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Stathmin 1 is highly expressed and associated with survival outcome in malignant adrenocortical tumours
Bárbara dos Santos Passaia 1 · Keli Lima2 · Jean Lucas Kremer1 · Barbara Brito da Conceição 1 · Beatriz Marinho de Paula Mariani3 . Jean Carlos Lipreri da Silva2 · Maria Claudia Nogueira Zerbini4 .
Maria Candida Barisson Villares Fragoso3 . João Agostinho Machado-Neto2 . Claudimara Ferini Pacicco Lotfi1 D
Received: 4 June 2019 / Accepted: 11 August 2019
C) Springer Science+Business Media, LLC, part of Springer Nature 2019Adrenocortical carcinoma (ACC) is an aggressive endocrine cancer with few molecular predictors of malignancy and survival, especially in paediatric patients. Stathmin 1 (STMN1) regulates microtubule dynamics and has been involved in the malignant phenotype of cancer cells. Recently, it was reported that STMN1 is highly expressed in ACC patients, and STMN1 silencing reduces the clonogenicity and migration of ACC cell lines. However, the prognostic significance of STMN1 and its therapeutic potential remain undefined in ACC. In the present study, STMN1 mRNA levels were significantly higher (p <0.05) in ACC patients, especially in an advanced stage, and correlated with BUB1B and PINK1 expression, the prognostic-related genes in ACC. In paediatric tumours, high STMN1 expression was observed in both adrenocortical carcinoma and adrenocortical adenoma patients. Among the adult malignant tumours, STMN1 level was an independent predictor of survival outcomes (overall survival: hazard ratio = 6.08, p = 0.002; disease-free survival: hazard ratio = 4.65, p <0.0001). Paclitaxel, a microtubule-stabilizing drug, reduces the activation of STMN1 and significantly decreases cell migration and invasion in ACC cell lines and ACC cells from secondary cell culture (all p<0.0001). In summary, STMN1 expression may be of great value to clinical and pathological findings in therapeutic trials and deserves future studies in ACC.
Keywords Adrenocortical carcinoma · Adrenocortical adenoma · Stathmin 1 · STMN1 . Paclitaxel
Abbreviations
ACA Adrenocortical adenomas ACC Adrenocortical carcinoma
João Agostinho Machado-Neto and Claudimara Ferini Pacicco Lotfi contributed equally to this work.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10637-019-00846-9) contains supplementary material, which is available to authorized users.
☒ Claudimara Ferini Pacicco Lotfi clotfi@usp.br
1 Department of Anatomy, Institute of Biomedical Science, University of São Paulo, Av. Prof. Lineu Prestes, 2415, São Paulo, SP 05508-000, Brazil
2 Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, São Paulo, Brazil
3 Adrenal Unit, Hormone and Molecular Genetic Laboratory/LIM42, Hospital of Clinics, School of Medicine, University of São Paulo, São Paulo, Brazil
4 Division of Anatomy Pathology, Laboratory of Liver Pathology/ LIM14, Hospital of Clinics, School of Medicine, University of São Paulo, São Paulo, Brazil
| BUB1B | Budding uninhibited by benzimidazoles 1 homologue beta |
| FBS | Foetal bovine serum |
| MTT | Methylthiazoletetrazolium |
| PINK | PTEN induced kinase 1 |
| qPCR | Quantitative PCR |
| STMN1 | Stathmin 1 |
| TCGA | The Cancer Genome Atlas. |
Introduction
Adrenocortical carcinoma (ACC) is a rare malignant neo- plasm that displays a poor prognosis. Moreover, the molecular events involved in the development and progression of this neoplasm remain incompletely understood, although signifi- cant progress should be acknowledged [1-5]. The current treatment for ACC patients is surgery, radiotherapy and adju- vant chemotherapy with mitotane and/or adjuvant systemic chemotherapy; however, the response rates remain unsatisfac- tory [6]. There is evidence that adult and paediatric adreno- cortical tumours are different pathophysiological entities in relation to proliferative potential and response to therapy. In
particular, for paediatric cases, there is a limitation to distin- guish between adrenocortical adenomas (ACA) and ACC and, consequently, a definition of prognosis [7, 8]. Thus, studies to understand the molecular basis to provide information rele- vant to the prognosis, diagnostic or response to therapy for ACC are a field of interest.
