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Long Non-Coding RNA H19 Expression Correlates with Autophagy Process in Adrenocortical Carcinoma
Pietro Di Fazio, Franziska D. Rusche, Silvia Roth, Anika Pehl, Sabine Wächter, Ioannis Mintziras, Detlef K. Bartsch & Katharina Holzer
To cite this article: Pietro Di Fazio, Franziska D. Rusche, Silvia Roth, Anika Pehl, Sabine Wächter, Ioannis Mintziras, Detlef K. Bartsch & Katharina Holzer (2022) Long Non-Coding RNA H19 Expression Correlates with Autophagy Process in Adrenocortical Carcinoma, Cancer Investigation, 40:3, 254-267, DOI: 10.1080/07357907.2021.2001483
To link to this article: https://doi.org/10.1080/07357907.2021.2001483
Published online: 16 Nov 2021.
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Long Non-Coding RNA H19 Expression Correlates with Autophagy Process in Adrenocortical Carcinoma
Pietro Di Fazioª* (D, Franziska D. Ruscheª*, Silvia Rothª, Anika Pehlb, Sabine Wächterª, Ioannis Mintzirasa, Detlef K. Bartschª and Katharina Holzera
ªDepartment of Visceral, Thoracic and Vascular Surgery, Philipps University Marburg, Marburg, Germany; bInstitute of Pathology, Philipps University Marburg, Marburg, Germany
ABSTRACT
Adrenocortical carcinoma (ACC) is characterized by poor prognosis and high mortality. The suppression of the long-non-coding RNA H19, counterbalanced by IGF2 over-expression, leads to down-regulation of the autophagy markers, high proliferation rate and metastatic potential in patients affected by ACC. The administration of the deacetylase inhibitors (DACi) panobinostat, trichostatin A (TSA) and SAHA affected the cell viability of H295R monolayer and spheroids and induced the over-expression of H19 and autophagy tran- scripts. H19 knock down in H295R cells was not able to modulate the expression level of autophagy transcripts. Instead, H19 knock down was able to impede the ability of DACi to modulate the protein level of the autophagy markers. Furthermore, the administration of higher concentration of DACi was able to down-regulate the protein level of Beclin1 and p62 and to induce the conversion of LC3B-I into the active LC3B-II form, thus confirming an active autophagic process. Neither the active protein level nor the activity of caspases 8 and 3 was prompted by the DACi, thus excluding the involvement of the executioners of apop- tosis in H295R decay. The DACi restore H19, the autophagy markers and trigger cell death in ACC cells. The re-activation of autophagy would represent a novel strategy for the treat- ment of patients affected by this severe malignancy.
ARTICLE HISTORY
Received 25 November 2020 Revised 23 September 2021 Accepted 29 October 2021
KEYWORDS
H19; autophagy; deacetylase inhibitors; adrenocortical carcinoma
Introduction
Human adrenal glands are affected by a rare malignancy of the cortex with an estimated inci- dence of 0.7-2.0 cases per million each year (1-3). The patients have poor prognosis and overall 5-year survival rates of 16-44% (4). The maternally imprinted long-non-coding RNA H19 is mapped contiguously with insulin growth fac- tor 2 (IGF2) on chromosome 11p15.5 (5) and it is supposed to be involved in tumor suppression (6). The low expression of H19 could contribute to adrenocortical carcinoma (ACC) tumorigenesis (7). H19 has been also found heterogeneously expressed in thyroid cancer and it is strongly restored in these cells by the treatment with deacetylases inhibitors, which trigger cell death (8). Recent findings have highlighted that 66 long-non-coding RNAs are associated with ACC
recurrence, including H19 (9) and, beyond them, the epitranscriptome, in terms of RNA methyla- tion (m6A) (10), could exert a key role for the classification of ACC (11). Nonetheless, muta- tions occurring at genes involved in histone mod- ifications have been identified in 38% of ACC included in a previous study (12). And, hyperme- thylation occurring at TERT promoter has been associated, as epigenetic alteration, with poor prognosis in patients affected by ACC (13). Autophagy is a catabolic process that, if pro- longed, can promote cell demise. Up to now, the direct correlation between H19 and autophagy has been found in breast cancer (14) and hepato- cellular carcinoma (15) cells. However, the correl- ation between H19 and autophagy is unknown in ACC.
*Authors’ equal contribution.
Interestingly, autophagy can be restored in ACC cells by treatment with peroxisome prolifer- ation-associated receptor y (PPARy) inhibitor, thus inducing cell death (16). Additionally, autophagy and necrosis can be induced in ACC cells by targeting estrogen-related receptor o. (ERRx) (17).
