Androgen-Induced MIG6 Regulates Phosphorylation of Retinoblastoma Protein and AKT to Counteract Non-Genomic AR Signaling in Prostate Cancer Cells

The bipolar androgen therapy (BAT) includes the treatment of prostate cancer (PCa) patients with supraphysiological androgen level (SAL). Interestingly, SAL induces cell senescence in PCa cell lines as well as ex vivo in tumor samples of patients. The SAL-mediated cell senescence was shown to be androgen receptor (AR)-dependent and mediated in part by non-genomic AKT signaling. RNA-seq analyses compared with and without SAL treatment as well as by AKT inhibition (AKTi) revealed a specific transcriptome landscape. Comparing the top 100 genes similarly regulated by SAL in two human PCa cell lines that undergo cell senescence and being counteracted by AKTi revealed 33 commonly regulated genes. One gene, ERBB receptor feedback inhibitor 1 (ERRFI1), encodes the mitogen-inducible gene 6 (MIG6) that is potently upregulated by SAL, whereas the combinatory treatment of SAL with AKTi reverses the SAL-mediated upregulation. Functionally, knockdown of ERRFI1 enhances the pro-survival AKT pathway by enhancing phosphorylation of AKT and the downstream AKT target S6, whereas the phospho-retinoblastoma (pRb) protein levels were decreased. Further, the expression of the cell cycle inhibitor p15INK4b is enhanced by SAL and ERRFI1 knockdown. In line with this, cell senescence is induced by ERRFI1 knockdown and is enhanced slightly further by SAL. Treatment of SAL in the ERRFI1 knockdown background enhances phosphorylation of both AKT and S6 whereas pRb becomes hypophosphorylated. Interestingly, the ERRFI1 knockdown does not reduce AR protein levels or AR target gene expression, suggesting that MIG6 does not interfere with genomic signaling of AR but represses androgen-induced cell senescence and might therefore counteract SAL-induced signaling. The findings indicate that SAL treatment, used in BAT, upregulates MIG6, which inactivates both pRb and the pro-survival AKT signaling. This indicates a novel negative feedback loop integrating genomic and non-genomic AR signaling.


Introduction
Prostate cancer (PCa) is an important age-related disease with the highest estimated incidence of new cancer cases [1]. Second only to lung cancer, it is one of the leading causes of cancer mortality in men in Western countries. The evidence shows that growth of the normal prostate tissue as well as the initial development of PCa relies on the activation of the androgen receptor (AR) [2]. Thus, the AR represents a major drug target in the treatment of PCa [3].
To inhibit AR signaling androgen deprivation therapy (ADT) and full blockade by AR antagonists are the major forms of PCa hormone therapy recommended for advanced for each experiment in cell culture plates. After 48 h of incubation, the cells were treated for 72 h with 1 pM R1881 (LAL), 1 nM R1881 (SAL), 10 µM bicalutamide (Bic), 10 µM enzalutamide (Enz) or 0.1% DMSO (Carl Roth, Karlsruhe, Germany) as solvent control (C) in a 5% CO 2 , humidified atmosphere at 37 • C.

RNA-Sequencing and Transcriptome Analysis
RNA-sequencing and transcriptome analysis of both LNCaP and C4-2 cell lines were previously described [9]. Cells were treated for 3 days with DMSO and the AR-specific agonist R1881, with and without the AKT inhibitor (1 µM) AKTi, prior to RNA isolation. The RNA-sequencing data is available in the gene expression omnibus (GEO) database under the accession numbers GSE162711, GSE155528, and GSE154755.

Cellular Senescence Assays
The assays were performed with 6-well plates, and the cells were seeded at 25,000 cells per well. The staining and detection were performed as described previously [15][16][17]. The percentage of SA-β-Gal positive cells was calculated by counting at least 3 × 200 cells per well and at least 2 wells per treatment under a light microscope (Zeiss, Oberkochen, Germany).

