Post-translational modification of retinoic acid receptor alpha and its roles in tumor cell differentiation

Journal Pre-proofs Commentary Post-translational modification of retinoic acid receptor alpha and its roles in tumor cell differentiation Aixiao Xu, Ning Zhang, Ji Cao, Hong Zhu, Bo Yang, Qiaojun He, Xuejing Shao, Meidan Ying PII: DOI: Reference:

S0006-2952(19)30395-8 https://doi.org/10.1016/j.bcp.2019.113696 BCP 113696

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Biochemical Pharmacology

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Please cite this article as: A. Xu, N. Zhang, J. Cao, H. Zhu, B. Yang, Q. He, X. Shao, M. Ying, Post-translational modification of retinoic acid receptor alpha and its roles in tumor cell differentiation, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113696

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Post-translational modification of retinoic acid receptor alpha and its roles in tumor cell differentiation Short title: Advanced differentiation therapy based on PTMs of RARα Aixiao Xu1, Ning Zhang2, Ji Cao1, Hong Zhu1, Bo Yang1, Qiaojun He1, Xuejing Shao1, * and Meidan Ying1, *

1, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. 2, Department of Orthopedics, The Second Affiliated Hospital of Zhejiang University, Zhejiang University, Hangzhou, China.

*, Corresponding authors: Meidan Ying, Ph.D., Room 115, Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China, 310058. E-mail: [email protected]. Xuejing Shao, Ph.D., Room109, Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China, 310058. E-mail: [email protected].

Conflict of interest: The authors declare no potential conflicts of interest.

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Post-translational modification of retinoic acid receptor alpha and its roles in tumor cell differentiation Abstract Retinoic acid (RA) is a well-known differentiation inducer that exerts its effects by binding to nuclear RA receptors. Retinoic acid receptor α (RARα), as an important nuclear RA receptor, is activated upon RA binding and facilitates the transcription of target genes related to differentiation, which ultimately initiates cell differentiation. Previous studies have found that the transcriptional activity of RARα is regulated by various post-translational modifications, which influence its DNA binding efficiency, transactivation ability and even lead to degradation. Post-translational modifications of RARα, as a consequence, play an important role in the RA-induced differentiation process. Therefore, in this review, we focus on recent advances in the understanding of how these modifications affect the activity of RARα as well as strategies to increase the differentiation effect of RA treatment in cancer cells based on RARαmodifications, which may promote the development of novel effective differentiation therapies for cancer treatment.

Keywords: Differentiation Therapy; Retinoic Acid; RARα; PTMs;

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Abbreviations ATRA, all-trans retinoic acid AML, acute myeloid leukemia APL, acute promyelocytic leukemia RAR, retinoic acid receptor RXR, retinoid receptor AF-1, activation function 1 AF-2, activation function 2 NTD, N-terminal domain DBD, DNA-binding domain LBD, ligand binding domain RARα, retinoic acid receptor alpha PTM, post-translational modification RARE, retinoic acid response element SK1, sphingosine kinase 1 PRAME, preferentially expressed antigen of melanoma PKA, protein kinase A MSK1, mitogen-and stress-activated protein kinase-1 CAK, cyclin-dependent kinase-activating kinase PKC, protein kinase C P38MAPK, p38 a mitogen-activated protein kinase JNK, c-Jun N-terminal kinase 3

GSK3, glycogen synthase kinase 3 MDM2, murine double minute-2 GRp58, glucose-regulated protein 58 ER, endoplasmic reticulum HACE1, HECT domain and ankyrin repeat containing E3 ubiquitin-protein ligase TRIM, tripartite motif FGF8f, fibroblast growth factor 8f ER+, estrogen-receptor-positive

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1. Introduction Differentiation therapy is a biology-based approach to cancer treatment that aims to restore malignant tumor cells to mature normal cells under differentiation-inducing agents [1]. Clinically, the first successful model of differentiation therapy was used in the treatment of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), and featured the introduction of all-trans retinoic acid (ATRA) in 1987 [2]. However, currently, ATRA is the only drug that is considered to be efficient for differentiation-based therapy for patients with APL, and many clinical trials show that ATRA therapy fails to induce the differentiation of non-APL AML and solid tumor cells. Therefore, research strategies seeking to extend the range of ATRA-affected malignancies beyond APL are key avenues of investigation.