Here, we proposed to analyze the basal expres- sion of H19, its contiguous mapped gene IGF2 and the autophagy markers in patients affected by ACC in order to identify a possible correlation between the long non-coding RNA and the autophagy process. Patients affected by benign Cushing’s adenoma were included in the study to get a comparison/normalization of the ACC patients characterized by Cushing’s syndrome and high level of cortisol (more than 45% of patients included in the study), according also to the most recent guidelines (18-21). Furthermore, the deacetylase inhibitors (DACi), well known for their ability to induce autophagy cell death (22,23), which have not been tested for the treat- ment of ACC yet, could be effective by triggering autophagy in ACC cells too. Therefore, we focused on identifying the effect of DACi in H295R cells-derived spheroids and their ability to control the expression of H19 and autoph- agy markers.
Materials and methods
Patients’ samples collection
The tumor probes were collected from patients underwent surgical resection of the adrenal gland at Marburg University Hospital between 2000 and 2019. A total of 21 patients were affected by ACC, eight by Cushing’s adenoma. Eight healthy adrenal cortex tissue probes, which were collected from the resected tumor tissue of patients under- gone ACC and Cushing’s adenoma surgical resec- tion, were used as control. Pathologist assessed ACC, CA and adrenal cortex probes about their tissue characteristics. The study was approved by the local ethic committee of the medical faculty of Philipps University of Marburg (Nr. 132/03 “Molekulargenetische Untersuchungen zur Entstehung und Progression von
Nebennierenrindentumoren”). All patients signed the informed consent.
Cells
The ACC cell line NCI-H295R (300483), repre- senting the only in vitro ACC model (24), was purchased from CLS (Cell Line Service GmbH, Eppelheim, Germany) and cultivated with DMEM: Ham’s F12 medium (1:1 mixture) (con- taining 15 mM HEPES, 0.00625 mg/ml insulin, 0.00625 mg/ml transferrin, 6.25 ng/ml selenium, 1.25 mg/ml bovine serum albumin, 0.00535 mg/ ml linoleic acid, 2.5% Nu-Serum I (355100, Corning, NY) and Corning®ITS + Premix (8211002, Corning, NY). To prevent bacterial contamination, the growth medium was supple- mented with penicillin (100 Units/ml) and streptomycin (100 µg/ml). The cells were kept at 37°℃ and humidified atmosphere with 5% CO2.
Compounds
Panobinostat was a kindly gift from Novartis (Basel, Switzerland). Trichostatin A (TSA) was purchased from Promega (G656A, Promega, Madison, WI) and SAHA (Vorinostat, Zolinza) from Invivogen (inh-saha, Invivogen, San Diego, CA). All compounds were dissolved in DMSO.
Real-time cell viability analysis
A total of 10,000 NCI-H295R cells were cultured on E-plates (05232368001, OLS, Bremen, Germany) and real-time cell viability was meas- ured after treatment with 1 nM - 1 µM of pano- binostat, TSA and 1 nM - 10 uM of SAHA by xCELLigence RTCA system (Roche, Basel, Switzerland). xCELLigence continuously meas- ured (120h) the impedance to quantify the adherence of the cells on the plate’s electrodes. Impedance was continuously measured every 15 min for 80 h.
Spheroids
NCI-H295R spheroids were formed on 50 ul 1.5% peqGOLD Universal Agarose (PEQLAB Biotechnology GmbH, Erlangen, Deutschland) in
a flat-bottom 96-well plate (SARSTEDT AG and Co. KG, Nümbrecht, Germany) for 6d without medium change. A total of 4,000 NCI-H295R cells were plated in 200 ul medium in a humidi- fied atmosphere containing 5% CO2 at 37 ℃, were placed on an orbital shaker with a shaking speed of 70 rpm overnight. Before treatment, 100 ul of complete growth medium were removed from each well containing a single spheroid. Of 100 ul medium with panobinostat, TSA or SAHA were added to the remaining 100 ul medium. The working concentration was 100 nM and 1 µM panobinostat, 100 nM and 1 uM TSA and 10 µM SAHA.
H19 transient knock down
H19 was transiently knocked down in H295R cells by 48h transfection with two specific siRNAs binding to H19 transcript. The oligos are commercially available and validated from Qiagen. Transfection was performed with HiPerfect (Qiagen, Hilden, Germany) by follow- ing the QIAGEN Fast Transfection Protocol as described by the manufacturer. AllStars Negative Control (Qiagen) oligonucleotides (NTC) were used as non-silencing control.
