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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Molecular Cancer 1/2017

TRIM8 restores p53 tumour suppressor function by blunting N-MYC activity in chemo-resistant tumours

Zeitschrift:
Molecular Cancer > Ausgabe 1/2017
Autoren:
Francesca Mastropasqua, Flaviana Marzano, Alessio Valletti, Italia Aiello, Giuseppe Di Tullio, Annalisa Morgano, Sabino Liuni, Elena Ranieri, Luisa Guerrini, Giuseppe Gasparre, Elisabetta Sbisà, Graziano Pesole, Antonio Moschetta, Mariano Francesco Caratozzolo, Apollonia Tullo
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12943-017-0634-7) contains supplementary material, which is available to authorized users.

Abstract

Background

TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway.

Methods

We used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays.

Results

We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8.

Conclusions

In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.
Zusatzmaterial
Additional file 1: Table S1. Main characteristics of patients affected by clear cell Renal Cell Carcinoma (ccRCC) and renal oncocytoma (RO). The anagraphic (gender and age) and clinical (Fuhrman grade) data are reported. (XLSX 65 kb)
12943_2017_634_MOESM1_ESM.xlsx
Additional file 2: Figure S1. (a) TRIM8 expression in ccRCC samples (T) and their paired non-tumour tissues (NT). The analysis was performed considering the Fuhrman grading of the tumour samples. Data are represented in box-and-whisker plots showing median and 10th, 25th, 75th and 90th percentiles for each category of sample. Expression data were measured respect to one normal sample chosen arbitrarily as calibrator and then normalized by the geometric mean of RPL13 and ACTB relative expression ratios. The bars represent the Standard Error of the Mean. (b) TRIM8 expression in three different renal cell lines: the human proximal tubular epithelial cells HK-2, the human renal cell carcinoma RCC-Shaw (p53wt) and the human renal carcinoma of BHD (Birt-Hogg-Dubè) origin UOK-257 cells (mutated-p53) and the colon cancer HCT116 (p53wt) cell line. Expression data were measured respect to HK-2 sample chosen as calibrator, and normalized by the expression levels of RPL13. **p-value < 0.005; *** p-value < 0.001. (c, d) Linear regression plots of miR-17-5p and miR-106b-5p expression levels compared with TRIM8 expression levels in ccRCC samples. On the x-axis the Ct of TRIM8 minus the arithmetic mean of Cts calculated for ACTB and RPL13 (used as housekeeping genes in RT-qPCR analysis) is reported, while the y-axis shows the Ct of miR-17-5p/miR-106b-5p minus the Ct of U6 snRNP (used to normalize miRNAs expression levels). The value of the correlation coefficient “r” and the p-value are indicated on the graph. Dots are coloured differentially according to the sample type: grey for non-tumour samples, green for Fuhrman Grade 1 samples, orange for Fuhrman Grade 2 samples and yellow for Fuhrman Grade 3 samples. (PDF 186 kb)
12943_2017_634_MOESM2_ESM.pdf
Additional File 3: Figure S2. (a) miR-106a-5p expression in ccRCC samples (T) and their paired non-tumour tissues (NT). The analysis was performed considering the Fuhrman grading of the tumour samples. Data are represented in box-and-whisker plots showing median and 10th, 25th, 75th and 90th percentiles for each category of sample. Expression data were measured respect to one normal sample chosen arbitrarily as calibrator and then normalized by the expression levels of U6 snRNA. The bars represent the Standard Error of the Mean. It is also reported the sequence alignment between the miR-106a-5p “seed” sequence and the TRIM8 3’UTR. (b) miR-106a-5p, miR-17-5p and miR-106b-5p expression in 4 oncocytoma samples (T) and their paired non-tumour tissues (NT). Data are represented in box-and-whisker plots showing median and 10th, 25th, 75th and 90th percentiles for each category of sample. Expression data were measured respect to one normal sample chosen arbitrarily as calibrator and then normalized by the expression levels of U6 snRNA. The bars represent the Standard Error of the Mean. (PDF 79 kb)
12943_2017_634_MOESM3_ESM.pdf
Additional file 4: Figure S3. (a, b) Luciferase assay. The HK-2 and the HCT116 cells were transfected with Negative Control miRNA Mimic, miR-17-5p, miR-106b-5p, anti-miR-17-5p or anti-miR-106b-5p, along with pMIR luciferase reporter construct containing p21 3’UTR. Cells were lysed and luciferase activity was determined as described in the Material and Methods section. Transfection efficacy was normalized by Renilla Luciferase activity. Data represent the averages of at least three independent experiments with their standard deviations. ** p-value < 0.005; *** p-value < 0.001. (PDF 64 kb)