2. RARα: A critical regulator of ATRA-based differentiation therapy ATRA is the active metabolite of vitamin A, and its biological effects are mediated by the activation of retinoic acid receptors (RARs), which contain three isotypes, RARα, RARβ and RARγ [3, 4]. They all have a conserved modular organization, composed of 6 regions of homology (A-F, from the N-terminal to the C-terminal) (Figure 1). In the absence of ligands, RARs bind to retinoic acid response elements (RAREs) in the form of heterodimers with retinoid receptors (RXRα, β, and γ) and interact with transcriptional corepressors to create a more condensed state of chromatin and inhibit transcription. In contrast, in the presence of ligands, such as ATRA, the binding of ligands leads to a conformational change of the 5

RAR/RXR heterodimers, causing the depolymerization of corepressors and the exposure of binding sites to coactivators, which ultimately leads to the transcription of target genes, such as genes related to differentiation [5, 6].

RARα, an isotype of RARs, is involved in various biological processes, such as embryonic patterning, reproduction, cell differentiation and homeostatic synaptic plasticity [7-11]. Accumulating evidence shows that RARα is the major RAR participating in the differentiation process induced by ATRA [12, 13]. In APL, pharmacological doses of ATRA induce disease remission in almost 90% of APL cases by inducing the degradation of PML/RARα and restoring the RARα signaling pathway [14-16]. In regard to cells, treatment of HL60 cells with a highly selective agonist of RARα, such as AGN195183 and AM580, was also sufficient to drive differentiation towards neutrophils [17, 18]. Furthermore, the RAR antagonists, ER27191 and Ro 41-5253, mostly blocked ATRA-induced differentiation in HL60 cells [19, 20]. In addition, a mutation in RARα (C411T) was shown to block the ATRA-induced differentiation of HL60 cells [21, 22], and the reintroduction of RARα into HL60R cells rendered these cells sensitive to ATRA [23]. In addition, ATRA has also shown its differentiation-inducing ability in some solid tumors, such as neuroblastoma [24, 25], osteosarcoma [26], breast cancer [27], glioma [28] and hepatocellular carcinoma [29]. Studies have shown that the response to ATRA is highly associated with the protein levels of RARα in different cancers [30-35]. Besides, a RARα antagonist inhibited the ATRA response in osteosarcoma cells, which weakened its differentiation effect [36]. Taken together, RARα plays an essential role in ATRA-induced solid tumor differentiation. 6

Innumerable preclinical studies have shown that ATRA exhibits differentiation-inducing abilities in different kinds of cancer cell lines, including non-APL AML cells, osteosarcoma cells, neuroblastoma cells and so on [25, 37, 38], however, clinical trials are often disappointing. Some clinical trials in non-APL AML utilized single agent ATRA in the 1980s, and almost all trials exhibit low response and high recurrence rates [39, 40]. Meanwhile, the results from some randomized clinical studies have shown that ATRA has little effect on the remission rate or survival when added to conventional therapy [41, 42]. In addition, a phase II trial of ATRA combined with interferon-alpha (IFN-alpha) was inactive in 16 children with refractory neuroblastoma [43]. As a potential mechanism, many studies have found that the expression of RARα is suppressed in tumors [44, 45], and/or the transcriptional activity of RARα is inhibited in cancers [46, 47], which may account for these disappointing results. The reason why RARα expression is suppressed in patients is mainly due to its aberrant degradation. In osteosarcoma patients, the E3 ligase of RARα, the human homolog of murine double minute-2 (MDM2), is abnormally amplified and overexpressed which leads to the degradation of RARα [48]. In addition, RARα expression in tumor tissues is lower than that in normal tissues and it decreases from early to late colorectal cancer stages [49]. MDM2 is also reported to be upregulated in colon cancers [50], which may lead to low expression of RARα. Meanwhile, the suppressed transcriptional activity of RARα may result from abnormal upstream signals, such as disrupted GSK3 [51, 52], SK1 [46], PRAME [47]and JNK signaling [53]. Therefore, restoring the expression and transcriptional activity of RARα in patients is essential for improving sensitivity to ATRA and developing effective therapeutic strategies 7