Quantitative RT-PCR
Total RNA was isolated with the RNeasy Mini Kit (74106, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Reverse Transcription of mRNA was performed with iScript™M cDNA Synthesis Kit (170-8891, BIORAD, Hercules, CA, USA) on FlexCycler (Analytik Jena AG, Jena, Deutschland). Qiagen primers for human lncRNA H19 (PPH05814B), IGF2 (QT01670802), BECN1 (QT00004221), UVRAG (QT00034328), MAP1LC3B (QT00055069), SQSTM1 (QT00095676), TFEB (QT00069951), GAPDH (QT01192646), RRN18S (QT00199367), PRKAA1_1 (QT000094), PRKAA2_1 (QT0004207) were used with GoTaq® qPCR Master Mix (Promega, Madison, WI) on RT-qPCR thermocycler CFX96™M Real-Time System (Bio-Rad Laboratories, Hercules, CA). Results were analyzed with the Bio-Rad CFX- Manager (Bio-Rad Laboratories) and normalized
with GAPDH mRNA content for each sample. Raw data were further analyzed with Rest2009 (relative Expression Software Tool version 2.0.13, Qiagen, Hilden, Germany).
DNA staining of spheroids
Spheroids treated with panobinostat or TSA were incubated with 100 nM PEqGreen (PEQLAB Biotechnology, GmbH) (dissolved 1:80 in PBS) the 7 d after treatment. The fluorescent signal of the DNA intercalating agent was visualized under fluorescence inverted microscope after 5 min incubation time with 100 nM PEqGreen.
Caspase assay
Caspase 3/7 and caspase 8 activity was measured in H295R spheroids after the administration of DACi by following the manufacturer instruction (Caspase Glo 3/7 and Caspase Glo 8 Assay System, Promega, Madison, WI, USA).
Western blot
Whole protein lysates were isolated from H295R cells treated with DACi and siH19. The protein content was isolated by the use of Jié’s Buffer (10 mM NaCl, 0.5% NonidetP40, 20 mM Tris-HCL pH7.4, 5mM MgCl2, 1 mM PMSF, Complete Protease Inhibitor and Phosphatase Inhibitor (Roche, Basel, Switzerland). The pro- teins were separated through SDS-Page (NP0342, Life Technologies, Carlsbad, CA) and transferred to nitrocellulose membranes (10600009, GE Healthcare Life science, Chicago, IL) by semi- dry-blotting with Trans-Blot®TurboTM Transfer System (Bio-Rad Laboratories). The membranes were further sliced according to the required molecular weight of the proteins of interest, blocked in 4% BSA (23208, Thermo Fisher Scientific, Waltham, MA) in TBS-Tween20 (0.5%) and incubated with primary antibodies against Beclin1 (ab92389, AbCam, Cambridge, UK), UVRAG (U7508. SIGMA-ALDRICH, St. Louis, USA), LC3B (ab51520, Abcam), SQSTM1 (p62) (ab96706, Abcam), Caspase 8 (ALX-804- 242, Enzo Life Sciences GmbH, Lörrach Germany), Caspase 3 (NB100-56708, Novus
Biologicals, Abingdon, UK) and ß-actin (A5441 SIGMA-ALDRICH, St Louis, MO). Bound pri- mary antibodies were detected by secondary horseradish-labeled goat anti-rabbit (A0545, Sigma-Aldrich) and goat anti-mouse (A9917, Sigma-Aldrich, St Louis, MO) antibodies and SuperSignal West Pico Chemiluminiscent Substrate (Thermo Fisher Scientific, Waltham, MA). The immuno-detection was quantified using Fusion image capture (VILBER LOURMAT Deutschland GmbH, Eberhardzell, Germany) and Bio-1D analysis System (VILBER LORUMAT Deutschland GmbH).
Immunofluorescence on paraffin-embedded tissue
Of 4 um thin sections of 4% formaldehyde fixed paraffin embedded ACC, Cushing’s adenoma and healthy adrenal cortex were cut, rehydrated and deparaffinized. Antigen retrieval was performed in citrate buffer (pH = 6) in a microwave with 480 Watt for 10 min. The endogenous peroxidase was blocked with 3% H2O2 for 10 min. The sec- tions were permeabilized by 0.5% Triton X-100 (Carl Roth Gmbh & Co. KG, Karlsruhe, Germany) in PBS Buffer (Life Technologies, Carlsbad, CA) for 10 min. Unspecific bindings were blocked through 30 min incubation in 10% immunized goat normal serum (Vectastain® Elite ABC-KIT, Burlingame, CA). The slides were then incubated with 2.5 µg/ml primary antibodies to TFEB (ab220695, Abcam, Cambridge, UK) and Beclin1 (ab114071, Abcam) in 1% BSA-PBS- 0.5%-Tween20 overnight at 4℃. The bound pri- mary antibodies were labeled with 2 ug/ml Alexa Fluor® 568 F(ab’)2 fragment of goat anti-rabbit IgG (H+L) (A21069, Invitrogen, Carlsbad, CA)/ Alexa Fluor® 488 goat anti-mouse IgG (H+L) secondary antibodies. Nuclei were stained with 1 µg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) in 1% BSA-PBST. After 90 min incubation with secondary antibodies and Hoechst, the tissue slides of tumor probes were processed with Vector® TrueVIEW™M Autofluorescence Quenching Kit (VECTOR Laboratories, Burlingame, CA) and mounted with VECTASHIELD® HardSet™M Antifade Mounting Medium (VECTOR Laboratories). LAS AF soft- ware (Leica microsystems, Wetzlar, Germany)
was used for the analysis of fluorescence images acquired with the wide-field fluorescence micro- scope LEICA DM 5500. FIJI (Image] version 1.52t, Wayne Rasbund, National Institutes of Health, Bethesda, MD) image processing software was used to further analyze the acquired images.