Stathmin 1 (STMN1, also known as oncoprotein 18 or OP18) is a microtubule destabilizer protein that regulates mi- crotubule dynamics, cell cycle progression, proliferation, and motility and is related to overall survival [9, 10]. STMN1 stands out as an important point in converging multiple path- ways of oncogenic signalling, and its role in the development and progression of cancer has been a subject of debate in recent years [9, 11, 12]. Studies in several solid tumours have shown that STMN1 is highly expressed and that its expression predicts worse clinical outcome [9, 13]. Additionally, in vitro and in vivo studies have shown that the genetic inhibition of STMN1 using siRNA or shRNA reduces proliferation, migra- tion, invasion, and tumourigenesis [11, 12]. Recently, Aronova et al. (2018) [14] reported that STMN1 is highly expressed in adult ACC and that the inhibition of STMN1 reduces the clonogenicity, resistance to serum starvation and migration of ACC cells. Although this study provides insights into the participation of STMN1 in the tumourigenesis of ACC [14], the clear prognostic significance of STMN1 and its therapeutic potential remain poorly explored in patients with ACC.
Herein, we aimed to investigate STMN1 expression in ACA and ACC tumour tissues from paediatric and adult patients. In addition, we correlated its expression with clinical outcomes and attempted to verify the effects of the microtubule stabilizer drug, paclitaxel, in ACC cell lines, NCI-H295R and SW13, and in a secondary cell culture from ACC patients.
Materials and methods
TCGA data
STMN1, BUB1B and PINK1 mRNA expression and clinical data from the RNAseq analysis of 79 ACC patients from the TCGA study were obtained through the cBioPortal for cancer genomics (http://www.cbioportal.org). Gene expression data were expressed as Z-scores.
Patients
The clinical features of patients with adrenocortical tumours are presented in Table 1. Samples of adrenocortical tumours were obtained from 74 patients, 57 adults (mean age 40.52 ± 14.3 years) and 17 paediatric (mean age 3.82 ±4.7 years) pa- tients. The final diagnosis of the patients was determined ac- cording to the score proposed by Lau and Weiss (2009) [15] and Wieneke et al. (2003) ☒ for adult and paediatric patients,
respectively, for which the tumours were classified as adeno- mas or carcinomas. The patients were evaluated at the Adrenal Unit in the School of Medicine, Hospital das Clinicas, the University of São Paulo. This study was approved by the Ethics Committees of the Hospital das Clinicas, Institute of Biomedical Sciences (#822/2016), São Paulo, Brazil. Written informed consent was obtained from all patients or from their parents.