Immunofluorescence Staining
LNCaP cells were seeded in RPMI 1640 medium containing 5% normal untreated FBS and cultured for 48 h. After 72 h of ligand treatment, cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton-X100/PBS [9] for 10 min at room temperature. After three washing steps in 1x PBS, a blocking solution (5% Normal Goat Serum/PBS) was added for 1 h. Primary antibodies were incubated in a humidified chamber overnight at 4 • C. Goat anti-rabbit secondary antibodies were incubated for 1 h at room temperature. After washing, cells were stained with Hoechst in 1x PBS followed by mounting with Flouremount G. Images were obtained with a confocal laser scanning microscope (Zeiss LSM 880, Oberkochen, Germany) with Airyscan in super resolution using a Plan-Apochromat 63x/1.4 oil DIC M27 objective confocal scanning fluorescence microscope. Fiji software (2.5.0) (https://fiji.sc/ Accessed on 20 January 2022) was used for analysis of the images.

Quantitative Reverse Transcription PCR (qRT-PCR)
The assays were performed with RNA isolated from 10-cm cell culture dishes, and the cells were seeded at 500,000 cells per dish. The total RNA extraction was performed using peqGOLD TriFast (Peqlab, Erlangen, Germany) according to the manufacturer's protocol. Two-step qRT-PCR was performed as described previously [16,17] with gene-specific primers. TBP and GAPDH mRNA served as the housekeeping gene for normalization. The primer sequences are listed (Table 1) as 5 →3 :

Statistical Analysis
For statistical analysis a two-tailed unpaired Student's t-test was performed using the GraphPad Prism 8.0 software, which was calculated from the mean, standard deviation (SD), standard error of mean (SEM) and number of replicates (n). A 95% confidence interval (p-value (p) < 0.05) was considered as statistically significant (*) between two subject groups. A 99.5% confidence interval (p < 0.05), 99% confidence interval (p < 0.01) and a 99.9% confidence interval (p < 0.001) were indicated by one (*) two (**) and three stars (***), respectively. A 99.99% confidence interval (p < 0.0001) was indicated by four stars (****). Western blotting analysis was performed for at least three biological replicates.

Results
3.1. RNA-seq Identifies ERRFI1 Being Upregulated by SAL in Both LNCaP and C4-2 Cells RNA-seq was performed and analyzed for an overlap of androgen-mediated upregulated genes in two cell lines, the androgen-dependent LNCaP and the castration-resistant C4-2 cell lines. In contrast to LAL treatment, SAL induces cellular senescence in both cell lines [9]. Interestingly, inhibition of AKT by AKTi represses androgen-induced cellular senescence. The top 100 genes with high score being specifically upregulated by SAL in each cell line were further analyzed for their common upregulation in both cell lines leading to 33 genes ( Figure 1A and Supplemental Figure S1). output_file". Peaks were annotated for promoters, exons, introns and intergenic regions by the script "annotatePeaks.pl" in software "HOMER" (v4.9.1, Sven Heinz, San Diego, CA, USA).

Statistical Analysis
For statistical analysis a two-tailed unpaired Student's t-test was performed using the GraphPad Prism 8.0 software, which was calculated from the mean, standard deviation (SD), standard error of mean (SEM) and number of replicates (n). A 95% confidence interval (p-value (p) < 0.05) was considered as statistically significant (*) between two subject groups. A 99,5% confidence interval (p < 0.05), 99% confidence interval (p < 0.01) and a 99.9% confidence interval (p < 0.001) were indicated by one (*) two (**) and three stars (***), respectively. A 99.99% confidence interval (p < 0.0001) was indicated by four stars (****). Western blotting analysis was performed for at least three biological replicates.

