SorCS3 suppresses adrenocortical carcinoma progression by enhancing IGF2R-mediated endocytic trafficking and signaling attenuation
Check for updates
Yanqiu Zhang1+, Shihui Cao2, Jingqi Nie3+, Shengmiao Yu3, Jinlai Gao4, Shi Zhang1, Feifei Zheng3, Shipeng Bo1, Nan Wang3, Haiyan Xu3, Yuchen Zhao1, Chao Shen4, Yang Li3* and Dongliang Li3*[D
Abstract
Background Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with limited treatment options and poor prognosis. Identifying novel molecular regulators is essential for improving therapeutic strategies. In this study, we identified Sortilin-related VPS10 domain-containing receptor 3 (SorCS3) as a novel tumor suppressor candidate in ACC.
Methods Through expression detection and functional validation, we investigated the role of SorCS3, a member of the VPS10p-domain receptor family, in ACC. Expression data from TCGA and clinical tissue samples were analyzed. Endocytic activity, protein interactions, and downstream signaling were examined using immunofluorescence, co-immunoprecipitation (Co-IP), and western blotting in ACC cell lines.
Results SorCS3, previously regarded as CNS-specific, was detected in ACC tissues and cell lines. Low SorCS3 expression was associated with reduced overall survival (OS) and disease-specific survival (DSS). Functional assays showed that SorCS3 enhanced endocytosis and colocalized with early endosomes. Co-IP revealed a physical or indirect interaction between SorCS3 and Insulin-like growth factor 2 receptor (IGF2R), a multifunctional receptor involved in IGF-II degradation and signal attenuation. SorCS3 overexpression (OE-SorCS3) increased IGF2R protein levels and suppressed PI3K/Akt and MAPK/Erk signaling. Blocking endocytosis partially reversed these effects, supporting a receptor trafficking-dependent mechanism.
Conclusions In this study, we identified SorCS3 as a novel tumor suppressor candidate in ACC. Although previously thought to be CNS specific, SorCS3 was found to be expressed in ACC tissues and cell lines. Low SorCS3 levels correlated with poor patient survival in the TCGA cohort. Functionally, SorCS3 enhanced endocytosis in ACC cells and
+Yanqiu Zhang, Shihui Cao, and Jingqi Nie have contributed equally to this work.
*Correspondence:
Yang Li 15303696823@163.com Dongliang Li
Full list of author information is available at the end of the article
BMC
@ The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
physically or indirectly interacted with IGF2R. OE-SorCS3 increased IGF2R protein levels and reduced phosphorylation of Akt/Erk, suggesting tumor-suppressive effects through IGF2R stabilization and signaling inhibition. Overall, our findings highlight the SorCS3-IGF2R axis as a previously unrecognized regulatory mechanism in ACC progression and suggest that receptor trafficking could be a viable therapeutic target in this malignancy.
Keywords SorCS3, Adrenocortical carcinoma, Biomarker, Prognosis
Introduction
ACC is a rare and aggressive endocrine malignancy char- acterized by poor prognosis, frequent metastasis, and resistance to conventional therapies [1]. Although sur- gical resection remains the primary treatment for local- ized disease, recurrence and progression are common in advanced-stage patients, with limited effective systemic options available [2]. The complex molecular landscape of ACC poses a significant barrier to the development of targeted therapies, highlighting the need for identifica- tion of novel biomarkers and mechanistic insights into ACC pathogenesis.
SorCS3 is a type I transmembrane protein that belongs to the VPS10P domain receptor family, primarily known for its roles in the nervous system [3, 4]. It participates in intracellular trafficking, synaptic plasticity, and recep- tor recycling [5-7]. Previous studies have revealed that SorCS3 negatively regulates BDNF signaling and modu- lates anxiety-like behaviors through TrkB internalization in neurons [8, 9]. In GBM, SorCS3 has been reported to suppress tumor proliferation and invasion by modulating the p75NTR signaling pathway [10]. Furthermore, High expression of SorCS3 as a signature was associated with poor prognosis and recurrent disease in GC [11]. These findings suggest that SorCS3 may exert context-depen- dent tumor suppressive functions beyond the nervous system, yet its role in adrenal cortical tumorigenesis has not been previously characterized.
In our analysis of TCGA, SorCS3 was significantly downregulated in ACC tissues compared to normal adre- nal samples, and its low expression correlated with poor overall and disease-free survival. These findings were validated at the mRNA and protein levels in clinical ACC samples and cell lines, suggesting a potential tumor-sup- pressive role for SorCS3 in this malignancy.
The IGF2R, also known as the cation-independent mannose-6-phosphate receptor, is a multifunctional traf- ficking receptor involved in lysosomal enzyme target- ing, growth factor clearance, and TGF-ß activation [12]. Unlike the insulin-like growth factor 1 receptor (IGF1R) [13], IGF2R lacks intrinsic kinase activity and is widely considered a tumor suppressor due to its ability to bind and degrade IGF2, a potent mitogen frequently overex- pressed in multiple cancers including ACC [14-16]. Loss of IGF2R expression or function leads to sustained IGF2 signaling and contributes to tumor progression, inva- sion, and angiogenesis via the PI3K/Akt and MAPK/
Erk pathways [17, 18]. However, IGF2R can also exhibit context-dependent pro-tumorigenic roles, particu- larly through interactions with intracellular trafficking machinery [19] and regulation of cell surface receptor recycling [20], which remain poorly defined in ACC. Preliminary studies have suggested that alterations in SorCS3 may be associated with cancer pathogenesis through mechanisms such as dysregulated cell signaling and impaired cellular trafficking [10]. Given the complex interplay of genetic factors in ACC [21], understand- ing the specific contributions of SorCS3 could provide valuable insights into the molecular landscape of this malignancy.
In this study, we demonstrate that SorCS3 physically interacts with IGF2R and promotes its stabilization and internalization, suggesting a novel mechanism through which SorCS3 may influence receptor trafficking and sig- nal attenuation in ACC cells. Functional assays revealed that OE-SorCS3 inhibited ACC cell proliferation, migra- tion, and survival, accompanied by downregulation of the PI3K/Akt and MAPK/Erk signaling pathways. These effects were reversed upon pharmacologic inhibition of endocytosis, indicating that SorCS3 exerts its tumor-sup- pressive functions in an endocytosis dependent manner.
Collectively, our findings identify SorCS3 as a novel tumor suppressor in ACC and uncover its functional interaction with IGF2R as a critical axis regulating intra- cellular signaling and tumor cell behavior. These results provide new mechanistic insights into the pathogenesis of ACC and suggest that targeting the SorCS3-IGF2R pathway may represent a promising therapeutic strategy for patients with this challenging malignancy.
Materials and methods
Cell culture and human ACC discard samples
ACC cell lines SW-13 and NCI-H295R were purchased from Wuhan Prichella Biotechnology Co. Ltd. These cells were cultured in DMEM medium (HyClone, SH3002201) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) and 1% (vol/vol) penicillin/streptomycin (Beyotime, C0222) in an incubator (37 ℃, 5% CO2).
