extfieldsofstudy: Biology Medicine

text:
SUMOylation Regulates Transcription by the Progesterone Receptor A Isoform in a Target Gene Selective Manner

Luminal breast cancers express estrogen (ER) and progesterone (PR) receptors, and respond to endocrine therapies. However, some ER+PR+ tumors display intrinsic or acquired resistance, possibly related to PR. Two PR isoforms, PR-A and PR-B, regulate distinct gene subsets that may differentially influence tumor fate. A high PR-A:PR-B ratio is associated with poor prognosis and tamoxifen resistance. We speculate that excessive PR-A marks tumors that will relapse early. Here we address mechanisms by which PR-A regulate transcription, focusing on SUMOylation. We use receptor mutants and synthetic promoter/reporters to show that SUMOylation deficiency or the deSUMOylase SENP1 enhance transcription by PR-A, independent of the receptors’ dimerization interface or DNA binding domain. De-SUMOylation exposes the agonist properties of the antiprogestin RU486. Thus, on synthetic promoters, SUMOylation functions as an independent brake on transcription by PR-A. What about PR-A SUMOylation of endogenous human breast cancer genes? To study these, we used gene expression profiling. Surprisingly, PR-A SUMOylation influences progestin target genes differentially, with some upregulated, others down-regulated, and others unaffected. Hormone-independent gene regulation is also PR-A SUMOylation dependent. Several SUMOylated genes were analyzed in clinical breast cancer database. In sum, we show that SUMOylation does not simply repress PR-A. Rather it regulates PR-A activity in a target selective manner including genes associated with poor prognosis, shortened survival, and metastasis.

Introduction
Actions of progesterone (P) are mediated by two progesterone receptor (PR) isoforms called PR-A and PR-B. They are co-expressed in the normal breast, but equimolarity may be disrupted in breast cancers [1]. The two isoforms have different physiological functions and regulate different sets of genes [1]. In animals, tissue-specific differences in their distribution patterns may account for dissimilar organ-specific P effects [2]. In the mouse mammary gland, PR-B is important for ductal side-branching and the labulo-alveolar development of pregnancy, while PR-A deficiency results in uterine and ovarian abnormalities and infertility. PR-A overexpression leads to abnormal mammary and acetylation (KxKK at aa 638-641) ( Figure 1A). PR-B SUMOylation is hormone-dependent and suppresses receptor-driven transcription on multiple PREs but not on a single PRE or on MMTV [15]. DeSUMOylation by mutation of PR-B Lys388 promotes transcriptional synergy with other factors on complex promoters. Since PR-A lacks the 165aa "B-upstream segment" (BUS) containing activation function 3 (AF-3), we conjectured that PR-A might not be subject to transcriptional synergy. We therefore compared effects of SUMOylation/deSUMOylation in HeLa cells transfected with WT PR-B or WT PR-A, and the PR-BK388R or PR-A K388R SUMOylation deficient mutants. All four were tested for ligand-independent (-) and ligand-dependent (+) transcription using PRE 2 -Luc ( Figure 1B) and MMTV-Luc ( Figure 1C) and the progestin R5020. There was no ligand-independent activity. In the presence of ligand, compared to their WT counterparts, both PR-B and PR-A SUMO-deficient mutants displayed increases in transcription on PRE 2 -luc (but not MMTV-Luc). Thus AF-3 of PR-B is not involved in de-repressing transcriptional activity mediated by SUMOylation. We confirmed that AF-3 was uninvolved using the PR-BdL140 BUS mutant in which we knocked out AF-3 function [12], by mutating its K388 to yield PR-Bdl140 K388R. This mutant nevertheless still demonstrated transcriptional upregulation, establishing on the full length PR-B molecule that AF-3 was unnecessary (data not shown). Note that while the activity of PR-A remains weak compared to PR-B it is nevertheless raised by deSUMOylation. Overall, based on use of synthetic promoters, this study demonstrates that SUMOylation suppresses transcription by PR-B and PR-A, which is relieved by deSUMOylation.

SUMOylation Suppresses PR Transcriptional Activity in a Promoter Dependent Manner
Downstream of aa165, PR-A and PR-B share post-translational modifications sites including ones for hormone-dependent phosphorylation (Ser294 and Ser345), SUMO conjugation (Lys388), and acetylation (KxKK at aa 638-641) ( Figure 1A). PR-B SUMOylation is hormone-dependent and suppresses receptor-driven transcription on multiple PREs but not on a single PRE or on MMTV [15]. DeSUMOylation by mutation of PR-B Lys388 promotes transcriptional synergy with other factors on complex promoters. Since PR-A lacks the 165aa "B-upstream segment" (BUS) containing activation function 3 (AF-3), we conjectured that PR-A might not be subject to transcriptional synergy. We therefore compared effects of SUMOylation/deSUMOylation in HeLa cells transfected with WT PR-B or WT PR-A, and the PR-BK388R or PR-A K388R SUMOylation deficient mutants. All four were tested for ligand-independent (-) and ligand-dependent (+) transcription using PRE2-Luc ( Figure 1B) and MMTV-Luc ( Figure 1C) and the progestin R5020. There was no ligand-independent activity. In the presence of ligand, compared to their WT counterparts, both PR-B and PR-A SUMO-deficient mutants displayed increases in transcription on PRE2-luc (but not MMTV-Luc). Thus AF-3 of PR-B is not involved in de-repressing transcriptional activity mediated by SUMOylation. We confirmed that AF-3 was uninvolved using the PR-BdL140 BUS mutant in which we knocked out AF-3 function [12], by mutating its K388 to yield PR-Bdl140 K388R. This mutant nevertheless still demonstrated transcriptional upregulation, establishing on the full length PR-B molecule that AF-3 was unnecessary (data not shown). Note that while the activity of PR-A remains weak compared to PR-B it is nevertheless raised by deSUMOylation. Overall, based on use of synthetic promoters, this study demonstrates that SUMOylation suppresses transcription by PR-B and PR-A, which is relieved by deSUMOylation.