RNA-seq Identifies ERRFI1 Being Upregulated by SAL in Both LNCaP and C4-2 Cells
RNA-seq was performed and analyzed for an overlap of androgen-mediated upregulated genes in two cell lines, the androgen-dependent LNCaP and the castrationresistant C4-2 cell lines. In contrast to LAL treatment, SAL induces cellular senescence in both cell lines [9]. Interestingly, inhibition of AKT by AKTi represses androgen-induced cellular senescence. The top 100 genes with high score being specifically upregulated by SAL in each cell line were further analyzed for their common upregulation in both cell lines leading to 33 genes ( Figure 1A and Supplemental Figure S1).  One of the prominently induced genes is ERRFI1, encoding MIG6, being upregulated significantly by SAL, whereas LAL did not upregulate ERRFI1 expression ( Figure 1B-D). AKTi treatment itself did not affect the basal mRNA expression of ERRFI1 ( Figure 1E). Since the combination treatment of SAL with AKTi did not show statistically significant downregulation, qRT-PCR experiments were used for both cell lines. The upregulation of ERRFI1 by SAL was confirmed and the downregulation of ERRFI1 by AKTi was revealed by qRT-PCR in both cell lines (Figure 2A,B). Notably, treatment with first and secondgeneration AR antagonists, enzalutamide and bicalutamide, did not induce the expression level of ERRFI1 (Supplemental Figure S2). To verify an upregulation of MIG6 at protein level, both cell lines were treated with LAL or SAL in the absence or presence of AKTi ( Figure 2C). The data indicate an upregulation of MIG6 at protein level by SAL treatment in both cell lines, whereas AKTi reduces MIG6 levels to a different extent comparing LNCaP with C4-2 cells. ChIP-seq experiments indicate the recruitment of AR up-and downstream of the ERRFI1 locus ( Figure 2D). More reads were obtained at this locus by One of the prominently induced genes is ERRFI1, encoding MIG6, being upregulated significantly by SAL, whereas LAL did not upregulate ERRFI1 expression ( Figure 1B-D). AKTi treatment itself did not affect the basal mRNA expression of ERRFI1 ( Figure 1E). Since the combination treatment of SAL with AKTi did not show statistically significant downregulation, qRT-PCR experiments were used for both cell lines. The upregulation of ERRFI1 by SAL was confirmed and the downregulation of ERRFI1 by AKTi was revealed by qRT-PCR in both cell lines (Figure 2A,B). Notably, treatment with first and second-generation AR antagonists, enzalutamide and bicalutamide, did not induce the expression level of ERRFI1 (Supplemental Figure S2). To verify an upregulation of MIG6 at protein level, both cell lines were treated with LAL or SAL in the absence or presence of AKTi ( Figure 2C). The data indicate an upregulation of MIG6 at protein level by SAL treatment in both cell lines, whereas AKTi reduces MIG6 levels to a different extent comparing LNCaP with C4-2 cells. ChIP-seq experiments indicate the recruitment of AR up-and downstream of the ERRFI1 locus ( Figure 2D). More reads were obtained at this locus by SAL treatment. The data suggest that ERRFI1 is a direct AR target gene.
One of the prominently induced genes is ERRFI1, encoding MIG6, being upregulated significantly by SAL, whereas LAL did not upregulate ERRFI1 expression ( Figure 1B-D). AKTi treatment itself did not affect the basal mRNA expression of ERRFI1 ( Figure 1E). Since the combination treatment of SAL with AKTi did not show statistically significant downregulation, qRT-PCR experiments were used for both cell lines. The upregulation of ERRFI1 by SAL was confirmed and the downregulation of ERRFI1 by AKTi was revealed by qRT-PCR in both cell lines (Figure 2A,B). Notably, treatment with first and secondgeneration AR antagonists, enzalutamide and bicalutamide, did not induce the expression level of ERRFI1 (Supplemental Figure S2). To verify an upregulation of MIG6 at protein level, both cell lines were treated with LAL or SAL in the absence or presence of AKTi ( Figure 2C). The data indicate an upregulation of MIG6 at protein level by SAL treatment in both cell lines, whereas AKTi reduces MIG6 levels to a different extent comparing LNCaP with C4-2 cells. ChIP-seq experiments indicate the recruitment of AR up-and downstream of the ERRFI1 locus ( Figure 2D). More reads were obtained at this locus by SAL treatment. The data suggest that ERRFI1 is a direct AR target gene. The dose-dependent treatment with androgen indicates that ERRFI1 mRNA expression is induced at 1 nM R1881 (Supplemental Figure S3A). Time-dependent androgen treatment indicates that MIG6 protein levels are enhanced at 48 and 72 h treatment with SAL (Supplementary Figure S3). In contrast, treatment with the AR antagonist Enz did not upregulate MIG6 protein levels at any time point. The dose-dependent treatment with androgen indicates that ERRFI1 mRNA expression is induced at 1 nM R1881 (Supplemental Figure S3A). Time-dependent androgen treatment indicates that MIG6 protein levels are enhanced at 48 and 72 h treatment with SAL (Supplementary Figure S3). In contrast, treatment with the AR antagonist Enz did not upregulate MIG6 protein levels at any time point.

