The KBTBD6/7-DRD2 axis regulates pituitary adenoma sensitivity to dopamine agonist treatment

Abstract

Pituitary adenoma (PA) is one of the most common intracranial tumors, and approximately 40% of all PAs are prolactinomas. Dopamine agonists (DAs), such as cabergoline (CAB), have been successfully used in the treatment of prolactinomas. The expression of dopamine type 2 receptor (DRD2) determines the therapeutic effect of DAs, but the molecular mechanisms of DRD2 regulation are not fully understood. In this study, we first demonstrated that DRD2 underwent proteasome-mediated degradation. We further employed the yeast two-hybrid system and identified kelch repeat and BTB (POZ) domain containing 7 (KBTBD7), a substrate adaptor for the CUL3-RING ubiquitin (Ub) ligase complex, as a DRD2-interacting protein. KBTBD6/7 directly interacted with, and ubiquitinated DRD2 at five ubiquitination sites (K221, K226, K241, K251, and K258). CAB, a high-affinity DRD2 agonist, induced DRD2 internalization, and cytoplasmic DRD2 was degraded via ubiquitination under the control of KBTBD6/7, the activity of which attenuated CAB-mediated inhibition of the AKT/mTOR pathway. KBTBD7 knockout (KO) mice were generated using the CRISPR-Cas9 technique, in which the static level of DRD2 protein was elevated in the pituitary gland, thalamus, and heart, compared to that of WT mice. Consistently, the expression of KBTBD6/7 was negatively correlated with that of DRD2 in human pituitary tumors. Moreover, KBTBD7 was highly expressed in dopamine-resistant prolactinomas, but at low levels in dopamine-sensitive prolactinomas. Knockdown of KBTBD6/7 sensitized MMQ cells and primary pituitary tumor cells to CAB treatment. Conversely, KBTBD7 overexpression increased CAB resistance of estrogen-induced in situ rat prolactinoma model. Together, our findings have uncovered the novel mechanism of DRD2 protein degradation and shown that the KBTBD6/7-DRD2 axis regulates PA sensitivity to DA treatment. KBTBD6/7 may thus become a promising therapeutic target for pituitary tumors.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China under Grant Number 81972339 and 81671371 (ZBW), Shanghai Municipal Science and Technology Commission 18XD1403400 (ZBW), Program of Shanghai Academic Research Leader (ZBW), and Shanghai Training and Support Program for Outstanding Young Medical Talents (ZBW). This work was supported by funding to RH from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000 and XDA12040323), the National Science Foundation of China (91853128, 31470770, and 81525019), the model animal project of Shanghai Science and Technology Commission (19140903500), the Ministry of Science and Technology of China (2019YFA082103 and 2018ZX10101004), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), and Shenzhen-Hong Kong Institute of Brain Science (NYKFKT2019006).

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YTL, FL, LC, and LX performed the experiments, analyzed the data, and co-wrote the manuscript. WTG, YZZ, and HT contributed to study of human pituitary adenoma samples. HY, YZ, WQX, BHR, ZHX, and YJN contributed to the data analysis. YW provided the specimens of pituitary tumours. ZBW and RH conceived the idea, designed and supervised the study, analyzed the data, and co-wrote the manuscript.

Corresponding authors

Correspondence to Ronggui Hu or Zhe Bao Wu.

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Supplementary file1 Suppl. Fig. 1 KBTBD6 directly interacts with and ubiquitinates DRD2. (a-b) KBTBD6 interacted with DRD2. Coimmunoprecipitation assays showed that tagged (a) or endogenous (b) KBTBD6 and DRD2 formed a complex in HEK293T cells. (c) Immunofluorescence microscopy analyses indicated that tagged KBTBD6 and DRD2 proteins colocalized in MMQ cells. Scale bar, 10 μm. (d) GST pulldown assays indicated that recombinant GST-tagged DRD2, but not GST, interacted with His-tagged KBTBD6. (e) KBTBD6 ubiquitinated DRD2. The level of DRD2 ubiquitination was increased after overexpression of KBTBD6 in HEK293T cells expressing KBTBD6-Myc, DRD2-flag and HA-Ub, as indicated (TIF 61057 kb)

Supplementary file2 Suppl. Fig. 2 KBTBD7 and KBTBD6 knockdown. (a-b) HEK293T cells were infected with efficient lentiviral KBTBD6 and KBTBD7 shRNA or a control shRNA. KBTBD6 and KBTBD7 mRNA levels were measured by qRT-PCR and normalized to those of actin. The data are presented as the means ± SEM. *n = 3, p < 0.05. (c-d) MMQ cells were infected with lentiviral KBTBD6 or KBTBD7 shRNA or a control shRNA. KBTBD6 and KBTBD7 mRNA levels were measured by qRT-PCR and normalized to those of actin. The data are presented as the means ± SEM. *n = 3, p < 0.05 (TIF 59340 kb)

Supplementary file3 Suppl. Fig. 3 KBTBD6 activity is dependent on its Kelch domain. (a) Schematic representation of the domain architecture of KBTBD6. (b) Three KBTBD6 deletion constructs interacted with the DRD2 protein. Co-IP experiments were performed with Flag-tagged DRD2 and different Myc-tagged KBTBD6 deletion mutants that were co-expressed in HEK293T cells. (c) Among the indicated plasmids co-expressed in HEK293T cells, deletion of the domain from aa 386-684 in KBTBD6 (KBTBD6-Δ3) significantly reduced the ubiquitination of DRD2 compared with that observed in cells expressing full-length KBTBD6 (TIF 60771 kb)