for non-APL cancers. 3. Post-translational modifications of RARα Post-translational modifications of proteins play an indispensable role in biological activities and the development of tumors [54, 55]. Their aberrancy results in altered functions of proteins and is closely associated with a variety of diseases, such as cancers and neurologic diseases [56]. Many studies have demonstrated that RARα harbors many conserved sites that are targets for several post-translational modifications, including phosphorylation, ubiquitination, sumoylation and so on. Furthermore, these modifications could alter its DNA binding efficiency and transcriptional activity and even lead to the degradation of RARα [57, 58] (Figure 2). Therefore, studying these modifications and analyzing how they affect the functions of RARα may help to identify novel strategies for cancer differentiation therapy. The details are further addressed in the following sections.

3.1 Phosphorylation of RARα Phosphorylation often occurs principally at serine, threonine and tyrosine residues [59]. Similarly, RARα is phosphorylated on Ser and Thr residues and some kinases that phosphorylate RARα have been recently identified (Figure 3). For instance, the cyclic AMP-dependent protein kinase (PKA) and MSK1 both phosphorylate RARα at Ser369, which is located in the LBD domain, and this is followed by the binding of TFIIH and the phosphorylation of RARα at Ser77 by cdk7/cyclin H, which ultimately increases the DNA binding efficiency of RARα-RXRα heterodimers [60, 61]. 8

In addition, RARα is phosphorylated by at least three kinases to regulate its transcriptional activity. For instance, cyclin-dependent kinase-activating kinase (CAK) has been shown to phosphorylate RARα at Ser77, which is located in the NTD [61]. Suppression of the CAK-induced phosphorylation of RARα is required for basal activation of transcription factors, therefore coordinating cell cycle G1 exit and transition into ATRA-induced cancer cell differentiation [62, 63]. Analogously, AKT phosphorylates RARα at Ser96 in the DBD, and this phosphorylation inhibits the transactivation of RARα-RXRα, rather than impairing the DNA binding or heterodimerization of RARα-RXRα [64]. Another kinase PKC can also phosphorylate RARα at Ser157 in the DBD, weakening the capacity of RARα to heterodimerize with RXRα and strongly decreasing the transcriptional activity of RARα [65, 66].

It has also been reported that RARα abundance is negatively regulated by the ubiquitin-proteasome pathway after phosphorylation by the Ser-Thr kinases. P38αMAPK, a downstream mediator of the MEK/ERK pathway, phosphorylates RARα at Ser369, which is followed by the degradation of RARα [67, 68]. Another study also found c-Jun N-terminal kinase (JNK) pathway is activated under oxidative stress, thus contributing to RARα ubiquitin-proteasomal degradation through phosphorylating the RARα residues at Thr181, Ser445 and Ser461 [53]. Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, also leads to the degradation of RARα by phosphorylating RARα at Ser443, Ser445 and Ser449 [69, 70]. 9

In conclusion, RARα can be phosphorylated in response to ATRA-related stimuli, which alters its DNA binding efficiency and transcriptional activity and even leads to the degradation of RARα. Thus, these studies led us to suggest that manipulating the activities of these kinases via pharmacologic inhibitors or activators, such as cAMP activators, AKT inhibitors and GSK3 inhibitors, could be a strategy to control the function of RARα.

3.2 Ubiquitination of RARα Ubiquitination has an essential role in targeting proteins for degradation by the proteasome, regulating cellular localization and/or activating specific signals at certain transcriptional stages [71, 72]. Previous studies have revealed that many regulators could regulate the ubiquitination of RARα to control its stability and/or transcriptional activity, thus affecting ATRA-based differentiation therapy.