Patient-derived discarded tissues were obtained from the Department of General Surgery, Jiaxing First Hos- pital, following relevant guidelines and after obtain- ing informed consent and institutional review board approval (LS2021-KY-006).
Public data mining and survival analyses
TCGA-ACC tumor RNA-seq expression data used in this study were retrieved via Home for Researchers (http s://www.home-for-researchers.com) and the GEPIA web server (http://gepia.cancer-pku.cn) on May 5, 2022. For comparisons with normal tissues, normal adrenal expres- sion data were obtained from GTEx as implemented in GEPIA, accessed on May 5, 2022. The underlying data originate from TCGA and GTEx.
Patients were dichotomized into SorCS3-high and SorCS3-low cohorts by median (or by maximally selected rank statistics where specified). OS and disease-free survival (DFS) were evaluated by Kaplan-Meier analy- sis with log-rank tests; hazard ratios (HRs) with 95% CIs were estimated using Cox proportional hazards models (univariate and multivariate where indicated). Time-dependent ROC curves were computed with the timeROC package to estimate AUC at 1-, 3-, and 5-year time points. Stage-stratified analyses (TNM) and sub- group analyses by radiotherapy status were pre-specified. Multiple testing for transcriptome-scale comparisons used Benjamini-Hochberg FDR control.
Transient overexpression of SorCS3
The full-length human SorCS3 ORF (RefSeq NM_014979) was cloned into pcDNA3.1(+) for transient expression, with a C-terminal 3xFLAG tag. All con- structs were verified by Sanger sequencing, and endo- toxin-free plasmid preparations were used.
Transfection procedure (Lipofectamine 3000). DNA- lipid complexes were prepared in Opti-MEM accord- ing to the manufacturer’s protocol. For a 6-well format, 2.5 µg pcDNA3.1-SorCS3 was mixed with 2 uL P3000 in 125 µL Opti-MEM; in a separate tube, 3.75 µL Lipo- fectamine 3000 was diluted in 125 µL Opti-MEM. After 10-15 min at room temperature, the mixtures were com- bined and incubated for 10 min, then added dropwise to cells in complete medium. Medium was refreshed after 6 h.
ASO-mediated knockdown of SorCS3
A chemically synthesized antisense oligonucleotide tar- geting SorCS3 (ASO-SorCS3; sequence 5’-CUUGGGA TGAAAGAGCAGGC-3’) and a non-targeting control ASO were transfected using Lipofectamine RNAiMAX (Thermo Fisher). For a 6-well format, complexes were prepared in Opti-MEM as follows: dilute 100 pmol ASO (to yield a 50 nM final concentration in 2 mL/well) in 125 uL Opti-MEM; in a separate tube dilute 4 AL RNAiMAX in 125 uL Opti-MEM. Incubate each for 10-15 min, combine, incubate 10 min, and add dropwise to cells in antibiotic-free complete medium. Replace medium after 6-8 h. Cells were harvested 24-48 h post-transfection for RNA and 48-72 h for protein and functional assays.
Knockdown efficiency was confirmed by qPCR and Western blot.
Transwell migration and invasion assays
NCI-H295R and SW-13 cells overexpressing SorCS3 (as described above) were serum-starved (0.5% FBS, 6-12 h) before assays. For migration, 24-well Transwell inserts (8-um pores) were used; 5x10^4 (NCI-H295R) or 4 x 10^4 (SW-13) cells in 100-150 uL serum-reduced medium were seeded in the upper chamber, with 600 AL complete medium (10% FBS) in the lower chamber, and incubated 12-16 h. For invasion, inserts were pre- coated with growth-factor-reduced Matrigel (1:8, 50 uL/insert, 30-45 min, 37 °℃). 5x10^5 (NCI-H295R) or 2×10^5 (SW-13) cells were seeded in serum-free/0.5% FBS medium; the lower chamber contained 10% FBS medium; incubation was 20-24 h. Cells received Dyna- sore (40 µM) or vehicle (0.1% DMSO) with a 120 min pretreatment prior to seeding; the same concentration was maintained in both chambers for the assay duration. After incubation, non-migrated/non-invaded cells were removed from the upper membrane surface. The mem- branes were fixed (4% PFA, 15 min) and stained (0.1% crystal violet, 30 min). Five random fields per insert were imaged at 200x, and cells were counted by a blinded operator.
Cell-cycle analysis by flow cytometry
Cells were harvested at ~70-80% confluence, washed twice with ice-cold PBS, and fixed in pre-chilled 75% eth- anol at-20 ℃ overnight. Fixed cells were washed twice with PBS to remove ethanol and incubated in RNase A (100 µg/mL, 30 min, 37 ℃) followed by propidium iodide (PI, 50 µg/mL) for 15 min at room temperature, pro- tected from light. Data were acquired on a benchtop flow cytometer (BD Biosciences) with linear area detection for PI. At least 10,000 single-cell events were collected per sample. Biological replicates (n≥3) were processed in parallel.
Apoptosis analysis by annexin V-FITC/PI staining
Cells were trypsinized without EDTA, pooled with float- ing cells, and washed twice with cold PBS. 5x 10^5 cells were resuspended in binding buffer, incubated with Annexin V-FITC for 10 min in the dark, followed by PI (5 µL; 50 µg/mL) for 5 min, then brought to 300-500 µL with binding buffer. At least 10,000 singlet events were recorded per sample; gating was applied uniformly across replicates (n ≥3).
Molecular docking and structural visualization
Protein-protein docking between SorCS3 and IGF2R was performed on the WeMol web platform with default parameters unless specified (WeMol, Wecomput
Technology Co., Ltd, ttps://wemol.wecomput.com). Briefly, input structures for the extracellular regions were prepared by removing signal peptides, transmembrane helices, heteroatoms, and alternate conformations; miss- ing side chains were completed and protonation states assigned at pH 7.4 during preprocessing. The WeMol protein-protein docking module generated and ranked candidate complexes; top-ranked poses were filtered by docking score and cluster size, and steric plausibility was verified by visual inspection. Three independent runs yielded consistent top clusters. Interface analysis (bur- ied surface area, hydrogen bonds/contacts, and distance measurements) and figure rendering were carried out in UCSF ChimeraX (v1.6). Publication images used ribbon representation with surfaces at the interface; residues at putative contact patches were labeled for clarity, and distances/hydrogen bonds were annotated using built-in ChimeraX tools.
Western blot and band densitometry
Total protein was extracted using RIPA buffer and pro- tein expression was analyzed by Western blotting as described previously. GAPDH served as an endogenous control. The antibodies information utilized in Western blotting has been listed in Supplementary File 1: Table S1. Chemiluminescent blots were imaged as 16-bit TIFF files without gamma adjustment using a digital imager, with exposure times chosen to avoid saturation. Band intensities were quantified in ImageJ using the Gel Ana- lyzer workflow. Rectangular ROIs of identical size were placed over each band; local background was removed, and Integrated Density was recorded.