SENP1 deSUMOylates PR-A, Which Enhances Its Transcriptional Activity
We analyzed SUMOylation in more detail focusing on PR-A. PR-B can be deSUMOylated by SENP1 [15]. Its catalytic function can be inactivated by mutation of Cys603 to yield mSENP1 [19]. We examined SENP1-mediated deSUMOylation of PR-A ( Figure 2A) in HeLa cells co-transfected with PR-A, GFP-SUMO1, and either wild type SENP1 or mSENP1, in the absence or presence of R5020. To be SUMOylated, PR-A must be liganded (lanes 1, 3, 5). Liganded PR-A are SUMOylated in the absence of SENP1 (lane 2), remain SUMOylated with mSENP1 (lane 6) but are deSUMOylated by SENP1 (lane 4) demonstrating a role for SENP1 in regulating SUMOylation of PR-A.
Based on Figure 1, this suggested that SENP1 would increase transcription by PR-A. To examine this, transcription of PRE2-Luc was measured in the presence of co-transfected SENP1 or mSENP1 when PR-A were liganded by the agonist R5020, by RU486 (a mixed agonist/antagonist on PR-B but a pure antagonist on PR-A), or by ZK98299, a pure antagonist on both PRs ( Figure 2B). In HeLa cells expressing SENP1 or mSENP1, agonist-occupied PR-A are poor trans-activators and the antagonists have no effects. However, PR-A deSUMOylation by SENP1 increases R5020-dependent transcription 10-fold. Remarkably, deSUMOylation exposes the partial agonist properties of RU486 on PR-A; properties that had been reported previously only on PR-B [20]. Note that ZK98299 remains a pure antagonist even on deSUMOylated PR-A. Thus SUMOylation control the agonist/antagonist activity of RU486 liganded PR-A. Parenthetically, this suggests that PR-A may provide a sensitive assay for screening new antiprogestins for any partial agonist activities. In PR-A positive breast cancer cells, co-transfection of SENP1 but not mSENP1 also enhances transcription demonstrating that this effect is not cell specific (not shown).
To confirm that the transcriptional effects in Figure 2B are due directly to PR-A deSUMOylation, a similar study was conducted using the SUMOylation deficient PR-AK388R mutant ( Figure 2C). K388R mutant PR-A are completely insensitive to SENP1 demonstrating that the enzymatic effects in Figure 2B require an intact PR-A SUMOylation site. Interestingly, since either PR-A deSUMOylation or mutation of the SUMOylation site expose the partial agonist activity of RU486, both modifications may globally impact PR-A protein structure, thereby altering co-regulator recruitment.

SENP1 deSUMOylates PR-A, Which Enhances Its Transcriptional Activity
We analyzed SUMOylation in more detail focusing on PR-A. PR-B can be deSUMOylated by SENP1 [15]. Its catalytic function can be inactivated by mutation of Cys603 to yield mSENP1 [19]. We examined SENP1-mediated deSUMOylation of PR-A ( Figure 2A) in HeLa cells co-transfected with PR-A, GFP-SUMO1, and either wild type SENP1 or mSENP1, in the absence or presence of R5020. To be SUMOylated, PR-A must be liganded (lanes 1, 3, 5). Liganded PR-A are SUMOylated in the absence of SENP1 (lane 2), remain SUMOylated with mSENP1 (lane 6) but are deSUMOylated by SENP1 (lane 4) demonstrating a role for SENP1 in regulating SUMOylation of PR-A.
Based on Figure 1, this suggested that SENP1 would increase transcription by PR-A. To examine this, transcription of PRE 2 -Luc was measured in the presence of co-transfected SENP1 or mSENP1 when PR-A were liganded by the agonist R5020, by RU486 (a mixed agonist/antagonist on PR-B but a pure antagonist on PR-A), or by ZK98299, a pure antagonist on both PRs ( Figure 2B). In HeLa cells expressing SENP1 or mSENP1, agonist-occupied PR-A are poor trans-activators and the antagonists have no effects. However, PR-A deSUMOylation by SENP1 increases R5020-dependent transcription 10-fold. Remarkably, deSUMOylation exposes the partial agonist properties of RU486 on PR-A; properties that had been reported previously only on PR-B [20]. Note that ZK98299 remains a pure antagonist even on deSUMOylated PR-A. Thus SUMOylation control the agonist/antagonist activity of RU486 liganded PR-A. Parenthetically, this suggests that PR-A may provide a sensitive assay for screening new antiprogestins for any partial agonist activities. In PR-A positive breast cancer cells, co-transfection of SENP1 but not mSENP1 also enhances transcription demonstrating that this effect is not cell specific (not shown).
To confirm that the transcriptional effects in Figure 2B are due directly to PR-A deSUMOylation, a similar study was conducted using the SUMOylation deficient PR-AK388R mutant ( Figure 2C). K388R mutant PR-A are completely insensitive to SENP1 demonstrating that the enzymatic effects in Figure 2B require an intact PR-A SUMOylation site. Interestingly, since either PR-A deSUMOylation or mutation of the SUMOylation site expose the partial agonist activity of RU486, both modifications may globally impact PR-A protein structure, thereby altering co-regulator recruitment. Transfected cells were treated with the agonist R5020 (R50-10 nM), the partial antagonist RU486 (RU-100 nM), or the pure antagonist ZK 98299 (ZK98-100 nM) for 24 h before being assayed for luciferase activity as described in Figure 1.

The PR DNA Binding Domain (DBD) Dimerization Interface is Unnecessary for SUMOylation or Transcriptional Control
Transcription by nuclear receptors is complex, involving post-translational modifications such as SUMOylation, a dimerization interface on the DBD [7,21], and for PR, interactions between the ligand binding domain (LBD) and N-termini to stabilize binding to DNA at multiple PREs [12]. To test the role of DBD dimerization, transcription was quantified on PRE2-luc using PR-A, the PR-A Cells were treated with 10 nM R5020 for 24 h. Cells were lysed and analyzed by western blot for PR-A using anti-PR1294 monoclonal antibody. (B) HeLa cells were transiently transfected with WT PR-A or PR-A K388R, ** p < 0.05 (C) together with the PRE 2 -luciferase reporter plasmids and 100 ng of Flag-SENP1 or Flag-SENP1m mutant or an empty vector control (−). Transfected cells were treated with the agonist R5020 (R50-10 nM), the partial antagonist RU486 (RU-100 nM), or the pure antagonist ZK 98299 (ZK98-100 nM) for 24 h before being assayed for luciferase activity as described in Figure 1