3.2.1 Lead to degradation Following ubiquitination, proteins often enter the ubiquitin-proteasome pathway [73]. Regulators mediating the ubiquitination of RARα to control its stability have been identified (Figure 5). Murine double minute-2 (MDM2), a ubiquitin E3 ligase, is an oncogene overexpressed in many human tumors [74]. A recent study demonstrated that the N-terminal domain of MDM2 (amino acids 1-109) binds to RARα and that MDM2 acts as an E3 ubiquitin ligase to target RARα for degradation [38]. Other E3 ligases, such as RNF41and TRIM24, have also been reported to regulate the steady state of RARα [75, 76], but whether 10

they act as an E3 ubiquitin ligase of RARα to mediate its degradation is still unknown. Researchers have also found thatE2F1, a member of the E2F family of transcription factors, specifically interacts with RARα and promotes ubiquitination-proteasome-mediated degradation of RARα [45]. Meanwhile, Glucose-regulated protein 58 (GRP58) may act as a molecular chaperone that mediates RARα nuclear import initiating the transcription of target genes and the subsequent proteasome-mediated degradation of RARα at the ER [77]. Of note, PIN1, a PPIase, induces ligand-independent degradation of RARα through the proteasome pathway via a phospho-serine at position 77 [78-80].

Recently, other proteins have been found to interact with RARα and inhibit the ubiquitination-dependent degradation of RARα. HECT domain and ankyrin repeat containing E3 ubiquitin-protein ligase (HACE1) has been shown to interact with N-terminus of RARα and inhibit the RA-dependent degradation of RARα, which also represses its transcriptional activity [81]. However, the mechanism involved is still unclear. Perhaps the interaction of HACE1 and RARα may interfere with the function of its usual E3 ubiquitin ligase, thus preventing its ubiquitination or interfering with a signal for RARα degradation, such as phosphorylation. It has also been reported that S100A3 interacts with the I396 residue of RARα,inhibits RARαdegradation, and acts as a RARα corepressor to decrease its transcriptional activity [82].

3.2.2 Enhance transcriptional activity On the other hand, ubiquitination of RARα may lead to enhanced transcriptional activity 11

instead of degradation. For instance, TRIM32, as well as TRIM24, also belongs to the tripartite motif (TRIM) family, the members of which are generally recognized as E3 ubiquitin-protein ligases [83]. TRIM32 is also found to interact with RARα and facilitate the ubiquitination of RARα through its RING domain. However, TRIM32-induced ubiquitination ultimately enhances RARα transcriptional activity and stabilizes the expression level of RARα instead of targeting RARα for degradation [84].

Based on the theory above, it is promising to improve the differentiation effect of ATRA by regulating the ubiquitination of RARα. Therefore, proteasome inhibitors [85], MDM2 inhibitors [86], E2F1 activity inhibitors, PIN 1 inhibitors or TRIM32 activators may restore the activity of RARα and show efficient synergy with ATRA to enhance differentiation therapies.

3.3 Sumoylation of RARα Small ubiquitin-related modifier alterations have been implicated in many important cellular processes, including cell cycle progression, apoptosis, and cellular proliferation [87, 88]. Higher eukaryotes possess at least three SUMO isoforms, namely, SUMO1-3 [89]. The sumoylation pathway resembles that of ubiquitin conjugation; however, the enzymes involved in these two processes are distinct, leading to distinct functional consequences [90]. Increasing studies have shown that RARα undergoes SUMO modifications, which affect its function. A previous study has reported that RARα is subject to SUMO-1 modification and that Lys399 is the major site for SUMO-1 conjugation of RARα [91]. SUMO-1 modification 12

increases the stability of RARα and facilitates heterodimerization of RARα with RXRα. Another study identified SUMO-2 modification at the Lys166 and Lys171 sites as another posttranslational regulatory mechanism controlling ATRA-dependent RARα subcellular localization and overexpressed SUMO-2 was shown to specifically suppress the transcriptional activity of RARα. Meanwhile, SUMO-specific protease 6 (SENP6) could deconjugate SUMO-2 from RARαto enhance its transcriptional activity [92].

3.4 Crosstalk among different post-translational modifications of RARα RARα harbors many post-translational modifications; in addition, different modifications of proteins can influence each other [93]. Previous studies have found that phosphorylation of RARα is required for its degradation in some conditions [78]. For example, PIN1, a PPIase, could specifically recognize phosphorylated proteins, altering their conformation; therefore, only a phospho-serine at position 77 of RARα can be regulated by PIN1, inducing the ligand-independent degradation of RARαvia the proteasome pathway [78, 80]. JNK also contributes to RARα dysfunction by phosphorylating the RARαThr181, Ser445, and Ser461 residues, which results in ubiquitination-dependent degradation, mainly because the creation of docking sites by phosphorylation is beneficial for E3 ubiquitin ligases to bind to RARα [53].