Statistical analysis
Statistical analysis was performed by RStudio ver- sion 1.2.5019 (2009-2019 RStudio, Inc., Boston, MA) and EXCEL 2016 (Microsoft, Albuquerque, NM) for the analysis of correlations within clin- ical groups as well as correlation analysis refer- ring to molecular and clinical variables. Kolmogorov-Smirnov test and Levene’s test have been applied to evaluate normal distribution and variance homogeneity. Significance was calculated using one-way-ANOVA, otherwise Kruskall- Wallis-Test was applied. In case of a significant result, further post-hoc-tests were performed for pairwise comparison of groups. Post-hoc-tests were pairwise t-test in case of normal distribution and variance homogeneity or pairwise Wilcoxon- rank-test. For correlation analysis, the Pearson- product-moment-correlation was performed. *p <. 05 were regarded as significant.
Results
Expression of IGF2, H19 and autophagy transcripts in Cushing’s adenoma, ACC and healthy adrenal gland cortex
IGF2, H19 and autophagy transcripts expression was detected in 7/8 probes of patients affected by CA, 17/21 patients affected by ACC (Table 1) and normalized vs. 8/8 samples of healthy adrenal
| Gender | ACC (n =21) 4 males/17 females | CA (n=8) 0 male/8 females |
|---|---|---|
| Age (years) | Median 46 (range: 15-84) | Median 46 (range: 19-66) |
| OS (months) | Median 70 (range: 5-298) | – |
| Weiss-score | Median 5 (range: 2-8) | – |
| Ki-67 | Median 20 (range: 2-40) | <1 |
| Tumor diameter (cm) | Median 10 (range: 4-21) | Median 3.5 (range: 2.4-4.5) |
| Cushing syndrome | 9 (3 males/6 females) | 8 |
| High cortisol | 9 (3 males/6 females) | – |
ACC: adrenocortical carcinoma (n=21) and CA: Cushing’s adenoma (n=8); OS: overall survival.
Mean relative engresson IGf 2 norm. GAPOH
IGF2
H19
TFEB
BECN1
A
w
Mean relative expression Pt? norm GAPOH
Mean relative expression TFED nom GAPOH
Mean relative expression Begint nom GAPCH
Figure 1
N
N
N
.
-
-
Mean rwdtve expression MAPTLC30 nom GAPO
MAP1LC3B
Mean relative expression SOSTMI nom GAPOP
SQSTM1
Meant rwatve expression UMRAG norm GAPOH
UVRAG
Meanswave expression PROKAA !! nimm GAPC:
PRKAA1_1
Meanswelive expression PROVAAZT nom GAPDR
PRKAA2_1
A
8
.
30
83
1
·
#
.
#
1
-
-
-
B
Mean rel expression Becint (ogto)
MAP1LC3B
Mean rel expression MAPILC30 (og10)
BECN1
Mean rel Expression TFEB (og10)
1.00
TFEB
UVRAG
Mean rel. expression UVRAG Dogte)
1
p<0.05
p<0.05
p<0.05
p<0.05
1
Mean rel. expression H19 (log10)
001
Mean rel. expression H19 (og10)
Mean rel Expression H19 (og10)
10:00
Mean rel. expression H19 (og10)
10:00
H19
gland cortex. The transcript level of IGF2, a standard marker for ACC and contiguously mapped to H19 (5), was significantly (p <. 05) over-expressed in CA and ACC tissue as well. The relative expression of H19 detected in CA was stable and comparable to the expression detected in healthy adrenal cortex tissue. Instead, the analysis of ACC tissue samples evidenced a significant (p <. 05, Anova-Bonferroni) down- regulation of H19 transcript (Figure 1).
The autophagy transcripts TFEB, BECN1, SQSTM1, UVRAG, PRKAA1_1 and PRKAA2_1 represent the key players of the autophagy pro- cess and encodes for the transcription factor (TF) responsible for autophagy genes modulation, for the proteins being part of autophagy vesicles and for the cAMP (cyclic-Adenosyl mono phosphate) kinase triggering autophagy (25-27). They were found stably expressed in CA tissue samples in comparison to healthy adrenal cortex tissue. Only MAP1LC3B transcript was significantly (p < .05, Anova-Bonferroni) over-expressed (Figure 1). The expression of all detected autophagy markers was significantly down-regulated (p <. 05,
Anova-Bonferroni) in ACC samples in compari- son to healthy adrenal cortex. MAP1LC3B was the only stably expressed transcript detected in ACC tissue samples (Figure 1).