The PR DNA Binding Domain (DBD) Dimerization Interface Is Unnecessary for SUMOylation or Transcriptional Control
Transcription by nuclear receptors is complex, involving post-translational modifications such as SUMOylation, a dimerization interface on the DBD [7,21], and for PR, interactions between the ligand binding domain (LBD) and N-termini to stabilize binding to DNA at multiple PREs [12]. To test the role of DBD dimerization, transcription was quantified on PRE2-luc using PR-A, the PR-A K388R Diseases 2018, 6, 5 7 of 16 mutant as control, and PR-A DX, an R606A mutant of the D-box in the second zinc finger of the DBD [7]. Figure 3A shows that unlike liganded PR-A K388R (lane 4), liganded PR-A DX (lane 6) are SUMOylated to levels equivalent to WT PR-A (lane 2). Thus, SUMOylation/deSUMOylation does not require an intact DBD or theoretically, DNA binding.
Transcription of PRE 2 by WT and PR mutants is shown in Figure 3B. SENP1-mediated deSUMOylation strongly activates transcription by R5020-occupied WT PR-A. Mutation of K388 exposes this synergy even in the absence of SENP1. Similar hyper-activation is seen with PR-A DX whether or not it is deSUMOylated by SENP1. This shows that SUMOylation of the DBD dimerization mutant does not inhibit its activity at compound PREs, indicating that DBD dimerization is required downstream of SUMOylation to unmask the inhibitory effects of SUMO. K388R mutant as control, and PR-A DX, an R606A mutant of the D-box in the second zinc finger of the DBD [7]. Figure 3A shows that unlike liganded PR-A K388R (lane 4), liganded PR-A DX (lane 6) are SUMOylated to levels equivalent to WT PR-A (lane 2). Thus, SUMOylation/deSUMOylation does not require an intact DBD or theoretically, DNA binding. Transcription of PRE2 by WT and PR mutants is shown in Figure 3B. SENP1-mediated deSUMOylation strongly activates transcription by R5020-occupied WT PR-A. Mutation of K388 exposes this synergy even in the absence of SENP1. Similar hyper-activation is seen with PR-A DX whether or not it is deSUMOylated by SENP1. This shows that SUMOylation of the DBD dimerization mutant does not inhibit its activity at compound PREs, indicating that DBD dimerization is required downstream of SUMOylation to unmask the inhibitory effects of SUMO.

Stimulation of PR-A Ligand-Independent Transcription by MEKK Is Independent of SUMOylation
To examine effects on transcription from the interplay between phosphorylation and SUMOylation, we compared transcription by WT PR-A, the K388R SUMOylation-deficient mutant, or a S294A phosphorylation-deficient mutant ( Figure 4A) in the presence or absence of constitutively active MEKK1. Low MEKK1 expression levels increased hormone-independent and hormone-dependent activities of WT and K388R PR-A. Thus, the stimulatory effect of MEKK1 occurs even on deSUMOylated PR and thus independently of SUMOylation. MEKK1 also stimulated hormone-independent and hormone-dependent transcription of the S294A mutant, demonstrating that MEKK is not acting through this phosphorylation site. Of note, stimulatory effects of MEKK1 are highly concentration dependent. The data in Figure 4B use physiological MEKK1. At pharmacologic concentrations MEKK1 inhibits hormone-dependent PR-A SUMOylation (data not shown); underscoring the importance of dose-response studies in such analyses.

Stimulation of PR-A Ligand-Independent Transcription by MEKK is Independent of SUMOylation
To examine effects on transcription from the interplay between phosphorylation and SUMOylation, we compared transcription by WT PR-A, the K388R SUMOylation-deficient mutant, or a S294A phosphorylation-deficient mutant ( Figure 4A) in the presence or absence of constitutively active MEKK1. Low MEKK1 expression levels increased hormone-independent and hormonedependent activities of WT and K388R PR-A. Thus, the stimulatory effect of MEKK1 occurs even on deSUMOylated PR and thus independently of SUMOylation. MEKK1 also stimulated hormoneindependent and hormone-dependent transcription of the S294A mutant, demonstrating that MEKK is not acting through this phosphorylation site. Of note, stimulatory effects of MEKK1 are highly concentration dependent. The data in Figure 4B use physiological MEKK1. At pharmacologic concentrations MEKK1 inhibits hormone-dependent PR-A SUMOylation (data not shown); underscoring the importance of dose-response studies in such analyses.

Histone Acetylation, SUMOylation and Transcription by PR-A
Trichostatin A (TSA) alters gene expression by impeding histone deacetylases (HDACs). HDACs have been implicated in transcriptional repression by SUMOylation [22]. To test whether HDACs influence SUMO regulation of PR, the transcriptional activities of WT PR-A or PR-A K388R were tested +/− TSA and +/− the SUMO de-conjugating enzyme SENP1 ( Figure 5A). TSA alone improved transcription by unliganded or liganded PR-A, but to a lesser extent than deSUMOylation by SENP1 (left). TSA plus SENP1 were additive or weakly synergistic, suggesting relatively independent mechanisms of action for acetylation and SUMOylation. This was tested directly with the SUMOylation-deficient mutant (right). Here too, TSA boosted ligand-independent and -dependent transcription. Interestingly at high concentrations ( Figure 5B), TSA induced SUMOylation of unliganded PR-A, but inhibited SUMOylation of liganded PR-A; a discordance which confirms that acetylation and SUMOylation are dissociated.
Diseases 2018, 6, 5 9 of 16 Trichostatin A (TSA) alters gene expression by impeding histone deacetylases (HDACs). HDACs have been implicated in transcriptional repression by SUMOylation [22]. To test whether HDACs influence SUMO regulation of PR, the transcriptional activities of WT PR-A or PR-A K388R were tested +/− TSA and +/− the SUMO de-conjugating enzyme SENP1 ( Figure 5A). TSA alone improved transcription by unliganded or liganded PR-A, but to a lesser extent than deSUMOylation by SENP1 (left). TSA plus SENP1 were additive or weakly synergistic, suggesting relatively independent mechanisms of action for acetylation and SUMOylation. This was tested directly with the SUMOylation-deficient mutant (right). Here too, TSA boosted ligand-independent and -dependent transcription. Interestingly at high concentrations ( Figure 5B), TSA induced SUMOylation of unliganded PR-A, but inhibited SUMOylation of liganded PR-A; a discordance which confirms that acetylation and SUMOylation are dissociated.

