1) (2024) 223-234.
[9] A. Berruti, S. Grisanti, A. Pulzer, M. Claps, F. Daffara, P. Loli, et al., Long-Term outcomes of adjuvant mitotane therapy in patients with radically resected adrenocortical carcinoma, J. Clin. Endocrinol. Metab. 102 (4) (2017) 1358-1365.
[10] A. Berruti, M. Terzolo, P. Sperone, A. Pia, S. Della Casa, D.J. Gross, et al., Etoposide, doxorubicin and cisplatin plus mitotane in the treatment of advanced adrenocortical carcinoma: a large prospective phase II trial, Endocr. Relat. Cancer 12 (3) (2005) 657-666.
[11] M. Fassnacht, M. Terzolo, B. Allolio, E. Baudin, H. Haak, A. Berruti, et al., Combination chemotherapy in advanced adrenocortical carcinoma, N. Engl. J. Med. 366 (23) (2012) 2189-2197.
[12] M. Lagana, S. Grisanti, D. Cosentini, V.D. Ferrari, B. Lazzari, R. Ambrosini, et al., Efficacy of the EDP-M scheme plus adjunctive surgery in the management of patients with advanced adrenocortical carcinoma: the brescia experience, Cancers 12 (4) (2020).
[13] V. Cremaschi, A. Abate, D. Cosentini, S. Grisanti, E. Rossini, M. Lagana, et al., Advances in adrenocortical carcinoma pharmacotherapy: what is the current state of the art? Expert Opin. Pharm. 23 (12) (2022) 1413-1424.
[14] S. Sigala, E. Rossini, A. Abate, M. Tamburello, S.R. Bornstein, C. Hantel, An update on adrenocortical cell lines of human origin, Endocrine 77 (3) (2022) 432-437.
[15] Y. Xie, N. Su, J. Yang, Q. Tan, S. Huang, M. Jin, et al., FGF/FGFR signaling in health and disease, Signal Transduct. Target Ther. 5 (1) (2020) 181.
[16] A. Giacomini, P. Chiodelli, S. Matarazzo, M. Rusnati, M. Presta, R. Ronca, Blocking the FGF/FGFR system as a “two-compartment” antiangiogenic/antitumor approach in cancer therapy, Pharm. Res. 107 (2016) 172-185.
[17] R. Porta, R. Borea, A. Coelho, S. Khan, A. Araujo, P. Reclusa, et al., FGFR a promising druggable target in cancer: molecular biology and new drugs, Crit. Rev. Oncol. Hematol. 113 (2017) 256-267.
[18] G.C. Ghedini, R. Ronca, M. Presta, A. Giacomini, Future applications of FGF/FGFR inhibitors in cancer, Expert Rev. Anticancer Ther. 18 (9) (2018) 861-872.
[19] Y. Loriot, A. Necchi, S.H. Park, J. Garcia-Donas, R. Huddart, E. Burgess, et al., Erdafitinib in locally advanced or metastatic urothelial carcinoma, N. Engl. J. Med. 381 (4) (2019) 338-348.
[20] G.K. Abou-Alfa, V. Sahai, A. Hollebecque, G. Vaccaro, D. Melisi, R. Al-Rajabi, et al., Pemigatinib for previously treated, locally advanced or metastatic cholangiocarcinoma: a multicentre, open-label, phase 2 study, Lancet Oncol. 21 (5) (2020) 671-684.
[21] L. Goyal, F. Meric-Bernstam, A. Hollebecque, J.W. Valle, C. Morizane, T.B. Karasic, et al., Futibatinib for FGFR2-rearranged intrahepatic cholangiocarcinoma, N. Engl. J. Med. 388 (3) (2023) 228-239.
[22] M. Tamburello, B. Altieri, I. Sbiera, S. Sigala, A. Berruti, M. Fassnacht, et al., FGF/ FGFR signaling in adrenocortical development and tumorigenesis: novel potential therapeutic targets in adrenocortical carcinoma, Endocrine 77 (3) (2022) 411-418.