Cell cultures and inhibitors
Human adrenocortical carcinoma cell lines NCI-H295R [16] and SW-13 [17] and the human T acute lymphoblastic leukae- mia cell line Jurkat cells [18] were obtained from ATCC (The ATCC Cell Biology Collection). NCI-H295R was cultured in
| Adults | n =57 | |||
| Characteristics | ACA (n=31) | ACC (n=26) | ||
| Mean Age (years) | 41.2±14.3 | 41.01 ± 14.3 | ||
| Female | 24 (77.4%) | 16 (61.5%) | ||
| Male | 7 (22.6%) | 10 (38.5%) | ||
| Cancer-related death* | 0 (0.0%) | 12 (46.1%) | ||
| Mean Follow-up (months) | 50.23 | 50.09 | ||
| Pediatrics | n= 17 | |||
| ACA (n=11) | ACC (n=6) | |||
| Mean Age (years) | 3.82 ±4.73 | 2.44±0.87 | ||
| Female | 9 (81.8%) | 4 (66.7%) | ||
| Male | 2 (18.2%) | 2 (33.3%) | ||
| Cancer-related death | 0 (0.0%) | 4 (66.7%) | ||
| Mean follow-up (months) | 74.87 | 61.22 | ||
*11 cases were not informed
RPMI medium supplemented with 2% foetal bovine serum (FBS) and 1% insulin-transferrin-selenium, Jurkat cells were cultured in RPMI medium supplemented with 10% FBS, and SW-13 cells were cultured with L-15 medium supplemented with 10% FBS. All carcinoma (ACC) and adenoma (ACA) secondary cell cultures from patients, namely, ACC-T36, ACC-T12, ACC-T218Ped, ACA-T101, ACA-T23 and ACA-T7Ped, were cultured in DMEM supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and obtained as described in Almeida et al. (2008) [19] and Franca et al. (2013) [20]. All cells were cultured at 37 ℃ in 95% air-5% CO2 in a fully humidified environment. The culture used was authenticated by STR DNA profiling analysis. Paclitaxel and mitotane (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichlo- roethane) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Quantitative PCR
Total RNA was obtained using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized from 1 µg of RNA using SuperScript III First-Strand Synthesis Supermix (Invitrogen). Quantitative PCR (qPCR) was performed on a 7500 Real Time PCR System Sequencer (Applied Biosystems, Foster City, CA, USA) with specific primers for beta-actin (ACTB, forward: CCTCGCCTTTGCCGATCC; reverse: CGCGGCGATATCATCATCC), 3-glucuronidase (GUSB, forward: AGCCAGTTCCTCATCAATGG; reverse: GGTAGTGGCTGGTACGAAA), Stathmin 1 (forward: AGCCCTCGGTCAAAAGAATC; reverse: TTCAAGAC CTCAGCTTCATGGG) and the TaqMan gene expression as- say for gene quantification according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The assay IDs were as follows: GUSB (Hs00939627_m1 ID); ACTB (Hs99999903_m1 ID); BUB1B (ID Hs01084828_m1) and PINK1 (ID Hs00260868_m1). ACTB and GUSB were used as the reference genes, and a normal adrenal pool sample was used as a calibrator (BioChain, USA). The relative quantification value was calculated using the eq. 2-AACT [21]. A negative ‘No Template Control’ was included for each primer pair. The dissociation protocol was performed at the end of each run to check for non-specific amplification. Three replicas were run on the same plate for each sample.
Western blot analysis
Equal amounts of protein were used as total extracts, followed by SDS-PAGE, Western blot analysis with the indicated anti- bodies and imaging using the SuperSignal™M West Dura Extended Duration Substrate System (Thermo Fisher Scientific, San Jose, CA, USA) and G:BOX Chemi XX6 gel doc systems (Syngene, Cambridge, UK). Antibodies against
phospho(p)-STMN1$16 (sc-12,948-R) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against STMN1 (#13655), acetyl (AC)-x- tubulinK40 (#5335), ß-actin (#3700) and x-tubulin (#2144) were obtained from Cell Signalling Technology (Danvers, MA, USA).
Cell viability assay
Cell viability was measured by methylthiazoletetrazolium (MTT) assay (Sigma Aldrich). SW-13 (5 × 103 cells/well), NCI-H295R cells (2.5x 103 cells/well) and ACC-T12 cells (5 × 103 cells/well) were cultured in a 96-well plate in the presence of graded concentrations of paclitaxel (5, 10, 25, 50, 100 and 250 nM) or mitotane (1, 2.5, 5, 10, 25 and 50 µM) for 72 h. For combined treatment analysis, SW-13 and NCI-H295R cells were treated with graded doses of pac- litaxel or mitotane alone or in combination with each other for 72 h, and the data were illustrated using multiple experiment viewer (MeV) 4.9.0 software (http://www.tm4.org/mev/).