SAL Treatment Enhances Cytosolic MIG6 Levels
In order to detect intracellular levels of MIG6 and the intracellular distribution of MIG6 in the absence or presence of SAL, high-resolution laser scanning microscopy was performed detecting endogenous expressed MIG6. LNCaP cells were cultured in normal serum and treated with or without SAL. Enhanced fluorescence signals were detected in the cytoplasm of LNCaP cells upon SAL treatment ( Figure 3). These data indicate that androgens induce the expression of MIG6, which is preferentially localized in the cytoplasm.

AKTi Reduces SAL-Enhanced p-AKT and p-S6 Levels
The increase of phosphorylation of AKT (p-AKT) at serine 473 (S473) by SAL treatment in LNCaP cells was shown previously [8]. Similarly, as shown before, LAL had no effect on the levels of p-AKT. To confirm that AKTi inhibits AKT signaling in both LNCaP and C4-2 cells, the AKT downstream target S6 was analyzed. SAL enhances in both cell lines the levels of p-AKT and p-S6 (Figure 4) indicating non-genomic activity of AR signaling by SAL. Interestingly, AKTi inhibits p-AKT levels in LNCaP more pronounced compared to C4-2 level, which might be one basis of castration-resistance. In contrast, p-S6 levels were more reduced in C4-2 cells compared to LNCaP (Figure 4). This indicates that the AR interacts not only with AKT but also directly or indirectly with S6 kinase(s) to regulate their activity in the presence of AKT inhibition.

AKTi Reduces SAL-Enhanced p-AKT and p-S6 Levels
The increase of phosphorylation of AKT (p-AKT) at serine 473 (S473) by SAL treatment in LNCaP cells was shown previously [8]. Similarly, as shown before, LAL had no effect on the levels of p-AKT. To confirm that AKTi inhibits AKT signaling in both LNCaP and C4-2 cells, the AKT downstream target S6 was analyzed. SAL enhances in both cell lines the levels of p-AKT and p-S6 (Figure 4) indicating non-genomic activity of AR signaling by SAL. Interestingly, AKTi inhibits p-AKT levels in LNCaP more pronounced compared to C4-2 level, which might be one basis of castration-resistance. In contrast, p-S6 levels were more reduced in C4-2 cells compared to LNCaP (Figure 4). This indicates that the AR interacts not only with AKT but also directly or indirectly with S6 kinase(s) to regulate their activity in the presence of AKT inhibition. Biomolecules 2022, 12, x FOR PEER REVIEW 9 of 15

Knockdown of ERRFI1 Increases SA-β-Gal Positive Cells
SAL is known to activate the AKT-mTOR pro-survival pathway [8]. To analyze whether MIG6 is also involved in or mediates the SAL-induced phosphorylation of AKT and S6, knockdown experiments of ERRFI1 were performed. According to the RNA-seq and qRT-PCR data, MIG6 encoded by ERRFI1 is induced by SAL and downregulated by AKTi in the presence of SAL. Since SAL enhances phospho-AKT (p-AKT) levels, the hypothesis was to analyze whether MIG6 also regulates p-AKT levels. To verify this hypothesis, siRNA pool for ERRFI1 and a non-targeting control pool (siControl), were transfected into LNCaP cells. The transfected LNCaP cells were treated for 72 h with SAL or DMSO as solvent control. siERRFI1 significantly reduced SAL-induced mRNA level of ERRFI1 ( Figure 5A). At the protein level, the knockdown of MIG6 was confirmed by Western blotting using siRNA or short hairpin-mediated knockdown ( Figure 5B,C). Of note, we did not detect upon knockdown of MIG6 an influence on AR protein level (Supplemental Figure S4A) or the expression of the direct AR target gene FKBP5 (Supplemental Figure S4B).
Since enhanced p-AKT and hypo-phosphorylated pRb levels are linked to the AR mediated induction of cellular senescence by SAL in PCa cells [8,9], we analyzed whether MIG6 is involved in SAL-mediated cellular senescence. Knockdown cells using siERRFI1 or shERRFI1 were generated ( Figure 5A-C). Interestingly, siERRFI1 or shERRFI1 transfected cells showed significantly higher basal SA-β-Gal positive cells compared to control transfected cells ( Figure 5D and Supplemental Figure S5 with si-mediated knockdown), indicating that MIG6 protects PCa cells to undergo cellular senescence. Still, SAL treatment enhances further cellular senescence with a slightly but significantly higher percentage of SA-β-Gal positive cells in ERRFI1-knockdown cells. It should be taken into account that the fold-enhancement of SA-β-Gal positive cell level in the ERRFI1knockdown cells is reduced. This may indicate that ERRFI1 is involved in protecting these cancer cells to undergo cellular senescence by SAL.