Immunohistochemistry (IHC)
FFPE sections of ACC and adjacent adrenal tissues (4 um) were deparaffinized, rehydrated, and subjected to heat- induced epitope retrieval (10 mM sodium citrate, pH 6.0, 15-20 min). Endogenous peroxidase was quenched with 3% H2O2 (10 min). After blocking, slides were incubated with primary antibodies against SorCS3 and IGF2R (dilu- tions/sources in Table S1) overnight at 4 ℃, followed by an HRP-polymer secondary (30 min, RT), DAB devel- opment, hematoxylin counterstain, dehydration, and mounting. Negative controls (primary antibody omitted) were included in each run. Staining was semi-quantified by H-score (0-300) by two blinded reviewers; discrepan- cies were resolved by consensus. Images were acquired under identical exposure settings.
5-FAM, SE-BSA internalization assay
5-FAM, SE (Invitrogen, C2210) is a fluorescent label- ling reagent that exists as a single isomer to bind pep- tides, proteins and nucleotides via chemical reactions. According to the manufacturer’s protocol, fluorescently
labelled bovine serum albumin (BSA, Sigma, A1933) was prepared by incubation in the dark. 5-FAM, SE-BSA collected from the cut-off column was filtered through a PVDF filter to remove bacteria. 5-FAM, SE-BSA was added to cultured glioma cells to observe the internaliza- tion ability under a fluorescence microscope.
Fluorescence quantification and statistics
Images were acquired under identical microscope set- tings (objective, exposure, gain, laser power) within each experiment while avoiding saturation. For each well, ≥4 fields were collected at random positions. Nuclei were counterstained with DAPI/Hoechst and segmented by adaptive thresholding with watershed separation to obtain the nuclei count per field. Background for the 5-FAM channel was estimated from≥3 cell-free regions in the same well and subtracted from each image prior to quantification. The 5-FAM signal was summarized as Integrated Density (background-corrected intensity summed over the field). Field-level fluorescence was then normalized to cell number as:
Normalized value = (Integrated density of 5-FAM)/ (Number of Nuclei).
Nude mice tumorigenicity assay
Our study was approved by the Ethics Committee of Harbin Medical University (Daqing). Nude mice were randomly grouped into the control group and the OE-SorCS3 group. Mice were inoculated subcuta- neously in the right flanks with 1x10^7 SW-13 cells transfected with the negative control vector or the LV-EFS> Kozak-SorCS3-CMV>EGFP/T2A/Puro.
Co-immunoprecipitation
Co-IP was performed using a Pierce Co-IP Kit (Thermo Scientific) according to the manufacturer’s protocol. According to experimental needs, a purified anti-Flag antibody was used to IP the antigen, and any co-immu- noprecipitated interacting proteins were immobilized directly onto an amine-reactive resin by covalent cou- pling. The final protein eluate was denatured and anal- ysed by western blotting.
Statistical analysis
Data are presented as mean+ SEM. Significance was determined by using Student’s t-test for comparison of two groups. All statistical tests were performed using Prism. Differences with P<0.05 were considered sig- nificant (*P<0.05, ** P<0.01, *** P<0.001). Analyses were conducted in GraphPad Prism v9.5 (GraphPad Software, San Diego, CA, USA).
Results SorCS3 is downregulated in ACC and correlates with poor prognosis
To investigate the expression pattern of SorCS3 in ACC, we analyzed transcriptomic data from TCGA. The results revealed that SorCS3 expression was significantly down- regulated in ACC tissues compared to normal adrenal
tissues (Fig. 1A). Kaplan-Meier survival analysis dem- onstrated that ACC patients with low SorCS3 expression exhibited significantly shorter OS and DFS compared to those with high SorCS3 expression (Fig. 1B-D). Further- more, time-dependent receiver operating characteristic (ROC) curve analysis showed that SorCS3 expression had a strong predictive value for patient survival, as
A
B
C
D
5-
High SORCS3
Low SORCS3
Overall Survival
Disease Free Survival
3.0
4-
100
3
100
Expression -log2 (PTM+1)
log2 (PTM+1)
SORCS3
SORCS3
2.5
2
+ SORCS3=High (n=39)
+ SORCS3=High (n=35)
1
Percent survival %
75
+ SORCS3=Low (n=40)
Percent survival %
80
+ SORCS3=Low (n=38)
2.0
0-
Alive
60
1.5-
Dead
10
50
1.0-
Time
40
Log-rank
5
25
0.5
P = 0.0026
20
Log-rank
p = 0.032
0.0-
P
A
0-
Normal (n = 77)
ACC
SORCS3
0
0
(n = 128)
4
Z-score of expression
0
1000
2000
3000
4000
5000
0
50
100
150
0 1 2 3
Days
Months
E
F
G
1.00-
pTNM Stage ¥
SorCS3
Status
SorCS3
Radiation
Status +
True Positive Fraction
0.75-
High Expression
1
High Expression
Alive
Alive
0.50-
=
NO
Low Expression
Low Expression
0.25-
E
Type
Dead
Dead
1-Years,AUC-0.649,95%C3(0.352-0.945)
3-Years,AUC-0.657,95%C3(0.53-0.784)
Yes
0.00
5-Years,AUC=0.737,95%C3(0.62-0.855)
≥
0.00
0.25
0.50
0.75
1.00
False Positive Fraction
H
J
N: #1
#2
#3
T: #1
#2
#3
Normal kidney tissue
Adrenocortical carcinoma tissue
Relative Expression Level of SORCS3 mRNA (fold change)
1.5
SORCS3
130KDa
GAPDH
36KDa
1.0-
Relative protein level
#1
1.000000
0.400394
1.2
0.5-
1.0
#2
1.213058
0.535824
0.8
0.0
#3
0.801358
0.338239
0.6
Normal kidney tissue
Adrenocortical carcinoma
0.4
50pm
Normal kidney tissue
Adrenocortical carcinoma
A
C
NCI-H295R
F
SW-13
Relative expression level of SorCS3(fold)
200-
Invasion
Invasion
150
100
2
1.
0
Negative Control
OE-SorCS3
Negative Control
OE-SorCS3
Negative Control
OE-SorCS3
D
Migration
G
Migration
4
B
SorCS3
130KDa
GAPDH
36KDa
Negative Control
OE-SorCS3
Negative Control
OE-SorCS3
Expression of protein (realtive to GAPDH)
E
H
15
*
Negative Control OE-SorCS3
Negative Control
OE-SorCS3
10
400-
300-
*
*
Number of cells
I
*
Number of cells
300
T
T
5
200
200
T
0
100
100
**
Negative Control
OE-SorCS3
T
0
0
-
Invasion
Migration
Invasion
Migration
NCI-H295R
J
K
SW-13
L
Negative Control
OE-SorCS3
Negative Control
OE-SorCS3
Scratch healing rate %
60
Scratch healing rate %
100-
**
OH
OH
40
80-
60-
200gm
20
200gm
40-
20
24H
0
Negative Control
OE-SorCS3
24H
0
Negative Control
OE-SorCS3
=
M
NCI-H295R
N
O
SW-13
P
3
Negative Control
3
OE-SorCS3
Early + Late Apoptosis Alive
Negative Control
OE-SorCS3
Early + Late Apoptosis Alive
PI Fluorescence
5.6%
11.1%
3
2.7%
3
9.4%
110-
110-
*
Apoptosis rate%
PI Fluorescence
100
T
Apoptosis rate%
1
“9
100
T
T
90
T
*2
*3
90
T
”
3
80
70
8
1
80
3
3
60
10
3
3
20
10
1
7
3
79.1%
14,9%
7
69.9%
18.2%
0-
=
93.9%
3.2%
~
88.7%
1,7%
0-
.