Migration and invasion assay
The migration assay was performed in a 10-well chemo- taxis chamber (Neuro Probe Inc., Maryland, USA) con- taining a polycarbonate membrane with 8 um pores. For the invasion assay, inserts and Matrigel® were used at 50 µL/cm2 (Corning, New York, USA), which was ap- plied 30 min before plating the cells. Approximately 1 × 105 cells were plated in culture medium supplement- ed with 0.1% FBS containing paclitaxel (100 nM or 250 nM) or vehicle DMSO as a control. In the lower compartment, complete culture medium was used as de- scribed above. The assay was performed at 37 ℃ in 95% air-5% CO2 in a fully humidified environment for 24 h (migration assay) or 48 h (invasion assay). After this period, the membrane was fixed for 30 min in 4% paraformaldehyde solution, stained with Giemsa’s azur eosin methylene blue solution (Merck KGaA, Darmstadt, Germany) for 2 h, washed and maintained in phosphate-buffered saline (PBS). The quantification was performed by optical microscope analysis, counting 5 fields per well, by using the NES-Nikon image anal- ysis program. At least three independent experiments were performed for each condition and cell type.
Immunocytochemistry
Approximately 5 × 104 cells were seeded onto Thermanox™M Coverslips (Nalge Nunc International) in DMEM supplement- ed with serum and then fixed with paraformaldehyde 4% for 30 min for the analysis of Stathmin1 protein. The anti- Stathmin 1 antibody [(1:50); (FineTest, FNab08310)] was
detected by immunoperoxidase staining using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine (DAB, Sigma, D8001) as described previously [13].
Statistical analyses
Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., San. Diego, CA, USA). The results are representative of three independent experiments. Statistical significance was determined using the Tukey- Kramer multiple comparisons test after analysis of variance (ANOVA). Both the Kruskal-Wallis test and Dunn’s post hoc test were used for the measured factors. The patients were dichotomized according to median gene expression for the survival analysis. The Kaplan-Meier method, Cox regression
analysis, and a log-rank test were used to estimate survival outcomes. A p value <0.05 was considered significant.
Results
STMN1 expression is associated with aggressiveness in ACC patients
Using the data from the TCGA study of ACC as a learning cohort, we observed that STMN1 mRNA levels were signifi- cantly increased in adult ACC patients with stage IV com- pared to stage I and II cancers (all p<0.05), suggesting that STMN1 expression is associated with aggressiveness in ACC (Fig. 1a). In the learning cohort, we also observed that high levels of STMN1 predict poor overall survival (p<0.0001; Fig. 1b) and disease-free survival (p<0.0001;
a
Adrenocortical Carcinoma TCGA cohort
b
Adrenocortical Carcinoma TCGA cohort
Relative levels of Stathmin 1 mRNA (Z-scores)
6-
*
100
- Low STMN1 (n=40)
Overall survival (%)
High STMN1 (n=39)
4.
80
60
2.
40-
0-
20
p<0.0001
-2
Stage I n=9
Stage II n=37
Stage III n=16
Stage IV n=15
0
0
50
100
150
200
Months
c
ACA and ACC USP cohort
d
Adrenocortical Carcinoma USP cohort
Relative levels of Stathmin 1 mRNA expression
100
100
- Low STMN1 (n=13)
**
Overall survival (%)
High STMN1 (n=13)
80-
10
60-
40-
1
20
p=0.03
0.1
ACA n=31
ACC
ACA
ACC n=06
0
0
100
200
300
400
n=26
n=11
Months
Adult patients
Pedriatric patients
| Overall survival | Disease free survival3 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Univariate | Multivariate | Univariate | Multivariate | |||||||||