Knockdown of ERRFI1 Increases SA-β-Gal Positive Cells
SAL is known to activate the AKT-mTOR pro-survival pathway [8]. To analyze whether MIG6 is also involved in or mediates the SAL-induced phosphorylation of AKT and S6, knockdown experiments of ERRFI1 were performed. According to the RNA-seq and qRT-PCR data, MIG6 encoded by ERRFI1 is induced by SAL and downregulated by AKTi in the presence of SAL. Since SAL enhances phospho-AKT (p-AKT) levels, the hypothesis was to analyze whether MIG6 also regulates p-AKT levels. To verify this hypothesis, siRNA pool for ERRFI1 and a non-targeting control pool (siControl), were transfected into LNCaP cells. The transfected LNCaP cells were treated for 72 h with SAL or DMSO as solvent control. siERRFI1 significantly reduced SAL-induced mRNA level of ERRFI1 ( Figure 5A). At the protein level, the knockdown of MIG6 was confirmed by Western blotting using siRNA or short hairpin-mediated knockdown ( Figure 5B,C). Of note, we did not detect upon knockdown of MIG6 an influence on AR protein level (Supplemental Figure S4A) or the expression of the direct AR target gene FKBP5 (Supplemental Figure S4B).
Since enhanced p-AKT and hypo-phosphorylated pRb levels are linked to the AR mediated induction of cellular senescence by SAL in PCa cells [8,9], we analyzed whether MIG6 is involved in SAL-mediated cellular senescence. Knockdown cells using siERRFI1 or shERRFI1 were generated ( Figure 5A-C). Interestingly, siERRFI1 or shERRFI1 transfected cells showed significantly higher basal SA-β-Gal positive cells compared to control transfected cells ( Figure 5D and Supplemental Figure S5 with si-mediated knockdown), indicating that MIG6 protects PCa cells to undergo cellular senescence. Still, SAL treatment enhances further cellular senescence with a slightly but significantly higher percentage of SA-β-Gal positive cells in ERRFI1-knockdown cells. It should be taken into account that the fold-enhancement of SA-β-Gal positive cell level in the ERRFI1-knockdown cells is reduced. This may indicate that ERRFI1 is involved in protecting these cancer cells to undergo cellular senescence by SAL.

ERRFI1 Knockdown Represses CCND1 and Induces the Expression of CDKN2B/p15 INK4b
SAL-mediated cellular senescence is associated with hypophosphorylation of pRb as well as decrease of the E2F target gene CCND1, encoding Cyclin D1. To investigate whether MIG6 is involved in this pathway, phosphorylation levels of pRb and CCND1 expression were analyzed in the knockdown background. The ERRFI1 knockdown leads to reduced pRb levels, which were further decreased in the presence of SAL ( Figure 6). In line with this and SAL-mediated induction of cellular senescence, the p-AKT and p-S6 levels were enhanced by ERRFI1 knockdown and further induced by SAL treatment (Figure 6).