-
A2
-
4
ES
72
OE-SorCS3
2
24 3
-
S
OS
72
Annexin V-FITC
Negative Control
72
Annexin V-FITC
Negative Control
OE-SorCS3
A
B
C
D
NCI-H295R
G0+G1
SW-13
G0+G1
Negative Control
OE-SorCS3
G2+S
Negative Control
OE-SorCS3
G2+S
*
G1: 74.55%
G2
11.55%
G1: 83.18% G2: 5.58%
Percentage of cells %
100
₸
1
G1: 52.38% G2: 23.40%
G1: 64.84%
G2: 9.90%
Percentage of cells %
100
-
₸
1000 2000 3000 4000 5000 6000 3000 A
S
13.90%
91
I-
S: 11.25%
S: 24.22%
S: 25.25%
Des
75-
發
Number
&
I
Number
8-
6
75.
50
.
.
g
₴
-
50
-
g
T
I
25
T
8
$
25
-
病
2
₿
.
19
15
30
25
Se
·
·
T
Não
20
30
0
·
0
18
20
30
40
50
0
1
50
20
30
1
0
DNA content (FL2-A)
Negative Control
OE-SorCS3
DNA content
(FL2-A)
Negative control
OE-SorCS3
E
NCI-H295R
F
G
SW-13
H
Negative Control
OE-SorCS3
Negative Control
OE-SorCS3
EdU
Relative Edu Positive Rate %
EdU
Relative Edu Positive Rate %
50
*
40
40-
30
T
Hoechst
30
Hoechst
20
20
T
10-
10
$:
0
0
Merge
Negative Control
OE-SorCS3
Merge
Negative Control
OE-SorCS3
J
K
NCI-H295R
SW-13
Negative Control
3.0-
OE-SorCS3
1.5
Negative Control
OE-SorCS3
CyclinD1
36KDa
I
OD (490 nm)
2.5-
*
OD(490nm)
I
1.0-
PCNA
L
*
29KDa
2.0·
I
0.5
T
Vimentin
57KDa
1.5.
I
1.0.
0.0
GAPDH
36KDa
Negative Control
2
3
OE-SorCS3
1
4
1
2
3
4
Days
Days
L
Negative Control
M
Relative protein expression level (fold)
OE-SorCS3
1.5.
*
*
*
Negative Control
1.0-
T
T
T
OE-SorCS3
0.5.
123456
7
0.0
Cyclin D1
Vimenin
PCNA
(See figure on previous page.)
Fig. 3 SorCS3 overexpression induces cell-cycle arrest and suppresses proliferation of adrenocortical carcinoma cells in vitro and in vivo. A-B Flow- cytometry analysis of cell-cycle distribution in NCI-H295R cells shows an increased G0/G1 fraction and a reduced S phase upon SorCS3 overexpression; quantification shown in (B). C and D Similar GO/G1 arrest is observed in SW-13 cells; quantification shown in (D). E-F EdU assay, NCI-H295R: EdU-positive nuclei (red) and Hoechst (blue); color legend is indicated on each panel. The EdU-positive rate (%) =EdU+/ Hoechst+x 100%; quantification shown in (F). G-H EdU assay, SW-13: EdU-positive nuclei (green) and Hoechst (blue) with the same palette and legend as in (E); quantification shown in (H). I-J CCK-8 assays show reduced cell viability over time in NCI-H295R (I) and SW-13 (J) upon SorCS3 overexpression. K-L WB of cell-cycle and mesenchymal markers in NCI-H295R cells. SorCS3 overexpression decreases Cyclin D1, PCNA, and Vimentin protein levels (K); densitometric quantification normalized to GAPDH and to the Negative Control is shown in (L). M Representative xenograft tumors derived from NCI-H295R cells in nude mice show smaller tumor size in the SorCS3-overexpression group than in the negative control. Data are mean ± SD from ≥3 independent experiments. * P<0.05, ** P <0.01, *** P <0.001
indicated by high area under the curve (AUC) values at various time points (Fig. 1E). Stratification by TNM stage indicated that SorCS3-high expression was more frequently observed in early-stage ACC, while patients with advanced-stage disease more commonly exhibited low SorCS3 expression (Fig. 1F). Notably, patients with high SorCS3 expression showed favorable prognosis even in the absence of radiotherapy, further supporting its potential protective role (Fig. 1G). To validate these findings, we performed IHC staining on three clinical ACC samples (Supplementary Fig. 1A), which confirmed markedly decreased SorCS3 protein expression in tumor tissues compared to adjacent normal adrenal tissues. Consistently, SorCS3 mRNA and protein levels were both significantly reduced in ACC samples (Fig. 1H-J and Supplementary Fig. 1B).
Taken together, these findings indicate that SorCS3 is frequently downregulated in ACC and its low expression is associated with poor prognosis, suggesting that SorCS3 may function as a potential tumor suppressor in ACC.
OE-SorCS3 suppresses proliferation, invasion, and migration of ACC cells
To investigate the biological function of SorCS3 in ACC, we established OE-SorCS3 models in two ACC cell lines, NCI-H295R and SW-13. qPCR and Western blot analyses confirmed a significant upregulation of SorCS3 expres- sion in the transfected cells (Fig. 2A, B). In Transwell assays, ectopic SorCS3 significantly reduced the num- bers of cells crossing the membrane in both NCI-H295R and SW-13, indicating diminished invasive and migra- tory capacities (Fig. 2C-H). Consistently, wound-healing assays showed a slower scratch-closure rate upon OE- SorCS3 in the two cell lines (Fig. 2I-L). Together, these data demonstrate that SorCS3 restrains ACC cell motil- ity and invasiveness in vitro. Western blotting confirmed efficient SorCS3 depletion by ASO-SorCS3 (Supplemen- tary Fig. 2A, B). In Transwell assays, SorCS3 knockdown increased the number of NCI-H295R and SW-13 cells traversing the membrane, indicating enhanced invasive and migratory capacities (Supplementary Fig. 2C-F). Consistently, wound-healing assays showed an acceler- ated scratch-closure rate upon SorCS3 loss in both lines (Supplementary Fig. 2G, H). These loss-of-function data phenocopy the inverse of the overexpression results and
further support a motility-suppressive role of SorCS3 in ACC cells. Further analysis of apoptosis using flow cytometry revealed a significantly increased propor- tion of early and late apoptotic cells in the OE-SorCS3 group compared to the control group (Fig. 2M-P), indi- cating that SorCS3 promotes apoptosis in ACC cells. Cell cycle analysis showed that OE-SorCS3 induced a marked G0/G1 phase arrest, with a corresponding reduc- tion in S-phase cell population (Fig. 3A-D). Additionally, EdU assays confirmed decreased DNA synthesis activ- ity in OE-SorCS3 cells (Fig. 3E-H and Supplementary Fig. 2I-J). CCK-8 assays demonstrated that OE-SorCS3 significantly inhibited the proliferation of NCI-H295R and SW-13 cells in vitro (Fig. 3I-J). We next examined the expression of key markers associated with cell prolif- eration and migration. Notably, OE-SorCS3 resulted in a marked reduction in the protein levels of PCNA, Cyclin D1, and Vimentin (Fig. 3K, L). Consistent with these findings, subcutaneous xenograft models in mice showed that tumors formed in the OE-SorCS3 group were signifi- cantly smaller in volume compared to those in the con- trol group (Fig. 3M), further supporting the in vivo tumor suppressive effect of SorCS3.