| Factors | HR1 | (95% C.I.) | 3 p3 | HR1 | (95% C.I.) | p 3 | HR1 | (95% C.I.) | p 3 | HR1 | (95% C.I.) | p 3 |
| Gender | ||||||||||||
| Male vs. female | 1.00 | 0.46-2.13 | 0.99 | - | - | – | 0.68 | 0.34-1.36 | 0.28 | - | - | – |
| Age at diagnosis2 | 1.01 | 0.98-1.03 | 0.37 | - | - | – | 1.00 | 0.98-1.02 | 0.79 | 1 - | - | – |
| Disease stage- | ||||||||||||
| IV vs. III vs. II vs. I | 2.91 | 1.85-4.56 | <0.0001 | 2.05 | 1.30-3.24 | 0.002 | 2.35 | 1.63-3.41 | <0.0001 | 2.02 | 1.38-2.95 | <0.0001 |
| STMN1 expression- | ||||||||||||
| High vs. low expression | 9.68 | 3.33-28.11 | <0.0001 | 6.08 | 1.96-18.78 | 0.002 | 5.68 | 2.74-11.75 | <0.0001 | 4.65 | 2.16-9.99 | <0.0001 |
TCGA The Cancer Genome Atlas; Statistically significant difference is highlighted in bold
1 Hazard ratios (HR) >1 indicate that increasing values for continuous variable or the first factor for categorical variable has the poorer outcome. Missing values were excluded in the calculation of p-values
2 Factors were analyzed as continuous variable
3 Adrenocortical carcinoma patients were stratified according Neoplasm Disease Stage American Joint Committee on Cancer Code
4 Factors were dichotomized by median
Supplementary Fig. 1). In the multivariate proportional hazard regression analysis correcting for confounders (age, gender, and tumour stage), STMN1 expression was an independent predictor of overall survival and disease-free survival (all p<0.01; Table 2). In our cohort (USP cohort), we found that
the STMN1 gene is more highly expressed in carcinomas than in adenomas (p<0.01; Fig. 1c). On the other hand, in a cohort of paediatric ACA and ACC patients, high expression of STMN1 was observed in both groups, suggesting a greater proliferative potential of paediatric adenomas. These results
a
Adrenocortical Carcinoma TCGA cohort
b
Adrenocortical Carcinoma TCGA cohort
Relative levels of BUB1B mRNA
4.
r=0.79
6-
r =- 0.52
p<0.0001
Relative levels of PINK1 mRNA
p<0.0001
4
2
2
0
0
-2
-2
-4
-2
0
2
4
6
-2
0
2
4
6
Relative levels of Stathmin 1 mRNA
Relative levels of Stathmin 1 mRNA
c
Adrenocortical Carcinoma USP cohort
d
Adrenocortical Carcinoma USP cohort
Relative levels of BUB1B mRNA
50
r=0.47
Relative levels of PINK1 mRNA
4-
r =- 0.19
p=0.02
p=0.34
40
3
30
2
20
1
10
0
0
0
5
10
15
0
5
10
15
Relative levels of Stathmin 1 mRNA
Relative levels of Stathmin 1 mRNA
IB:STMN1 18kDa
IB:ß-actin 42kDa
a
Jurkat
ACC-T36
ACA-T101
ACC-T218Ped
H295R
SW-13
ACA-T7Ped
b
ACC and ACA cells
8
STMN1/B-actin expression
p<0.0001
6
4
2.
0
Jurkat .
H295R
SW-13
ACC-T36
ACA-T101
ACA-T7Ped
ACC-T218Ped
reinforce the hypothesis that adult and paediatric adrenocorti- cal tumours are different entities from a biological point of view. In the ACC adult cohort (validation cohort), we con- firmed that a high level of STMN1 predicts poor survival out- comes (p<0.03; Fig. 1d).
STMN1 expression is correlated with known prognostic-related genes, BUB1B and PINK1, in adult ACC patients
Since BUB1B and PINK1 are recognized prognostic-related genes in ACC [22, 23] and BUB1B and STMN1 play impor- tant roles in mitosis, we verified the correlation between STMN1 and BUB1B or PINK1 expression. In both the learning and validation cohorts, STMN1 was positively correlated with BUB1B expression (learning cohort: r = 0.79, p < 0.0001; val- idation cohort: r= 0.47, p=0.02) and negatively correlated
with PINK1 expression (learning cohort: r =- 0.52, p<0.0001; validation cohort: r =- 0.19, p> 0.05) (Fig. 2).