ERRFI1 Knockdown Represses CCND1 and Induces the Expression of CDKN2B/p15 INK4b
SAL-mediated cellular senescence is associated with hypophosphorylation of pRb as well as decrease of the E2F target gene CCND1, encoding Cyclin D1. To investigate whether MIG6 is involved in this pathway, phosphorylation levels of pRb and CCND1 expression were analyzed in the knockdown background. The ERRFI1 knockdown leads to reduced pRb levels, which were further decreased in the presence of SAL ( Figure 6). In line with this and SAL-mediated induction of cellular senescence, the p-AKT and p-S6 levels were enhanced by ERRFI1 knockdown and further induced by SAL treatment (Figure 6).
These data confirm induction of cellular senescence by ERRFI1 knockdown using a similar pathway as the AR. In accordance with that, CCND1 expression was repressed, and this effect was further enhanced in the SAL treated ERRFI1 knockdown cells ( Figure 7A). In addition, the expression of the cell cycle inhibitor CDKN1A, encoding p21, shows an expression pattern being in line with the induction of cellular senescence by knockdown. The expression further increased by the combination of SAL and ERRFI1 knockdown ( Figure 7B). Recently, it was shown that the cell cycle inhibitor p15 INK4b mediates SALinduced cellular senescence [9]. To analyze whether MIG6 also regulates the expression of this factor, knockdown cells were analyzed. Upon SAL treatment, the expression of CDKN2B mRNA, encoding p15 INK4b , was enhanced and further upregulated in the ERRFI1 knockdown cells ( Figure 7C). Accordingly, at protein level p15 INK4b is potently enhanced in the SAL-treated knockdown cells ( Figure 7D). These data confirm induction of cellular senescence by ERRFI1 knockdown using a similar pathway as the AR. In accordance with that, CCND1 expression was repressed, and this effect was further enhanced in the SAL treated ERRFI1 knockdown cells ( Figure  7A). In addition, the expression of the cell cycle inhibitor CDKN1A, encoding p21, shows an expression pattern being in line with the induction of cellular senescence by knockdown. The expression further increased by the combination of SAL and ERRFI1 knockdown ( Figure 7B). Recently, it was shown that the cell cycle inhibitor p15 INK4b mediates SAL-induced cellular senescence [9]. To analyze whether MIG6 also regulates the expression of this factor, knockdown cells were analyzed. Upon SAL treatment, the expression of CDKN2B mRNA, encoding p15 INK4b , was enhanced and further upregulated in the ERRFI1 knockdown cells ( Figure 7C). Accordingly, at protein level p15 INK4b is potently enhanced in the SAL-treated knockdown cells ( Figure 7D).
Taken together, these data indicate that MIG6 regulates the key factors associated with and mediating induction of cellular senescence by SAL treatment.  These data confirm induction of cellular senescence by ERRFI1 knockdown using a similar pathway as the AR. In accordance with that, CCND1 expression was repressed, and this effect was further enhanced in the SAL treated ERRFI1 knockdown cells ( Figure  7A). In addition, the expression of the cell cycle inhibitor CDKN1A, encoding p21, shows an expression pattern being in line with the induction of cellular senescence by knockdown. The expression further increased by the combination of SAL and ERRFI1 knockdown ( Figure 7B). Recently, it was shown that the cell cycle inhibitor p15 INK4b mediates SAL-induced cellular senescence [9]. To analyze whether MIG6 also regulates the expression of this factor, knockdown cells were analyzed. Upon SAL treatment, the expression of CDKN2B mRNA, encoding p15 INK4b , was enhanced and further upregulated in the ERRFI1 knockdown cells ( Figure 7C). Accordingly, at protein level p15 INK4b is potently enhanced in the SAL-treated knockdown cells ( Figure 7D).
Taken together, these data indicate that MIG6 regulates the key factors associated with and mediating induction of cellular senescence by SAL treatment.

Discussion and Conclusions
SAL treatment induces cellular senescence in PCa cells. Treatment with AKTi abrogates SAL-induced cellular senescence, indicating that SAL-induced cellular senescence is mediated in part by non-genomic signaling. SAL treatment induces AR-AKT Taken together, these data indicate that MIG6 regulates the key factors associated with and mediating induction of cellular senescence by SAL treatment.