SUMOylation Differentially Regulates Endogenous Progestin Target Genes in Breast Cancer Cells
The above synthetic promoter studies support the conclusion that SUMOylation exerts similar repressive effects on PR-A and PR-B. Can this conclusion be applied to endogenous genes? To answer this, we generated sublines from our previously generated PR-negative T47D-Y breast cancer line, that stably express WT PR-A or K388R PR-A (AKR). Figure 6C shows that T47D-Y are PR-; that the

SUMOylation Differentially Regulates Endogenous Progestin Target Genes in Breast Cancer Cells
The above synthetic promoter studies support the conclusion that SUMOylation exerts similar repressive effects on PR-A and PR-B. Can this conclusion be applied to endogenous genes? To answer this, we generated sublines from our previously generated PR-negative T47D-Y breast cancer line, that stably express WT PR-A or K388R PR-A (AKR). Figure 6C shows that T47D-Y are PR-; that the two modified cell lines express similar levels of PR in the absence of R5020; and that receptors in both cells undergo normal ligand-dependent down-regulation [16]. Further, both cell lines appropriately activate transcription of PRE 2 -Luc [16]. Transcript levels of the three untreated, or 24 h R5020-treated, cells were quantified using U133 Plus 2 chips [23]. Unliganded and progestin-regulated genes were grouped by hierarchical clustering into upregulated (red) and down-regulated (blue) genes ( Figure 6A). Despite the complexity, statistical analyses resolved 7 distinct gene clusters ( Figure 6A). In general Cluster 1 genes are upregulated uniquely by liganded WT PR-A; Cluster 2 genes are upregulated by WT and AKR mutant PR-A; Cluster 3 genes and to a lesser extent Class 4 genes are upregulated mainly by AKR mutant PR-A; Class 5 genes are interesting, apparently down-regulated by both unliganded and liganded WT PR-A; Class 6 genes tend to be down-regulated by liganded WT and mutant PR; Class 7 genes are demonstrably down-regulated mainly by the liganded AKR PR mutants.
Venn diagrams ( Figure 6B) quantify progestin-regulated (red and blue) and ligand independent (yellow and grey) genes by WT PR-A or the SUMO-deficient AKR mutant. Four-hundred forty-five (445) genes were R5020-upregulated by both WT and mutant PR-A, 902 were upregulated only by WT PR-A, and 806 were upregulated only by the mutant. R5020-bound WT PR-A uniquely down-regulated 962 genes, the mutant down-regulated 276, and both receptors down-regulated 206. These diagrams clearly demonstrate the variable responses of endogenous genes to progestin-bound PR-A. The lower diagrams in Figure 2B quantify genes down-or upregulated by unliganded PR-A or PR-AK388R. These are of course also of considerable interest as they may modify tumor biology even in the absence of progesterone.
Expression profiling clearly shows that endogenously, SUMOylation modulates PR-A function in a target gene selective fashion, and not by general repression of PR-A activity. While some genes, such as SGK1 and PDK4 are unaffected by SUMOylation, others, such as CDKN1c and Claudin 8 (Figure 7) are only expressed in cells capable of SUMOylating WT PR-A. Expression of other genes, such as NDRG1, is enhanced only in the de-repressed K388R mutant cells, indicating that in WT cells SUMOylation represses NDRG1 activity. We used Ingenuity Pathway Analysis (IPA) to identify cellular functions and pathways significantly enriched by PR-A SUMOylation. The top five diseases were cancer, organismal injury and abnormalities, gastrointestinal disease, reproductive system disease and respiratory disease. The top five molecular and cellular functions were cell cycle, cellular assembly and organization, DNA replication, recombination and repair, cell death or survival, and cellular development ( Figure 6D).
Several genes were selected using the Oncomine database, to assess their possible role in patients with breast cancer (Figure 7). ATAD2 and SPINT3 are upregulated by ligand in K388R mutant cells indicating that their expression is normally suppressed by SUMOylated PR (Figure 7(Ai)). Both genes are upregulated in ductal carcinomas (Figure 7(Aii)). Using Kaplan-Meier survival analysis (http://kmplot.com/analysis), we compared tumors that highly express these genes (red) to ones with low expression (black), in ER+/PR+ (Figure 7(Aiii)) or ER−/PR− disease (Figure 7(Aiv)). Significance data and p-values are shown in the upper right corner of each box. It is clear that upregulated genes may significantly (p ≤ 0.05) correlate with survival in some tumors. CDKN1c and Claudin 8 are strongly upregulated by progestin-occupied, SUMOylated WT PR-A. Receptor deSUMOylation suppresses this regulation. Interestingly, these genes are strikingly suppressed in ductal carcinomas and their expression may also correlate with tumor behavior. In general, we hypothesize that besides acting as markers, PRs and their SUMOylation/deSUMOylation state may play a functional role in tumor survival.