Pharmacological inhibition of microtubule dynamics slightly reduces cell viability in contrast with the effect on the migration and invasion of ACC cells
We first investigated the expression of STMN1 in a panel of ACA and ACC cell cultures. STMN1 was highly expressed in adult ACC secondary cultures and cell lines (SW-13 and NCI- H2915R) compared to adult ACA secondary cultures. However, secondary culture of the cells from paediatric ACA and ACC patients showed slight STMN1 expression (Fig. 3 and Supplementary Fig. 2). Next, using SW-13, NCI- H2915R, and ACC-T12 cells, we investigated the effects of the microtubule stabilizer drug paclitaxel on cell viability and migration. In SW13 and ACC-T12, but not in NCI-H2915R, paclitaxel reduces cell viability (p<0.05; Fig. 4a). Mitotane at 10, 25 and/or 50 µM reduced the viability of SW13 and NCI- H2915R cells but not ACC-T12 cells (p<0.05; Fig. 4b). Synergism analyses between paclitaxel and mitotane indicated that paclitaxel treatment did not impair the cytotoxic effects of mitotane in the ACC cell lines tested (Fig. 4c). Paclitaxel treatment increased STMN1 phosphorylation at serine 16 (an inhibitory site) and microtubule stability, as observed by increased levels of «-tubulin acetylation (Fig. 4d).
Paclitaxel at 100 nM and 250 nM reduced the migration of SW-13, NCI-H295R and ACC-T12 cells by 96.2±1.7%, 97.3±1.2%, 91.1±1.7%, respectively, and reduced invasion by 98.1 ±1.1%, 94.8±6.1%, 87.7±2.5%, respectively, com- pared to that of control cells (p<0.0001; Fig. 5).
Discussion
Although data from The Cancer Genome Atlas Project [2] have identified genomic clusters in ACC from adult pa- tients that have been defined as differential markers of survival, such as IGF2 overexpression, Wnt/ß-catenin signalling pathway activation, and deficient cell cycle control, other molecular predictors of malignancy and sur- vival of ACC should be involved and need to be investi- gated. In the present study, high STMN1 expression pre- dicted a worse clinical outcome in adult patients with ACC in two independent cohorts. Of note, STMN1 ex- pression was an independent predictor of survival out- comes in the TCGA ACC cohort. The clinical impact of STMN1 expression has been extensively addressed in many types of human cancers, and in general, the in- creased STMN1 expression confers a poor prognosis [9, 11, 13]. However, to our knowledge, this study is the first that defines the prognostic value of STMN1 expression in
a
SW-13 cells
NCI-H295R cells
ACC-T12 cells
120-
120-
120-
Cell viability (% of control)
Cell viability (% of control)
Cell viability (% of control)
100
100
T
100-
*
**
80
80-
80-
60
60-
60-
40
40-
40-
20-
20-
20-
0
0
5
10
25
50
100
250
0
Ø
5
10
25
50
100
250
0
Ø
5
10
25
50
100
250
Paclitaxel (nM)
Paclitaxel (nM)
Paclitaxel (nM)
b
SW-13 cells
NCI-H295R cells
ACC-T12 cells
120-
120-
Cell viability (% of control)
Cell viability (% of control)
120-
Cell viability (% of control)
100
100
T
100
4
**
80-
**
80-
80-
60
60-
60-
40-
40-
40-
20
20
20-
0
0
0
1
2.5
5
10
25
50
Ø
1
2.5
5
10
25
50
0
Ø
1
2.5
5
10
25
50
Mitotane (u M)
Mitotane (u.M)
Mitotane (LM)
c
SW-13 cells
NCI-H295R cells
ACC-T12 cells
Paclitaxel (nM)
Paclitaxel (nM)
Paclitaxel (nM)
5
10
25
50
100
250
5
10