Correlation between H19 and autophagy markers
The expression of H19 was statistically compared with the expression of all detected autophagy transcripts. Interestingly, Pearson-product- moment-correlation test evidenced that the expression of H19 directly and significantly corre- lates with the expression of BECN1, MAP1LC3B, TFEB and UVRAG (Figure 1(B)) in patients affected by ACC, according to Anova-Bonferroni test. No significant correlation was found between H19 and SQSTM1, PRKAA1_1, PRKAA2_1 (data not shown).
TFEB and Beclin1 proteins detection in CA and ACC tissue samples
TFEB is a TF responsible for the regulation of the expression of autophagy and lysosomal genes
A
Adrenal cortex
Cushing’s Adenoma
Adrenocortical carcinoma
Figure 2
TFEB
Beclin1
TFEB
Beclin1
TFEB
Beclin1
E
Adrenal cortex
Cushing’s Adenoma
ACC
B
1000
C
1000
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1.00
density of fluorescence intensity TFEB.
density of fluorescence intensity Becint
750
750
0.75
r. TFEB & Becint
500
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:
0
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Adenoma
ACC
Normal Baque
Adenoma
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Normal Tissue
Adenoma
ACC
Normal Esque
and, therefore, sustaining the autophagy process (27). The detection of its expression in the cellu- lar compartment is indicative of the autophagy process of the cells. TFEB was detected in tissue slides of eight healthy adrenal cortex tissue probes, eight CA and 18 ACC (Figure 2(A)). Its green fluorescent signal was detectable in the healthy tissue, in Cushing’s adenoma and ACC tissue as well. Its detection was lower in CA and further less (p <. 05, statistically significant)) in ACC samples (Figure 2(B)). Beclin1, a well- known autophagy marker responsible for the autophagosome vesicle nucleation (25), was also investigated by immunofluorescence in the same samples used for TFEB detection. As shown in Figure 2(A), its expression is weak but detectable as red fluorescence in normal adrenal cortex and slightly stronger in CA. Instead, its fluorescent signal was almost no detectable in ACC tissue (Figure 2(C)). Anova-Bonferroni analysis of the densitometry of TFEB and Beclin1 fluorescent signal (Figure 2(D-E)) and analysis of their
correlation confirmed their homogeneous distri- bution and co-localization in adrenal cortex and CA. However, both autophagy markers were sig- nificantly (p <. 05) reduced in ACC, thus affect- ing their co-localization.
Analysis of H295R cell viability after treatment with deacetylase inhibitors
H295R ACC cells were incubated with several different concentrations of panobinostat, TSA and SAHA and the cell growth were monitored for 80 h. As shown in Figure 3(A), the treatment with panobinostat caused a reduction of cell growth at 1 and 10 nM concentration. The cyto- toxic effect of higher concentrations, 100 nM and 1 µM, was characterized by a significant (p <. 05, t-test) block of cell growth after 50 h of treatment (Figure 3(A)). Instead, treatment with TSA caused no reduction of cell growth. Treatment with 100 nM of SAHA caused a reduction of H295R cell growth. However, the block of cell
A
40
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growth was observed after incubation with 10 uM of SAHA (Figure 3(A)). The measurement of the diameter of H295R spheroids under visible light microscope after 13d of treatment with 100 nM of panobinostat, 10 uM of SAHA and 100 nM of TSA highlighted a significant reduction of their size after the administration of panobinostat and SAHA. Instead, the spheroids treated with TSA could recover their size, which was comparable to the untreated spheroids (Figure 3(B)).