Sumoylation may crosstalk with ubiquitination on many proteins [71, 94]. Similarly, RARα possesses a possible dynamic balance mechanism between sumoylation and ubiquitination. SUMO-1 modification of RARα acts as an important gatekeeper in abrogating its 13

ubiquitination and increasing its stability, meaning that it may provide an important regulatory mechanism that controls the stability and activity of RARα during ATRA-induced cell differentiation [91].

In conclusion, different modifications of RARα can influence each other, thus we can regulate one modification by controlling another modification. For example, some phosphorylated residues of RARα are essential for ubiquitination-dependent degradation, which leads to the possibility to regulate ubiquitination-mediated degradation of RARα via regulating its phosphorylation. Therefore, this crosstalk provides more pathways through which to regulate RARα degradation, especially those pathways affected by several available kinase inhibitors. In addition, inhibiting the activity of certain kinases could not only improve the transcriptional activity of RARα but also decrease its degradation, which are both beneficial for ATRA-induced differentiation. Therefore, utilizing cross-regulation among different modifications would be a more potent strategy to control the function of RARα in ATRA-based differentiation therapy.

4. Strategies to expand the indications of ATRA based on the post-translational modifications of RARα On the basis of the mechanisms underlying the post-translational regulation of RARα, many combination therapies have been proposed and have shown more potent efficiency than single agent ATRA not only in APL cells, but also in non-APL AML, osteosarcoma cells, neuroblastoma cells and so on (Table 1). Some studies described herein have yielded strong 14

evidence for translating their basic research findings into clinical trials.

4.1 Decreasing the ubiquitination-dependent degradation of RARα Previous studies have shown that ATRA promotes the ubiquitination-based degradation of RARα, which weakens its effect on differentiation. Thus, proteasome inhibitors such as bortezomib could be drug candidates to synergize with ATRA to enhance differentiation, which would provide new hope for cancer therapy [37, 95].To date, three specific proteasome inhibitors, bortezomib (2003), carfilzomib (2012) and ninlaro (2015), have been approved by the U.S. Food and Drug Administration for multiple myeloma and mantle cell lymphoma treatment. Many preclinical studies have also assessed their efficiency in different cancers [96]. Other proteasome inhibitors, including oprozomib (NCT02227914), marizomib (NCT03345095) and delanzomib (NCT00572637), have also entered clinical trials. Therefore, the combination of these proteasome inhibitors with ATRA may be a potent differentiation therapy for several cancers, especially in patients with high proteasome activity.

MDM2 lessens the differentiation induced by ATRA by acting as an E3 ligase of RARα; thus, the combination of MDM2 inhibitors with ATRA may also increase ATRA effects on tumor differentiation. Our group found that inhibitors of the MDM2 ubiquitin ligase, HLI373 and nutlin-3, both synergized with ATRA to enhance the differentiation of osteosarcoma cells and primary osteosarcoma blasts via reducing the degradation of RARα[38]. Until now, several MDM2 inhibitors, such as DS-3032b (NCT01877382), AMG-232 (NCT03107780), APG-115 (NCT03781986), milademetan (NCT03671564) and ALRN-6924 (NCT03654716) have 15

entered clinical trials. Therefore, the combination of these inhibitors with ATRA is more likely to enter clinical trials.

4.2 Regulating the phosphorylation of RARα to improve its activity 4.2.1 Non-APL AML Due to the phosphorylation of RARα at Ser369, drugs that elevate cAMP levels, such as piclamilast, synergized with RA to trigger the differentiation of APL cells both in vitro and in vivo [97]. Pharmacological inhibitors of P38α such as PD169316 and SB203580 enhanced the retinoid-dependent growth inhibition and differentiation of AML cells by inhibiting Ser369 phosphorylation of RARα, which expands the treatment possibilities of ATRA [98]. Gupta et al. utilized GSK3 inhibitors to regulate the phosphorylation of RARα to synergize with ATRA in the induction of differentiation in AML cell lines [69]. More importantly, this combination therapy includingGSK3 inhibitor, lithium, and ATRA has entered clinical trial in patients with relapsed or refractory AML (NCT01820624).

