Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors
Abstract
:1. Background
2. Results
2.1. The KEAP1 Silencing Affects the NRF2 and Expression Levels of TXNRD1, AKR1C1 and NQO1 in Carcinoid Lines
2.2. Restoration of KEAP1 Expression Correlates with KEAP1 P1 Region Demethylation by 5-aza-dC Treatment in Carcinoid Cell Lines
2.3. KEAP1 Promoter Region Hypermethylation are Frequent Epigenetic in Lung Carcinoids, Whereas Point Mutations are Absent
3. Discussion
4. Methods
4.1. Cell Lines
4.2. KEAP1 Silencing by siRNA
4.3. Western Blot Analysis
4.4. In Vitro 5-Aza-2′-deoxycytidine (5-aza-dC) Treatment
4.5. RNA Extraction, Reverse Transcription and Quantitative Real-Time PCR (qRT-PCR)
4.6. Patients and Tissue Samples
4.7. Bisulfite Conversion and Quantitative Methylation Specific-PCR Analysis (qMSP)
4.8. Mutation Analysis And Loss of Heterozygosity Analysis (LOH)
4.9. Immunohistochemistry (IHC)
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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ID Sample | Histotypes | Gender | KEAP1 (Meth and/or LOH 1) | Years at Diagnosis | UICC Stage | Progression (Y/N) | Status (D/A) | DFS (mos) | OS (mos) |
---|---|---|---|---|---|---|---|---|---|
LCCH01-322 | AC | M | Meth | 52 | IIA | Y | A | 84 | 120 |
LCCH01-328 | AC | M | LOH | 47 | IIA | N | A | 67 | 67 |
LCCH01-329 | AC | M | LOH | 49 | IA | N | A | 124 | 204 |
LCCH01-330 | AC | F | Meth | 67 | IB | N | A | 142 | 142 |
LCCH01-378/85 | AC | M | Meth | 71 | IA | Y | D | 12 | 23 |
UD-3 | AC | M | Meth | 44 | IB | Y | D | 60 | 132 |
UD-7 | AC | M | Meth | 61 | IB | N | A | 84 | 84 |
UD-18 | AC | F | Meth | 35 | IB | Y | A | 36 | 120 |
UD-20 | AC | F | Meth | 69 | IIIA | N | A | 108 | 108 |
UD-24 | AC | F | Meth | 69 | IB | Y | D | 12 | 28 |
LCCH01-315 | TC | M | Meth, LOH | 54 | IA | N | A | 203 | 288 |
LCCH01-377/35 | TC | F | Meth, LOH | 35 | IB | N | A | 52 | 52 |
LCCH01-313 | TC | F | LOH | 57 | IA | N | A | 36 | 36 |
LCCH01-327 | TC | M | Meth, LOH | 69 | IA | N | A | 66 | 66 |
LCCH01-320 | TC | F | Meth | 29 | IB | N | A | 157 | 157 |
LCCH01-323 | TC | F | Meth, LOH | 70 | IA | N | A | 132 | 132 |
LCCH01-325 | TC | F | Meth | 36 | IA | N | A | 124 | 124 |
LCCH01-327 | TC | M | Meth | 69 | IA | N | A | 66 | 66 |
LCCH01-321bis/380 | TC | M | Meth | 48 | IB | N | A | 172 | 172 |
LCCH01-333 | TC | M | Meth | 71 | IA | N | A | 108 | 108 |
LCCH01-311 | TC | M | Meth | 50 | IA | N | A | 91 | 91 |
LCCH01-54 | TC | M | Meth, LOH | 70 | IA | N | A | 47 | 47 |
UD-9 | TC | M | Meth | 64 | IA | N | A | 72 | 72 |
UD-10 | TC | M | Meth | 74 | IA | Y | D | 48 | 60 |
UD-11 | TC | F | Meth | 63 | IA | N | A | 60 | 60 |
UD-19 | TC | F | Meth | 65 | IB | N | A | 48 | 48 |
UD-25 | TC | M | Meth | 65 | IA | N | A | 24 | 24 |
Characteristics | Category | All subjects (N = 47) |
---|---|---|
Age at diagnosis (years, median, IQR) | - | 63, 19.75 |
Gender (n,%) | Female | 26 (55.3%) |
Male | 21 (44.7%) | |
Histotype subclassification (n,%) | Typical Carcinoid | 30 (63.8%) |
Atypical Carcinoid | 17 (36.2%) | |
T (n,%) | T1 | 31 (67%) |
T2 | 15 (33%) | |
N (n,%) | N0 | 41 (89.1%) |
N1 | 3 (6.5%) | |