Macroscopic alterations of H295R spheroids treated with deacetylase inhibitors
Visible light micrographs of H295R spheroids treated for 8 d with 100 nM and 1 µM of panobino- stat, 100 nM and 1 µM of TSA and 10uM of SAHA (Figure 3(C)) highlighted that the size of untreated spheroids was slightly increased, their
morphology was unchanged and they looked bril- liant under light contrast microscope. Two days of treatment with 100 nM of panobinostat caused a significant reduction of spheroids diameter and they got a dark grey color, typical of dead cells (Figure 3(C)). Similar effects have been observed with 1 µM of panobinostat and with 10 uM of SAHA (Figure 3(C)). The effect of treatment with TSA was characterized by an absent reduction of the size but the spheroids assumed a dark grey color typical of dead cells (Figure 3(C)). Detection of the fluorescent signal of the cellular DNA high- lighted a peripheral luminescence of untreated H295R spheroids. Instead, spheroids treated for 7 d with 1 µM of panobinostat or 1 µM of TSA were characterized for a fluorescent central region, whereas the outer region of the spheroids, which is completely dismantled, was not fluorescent (Figure 3(D)). This result could be explained by the
AllStar
siH19
A
10000
100 nM TSA
C
mean log realtive expression normalised to GAPDH
100 nM panobinostat
1000
1 µM pa nobinostat
10 μM SAHA
100 nM panobinostat
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LC38-1
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16 kDa
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Beclin
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0,01
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90 kDa
UVRAG
100000
TSA 100 nM
mean log relative expression normalised to GAPDH
10000
TSA 1 µM
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Beta-Actin
1000
0,35
LC3 B-I
100
LC3 B-II
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mean relative densitometry normalised to beta-actin
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mean log relative expression normalised to GAPDH
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0,1
H19
TFEB
BECN1
MAP1LC38
UVRAG
SOSTM1
PRKAA1_1
PRKAA2_1
0,05
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10
siH 19 2
siH19 3
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mean log relative expression normalised to RAN185
AllStar
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impermeable tight structure of the outer region of vital spheroids that limits the access of the DNA- binding fluorochrome in the inner spheroid com- partment. Instead, the cytotoxic effect of the his- tone DACi caused the dismantling of the spheroid outer membrane and the cellular membrane allow- ing the DNA-binding fluorescent dye to permeate the cell membranes and to bind the H295R nucleic acid, thus highlighting the lethal effect of 1 uM panobinostat and of 1 µM TSA.
Transcripts expression in treated H295R spheroids
Spheroids were treated for 48 h with 100 nM and 1 µM of panobinostat or of TSA, and 10 uM of
SAHA (Figure 4(A)). Treatment with 100 nM of panobinostat caused a significant down-regula- tion of H19 and PRKAA2_1. Instead, 1 µM pano- binostat was able to induce a significant (p <. 05, t-test) over-expression of H19, MAP1LC3B and PRKAA2_1 and a significant down-regulation of TFEB and SQSTM1. The other analyzed autoph- agy markers were stably expressed after treatment with both concentrations. 100 nM of TSA was responsible for the significant over-expression of H19 and PRKAA2_1. The higher concentration caused a significant (p <. 05, t-test) over-expres- sion of H19, TFEB, BECN1 and PRKAA2_1 and a significant down-regulation of SQSTM1. 10 uM SAHA, despite its 10-times higher concentration,
5
A
1
2
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55 kDa
Beclin1
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100 nM panobinostat
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18 kDa
3.
cleaved caspase 3
Beta-Actin
36 kDa
p62
4.
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uncleaved caspase 8
44 kDa
Beta-Actin
6.
10 µM SAHA
55 kDa
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mean relative dersitometry normalised to beta-actin
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*
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Caspase 3
uncleaved
6
Caspase 8
uncleaved
800
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cleaved
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43-41 kDa cleaved
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*
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*
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1
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*
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8
300
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mean relativ densitometry normalised to beta-actin
mean relative dersitometry normaised to beta-actin
mean relative dersitometry normalised to beta-actin
Beclin1
200
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*
*
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E
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6
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Caspase 3/7 activity
24h
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Caspase 8 activity
24h
48h
48h
T
T
2500
2000
*
2
T
2000
T
1500
*
0
300
1500
*
*
p62
1000
1
mean relative dersitontry normalised to beta-actin
mean relative luminescence activity
mean relative luminescence activity
250
T
*
1000
200
*
*
500
I
JA
500
150
L
*
0
0
100
1
2
3
4
5
6
1
2
3
4
5
6
50
*
I
L
0
1
2
3
4
5
6
caused a significant over-expression of only PRKAA2_1. The other autophagy markers were stable (Figure 4(A)).
Expression of autophagy markers after H19 knock down
H295R cells were transiently knocked down for H19. As shown in Figure 4(B), H19 expression was significantly down-regulated after 48 h administration of two different short interference RNAs. The expression of the autophagy markers TFEB, BECN1, MAP1LC3B, UVRAG, SQSTM1, PRKAA1_1 and PRKAA2_1 was stable after H19
knockdown. The protein level of LC3B, Beclin1, DRAM1 and UVRAG was detected after adminis- tration of 100 nM of TSA, 10 uM of SAHA and 100 nM of panobinostat in H295R cells knocked down for H19. As shown in Figure 4(C), the knock down of H19 caused an accumulation of the autophagy markers, which was significantly higher than the protein level detected in H295R treated with DACi. The knock down of H19 was even able to cause an accumulation of almost all autophagy markers in the cells treated with 100 nM of panobinostat and 10 uM of SAHA. However, this accumulation could not be observed after the administration of TSA, which
caused a significant down-regulation, probably acting independently of the H19 status. These results, confirmed by the densitometry and statis- tical (t-test) analysis (Figure 4(D)), highlighted that the expression of H19 contributes to the autophagy process. Its knock down most prob- ably causes an accumulation of the autophagy proteins, which represents a block of the auto- phagic process.