25
50
100
250
5
10
25
50
100
250
5
1
1
5
1
1
5
1
1
10
LEPA
Paclitaxel (nM)
2.5
Mitotane (UM)
1025 1025
2.5
Mitotane (M)
Paclitaxel (nM)
Mitotane (UM)
Mitotane (UM)
Paclitaxel (nM)
1025 1025
2.5
2.5
Mitotane (UM)
Mitotane (pM)
25
5
5
5
5
5
5
JO
10
10
50
10
10
50
10
10
100
25
25
25
25
100
25
25
250
50
50
250
50
50
250
50
50
Cell viability (% of control)
Cell viability (% of control)
Cell viability (% of control)
100
50
0
100
50
0
100
50
0
d
SW-13 cells
NCI-H295R cells
ACC-T12 cells
Paclitaxel (nM)
Ø
50
100
250
Paclitaxel (nM)
Ø
50
100
250
Paclitaxel (nM)
Ø
50
100
250
ratio:
1.00
6.23
6.18
6.50
ratio:
1.00
1.08
1.98
6.06
ratio:
1.00
17.67
15.09
16.19
IB: p-STMN1$16
IB: p-STMN1$16
(18 kDa)
(18 kDa)
IB: p-STMN1$16
(18 kDa)
IB: STMN1 (18 kDa)
IB: STMN1 (18 kDa)
IB: STMN1 (18 kDa)
ratio:
1.00
23.98
26.40
27.19
ratio:
1.00
6.14
8.43
17.61
ratio:
1.00
5.94
6.81
7.75
IB: Ac-a-tubulin₭40 (55 kDa)
IB: Ac-a-tubulinK40 (55 kDa)
IB: Ac-a-tubulinK40 (55 kDa)
IB: a-tubulin (55 kDa)
IB: a-tubulin (55 kDa)
IB: a-tubulin (55 kDa)
ACC. Regarding STMN1 expression, adult ACC present- ed higher levels than adult ACA, which confirmed the
concentrations of paclitaxel (5, 10, 25, 50, 100 and 250 nM) and mitotane (1, 2.5, 5, 10, 25 and 50 uM) alone or in combination with each other for 72 h. The values are expressed as the percentage of viable cells for each condition relative to untreated controls. The results are shown as the mean of at least four independent experiments. (D) Western blot analysis for phospho (p)-STMN116/STMN1 and AC-x- tubulinK40/x-tubulin is indicated
previous finding from Aronova et al. (2018) [14]. Both ACA and ACC paediatric samples presented increased
a
SW-13 cells - Migration
SW-13 cells - Invasion
Paclitaxel (nM)
Migration (Cell number/field)
30-
Invasion (Cell number/field)
25-
Ø
Ø
20
20
Paclitaxel (nM)
15-
100
100
10
10-
5
250
0
250
Ø
100
250
0
Ø
100
250
Paclitaxel (nM)
Paclitaxel (nM)
b NCI-H295R cells - Migration
NCI-H295R cells - Invasion
Migration (Cell number/field)
60-
Paclitaxel (nM)
Paclitaxel (nM)
Invasion (Cell number/field)
20
Ø
Ø
15-
40
100
100
10
20-
5-
250
250
0
Ø
100
250
0
Ø
100
250
Paclitaxel (nM)
Paclitaxel (nM)
c
ACC-T12 cells - Migration
ACC-T12 cells - Invasion
Migration (Cell number/field)
40-
Invasion (Cell number/field)
25
Ø
Ø
30-
20
Paclitaxel (nM)
Paclitaxel (nM)
15-
100
20-
100
10-
10-
5-
250
0
250
Ø
100
250
0
Ø
100
250
Paclitaxel (nM)
Paclitaxel (nM)
STMN1 levels at similar levels of adult ACC. Indeed, STMN1 is highly expressed in proliferating cells [9, 24], and there is evidence that paediatric ACA presents higher proliferative potential than adult ACA, which represents a limitation for the definition between the diagnosis of ACA or ACC in paediatric patients [7, 8]. In addition, high STMN1 expression has previously been associated with the presence of TP53 mutations in solid tumours [25, 26]; thus, the high prevalence of TP53 mutations in Brazilian children with ACA and ACC may partially ex- plain our findings and deserve future investigation [27]. Taken together, our results provide novel molecular in- sights and corroborate that paediatric adenomas display increased proliferative potential and are biologically dis- tinct from adult adrenocortical tumours.