Taken together, these results indicate that SorCS3 exerts potent tumor-suppressive functions in ACC by inducing apoptosis, causing cell cycle arrest, and inhibit- ing cell invasion, migration, and proliferation, highlight- ing its potential as a therapeutic target in ACC.
SorCS3 has an internalization function in ACC
To explore the biological role of SorCS3 in ACC cells, we performed Albumin-5, FAM uptake assay and colo- calization analysis. In OE-SorCS3 cells, we observed a significant increase in the intracellular accumulation of fluorescently labeled albumin compared with the nega- tive control, which was observed by confocal microscopy (Fig. 4A). Fluorescence intensity quantitative analysis showed that albumin uptake was significantly increased in OE-SorCS3 cells (Fig. 4B), indicating enhanced inter- nalization. To further evaluate the endosomal local- ization of SorCS3, we detected its colocalization with the early endosomal marker EEA1. In the PBS-treated OE-SorCS3 group, partial colocalization of SorCS3 and EEA1 was detected (Fig. 4C, D), while albumin stimula- tion significantly enhanced this colocalization, which was
A
B
Negative Control
Inset
5FAM,SE Integrated Density Per Cell
Albumin-5FAM, SE
a
F-actin
Hoechst
Merge
600
C
0
0
9
400
200
OE-SorCS3
Inset
Albumin-5FAM, SE
-actin
Hoechst
Merge
C
O
0
Negative Control
OE-SorCS3
O
&
O
G
=
2
1
1
C
Negative Control + PBS
D
OE-SorCS3 + PBS
EEA1
SorCS3
Hoechst
Merge
SorCS3
EEA1
Hoechst
Merge
4
O
0
225
2
=
Inset
Inset
Inset
Inset
Inset
Inset
Green-EEA1
200
Red-SorCS3
250
Red-SorCS3
Green-EEA1
Intensity (a.u.)
150-
Intensity (a.u.)
200
150-
100-
100
50-
50-
0
0
0
20
40
60
80
100
0
200
400
600
E
Distance
F
Distance
Negative Control + Albumin
OE-SorCS3 + Albumin
EEA1
SorCS3
Hoechst
Merge
SorCS3
EEA1
Hoechst
Merge
C
G
9
2
2
2
=
Inset
Inset
Inset
Inset
Inset
Inset
Green-EEA1
Red-SorCS3
200-
Red-SorCS3
300
Green-EEA1
Intensity (a.u.)
Intensity (a.u.)
150
200
100-
100
50-
0
0
0
25
50
75
100
0
200
400
600
Distance
Distance
(See figure on previous page.)
Fig. 4 SorCS3 regulates specific ligand internalization in ACC. A Representative confocal images showing albumin (green), F-actin (red), and nuclei (blue) in negative control and OE-SorCS3 NCI-H295R cells. Scale bar = 17 um; inset magnification on the right. B Quantification of relative fluorescence intensity (Integrated Density of 5-FAM)/(Number of Nuclei), showing significantly increased albumin uptake in OE-SorCS3 cells ( **** P <0.0001). C and D Confocal images of Negative Control (C) and OE-SorCS3 (D) cells treated with PBS, showing partial colocalization of SorCS3 (red) with early endosome marker EEA1 (green); nuclei stained with Hoechst (blue). Inset shows higher magnification of colocalized puncta (arrowheads). Line-scan analysis of fluorescence intensity along a selected region from (C-D), demonstrating low overlap between SorCS3 and EEA1 signals. E-F Confocal images of Negative Control (E) and OE-SorCS3 (F) cells treated with albumin showing increased colocalization of SorCS3 (red) with EEA1 (green). Insets highlight more frequent colocal- ized puncta. Line-scan analysis from (E-F) reveals enhanced colocalization between SorCS3 and EEA1 following albumin stimulation. a.u., arbitrary units. Data are presented as mean ± SD
confirmed by the increase in overlapping fluorescence signals and intensity distribution analysis (Fig. 4E, F). These results indicate that SorCS3 is involved in inter- nalization trafficking and may promote cargo delivery to early endosomes.
Taken together, these results suggest that SorCS3 has internalization capacity in ACC cells and may play a role in mediating the intracellular trafficking of specific ligands.
SorCS3 interacts with IGF2R and regulates its expression in ACC
To further investigate the molecular mechanisms under- lying the tumor-suppressive function of SorCS3 in ACC, we explored potential interacting proteins. In our previ- ous study, mass spectrometry analysis following Co-IP/ MS identified IGF2R as a potential binding partner of SorCS3. Structural modeling suggested a direct protein- protein interaction interface between SorCS3 and IGF2R (Fig. 5A). Co-IP assays in ACC cells confirmed the inter- action between SorCS3 and IGF2R. In the OE-SorCS3 group, IGF2R was efficiently pulled down by the anti- SorCS3 antibody (Fig. 5B). Moreover, analysis of endoge- nous protein complexes further validated this interaction, as SorCS3 was successfully co-precipitated using an anti- IGF2R antibody (Fig. 5C). Furthermore, immunofluores- cence analysis revealed a clear co-localization of SorCS3 and IGF2R within ACC cells (Fig. 5D and Supplementary Fig. 5). Western blot analysis demonstrated that OE- SorCS3 significantly increased IGF2R protein levels in ACC cells (Fig. 5E), suggesting that SorCS3 may stabilize or positively regulate IGF2R expression. Consistent with these in vitro findings, IHC staining of clinical samples showed markedly lower IGF2R expression in ACC tissues compared to adjacent normal tissues (Fig. 5F).