Further analysis of the autophagy proteins revealed that higher concentrations of the DACi were able to prompt more significantly (p <. 05, t-test) the autophagic process by causing a stron- ger down-regulation of the autophagy proteins, which represents a positive marker of active autophagy in H295R cells, thus confirmed by densitometry and statistical (t-test) analysis. In particular, the administration of 1 uM of panobi- nostat and TSA caused a significant down-regula- tion of LC3B-I and its conversion into LC3B-II and a significant down-regulation of p62, thus confirming the previous results and suggesting that a further increase of the concentration is suf- ficient to strongly promote autophagy in ACC cells, which are probably less sensitive than other solid cancer cells (8,22,26). The protein level of Beclin1 was down-regulated only after treatment with 1 µM of panobinostat (Figure 5(A)).
Modulation of caspases in H295R cells treated with DACi
H295R derived spheroids were treated for 48 h with 100 nM and 1 µM of panobinostat, TSA and 10 µM of SAHA. As shown in Figure 5(B), the administration of both concentrations of panobi- nostat was responsible for the over-expression of the protein level of both uncleaved and cleaved form of Caspase 3. Instead, TSA and SAHA caused a significant down-regulation of the pro- tein level of both uncleaved and cleaved Caspase 3. Similar effect could be observed after the detection of Caspase 8 in H295R cells treated with panobinostat, TSA and SAHA. Additionally, the 18 kDa cleaved active form of Caspase 8 was not detectable in all samples.
To prove further the modulation of caspases and their involvement in the mechanism induced by the DACi in H295R cells, the activity of Caspase 3/7 (Figure 5(C)) and Caspase 8 (Figure
5(D)) was detected by luminescence after 24 and 48 h of treatment. Interestingly, only the shorter administration (24h) of 1 uM of panobinostat was able to slight increase the activity of the exe- cutioner caspase 3/7. Instead, all other treatments and exposures caused no change in the activity or were even able to downregulate significantly (p <. 05, t-test) not only caspase 3/7 activity but also the activity of Caspase 8. Despite the up- regulation of the activity of the Caspase 3 caused by an over-expression of the whole content of the Caspase 3 after administration of panobinostat, it becomes clear that the treatment with DACi sup- presses caspases in H295R and these compounds exerts a pro-autophagy activity excluding any involvement of the final executioners of the apoptotic process.
Discussion
ACC, characterized by a poor prognosis and a lack of effective therapy, has a high local recur- rence and high metastatic potential that gain the lethality rate of the patients (28). Current ther- apy, mainly based on surgical resection and adju- vant therapy with mitotane (29,30), is disappointing and the most recent development, including the allowance of nivolumab for clinical trials, has shown a poor outcome (31).
The maternally imprinted long non-coding RNA H19 down-regulation has been identified has a prognostic factor for the poor overall sur- vival of patients affected by ACC (7). Because of its gene locus, H19 could interfere with IGF2 expression thus inhibiting tumor progression (5). This study highlighted that a significant suppres- sion of H19 occurred in patients affected by ACC, thus confirming the previous findings (32). Additionally, H19 expression was found stable in patients affected by benign Cushing’s adenoma, which were included in order to get a compari- son to ACC patients affected by Cushing’s syn- drome and high level of cortisol. The transcript of IGF2 was found significantly over-expressed in ACC and CA included in our study, thus con- firming the previous findings in mice human tumor xenograft and cancer tissue (5,6,33). Despite the discovery of a significant down-regu- lation of H19 in ACC, no evidence of an existing
correlation has been found between H19 expres- sion and cell death mechanism yet. Previous studies have identified autophagy has a target for inducing cell death in ACC cells (16,17,34). Analysis of patients affected by ACC evidenced, in this study, a significant down-regulation of the autophagy markers. Instead, patients affected by benign CA are characterized by stable TFEB, BECN1, UVRAG, SQSTM1, PRKAA1_1 and PRKAA2_1 transcripts and over-expressed MAP1LC3B. These findings highlighted an exist- ing correlation between the suppression of H19 and the significant down-regulation of the autophagy transcripts that need to be further clarified. Nonetheless, analysis of the protein level of TFEB evidenced that its expression is lowered in Cushing’s adenoma and further significantly lower in ACC. Additionally, Beclin1 protein, detectable at low level in normal tissue and CA, reduced significantly in ACC and it was almost no detectable. Thus, sustaining that the autoph- agy process is impaired in ACC by the significant down-regulation of the protein level of TFEB, the main regulator of autophagy genes transcription (27), and of the almost absent protein level of Beclin1, one of the main regulator of autophago- some vesicles maturation (25).