Discussion and Conclusions
SAL treatment induces cellular senescence in PCa cells. Treatment with AKTi abrogates SAL-induced cellular senescence, indicating that SAL-induced cellular senescence is mediated in part by non-genomic signaling. SAL treatment induces AR-AKT signaling pathway. MIG6 is known as the mitogen-inducible gene and is mainly known for its feedback inhibitor function of ERBB-2 mitogenic and transforming signals indicating oncogenic function [18][19][20]. However, MIG6 also exhibits tumor suppressor activity presumably in a cell type and context-dependent manner [21][22][23]. In line with this, both MIG6-mediated inhibition and stimulation of AKT phosphorylation were reported [24]. In endometrial epithelial cells MIG6 suppresses proliferation by inhibiting p-AKT [25]. MIG6 expression decreased migration and invasion of MEK-inhibited mutant NRAS melanoma, especially in response to epidermal growth factor stimulation, also indicating a tumor suppressive role in other cancers [26].
Since inhibition of AKT reduced SAL-mediated induction of cellular senescence in PCa cells, it indicates that AKT-signaling pathway is partly essential for SAL-mediated induction of cellular senescence [8,9]. Interestingly, the knockdown of ERRFI1 induces cellular senescence as well as the levels of p-AKT and downstream target p-S6, suggesting an activation of AKT-signaling by knockdown. This is in line with the suggestion that SAL increases p-AKT levels and downstream AKT signaling in order to enhance a pro-survival pathway and to induce cellular senescence [7,27]. The knockdown of ERRFI1 further increased SAL-mediated induction of cellular senescence but reduced the fold induction of cellular senescence compared to control transfected cells. The knockdown experiments suggest that MIG6 inhibits AKT phosphorylation and represses the expression of p15 INK4b , in order to suppress the induction of cellular senescence by SAL. This may suggest that the knockdown of ERRFl1 causes attenuation of the increased number of SA-Gal positive cells when compared to the increase upon SAL treatment in shLuc cells, and therefore, MIG6 might be involved in the SAL inhibition of tumor progression. These findings also suggest that SAL treatment enhances in addition to p-AKT levels, MIG6 expression. MIG6 subsequently represses phosphorylation of AKT. The data suggest that SAL treatment induces, through MIG6, a negative feed-back loop to reduce p-AKT levels.
In line with our findings, Mig-6 was shown to be upregulated during the oncogeneinduced senescence process and overexpressing of Mig-6 enhances cellular senescence levels in human embryonic lung diploid fibroblast (2BS cells), whereas knockdown of Mig-6 delayed the initiation of Ras-induced cellular senescence [28,29]. Also, it was shown that overexpression of Mig-6 is sufficient to trigger premature cellular senescence of early passage of human diploid lung fibroblasts (WI-38 cells). Notably, the induction of ERRFI1 mRNA and recruitment of AR by SAL was recently also described for VCaP cells [30]. Furthermore, SAL was shown to enhance ERRFI1 mRNA levels in patient-derived mouse xenografts treated with vehicle or high-dose androgen [31] (supplemental Figure S6). Based on the high degree of tumor heterogeneity of PCa [27], a generalized conclusion cannot be drawn. Therefore, further experiments using different PCa cell lines have to be performed to get more insights into the pathways activated by SAL treatment using in the BAT therapy.
Taken together, these results suggest that MIG6 induced by genomic AR signaling regulates AKT non-genomic AR signaling in LNCaP cells thus integrating the genomic and non-genomic androgen response.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom12081048/s1, Figure S1, List of the 33 genes upregulated by SAL; Figure S2, Treatment with AR antagonists do not induce ERRFI1 expression; Figure S3, Expression change of ERRFI1 by androgen dose and incubation time; Figure S4, AR protein level and the AR target gene FKBP5 are not affected by knockdown of ERRFI1/MIG6; Figure S5, Similar to sh-mediated knockdown the si-mediated knockdown of ERRFI1 induced cellular senescence; Figure S6, The expression of ERRFI1 is upregulated upon high-dose of testosterone treatment of mice in patient-derived xenografts using the LuCaP 35CR model system.