To evaluate the clinical significance of IGF2R in ACC, we analyzed patient survival data. Although the differ- ences were not statistically significant, patients with higher IGF2R expression exhibited a trend toward shorter disease-free and overall survival compared to those with lower expression levels (Supplementary Fig. 3A-C). In addition, Western blot analysis of three paired ACC and adjacent normal adrenal tissues confirmed a signifi- cant downregulation of IGF2R in tumor tissues (Fig. 5G, H). Functional assays further demonstrated that IGF2R
overexpression in ACC cells significantly inhibited both migration and proliferation (Fig. 5I, J), supporting a potential role for IGF2R in modulating tumor behavior. Previous studies have shown that loss of IGF2R expres- sion can result in sustained activation of IGF2 signaling [17, 22]. Accordingly, we also examined IGF2 expression in ACC tissues (Supplementary Fig. 4A, B), and found that OE-SorCS3 altered IGF2 expression levels (Supple- mentary Fig. 4C, D).
Collectively, these findings indicate that SorCS3 physi- cally interacts with IGF2R and modulates its expression, potentially stabilizing IGF2R in ACC cells. The regulatory effect on IGF2R may contribute, at least in part, to the tumor-suppressive role of SorCS3, and highlights IGF2R as a candidate biomarker and potential therapeutic target in ACC.
The endocytic inhibitor dynasore reverses the tumor- suppressive effects of SorCS3 in ACC cells
To further investigate whether the tumor suppressive effects of SorCS3 are dependent on endocytic processes, we treated ACC cells with Dynasore, a specific inhibi- tor of dynamin-dependent endocytosis. CCK-8 assays demonstrated that OE-SorCS3 cells treated with Dyna- sore exhibited no significant difference in cell prolifera- tion compared to control cells, suggesting that Dynasore effectively blocked SorCS3-mediated growth inhibition (Fig. 6A). Flow cytometry analysis revealed that Dyna- sore treatment abolished the SorCS3-induced G1 phase arrest, as no significant differences in the distribution of cell cycle phases were observed between OE-SorCS3 and control groups under Dynasore treatment (Fig. 6B, C). Consistently, transwell migration and invasion assays showed that the inhibitory effects of SorCS3 on cell migration and invasion were also diminished in the pres- ence of Dynasore (Fig. 6D, F and Supplementary Fig. 6). Moreover, Annexin V/PI staining showed no significant increase in apoptosis in OE-SorCS3 cells treated with Dynasore compared to control cells (Fig. 6G, H), sug- gesting that Dynasore reverses SorCS3-mediated pro- apoptotic effects. Western blot analysis further revealed that OE-SorCS3 significantly suppressed the phosphory- lation of Akt and Erk1/2; however, this inhibitory effect was abolished by Dynasore treatment (Fig. 6I-K). These results indicate that SorCS3 suppresses cell proliferation,
A
B
IP
OE-SorCS3
SorCS3
IGF2R
WCL
lgG
+ 274KDa
IGF2R
SorCS3
+ 130KDa
GAPDH
+ 36KDa
C
IP
WCL
IGF2R
D
lgG
SorCS3
IGF2R
250
Red-IGF2R
SorCS3
+130KDa
Green-SorCS3
200
Intensity ( a.u.)
150
Hoechst
Merge
100
IGF2R
+ 274KDa
50-
0
0
20
40
60
GAPDH
+36KDa
Distance
5 pm
E
Relative protein expression
2.5-
**
level of SorCS3 (fold)
Relative protein expression
F
Adjacent normal tissue
SorCS3
130KDa
2.5-
*
2.0-
level of IGF2R(fold)
IGF2R
2.0-
IGF2R
274KDa
1.5-
1.5-
1.0-
1.0-
GAPDH
36KDa
0.5
0.5
Negative Control
OE-SorCS3
0.0
Negative Control
OE-SorCS3
0.0
Negative Control
OE-SorCS3
50um
Adrenocortical carcinoma tissue
G
H
Relative protein level
1.0
#1
N: #1
1.000000
0.355136
#2
#3 T: #1
#2
#3
0.8
IGF2R
274KDa
#2
0.712460
0.205118
0.6
GAPDH
36KDa
#3
0.810164
0.192918
0.4
Normal
Tumor
0.2
50pm
J
Negative Control
Negative Control
Relative Edu Positive Rate %
80
**
60-
40
20
OE-IGF2R
OE-IGF2R
0
Negative Control
OE-IGF2R
80
(See figure on previous page.)
Fig. 5 SorCS3 interacts with and upregulates IGF2R expression in ACC. A Predicted structural model of the interaction between SorCS3 (brown) and IGF2R (purple), with a zoomed-in view showing the predicted binding interface. B Co-IP assay demonstrating the interaction between overexpressed SorCS3 and endogenous IGF2R in ACC cells. IgG and whole cell lysate (WCL) served as controls. C Co-IP assay demonstrating the interaction between endogenous SorCS3 and endogenous IGF2R in ACC cells. IgG and WCL served as controls. D Immunofluorescence staining of SorCS3 (green), IGF2R (red), and nuclei (Hoechst, blue) in ACC cells. Merged images show subcellular co-localization of SorCS3 and IGF2R. Scale bar=5 um. Fluorescence intensity profile along the line drawn in (D), showing overlapping signal peaks of SorCS3 and IGF2R. E Western blot analysis showing increased expression of both SorCS3 and IGF2R in OE-SorCS3 NCI-H295R cells compared to negative control. GAPDH was used as the internal loading control. F Representative IHC images of IGF2R expression in adjacent normal adrenal tissue and ACC tumor tissue, showing reduced IGF2R expression in tumors. Scale bar=50 um. G and H Quantification of SorCS3 (F) and IGF2R (G) protein expression levels in paired normal kidney and ACC tissues. I Transwell invasion assay comparing negative control and OE-IGF2R ACC cells. J EdU incorporation assay showing decreased proliferation in OE-IGF2R cells compared to control. Quantifica- tion of EdU-positive cells is shown. Data are presented as mean ± SD; * P < 0.05, ** P < 0.01
migration, invasion, and survival through endocyto- sis-dependent inhibition of the Akt and Erk signaling pathways.
Taken together, these findings suggest that the tumor- suppressive functions of SorCS3 in ACC are mediated, at least in part, through internalization mechanisms involv- ing downregulation of the PI3K/Akt and MAPK/Erk pathways.
Discussion
ACC is a rare but aggressive malignancy with limited therapeutic options and poor prognosis [23, 24]. In this study, we identified SorCS3, a member of the VPS10p- domain receptor family previously thought to be brain- specific, as a novel tumor suppressor candidate in ACC. We demonstrated that SorCS3 is expressed in both ACC tissues and cell lines, and its low expression was signifi- cantly associated with poorer OS and DSS in the TCGA cohort, underscoring its potential clinical relevance (Fig. 1).
SorCS3 is known to regulate receptor trafficking and endocytosis in neurons [25, 26], and our findings extend this function to ACC cells. Specifically, SorCS3 overex- pression enhanced endocytic activity and colocalized with early endosomes, as indicated by EEA1 staining (Fig. 4). Importantly, SorCS3 interacts with the IGF2R a multifunctional receptor involved in IGF2 degradation and known to suppress tumor progression by dampen- ing PI3K/Akt and MAPK/Erk signaling pathways. We observed that SorCS3 overexpression increased IGF2R protein levels and concurrently reduced the phosphory- lation of Akt and Erk, suggesting suppression of key oncogenic signaling cascades (Figs. 5 and 6). These find- ings support a model in which SorCS3 stabilizes IGF2R via enhanced endocytic trafficking, contributing to its tumor-suppressive effect in ACC.