Based on the previous findings that the DACi play a central role by triggering autophagic cell death (22,23,25) and modulating H19 in cancer cells (8), H295R ACC cells were treated with panobinostat, TSA and SAHA. Panobinostat and SAHA caused a block of cell viability in cells grown as monolayer. Interestingly, the treatment of H295R spheroids with all three compounds caused a reduction of their size and the disman- tling of their outer membrane and their morph- ology. Only the spheroids treated with TSA were able to recover their size after long time treat- ment. The three DACi administered in H295R cells have shown cytotoxicity at different dosage in thyroid cancer cells too (8). Here, they further confirmed a similar behavior but a lower potency in terms of cytotoxicity, specially the administra- tion of TSA. However, they acted by modulating the same molecular mechanisms. The administra- tion of the higher dosage of panobinostat and TSA were responsible for the significant over- expression of H19 and the autophagy markers.
SAHA caused only the over-expression of PRKAA2_1. Similar effects on the autophagy pro- cess were previously highlighted by the treatment with rosiglitazone and tauroursodeoxycholic acid (16,34). However, the knock down of H19, per- formed to prove any direct correlation between H19 expression and autophagy markers in H295R cells, caused no significant variation of the autophagy transcripts expression in the in vitro model of ACC. Instead, it was observed that the knocking down of H19 not only caused an accumulation of the autophagy proteins LC3B-I, LC3B.II, Beclin1 DRAM1 and UVRAG, but also impeded the reduction of their protein level mediated by the treatment with the DACi. Interestingly, the administration of DACi was responsible for the significant reduction of the protein level of Beclin1, p62 and the conversion of LC3B-I into LC3B-II (35), which clearly high- lights an active autophagy process in H295R cells. Thus supporting that the treatment with DACi is the main responsible to prompt the over-expres- sion of both H19 and the autophagy markers and to lead to an active catabolic autophagic process causing ACC cell decay.
Although the involvement of the executioner caspases and their activation is a clear evidence of apoptotic cell death (36), it can exert an anti- autophagy function by degrading the autophagy proteins and thus inhibiting the maturation proc- esses of autophagy (37). The protein level and the activity of Caspases 3 and 8 were monitored in H295R monolayer and spheroids evidencing that neither the protein level of both caspases nor their activity was profoundly modulated by DACi. Panobinostat was the only DACi respon- sible for inducing the over-expression of the cleaved form of Caspase 3 and the increase of its activity, which could be attributed to an over- expression of the total protein content of Caspase 3, thus confirmed by the accumulation of the uncleaved Caspase 3. In contrast to the previous study describing a strategy against ACC based on the inhibition of autophagy (38), this study high- lighted that DACi are responsible for the promo- tion of autophagy and for the suppression of their counterparts’ apoptotic executioner caspases. Thus leading to autophagy-mediated reduction of cell viability and furthermore decay of ACC cells.
In particular, the treatment with DACi could restore, by inducing epigenetic modifications, the expression of H19, which is extremely low in ACC (9), and of the autophagy markers thus trig- gering cell death of ACC cells. However, the molecular mechanisms prompting the restoration of H19 are not clear in the proposed model yet. It could be dependent of the hyper acetylation and the de-methylation of the IGF-II/H19 pro- moter (32,39), which could trigger the transcrip- tion of H19 as it has been already shown for RASSF1A (Ras association domain family 1 iso- form A) and Adenomatous Polyposis Coli (APC) promoters in liver cancer cells (40).
Recently, it has been found that the adminis- tration of the DACi is responsible for the alter- ation in the glycome and the gene PTTG1, encoding for securin in ACC cells (41,42). Thus, highlighting other druggable targets that could be included, in combination with DACi, as potential new treatment.
In conclusion, the administration of DACi to restore autophagy and the expression of the long- non-coding-RNA H19 in ACC could further rep- resent a valid option for the treatment of this aggressive disease.
Acknowledgments
The authors are thankful to Katrin Roth (Center for Tumor Biology and Immunology, Philipps University Marburg) for the microscopy advices and support, to Carmen Bollmann (Department of Visceral, Thoracic and Vascular Surgery, Philipps University Marburg) for the technical assistance, to Uwe Schlomann (Department of Visceral, Thoracic and Vascular Surgery, Philipps University Marburg) for reading the manuscript and his meaningful advices.
Author contributions
PD, KH conceived and designed the study. FR, SR, PD, AP performed the experiments. PD, FR, IM analyzed the data. PD, FR wrote and revised the manuscript. SW, KH, DKB provided study material, supervised and supported analyses and edited the manuscript. All authors critically reviewed the manuscript.
Disclosure statement
All authors declare no conflict of interest.
Funding
The authors reported there is no funding associated with the work featured in this article.
ORCID
Pietro Di Fazio ID http://orcid.org/0000-0003-0091-8498
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