12943_2017_634_MOESM4_ESM.pdf
Additional file 5: Figure S4. (a, b) Expression levels of miR-17-5p and miR-106b-5p were measured by qPCR in HK-2, RCC-Shaw, UOK-257 and HCT116, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p. Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression level of U6 snRNA. The bars represent the Standard deviation of the Mean. (c) Colony suppression assays. Cell growth was measured in HK-2, RCC-Shaw, UOK-257 and HCT116 cells transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p. (PDF 1350 kb)
12943_2017_634_MOESM5_ESM.pdf
Additional file 6: Figure S5. (a-b) Densitometry analysis of p53, Trim8, N-MYC and p21 proteins in HK-2, UOK-257, RCC-Shaw and HCT116 cells transfected with negative control miRNA Mimic, anti-miR-17-5p (a) or anti-miR-106b-5p (b). The relative amounts of proteins, reported in the table below the western blotting, have been calculated normalizing to actin and calibrating to the respective control. (PDF 925 kb)
12943_2017_634_MOESM6_ESM.pdf
Additional file 7: Figure S6. (a-f) Expression levels of miR-17-5p and miR-106b-5p were measured by RT-qPCR in HK-2, RCC-Shaw and UOK-257 cells, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression level of U6 snRNA. ** p-value < 0.005 (g-i) Colony suppression assay. Cell growth were measured in HK-2, RCC-Shaw and UOK-257 cells transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). (PDF 380 kb)
12943_2017_634_MOESM7_ESM.pdf
Additional file 8: Figure S7. (a-i) Expression levels of TRIM8, p21 and miR-34a were measured by RT-qPCR in HK-2, RCC-Shaw and UOK-257 cells, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression levels of RPL13 for TRIM8 and p21, and by the expression level of U6 snRNA for miR-34a. * p-value < 0.05; ** p-value < 0.005; *** p-value < 0.001. (PDF 220 kb)
12943_2017_634_MOESM8_ESM.pdf
Additional file 9: Figure S8. (a, b) Expression levels of miR-17-5p and miR-106b-5p were measured by RT-qPCR in HCT116 cells, transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression level of U6 snRNA. (c-e) Expression levels of TRIM8, p21, miR-34a and miR-17-5p were measured by RT-qPCR in the RCC-Shaw cells transfected with Negative Control miRNA Mimic or anti-miR-17-5p plus control short hairpin-RNA (control shRNA) or specific short hairpin against TRIM8 (shRNA-TRIM8). After transfection the cells were treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression levels of RPL13 for TRIM8 and p21, and by the expression level of U6 snRNA for miR-34a and miR-17-5p. * p-value < 0.05; ** p-value < 0.005; *** p-value < 0.001 (f) Colony suppression assay. Cell growth were measured in HCT116 cells transfected with Negative Control miRNA Mimic, anti-miR-17-5p or anti-miR-106b-5p, and treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). (PDF 531 kb)
12943_2017_634_MOESM9_ESM.pdf
Additional file 10: Figure S9. (a-d) Expression levels of TRIM8, p21, miR-34a and miR-17-5p were measured by RT-qPCR in the RCC-Shaw cells transfected with Negative Control miRNA Mimic or anti-miR-17-5p plus control short hairpin-RNA (control shRNA) or specific short hairpin against TRIM8 (shRNA-TRIM8). After transfection the cells were treated for 24 h with Nutlin-3 (N) (10 μM), Cisplatin (C) (7.5 μM), Sorafenib (S) (10 μM), Axitinib (A) (10 μM) or drug-untreated cells (-). Relative expression ratios were measured respect to the sample transfected with the Negative Control miRNA Mimic and normalized by the expression levels of RPL13 for TRIM8 and p21, and by the expression level of U6 snRNA for miR-34a and miR-17-5p. ** p-value < 0.005; *** p-value < 0.001. (PDF 150 kb)
12943_2017_634_MOESM10_ESM.pdf
Additional file 11: Figure S10. (a) Representative xenograft tumours at the moment of the excision. (b) RT-qPCR demonstrating the expression of the exogenous TRIM8 or RING-TRIM8. The histogram shows the ratio between exogenous TRIM8 or RING-domain and the endogenous one. The analysis was conducted measuring the expression of the HA epitope and the expression of the total amount of RING domain in Control and Treated samples. The bars represent the Standard Error of the Mean. **p-value < 0.005. (PDF 650 kb)
12943_2017_634_MOESM11_ESM.pdf
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