Discussion
ER+/PR+ tumors respond better to endocrine therapies than ER+/PR-ones but PR levels are not necessarily related to benefit, raising questions about appropriate PR "cut-off" levels in clinical assays. Additionally, besides simply acting as markers of hormone responsiveness, PR signaling undoubtedly has functional consequences in both liganded and unliganded states. The receptors are extensively modified post-translationally, which influences their gene regulatory capacity including crosstalk with ER [24,25]. For instance it is not always appreciated that PR can inhibit ER transcriptional activity [11,24].
Moreover, all P target tissues express two PR isoforms, which regulate different sets of genes with different downstream sequelae [4]. PR-B but not PR-A are required for mammary gland development and expansion [26]. RANKL, a gene activated only by PR-B [27], plays an important role in the pregnant mammary gland; in initiation and progression of P-induced breast cancer [28]; and for maintenance and expansion of mammary stem cells [29]. The structural and functional mechanisms underlying differences between PR-A and PR-B are under active investigation. With respect to structure, we find that we can completely destroy the AF-3 transcriptional function unique to the N-terminal region of PR-B, without converting the mutant to PR-A. Differences in function are unrelated to DNA binding affinity since both receptors bind DNA equally well. Rather, it appears that functional differences are dependent on recruitment of different coactivators to the two DNA-bound receptors [30]. This recruitment in turn is controlled by post-translational modifications of the receptors, including their phosphorylation, ubiquitination, acetylation and SUMOylation states. Here we focus on SUMOylation of PR-A, and examine phosphorylation and acetylation, to analyze the consequences of these modifications on transcription of synthetic promoter/reporters, and also on transcription of endogenous genes in human breast cancer cells. The latter have rarely been assessed.
We use PR-negative human breast cancer cells that we isolated, then modified to stably express WT PR-A, a SUMOylation deficient PR-A K388R mutant to model SUMOylation failure, plus other mutants. We also use various concentrations of enzymes that de-SUMOylate the receptors or drugs that target phosphorylation or acetylation. With regard to SUMOylation, our analyses indicate that this modification does not simply attenuate PR activity on all target genes as has been concluded from use of synthetic promoters. Instead endogenously, PR SUMOylation controls whether the receptors either liganded or unliganded, up-or down-regulate genes, or lose regulatory capacity altogether. This endogenous complexity is consistent with previous reports for PR-B, and for glucocorticoid (GR) and androgen (AR) receptors, which also show SUMO-sensitive target-selectivity [31].
We report pathway analysis to identify genes differentially regulated by SUMOylated vs. deSUMOylated PR-A, and find that the dominant genes are involved in molecular and cellular functions of the cell cycle, cellular assembly and organization, DNA replication, recombination and repair, cell death/survival, and cellular development. The top genes upregulated by liganded but deSUMOylated PR-A (i.e., genes that are normally repressed by receptor SUMOylation) are associated with brain metastasis, poor overall survival and poor prognosis. Among these are: ATAD2, ADAMTS8, DIO2, KCNMA, and NOK1. Briefly, ATAD2 is a nuclear coactivator of ER and AR crucial for assembly of chromatin-modifying complexes and proliferation of hormone-responsive cancer cells [32]. Its overexpression correlates with aggressiveness of breast and endometrial cancers [33] via recruitment of the coactivator SRC3 [34]. ADAMTS8 is also a predictor of poor overall survival [35]. DIO2 activates thyroid hormone and plays a role in P/PR-induced proliferation [36]. KCNMA1 is a pore forming α-subunit of the large-conductance calcium-and voltage-activated potassium channel linked to heightened proliferation and brain metastases [37]. NOK is a potent oncogene expressed in multiple cancers [38], which activates signaling pathways including MAPK and PI3K by phosphorylating them [39]. Clearly, all these genes are important and subject to complex regulation, among which are the "repressor" effects of SUMOylated PR-A.
Then, we find genes that require liganded SUMOylated PR-A for their upregulation. One is CDKN1C-p57Kip2 (Figure 7), a protein reduced or lost in the majority of breast cancers, possibly through ER-dependent mechanisms. Similarly, Claudin 8 (CLDN8), a tight junction protein dysregulated in a number of cancers [40], is down-regulated in ductal carcinomas compared to the normal breast ( Figure 7) and is associated with differential survival of patients with ER+/PR+ vs. ER−/PR− disease. Another interesting gene in this category is C/EBPδ, an inflammatory response gene and tumor suppressor associated with metastasis [41]. Other genes uniquely upregulated by SUMOylated PR-A include ALDH1A3 (4.5-fold), Annexin A1 (5.6-fold) and ERRγ, an estrogen-related receptor induced 26.2-fold. All are associated with poor patient survival, triple negative disease and tamoxifen resistance, and all are examples of genes that are not suppressed by PR-A SUMOylation.
In sum, our studies address PR structural regions and signaling pathways involved in post-translational PR-A modifications, focused on transcriptional regulation by SUMOylation. We demonstrate that the mixed progestin/antiprogestin RU486, used to terminate early pregnancy by blocking P-activated PRs, loses its antagonist properties on de-SUMOylated receptors. This is an interesting example of how a receptor's SUMOylation state modifies the target specificity of a bound hormone. Parenthetically, this suggests use of SUMO-deficient PR-A as a sensitive assay for screening new antiprogestins for their partial agonist activities. Only a pure antiprogestin would be inactive on de-SUMOylated PR-A. We also show that the PR-A SUMOylation state controls not only the transcriptional potency of these receptors, but also the direction, both up and down, in which endogenous genes are progestin-regulated through PR-A. These issues need to be understood, especially in premenopausal women, as SUMO-modifying drugs enter mainstream medical practice for treatment of cancers, cardiac disease, neurodegenerative diseases, viral infections, and the like [42].


s2fieldsofstudy: Biology Medicine

text:
p62‐mediated autophagy affects nutrition‐dependent insulin receptor substrate 1 dynamics in 3T3‐L1 preadipocytes

Abstract Aims/Introduction Previous studies have shown that an organism's nutritional status changes the protein levels of insulin receptor substrate 1 (IRS‐1) in a tissue‐specific manner. Although the mechanisms underlying the regulation of IRS‐1 in the nutrient‐rich conditions associated with diabetes and insulin resistance have been well studied, those under nutrient‐poor conditions remain unknown. The aim of the present study was to investigate how IRS‐1 protein levels change depending on the nutritional status of 3T3‐L1 preadipocytes. Materials and Methods 3T3‐L1 preadipocytes were treated with glucose‐, amino acid‐ and serum‐free medium for starvation. IRS‐1 protein levels were detected by western blot. Autophagy activity was observed by western blot and fluorescence microscopy. The effect of autophagy and p62, an adaptor for selective autophagy, on IRS‐1 protein levels under starvation conditions was examined by western blot and immunocytochemistry. Results We showed that the levels of IRS‐1, but not those of insulin receptor and protein kinase B, decreased when starvation activated autophagy. The inhibition of autophagy by chloroquine or autophagy‐related 7 (Atg7) ribonucleic acid interference counteracted the starvation‐induced decrease of IRS‐1. Additionally, Atg7 knockdown increased insulin‐stimulated phosphorylation of protein kinase B under starvation conditions. Furthermore, p62 colocalized with IRS‐1 under starvation conditions, and p62 knockdown counteracted the starvation‐induced degradation of IRS‐1. Conclusions Autophagy through p62 plays an important role in regulating IRS‐1 protein levels in response to nutritional deficiency. The present findings suggest that autophagy might function as energy depletion‐sensing machinery that finely tunes insulin signal transduction.