[23] I. Sbiera, S. Kircher, B. Altieri, K. Lenz, C. Hantel, M. Fassnacht, et al., Role of FGF receptors and their pathways in adrenocortical tumors and possible therapeutic implications, Front. Endocrinol. 12 (2021) 795116.
[24] R. Ronca, A. Giacomini, E. Di Salle, D. Coltrini, K. Pagano, L. Ragona, et al., Long- pentraxin 3 derivative as a small-molecule FGF trap for cancer therapy, Cancer Cell 28 (2) (2015) 225-239.
[25] R. Castelli, A. Giacomini, M. Anselmi, N. Bozza, F. Vacondio, S. Rivara, et al., Synthesis, structural elucidation, and biological evaluation of NSC12, an orally available fibroblast growth factor (FGF) ligand trap for the treatment of FGF- dependent lung tumors, J. Med. Chem. 59 (10) (2016) 4651-4663.
[26] W.E. Rainey, K. Saner, B.P. Schimmer, Adrenocortical cell lines, Mol. Cell. Endocrinol. 228 (1-2) (2004) 23-38.
[27] I C. Hantel, I. Shapiro, G. Poli, C. Chiapponi, M. Bidlingmaier, M. Reincke, et al., Targeting heterogeneity of adrenocortical carcinoma: evaluation and extension of preclinical tumor models to improve clinical translation, Oncotarget 7 (48) (2016) 79292-79304.
[28] R. Ronca, A. Giacomini, M. Rusnati, M. Presta, The potential of fibroblast growth factor/fibroblast growth factor receptor signaling as a therapeutic target in tumor angiogenesis, Expert Opin. Ther. Targets 19 (10) (2015) 1361-1377.
[29] R. Ronca, G.C. Ghedini, F. Maccarinelli, A. Sacco, S.L. Locatelli, E. Foglio, et al., FGF trapping inhibits multiple myeloma growth through c-Myc degradation- Induced mitochondrial oxidative stress, Cancer Res. 80 (11) (2020) 2340-2354.
[30] A. Sacco, C. Federico, A. Giacomini, C. Caprio, F. Maccarinelli, K. Todoerti, et al., Halting the FGF/FGFR axis leads to antitumor activity in Waldenstrom macroglobulinemia by silencing MYD88, Blood 137 (18) (2021) 2495-2508.
[31] A. Giacomini, S. Taranto, S. Rezzola, S. Matarazzo, E. Grillo, M. Bugatti, et al., Inhibition of the FGF/FGFR system induces apoptosis in lung cancer cells via c-Myc downregulation and oxidative stress, Int. J. Mol. Sci. 21 (24) (2020).
[32] S. Taranto, R. Castelli, G. Marseglia, L. Scalvini, F. Vacondio, A. Gianoncelli, et al., Discovery of novel FGF trap small molecules endowed with anti-myeloma activity, Pharm. Res. 206 (2024) 107291.
[33] A. Giacomini, S. Taranto, G. Gazzaroli, J. Faletti, D. Capoferri, R. Marcheselli, et al., The FGF/FGFR/c-Myc axis as a promising therapeutic target in multiple myeloma, J. Exp. Clin. Cancer Res. 43 (1) (2024) 294.
[34] M. Pennanen, J. Hagstrom, I. Heiskanen, T. Sane, H. Mustonen, J. Arola, et al., C- myc expression in adrenocortical tumours, J. Clin. Pathol. 71 (2) (2018) 129-134.
[35] S. Zhang, Y. Li, Y. Wu, K. Shi, L. Bing, J. Hao, Wnt/beta-catenin signaling pathway upregulates c-Myc expression to promote cell proliferation of P19 teratocarcinoma cells, Anat. Rec. 295 (12) (2012) 2104-2113.
[36] J. Liu, Q. Xiao, J. Xiao, C. Niu, Y. Li, X. Zhang, et al., Wnt/beta-catenin signalling: function, biological mechanisms, and therapeutic opportunities, Signal Transduct. Target Ther. 7 (1) (2022) 3.
[37] F. Tissier, C. Cavard, L. Groussin, K. Perlemoine, G. Fumey, A.M. Hagnere, et al., Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors, Cancer Res. 65 (17) (2005) 7622-7627.