A point of complexity is the discrepancy between our findings and previously published data regarding IGF2R expression in ACC [15]. This study demonstrates the presence of a high IGF2R protein expression in most ACCs, suggesting that a high level of IGF2R protein
might counteract the growth-stimulating effects of IGF2 in adrenocortical tumorigenesis [15]. In contrast, our analysis of clinical specimens revealed a reduction in IGF2R protein expression, paralleling that of SorCS3 (Fig. 1) and 5F-G). This inconsistency may reflect dif- ferences in detection levels (mRNA vs. protein), post- transcriptional regulation, tumor stage, or sample heterogeneity. Receptor protein levels are often influ- enced by degradation mechanisms, trafficking efficiency, and microenvironmental factors, all of which are highly dynamic in cancer [27, 28]. Moreover, recent studies emphasize the context-dependent dual roles of IGF2R in cancer [29, 30]. While commonly viewed as a tumor sup- pressor by degrading IGF2 as in hepatocellular [29] and bladder cancers [31] IGF2R has also been implicated in tumor progression. For instance, it can promote breast cancer invasion via «Vß3 integrin processing and medi- ate immune evasion in lung adenocarcinoma through TGF-ß activation [20].
Collectively, our study expands the functional reper- toire of SorCS3 beyond the nervous system and high- lights endocytic trafficking as a critical regulatory layer of receptor-mediated signaling in ACC. The identifica- tion of a potential SorCS3-IGF2R-Akt/Erk axis under- scores the importance of vesicular transport in tumor biology and opens new avenues for therapeutic interven- tion aimed at modulating receptor fate and downstream signaling.
Nonetheless, this study has several limitations. First, the number of clinical ACC tissue samples analyzed was relatively limited, which may affect the statistical power and generalizability of our findings. Second, although our study proposes SorCS3 as a potential therapeutic target in ACC, we have not yet conducted pharmacological evaluations to validate its druggability. The current find- ings remain at the mechanistic level, without exploration of small-molecule modulators, peptide mimetics, or in vivo efficacy assessments. Future studies should focus on structure-based drug design and targeted delivery strate- gies to assess the feasibility of translating SorCS3 modu- lation into clinical applications.
A
Dynasore+Negative Control
B
Negative Control + Dynasore
C
OE-SorCS3
GO+G1
Dynasore+OE SorCS3
+ Dynasore
G2+S
1.5
Percentage of cells %
ns
G1: 67.59%
100
₸
-
G2:
13.64%
1200
G2: 12.60%
G1: 71.72%
OD (490 nm)
1.0-
4
S:
19.81%
8
S:
14.64%
75
I
Number
1000
:
50-
I
0.5
17
$
.
1
8
25-
T
-
:
:
:
0-
0.0
₹
.
Negative Control + OE-SorCS3
-
-
T
A
.
-
+
1
2
3
4
0
50
100
150
200
250
%
50
100
150
200
+
+
Days
DNA content (FL3-A)
Dynasore
D
E
F
Migration
Invasion
Dynasore+Negative Control Dynasore+OE SorCS3
+
80
4
Number of cells
ns
:
60
ns
T
40-
T
20
100pm
.100pm
100pm
100g
Negative Control + Dynasore
OE-SorCS3 + Dynasore
Negative Control + Dynasore
OE-SorCS3 + Dynasore
0-
Migration
Invasion
G
H
PI Fluorescence
Negative Control + Dynasore
OE-SorCS3 + Dynasore
Eratly + Late Apoptosis Alive
8.8
5.0%
A0.8
E
3.1%
9
110-
9
Apoptosis rate%
ns
100
T
T
*%
%
LLILLE
T
T
90
%
%
LILI
80
3
3
20
2.3
91.7%
1.8%
2.3
91.6%
5.1%
10.
2.9
7.2
12.9
T’TIMTIM
30 4
10 7.2
0
Annexin V-FITC
Negative Control OE-SorCS3 Dynasore
r
+
-
-
+
+
+
I
J
K
P-Akt / Akt (fold of control)
p-Erk1/2 / Erk1/2 (fold of control)
*
Akt
56KDa
1.5
ns
*
1.5
ns
p-Akt
ns
56KDa
*
1.0-
1.0-
I
Erk1/2
40KDa
T
T
p-Erk1/2
40KDa
0.5
T
0.5-
GAPDH
36KDa
0.0
T
T
T
0.0
Negative Control OE-SorCS3 Dynasore
+
-
+
Negative Control OE-SorCS3 Dynasore
-
+
-
+
-
Negative Control OE-SorCS3 Dynasore
+
-
+
-
-
+
-
+
-
+
-
+
-
+
-
+
-
-
+
+
-
-
+
+
-
-
+
+
Supplementary Information
The online version contains supplementary material available at https://doi.or g/10.1186/s12967-025-07146-2.
Supplementary Material 1.
Supplementary Material 2.
Supplementary Material 3.
Supplementary Material 4.
Supplementary Material 5.
Supplementary Material 6.
Supplementary Material 7.
Supplementary Material 8.
Supplementary Material 9.
Supplementary Material 10.
Supplementary Material 11.
Author contributions
Y. Zhang, D. Li, J. Nie, S. Zhang designed the research. D. Li, J. Nie, S. Zhang, S. Yu, S. Bo, Y. Li, F. Zheng, N. Wang, Y. Zhang performed the research. D. Li, J. Nie, S. Zhang, S. Yu, S. Cao and Y. Zhang analyzed data. D. Li, and S. Cao wrote the article. J. Gao and C. Shen provided ACC patient tissue samples.
Funding
This work was supported by the National Natural Science Foundation of China (NSFC) [82203734]; the Natural Science Foundation of Heilongjiang [YQ2023H006]; Fundamental Research Funds for the Provincial Universities [JFJQPY202401]; Research Training program (SRT) of Jiaxing University [NO.8517241030].
Data availability
Data will be made available on request.
Declarations
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.
Author details
1From the Department of Basic Medical College, Harbin Medical University (Daqing), Daqing, China
2Tianjin Medical University Cancer Institute and Hospital, Tianjin, China 3The College of Medical Laboratory Science and Technology, Harbin Medical University (Daqing), Daqing, China
4College of Medical, Jiaxing University, Jiaxing, China
Received: 2 July 2025 / Accepted: 11 September 2025
Published online: 21 October 2025
References
1. Allolio B, Fassnacht M. Clinical review: adrenocortical carcinoma: clinical update. J Clin Endocrinol Metab. 2006;91(6):2027-37.
2. Sisman P, Sahin AB, Peynirci H, Cander S, Gul OO, Erturk E, et al. Adrenocortical carcinoma: single center experience. Turk J Urol. 2017;43(4):462-9.