INTRODUCTION
Insulin receptor substrate (IRS)-1 is one of the most widely distributed members of the IRS family, an important class of intracellular molecules that mediate insulin signaling [1][2][3] . IRS-1 is mainly localized in the skeletal muscle and adipose tissue, and it plays an important role in glucose metabolism. Irs-1 knockout mice are insulin-resistant, showing that IRS-1 is essential for insulin signal transduction 4 .
The nutritional status changes IRS-1 protein levels. In animal models, obesity is associated with decreased IRS-1 levels in adipocytes 13 . However, it is still not fully understood how IRS-1 protein levels are regulated in response to nutritional change. Additionally, little is known about the mechanisms that control the expression of IRS-1 in adipocytes under nutrient-poor conditions.
Macroautophagy (hereafter referred to as autophagy) and the ubiquitin-proteasome pathway play important roles in protein degradation in eukaryotes, and are activated by starvation 14 . In autophagy, double-membrane vesicles, called autophagosomes, deliver large cytoplasmic components to the lysosomes for degradation 15 . Autophagy degrades proteins and entire organelles, and thereby maintains intracellular homeostasis. In addition, autophagy supplies nutrient sources for cellular renovation during cell differentiation under starvation conditions 16 . Autophagy was initially considered a non-selective degradative pathway 17 . Recent studies, however, have shown that autophagy selectively degrades targeted proteins 18 . For example, selective autophagy is involved in the removal of aggregation-prone proteins, damaged organelles and microbes [19][20][21] . Although earlier studies reported that autophagy plays important roles in diabetes and insulin resistance [22][23][24] , the role of autophagy in the alteration of insulin signaling is unclear.
p62 is one of the best-known autophagy substrates 25,26 . The domain structure of p62 includes a region interacting with the autophagosomal protein, microtubule-associated protein 1 light chain 3 (LC3), and a ubiquitin-associated domain. p62 recognizes ubiquitinated substrates and links them to LC3 27 . Furthermore, p62 directly interacts with IRS-1 28 . p62 has the SH2 domain at the amino terminus, which interacts with IRS-1 through its phosphotyrosine (YXXM) motifs at Tyr-608, Tyr-628 and/or Tyr-658 in a manner similar to the interaction between IRS-1 and p85 of phosphoinositol 3-kinase: this interaction plays an important role in insulin signaling 28 .
In the present study, we hypothesized that autophagy is involved in the degradation of insulin-signaling molecules in 3T3-L1 preadipocytes in response to nutritional changes. Our results show that autophagy is involved in the degradation of IRS-1, but not of the insulin receptor (IR) or protein kinase B (Akt), under starvation conditions induced by lack of glucose, amino acids and serum, and that inhibition of autophagy increases insulin-stimulated phosphorylation of Akt. Furthermore, our data show that p62 is involved in the starvationinduced degradation of IRS-1. These findings indicate that p62 and autophagy play an important role in the changes in IRS-1 levels in response to nutritional deficiency.

Materials
The antibodies against IR, IRS-1, Akt, b-actin and Akt (phosphorylated on serine 473) were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against LC3 was purchased from Cell Signaling Technology and Thermo Fisher Scientific Inc. (Waltham, MA, USA). The antibody against p62/SQSTM1 (C terminus) was purchased from Progen (Heidelberg, Germany). The antibodies against Atg7 were purchased from Wako (Osaka, Japan). Chloroquine and digitonin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Animals
Male C57BL/6J mice were obtained from CLEA Japan, Inc. (Tokyo, Japan). All animals were maintained under a 12-h light-dark cycle, fed with a regular diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan) for up to 12 weeks and provided with water ad libitum. For fasting, mice were fed with a 60% highfat diet (D12492; Research Diets, Inc., New Brunswick, NJ, USA) for 2 weeks, followed by fasting for 48 h. Mice were then anesthetized and killed to allow for the isolation of epididymal fat tissue. The study protocol was reviewed and approved by the animal care and use committee of Kanazawa University.

Cell culture and treatments
The mouse fibroblast cell line 3T3-L1 was obtained from the National Institute of Biomedical Innovation, Health and Nutrition (Osaka, Japan). Cells were cultured in the growth medium (GM), consisting of Dulbecco's modified Eagle's medium (Thermo Fisher Scientific Inc.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc.), 2 mmol/L L-glutamine (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 100 units/mL penicillin and 0.1 mg/mL streptomycin (Wako Pure Chemical Industries, Ltd.). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . For starvation, the cells were treated with the glucose-, amino acid-and serum-free starvation medium (SM), which was prepared by mixing 5.81 g of Nutrition-Free Dulbecco's modified Eagle's medium (Nacalai Tesque, Inc., Kyoto, Japan), 1.85 g of NaHCO 3 and 5 mL of 0.1 mol/L sodium pyruvate in 500 mL Milli-Q water, and then sterilizing with a 0.22-lm filter (Millipore, Billerica, MA, USA). For pharmacological inhibition of autophagy, the cells were treated with chloroquine (50 lmol/L).

Induction of adipocyte differentiation
Induction of adipocyte differentiation was carried out as previously described 29 .