Supplementary file4 Suppl. Fig. 4 Knockdown of KBTBD6/7 enhances the inhibitory effect of CAB on cAMP levels. (a) HEK293T cells were infected with efficient lentiviral KBTBD6 and KBTBD7 shRNA or a control shRNA and treated with CAB (20 μM) for another 2 h. The data are presented as the means ± SEM. **n = 3, p < 0.01 (TIF 58770 kb)

Supplementary file5 Suppl. Fig. 5 KBTBD7 knockout increases DRD2 protein expression in KBTBD7-/- mice. (a) Representative IHC staining images showed that DRD2 was increased in the hearts, lungs, livers and stomachs of KBTBD7-/- mice compared with WT mice. Scale bar, 100 μm. (b) Representative IHC staining images of samples from the spleen and large intestine showed that DRD2 expression was not significantly different between WT and KO mice. Scale bar, 100 μmSuppl. Fig. 5 KBTBD7 knockout increases DRD2 protein expression in KBTBD7-/- mice. (a) Representative IHC staining images showed that DRD2 was increased in the hearts, lungs, livers and stomachs of KBTBD7-/- mice compared with WT mice. Scale bar, 100 μm. (b) Representative IHC staining images of samples from the spleen and large intestine showed that DRD2 expression was not significantly different between WT and KO mice. Scale bar, 100 μm (TIF 76775 kb)

Supplementary file6 Suppl. Fig. 6 Knockdown of KBTBD6/7 upregulates the expression of DRD2 and increases the sensitivity of MMQ cells to CAB. (a) Knockdown of KBTBD6/7 increased the stability of DRD2 in MMQ cells. Cells transfected with KBTBD6/7 shRNA or a control shRNA were cultured for 48 h. (b) Knockdown of KBTBD6/7 increased the sensitivity of MMQ cells to CAB. MMQ cells were infected with lentiviral KBTBD6/7 shRNA or a control shRNA and cultured in the presence or absence of 50 μM CAB for 48 h, after which cell survival was determined by CCK8 assay. The data are presented as the means ± SEM. ***n = 4, p < 0.001. (c) Colony formation assays showed that KBTBD6/7 double knockdown inhibited the colony formation of MMQ cells. One thousand cells per well were seeded in six-well plates and cultured for 12 days, which was followed by crystal violet staining and colony counting. Scale bar, 1 cm (TIF 60052 kb)

Supplementary file7 Suppl. Fig. 7 KBTBD6/7 knockdown increases the sensitivity of primary pituitary tumor cells to CAB treatment. (a-b) The representative results for KBTBD6/7 knockdown increased the sensitivity of primary cells from NFPAs (a) and GH-secreting adenomas (b) to CAB treatment. Cells were infected with adenovirus at 10 MOI to decrease KBTBD6/7 expression and then treated with CAB, after which cell viability was detected by CCK8 assay. The data are presented as the means ± SEM. ***n = 5, p < 0.001 (TIF 59556 kb)

Supplementary file8 Suppl. Fig. 8 KBTBD6/7 knockdown increases the inhibitory effect of PRL levels to CAB treatment. (a) Overexpression of KBTBD7 was observed to increase PRL levels following CAB treatment in MMQ cells. MMQ cells were infected with adenovirus Myc-KBTBD7 in the presence of CAB at different concentration. Then, the supernatants were collected, and PRL levels were determined by ELISA. The data are presented as the means ± SEM. ***n = 3, p < 0.001. (b-c) MMQ cells (b) and seven primary prolactinoma cells (c) were infected with shKBTBD6/7 lentivirus or adenovirus and then treated with DMSO or CAB. The supernatants were collected, and PRL levels were determined by ELISA. The data are presented as the means ± SEM. **n = 3, p < 0.05 (TIF 61247 kb)

Supplementary file9 Suppl. Fig. 9 Quantitative real-time PCR analysis of the expression of DRD2 in DA-sensitive and DA-resistant prolactinomas. (a) DRD2 mRNA levels were not significantly different between DA-resistant (n=7) and DA-sensitive (n=6) pituitary tumors. The data are presented as the means ± SEM. p > 0.05 (TIF 58718 kb)

Supplementary file10 Suppl. Fig. 10 Correlation between KBTBD6/7 and DRD2. (a) IHC analysis of the indicated proteins in human pituitary tumors. Scale bars, 100 μm. (b) Quantification of the staining intensities from the sections in (a). KBTBD7 and KBTBD6 expression levels were negatively corrected with DRD2 expression in patients. *** n = 109, p < 0.001. (c) KBTBD7 and KBTBD6 expression levels were negatively corrected with DRD2 expression in prolactinomas. *** n = 25, p < 0.001 (TIF 64197 kb)

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Liu, Y.T., Liu, F., Cao, L. et al. The KBTBD6/7-DRD2 axis regulates pituitary adenoma sensitivity to dopamine agonist treatment. Acta Neuropathol (2020). https://doi.org/10.1007/s00401-020-02180-4

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Keywords

  • Pituitary tumor
  • DRD2
  • KBTBD7
  • KBTBD6
  • Ubiquitin degradation