3. Hermey G. The vps10p-domain receptor family. Cell Mol Life Sci. 2009;66(16):2677-89.
4. Salasova A, Monti G, Andersen OM, Nykjaer A. Finding memo: versatile interactions of the VPS10p-domain receptors in Alzheimer’s disease. Mol Neurodegener. 2022;17(1):74.
5. Lane RF, St George-Hyslop P, Hempstead BL, Small SA, Strittmatter SM, Gandy S. Vps10 family proteins and the retromer complex in aging-related neurode- generation and diabetes. J Neurosci. 2012;32(41):14080-6.
6. Breiderhoff T, Christiansen GB, Pallesen LT, Vaegter C, Nykjaer A, Holm MM, et al. Sortilin-related receptor SORCS3 is a postsynaptic modulator of synaptic depression and fear extinction. PLoS ONE. 2013;8(9):e75006.
7. Oetjen S, Mahlke C, Hermans-Borgmeyer I, Hermey G. Spatiotemporal expression analysis of the growth factor receptor SorCS3. J Comp Neurol. 2014;522(15):3386-402.
8. Subkhangulova A, Malik AR, Hermey G, Popp O, Dittmar G, Rathjen T, et al. SORCS1 and SORCS3 control energy balance and orexigenic peptide produc- tion. EMBO Rep. 2018. https://doi.org/10.15252/embr.201744810.
9. Westergaard UB, Kirkegaard K, Sorensen ES, Jacobsen C, Nielsen MS, Petersen CM, et al. SorCS3 does not require propeptide cleavage to bind nerve growth factor. FEBS Lett. 2005;579(5):1172-6.
10. Zhang Y, Li Y, Fan Y, Zhang X, Tang Z, Qi J, et al. SorCS3 promotes the internal- ization of p75(NTR) to inhibit GBM progression. Cell Death Dis. 2022;13(4):313.
11. Elizazu J, Artetxe-Zurutuza A, Otaegi-Ugartemendia M, Moncho-Amor V, Moreno-Valladares M, Matheu A, et al. Identification of a novel gene signa- ture related to prognosis and metastasis in gastric cancer. Cell Oncol (Dordr). 2024;47(4):1355-73.
12. De Souza AT, Hankins GR, Washington MK, Orton TC, Jirtle RL. M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozy- gosity. Nat Genet. 1995;11(4):447-9.
13. Ma D, Zhang Q, Duan Q, Tan Y, Sun T, Qi C, et al. Identification of IGF1R muta- tion as a novel predictor of efficacious immunotherapy in melanoma. J Transl Med. 2022;20(1):172.
14. Luo W, Pryzbyl KJ, Bigio EH, Weintraub S, Mesulam MM, Redei EE. Reduced hippocampal and anterior cingulate expression of antioxidant enzymes and membrane progesterone receptors in Alzheimer’s disease with depression. J Alzheimers Dis. 2022;89(1):309-21.
15. De Martino MC, van Koetsveld PM, Feelders RA, de Herder WW, Dogan F, Janssen JA, et al. IGF and mTOR pathway expression and in vitro effects of linsitinib and mTOR inhibitors in adrenocortical cancer. Endocrine. 2019;64(3):673-84.
16. Williams C, Hoppe HJ, Rezgui D, Strickland M, Forbes BE, Grutzner F, et al. An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution. Science. 2012;338(6111):1209-13.
17. Liu SB, Zhou LB, Wang HF, Li G, Xie QP, Hu B. Loss of IGF2R indicates a poor prognosis and promotes cell proliferation and tumorigenesis in bladder cancer via AKT signaling pathway. Neoplasma. 2020;67(1):129-36.
18. Li T, Wang J, Liu P, Chi J, Yan H, Lei L, et al. Insulin-like growth factor 2 axis supports the serum-independent growth of malignant rhabdoid tumor and is activated by microenvironment stress. Oncotarget. 2017;8(29):47269-83.
19. Kang JX, Bell J, Leaf A, Beard RL, Chandraratna RA. Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes. Proc Natl Acad Sci U S A. 1998;95(23):13687-91.
20. Schiller HB, Szekeres A, Binder BR, Stockinger H, Leksa V. Mannose 6-phos- phate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase- type plasminogen activator receptor. Mol Biol Cell. 2009;20(3):745-56.
21. Huang B, Abedi M, Ahn G, Coventry B, Sappington I, Tang C, et al. Designed endocytosis-inducing proteins degrade targets and amplify signals. Nature. 2025;638(8051):796-804.
22. Tian Z, Yao G, Song H, Zhou Y, Geng J. IGF2R expression is associated with the chemotherapy response and prognosis of patients with advanced NSCLC. Cell Physiol Biochem. 2014;34(5):1578-88.
23. Lebastchi AH, Kunstman JW, Carling T. Adrenocortical carcinoma: current therapeutic state-of-the-art. J Oncol. 2012;2012:234726.
24. 4. Alyateem G, Nilubol N. Current status and future targeted therapy in adreno- cortical cancer. Front Endocrinol (Lausanne). 2021;12:613248.
25. Schmainda KM, Prah MA, Hu LS, Quarles CC, Semmineh N, Rand SD, et al. Moving toward a consensus DSC-MRI protocol: validation of a low-flip angle single-dose option as a reference standard for brain tumors. AJNR Am J Neuroradiol. 2019;40(4):626-33.
26. Hsieh LT, Gruber MJ, Jenkins LJ, Ranganath C. Hippocampal activity patterns carry information about objects in temporal context. Neuron. 2014;81(5):1165-78.
27. Jin Y, Deng Z, Zhu T. Membrane protein trafficking in the anti-tumor immune response: work of endosomal-lysosomal system. Cancer Cell Int. 2022;22(1):413.
28. Zhang X, Meng T, Cui S, Liu D, Pang Q, Wang P. Roles of ubiquitination in the crosstalk between tumors and the tumor microenvironment (review). Int J Oncol. 2022. https://doi.org/10.3892/ijo.2022.5374.
29. Zhu XY, Liu WT, Hou XJ, Zong C, Yu W, Shen ZM, Qu SP, Tao M, Xue MM, Zhou DY, Bai HR, Gao L, Jiang JH, Zhao QD, Wei LX, Yang X, Han ZP, Zhang L. CD34(+)CLDN5(+) tumor associated senescent endothelial cells through IGF2-IGF2R signaling increased cholangiocellular phenotype in hepatocel- lular carcinoma, J Adv Res. 2024.
30. Cai X, Sun R, Yang L, Yao N, Sun Y, Zhang G, et al. Proteomic analysis reveals modulation of key proteins in follicular thyroid cancer progression. Chin Med J (Engl). 2025. https://doi.org/10.1097/CM9.0000000000003645.
31. Zhang Z, Mou Z, Xu C, Wu S, Dai X, Chen X, et al. Autophagy-associated circu- lar RNA hsa_circ_0007813 modulates human bladder cancer progression via hsa-miR-361-3p/IGF2R regulation. Cell Death Dis. 2021;12(8):778.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.