Western blotting
Western blotting was carried out as previously reported 30 . Densitometry analysis of the blotted membranes was carried out using ImageJ software (National Institutes of Health, Rockville, MD, USA).
Quantitative polymerase chain reaction Total RNA isolation, complementary deoxyribonucleic acid synthesis and quantitative polymerase chain reaction analysis were performed as previously described 30 . Quantitative polymerase chain reaction was carried out using the Step One Plus Real-Time PCR System (Thermo Fisher Scientific Inc.) and the following TaqMan (Applied Biosystems, Carlsbad, CA, USA) probes: actin B, 4352341E_m1; IRS-1: Mn01278327_m1; autophagy related 7 (Atg7), Mn00512209_m1; and p62: Mm00448091_m1.
Colocalization studies of IRS-1 and LC3 3T3-L1 preadipocytes grown on four-chamber slides were incubated in GM or SM for 60 min, with or without chloroquine (50 lmol/L), and then washed with phosphate-buffered saline (PBS) and fixed in 4% formaldehyde at room temperature for 15 min. After washing with PBS, the cells were permeabilized with 500 lL of a digitonin solution (100 lg/mL) at room temperature for 15 min. After washing with PBS, non-specific antibody binding sites were blocked with Protein Block Serum-Free Ready-to-Use (Agilent Technologies, Santa Clara, CA, USA) at room temperature for 10 min. The cells were then washed with 0.01% TWEEN 20 in PBS (PBS-T) and incubated at 4°C overnight with the anti-IRS-1 antibody (diluted 1:100 in PBS-T). After washing, the cells were incubated at room temperature for 60 min with goat anti-rabbit immunoglobulin G conjugated with Alexa Fluor 488 (Abcam, Cambridge, UK) diluted in PBS-T. Subsequently, the anti-LC3 antibody (Thermo Fisher Scientific Inc.) labeled with HiLyte Fluor 555 was applied to the cells at room temperature for 60 min. After washing, the cells were incubated in Fluoroshield Mounting Medium with 4 0 ,6-diamidino-2-phenylindole (Abcam). Images were obtained by optical sectioning observation by structured illumination microscopy using a BZ-X700 microscope (Keyence).
Colocalization studies of IRS-1 and P62 3T3-L1 preadipocytes grown on four-chamber slides were incubated in GM or SM for 30 min, washed with PBS and fixed in 100% methanol at -20°C for 30 min. After fixation, the cells were washed with PBS-T for 15 min, and non-specific antibody binding sites were blocked with Protein Block Serum-Free Ready-to-Use at 4°C overnight. Cells were then incubated at 4°C overnight with the anti-IRS-1 and anti-p62 antibodies (both diluted 1:100 in PBS-T). After washing, the cells were incubated for 60 min with goat anti-rabbit immunoglobulin G conjugated with Alexa Fluor 488 and goat anti-guinea pig immunoglobulin G conjugated with Alexa Fluor 594 (Abcam) diluted in PBS-T. After washing, cells were incubated in Fluoroshield Mounting Medium with 4 0 ,6-diamidino-2-phenylindole. Images were obtained by optical sectioning observation by structured illumination microscopy using a BZ-X700 microscope (Keyence).

Statistical analysis
Data from the experiments are shown as box plots with individual points 31 , and graphics were prepared using R software (https://www.r-project.org). Significance was compared by unpaired two-tailed Student's t-test or one-way analysis of variance, and differences were considered statistically significant for P < 0.05.

IRS-1 is degraded under starvation conditions
We examined the effect of starvation on the levels of insulinsignaling molecules in 3T3-L1 preadipocytes incubated in GM or SM. SM decreased the levels of IRS-1, but not those of IR and Akt over time (Figure 1a,b). In contrast, SM temporally increased the expression of Irs-1 (Figure 1c). These results show that, in 3T3-L1 preadipocytes, the levels of IRS-1 decrease under starvation because of protein degradation. In addition, we examined the effect of starvation conditions on the protein levels of IRS-1 in 3T3-L1 mature adipocytes and mouse epididymal adipose tissue. First, we assessed the protein levels of insulin-signaling molecules in 3T3-L1 adipocytes. In 3T3-L1 adipocytes, as well as in 3T3-L1 preadipocytes, SM time dependently decreased IRS-1, although it did not alter insulin receptors or Akt ( Figure 1d). Next, to examine the effect of fasting on protein levels of IRS-1 in the epididymal adipose tissue of mice, we fed or fasted C57BL/6J mice for 48 h. Fasting decreased the protein levels of IRS-1, but not those of insulin receptors or Akt (Figure 1e). These findings suggest that the starvation-induced degradation of IRS-1 by autophagy might occur in 3T3-L1 adipocytes and mouse epididymal adipose tissue.
Starvation induces autophagy in 3T3-L1 preadipocytes Next, we evaluated whether autophagy is activated by starvation. First, we examined autophagy flux by evaluating the immunofluorescence associated with autophagic vacuoles. When the cells were incubated in GM, they did not show specific fluorescence (Figure 2a). In contrast, after culture in SM for 60 min, 3T3-L1 preadipocytes showed significant green fluorescence, indicating the formation of pre-autophagosomes, autophagosomes and autolysosomes. Additionally, treatment with chloroquine, an autophagy inhibitor, increased green fluorescence in cells that were incubated in SM. Furthermore, we tested the autophagy flux by analyzing LC3 turnover 32 . During autophagy, cytosolic LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-II, and the two forms can be distinguished by western blot. We treated 3T3-L1 preadipocytes with GM or SM for 30 min in the presence or absence of 50 lmol/L chloroquine and assessed the difference in LC3-II protein levels. SM significantly increased LC3-II levels in the presence of chloroquine, indicating an increase of autophagy flux (Figure 2b,c). These findings show that autophagy is induced by starvation. Autophagy activated by starvation decreases IRS-1 levels We carried out experiments to elucidate the mechanism underlying the degradation of IRS-1 under starvation. To assess the involvement of autophagy in this process, we investigated the effect of the inhibition of autophagy on IRS-1 levels by treating the cells with chloroquine. The inhibition of autophagy counteracted the decrease in IRS-1 levels in cells incubated in SM (Figure 3a,b). Similarly, 10 lg/mL pepstatin A and 10 lg/mL E64-d, which are lysosomal inhibitors, counteracted the decrease in IRS-1 levels in cells incubated in SM ( Figure S1). Furthermore, we genetically blocked autophagy through RNA interference against Atg7 (Figure 3c). Knockdown of Atg7 was associated with increased IRS-1 levels in cells incubated in SM (Figure 3d,e). Next, we assessed the localization of IRS-1 and LC3 in the cells by immunocytochemistry. 3T3-L1 preadipocytes treated with GM or SM for 30 min in the presence or absence of chloroquine were immunostained for IRS-1 and LC3 (Figure 3f). In cells incubated in SM and in the presence of chloroquine, some of the IRS-1-positive puncta colocalized with the LC3-positive puncta, suggesting that these two proteins form the same complexes. Next, we tested the effect of autophagy on insulin signaling. We treated control and Atg7-knockdown cells with SM for 30 min, followed by stimulation with or without 1 lg/ mL insulin for 10 min. Knockdown of Atg7 significantly increased insulin-stimulated Akt phosphorylation under starvation conditions (Figure 3g,h). In addition, we treated 3T3-L1 preadipocytes with SM in the presence or absence of chloroquine for 30 min, followed by stimulation with or without 1 lg/mL insulin for 10 min. Chloroquine also significantly increased insulin-stimulated Akt and extracellular signalregulated kinase phosphorylation under starvation conditions ( Figure S2). Because Akt and extracellular signal-regulated kinase is a downstream effector of IRS-1, these findings suggest that starvation-induced autophagy induces IRS-1 degradation, leading to decreased insulin-stimulated Akt phosphorylation.  the cytosol of cells incubated in SM (Figure 4a). Next, we silenced p62 by RNA interference (Figure 4b,c). Knockdown of p62 counteracted the starvation-induced decrease of IRS-1, which was consistent with the inhibition of autophagy (Figure 4c,d). These findings suggest that IRS-1 might be degraded through autophagy on p62 binding.