The correlation intensities of STMN1/BUB1B and STMN1/PINK1 varied between the TCGA and USP co- horts, but the TCGA cohort has a larger number of ACC patients and the same trend was observed for both cohorts. BUB1B is more highly expressed in ACC than in ACA, and its combined expression with TCF21/ POD-1 (transcription factor 21) is considered a predictor of overall survival in adult ACC [28]. The positive cor- relation between STMN1 and BUB1B expression in adult ACC, which are both important to the mitotic phase of the cell cycle, reinforces the importance of these two factors in proliferative potential and carcinoma aggres- siveness. Although PINK1 expression was similar among adult tumours, the combined expression of BUB1B and PINK1 was demonstrated as an important
molecular marker in adult ACC in different cohorts [22, 23]. However, the relationship between PINK1 and STMN1 deserves to be explored.
Based on previous evidence that paclitaxel inhibits STMN1 activity and induces microtubule stability [29-31], this drug was used as a pharmacological inhib- itor of microtubule dysfunctional dynamics. Indeed, in our study, paclitaxel treatment reduced STMN1 expres- sion and induced the phosphorylation of an inhibitory site (serine 16) in ACC cell lines. Aronova et al. (2018) [14] reported that STMN1 silencing by siRNA did not modulate cell viability but modulates migration, which agrees with our results using paclitaxel in ACC cell lines. Other studies reported that paclitaxel reduces cell viability and tumour burden from SW-13, but not NCI- H295R cells [32, 33]. A phase II clinical trial failed to demonstrate the beneficial effects of paclitaxel as a second/third-line therapy for advanced ACC patients [34]. These findings agree with our results that inhibi- tion of microtubule dynamics has limited efficacy in advanced ACC models. On the other hand, our results also suggest that microtubule inhibitors as adjuvant ther- apy may be useful in the early stages of the disease since they do not alter the response to current therapy (mitotane) but significantly reduce cell migration and invasion in vitro.
In summary, our data provide new insights into the tumourigenesis of paediatric ACC and indicate that STMN1 expression may be of great value as a diagnostic tool and risk stratification of adult ACC patients. Nevertheless, its potential therapeutic value deserves further study.
Acknowledgements We are grateful to Cintia Fridman, Department of Legal Medicine and Medical Ethics, School of Medicine, University of São Paulo, for STR DNA profiling analysis in cell cultures. The authors would like to acknowledge all the research participants contributing to The Cancer Genome Atlas (TCGA) resource for providing high-quality data for analyses.
Author contributions BSP, KL, JLK, and BBC performed the experi- ments, data analysis and interpretation, and manuscript editing. BMPM processed patient samples and performed manuscript editing. JCLS per- formed multivariate statistical analysis, data interpretation, and manu- script editing. MCBVF provided the patient samples and clinical follow-up and performed manuscript editing. MCZ selected the patients and performed manuscript editing; JAMN and CFPL idealized the study, analysed the data and wrote the manuscript.
Funding BSP and JLK are recipients of a scholarship from FAPESP (nº 2016/12381-7 / nº 2016/17285-6), the São Paulo State Research Foundation (FAPESP); BBC is the recipient of a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); JAM-N received funding from FAPESP (nº 2017/24993-0) and from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CFPL received funding from FAPESP (nº 2015/014199-9; 2018/19035-2) and from CNPq. This study was fi- nanced in part by CAPES- Finance Code 001.
Compliance with ethical standards
Ethical approval Informed consent was obtained from all individual participants included in the study prior to sample collection, and the study was approved by the Institutional Review Board.
Conflict of interest The authors declare that there is no conflict of inter- est that could be perceived as prejudicing the impartiality of the research reported.
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