N2 | 2 (4.4%) | |
M (n,%) | M0 | 47 (100.00%) |
Tumour stage (n,%) | IA - IB | 42 (89.4%) |
IIA - IIB - IIIA | 5 (10.6%) |
Characteristics | Category | UM | M | p-Value * |
---|---|---|---|---|
Age at diagnosis (years, median (range)) | - | 63 (30–76) | 64 (29–74) | 0.755 |
Tumor_size (mm, median (range)) | - | 25 (10–40) | 20 (10–45) | 0.775 |
Gender | Female | 11 (73%) | 6 (40%) | 0.07 |
Male | 4 (27%) | 9 (60%) | ||
T | T1 | 10 (71%) | 11 (73%) | 1.000 |
T2 | 4 (29%) | 4 (27%) | ||
N | N0 | 13 (93%) | 15 (100%) | 0.483 |
N1 | 1 (7%) | 0 (0%) | ||
M | M0 | 15 (100%) | 15 (100%) | n.a. |
Tumor stage | IA - IB | 14 (93%) | 15 (100%) | 1.000 |
IIA-IIB-IIIA | 1 (7%) | 0 (0%) |
Characteristics | Category | UM | M | p-Value * |
---|---|---|---|---|
Age at diagnosis (years, median (range)) | --- | 53 (40–70) | 64 (35–71) | 0.736 |
Tumor size (mm, median (range)) | --- | 20 (10–48) | 32.5 (20–60) | 0.132 |
Gender | Female | 5 (56%) | 4 (50%) | 1.000 |
Male | 4 (44%) | 4 (50%) | ||
T | T1 | 7 (78%) | 3 (38%) | 0.153 |
T2 | 2 (22%) | 5 (62%) | ||
N | N0 | 7 (78%) | 6 (75%) | 0.133 |
N1 | 2 (22%) | 0 (0%) | ||
N2 | 0 (0%) | 2 (25%) | ||
M | M0 | 9 (100%) | 8 (100%) | n.a. |
Tumor stage | IA-IB | 7 (%) | 6 (%) | 0.893 |
IIA-IIB-IIIA | 2 (%) | 2 (%) |
Protein Name (symbol) | Reference |
---|---|
NAD(P)H dehydrogenase (quinone 1) (NQO1) | [21], this report |
Aldo-Keto Reductase Family 1 (AKR1) (AKR1C1) | [22], this report |
Hemoglobin subunit beta (HBB) | [23,24] |
Ferritin light chain (FTL) | [24,25] |
Ferritin heavy chain (FTH) | [24,26,27,28,29] |
Superoxide dismutase (SOD) | [24,30,31,32,33,34,35] |
Extracellular Superoxide dismutase (SODE) | [24,36,37] |
Carbonic Anhydrase 1 (CAH1) | [24,38] |
Annexin V (ANXA5) | [24,39,40] |
Peroxiredoxin-2 (PRX2) | [24,41,42,43] |
Peroxiredoxin-3 (PRX3) | [24,42,44,45] |
Transthyretin (TTR) | [24,46] |
Hemopexin (HPX) | [24,47] |
Transaldolase (TALDO) | [24,48] |
Heterogenous Nuclear Ribonucleo protein K (HNRPK) | [24,49,50] |
Histone H4 | [24,51,52,53,54] |
Heat Shock Protein Hsp90-alpha (HSP90) | [24,55,56,57] |
Elongation Factor 1-alpha1 (EF1A1) | [24,58] |
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Sparaneo, A.; Fabrizio, F.P.; la Torre, A.; Graziano, P.; Di Maio, M.; Fontana, A.; Bisceglia, M.; Rossi, A.; Pizzolitto, S.; De Maglio, G.; et al. Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors. Int. J. Mol. Sci. 2019, 20, 2531. https://doi.org/10.3390/ijms20102531
Sparaneo A, Fabrizio FP, la Torre A, Graziano P, Di Maio M, Fontana A, Bisceglia M, Rossi A, Pizzolitto S, De Maglio G, et al. Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors. International Journal of Molecular Sciences. 2019; 20(10):2531. https://doi.org/10.3390/ijms20102531
Chicago/Turabian StyleSparaneo, Angelo, Federico Pio Fabrizio, Annamaria la Torre, Paolo Graziano, Massimo Di Maio, Andrea Fontana, Michele Bisceglia, Antonio Rossi, Stefano Pizzolitto, Giovanna De Maglio, and et al. 2019. "Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors" International Journal of Molecular Sciences 20, no. 10: 2531. https://doi.org/10.3390/ijms20102531