DISCUSSION
The present study provides evidence that p62 is involved in the degradation of IRS-1 through autophagy in 3T3-L1 preadipocytes under starvation conditions. We showed that starvation, induced by the absence of glucose, amino acids and serum, activated autophagy and was associated with lower IRS-1 protein levels, whereas inhibition of autophagy counteracted the starvation-induced degradation of IRS-1. Furthermore, p62 interacted with IRS-1 in starved 3T3-L1 preadipocytes, and knockdown of p62 counteracted the starvation-induced decrease of IRS-1. These studies suggest that autophagy plays a major role in the regulation of insulin-signaling molecules in 3T3-L1 preadipocytes in response to nutritional changes. Notably, pharmacological and genetic inhibition of autophagy also increased IRS-1 levels in cells incubated in GM (Figure 3a-e), indicating that autophagy constitutively degrades IRS-1 to maintain constant protein levels in growth conditions, and additionally induces IRS-1 degradation under starvation conditions. It is interesting to note that starvation temporally increased Irs-1 messenger RNA levels (Figure 1c). Meng et al. 33 showed that the transcription factor activating enhancer-binding protein-2b, which increases during the maturation of adipocytes, negatively regulates IRS-1 gene expression. Starvation conditions might decrease activating enhancer-binding protein-2b, leading to induce Irs-1 messenger RNA expression. These findings suggest that there exists a dynamic equilibrium between IRS-1 synthesis and degradation to fine-tune its protein levels in response to nutritional changes.
Autophagy was originally considered an unselective bulk degradation pathway. However, recent studies have shown that starvation induces selective autophagy. Onodera et al. 34 reported that cytosolic acetaldehyde dehydrogenase is selectively degraded by autophagy under nitrogen starvation in yeast. In addition, Kristensen et al. 35 analyzed global protein dynamics in human MCF7 breast carcinoma cells under amino acid starvation conditions by quantitative mass spectrometry, suggesting that starvation-induced degradation through autophagy is selective. In the present study, we found that the levels of IRS-1 decreased in 3T3-L1 preadipocytes under glucose-, amino acid-and serumstarvation conditions, although the levels of IR and Akt did not change (Figure 1a,b). Furthermore, we showed that p62, which is an adaptor for selective autophagy, mediated the degradation of IRS-1 by autophagy in starved preadipocytes (Figure 4). These results suggest the possibility that IRS-1, on interaction with p62, might be selectively degraded by autophagy.
Cellular models of insulin resistance and in vivo studies suggest an association between insulin resistance and changes in IRS-1 levels [7][8][9] . Decreased IRS-1 protein levels have been observed in animal models of insulin resistance 5,36 and in vitro models in which cells are treated with tumor necrosis factor-a or chronically stimulated with insulin 12,13,37 . We found that inhibition of autophagy counteracted the starvation-induced degradation of IRS-1, leading to increased insulin-stimulated phosphorylation of Akt (Figure 3g,h). These data suggest that the starvation-induced degradation of IRS-1 through autophagy might cause insulin resistance in response to nutritional deficiencies. Indeed, previous studies have shown that starvation causes insulin resistance in lean and obese participants with or without diabetes [38][39][40][41] . This is the physiological adaptation to fasting, leading to a shift in energy source from glucose to fat. The starvation-induced degradation of IRS-1 through autophagy might be associated with this adaptation.
Interestingly, Zhang et al. 42 found that inhibition of autophagy decreased insulin-stimulated phosphorylation of Akt under serum-starvation conditions in 3T3-L1 adipocytes. Our studies differ in that we used glucose-, amino acid-and serumstarvation conditions for a shorter time. These opposing observations raise the interesting notion that different starvation conditions can cause different insulin sensitivity.
We acknowledge there were several limitations to the present study. First, we assessed the protein levels of IR, IRS-1 and Akt under starvation conditions; however, it is still unknown whether starvation affects the levels of other insulin signaling molecules. We have just started a comprehensive analysis of the changes in such molecules under starvation conditions by proteomics. Additionally, the degradation of IRS-1 through autophagy under starvation conditions was only shown in vitro. Although Zhang et al. 24 have shown that an adipocyte-specific mouse knockout of Atg7 decreases adipose mass and increases insulin sensitivity by increasing beige cells in white adipose tissue, it remains unclear how starvation conditions affect IRS-1 protein levels and insulin signaling in the mice. We have observed that IRS-1 levels decrease in starved 3T3-L1 mature adipocytes and epididymal adipose tissue of C57BL/6J mice fasted for 48 h (Figure 1d,e). However, a detailed in vivo analysis is required to understand the significance of the starvationinduced degradation of IRS-1 in the adipose tissue.
In conclusion, the present study highlights a novel mechanism for the nutrition-dependent dynamics of IRS-1. This new finding suggests the possibility that autophagy is involved in the degradation of proteins on their interaction with an autophagy adaptor, such as p62, and might therefore play an important role in the regulation of insulin signaling. The elucidation of this process might help to improve the current understanding of the dynamics of insulin signaling.

ACKNOWLEDGMENTS
We thank Maki Kawamura (Kanazawa University) for technical assistance, Kenji Takehana (EA Pharma) for providing Nutrition-Free Dulbecco's modified Eagle's medium Zero medium, Nobuyuki Yoshioka (Keyence) for support of the fluorescence