4.2.2 Solid tumors Studies have shown that ATRA-suppressed phosphorylation of RARα by CAK at Ser77 induced FGF8f expression to mediate the differentiation response pathway in U2OS osteosarcoma cells [63]. Because inhibition of AKT, which phosphorylates RARα at Ser96, enhances RARα activity and exhibits growth-inhibitory effects related to retinoids in non-small cell lung carcinoma (NSCLC), AKT inhibitors such as perifosine and GSK690693 may be useful for investigating the effects in differentiation therapy [64]. Yoshiko et al. found 16

that SP600125, an inhibitor of JNK, could enhance RARα levels by suppressing its phosphorylation, leading to ligand-induced activation of RARα-RXRα dimers and growth inhibition by ATRA in a human lung cancer cell line [99]. Our group also showed that the proteasome inhibitor bortezomib enhanced the ATRA-induced differentiation of neuroblastoma cells via the JNK mitogen-activated protein kinase pathway [25]. In summary, the concept that inhibition of these pathways can lead to activation of RARα via regulation of phosphorylation is worth in-depth investigation.

5. Future Directions In recent years, due to the ability of ATRA to induce tumor stem cell differentiation [100-102], ATRA has been considered an agent for overcoming drug resistance and/or preventing metastasis to improve patient outcomes when combined with chemotherapeutic drugs or target drugs [103, 104]. Based on this idea, some combination therapies have entered clinical trials. For example, a clinical trial assessing the combination effect between dasatinib and ATRA for relapsed/refractory patients with AML or myelodysplastic syndrome has begun (NCT00892190). Therefore, these studies stimulated us to pave the way for these ATRA-based combination strategies. On the one hand, these inhibitors may enhance the transcriptional activity of RARαby regulating phosphorylation or degradation, leading to a more effective induction of differentiation than is seen with ATRA alone. On the other hand, these inhibitors could exhibit apoptotic effects in majority tumor cells while ATRA induces the remaining tumor stem cells to differentiate [105]. As mentioned above, the clinical trial assessing the synergy of lithium and ATRA for relapsed or refractory AML patients may be 17

promising (NCT01820624). Therefore, these results indicate that future combinations of ATRA and anti-tumor agents that regulate RARα activity hold promise to enhance and improve anti-carcinogenic therapies.

Although studies on

ATRA-based solid tumor differentiation have mainly focused on

osteosarcoma [38], neuroblastoma [106, 107] and lung cancer [108, 109], some studies have also shown that ATRA modulates plasticity and inhibits the motility of breast cancer cells [110]. RARα overexpression sensitized retinoid-resistant MDA-MB453 cells to the antiproliferative effects of ATRA [31]. In breast cancer cells, a high level of estrogen receptor was observed in estrogen-receptor-positive (ER+) tumors, and its expression was related to responsiveness to ATRA via the control of RARα expression [111]. Therefore, the combination approaches developed in several cancers, such as osteosarcoma, may also be applicable for other solid tumors, including breast cancer, and especially for tumors with low RARα expression or weak RARαactivation.

The biological effects of ATRA are mediated by two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Studies have also found that these receptors also harbor many sites for post-translational modifications. In terms of ubiquitination, in addition to RARα, ATRA also induced the ubiquitin-dependent degradation of RARβ [112], RARγ [113, 114] and RXRα [115]. In terms of phosphorylation, RARγ can be phosphorylated by CDK7 [116] and MAPK [117]; RXRα was reported to be phosphorylated by MAPK [118], JNK [119], MER [120] and MKK4 [121]. In addition, 18

SUSP1 promoted the modification of RXRα by SUMO-1 [122]. Although the RARs and RXRs share some signaling pathways to regulate their post-translational modifications, the expression levels varied among the different isoforms in the same cells. Furthermore, RARs and RXRs were distributed inconsistently across different tissues. In humans, RARα is expressed in most tissues, while RARβ expression is prevalent in neural tissues, and RARγ is expressed predominantly in the skin [123]. Therefore, organ-selective drug delivery systems and determination of the appropriate tumor types would be beneficial for improving the specificity of these combination approaches and decreasing the potential adverse effects. Certainly, further studies are needed to understand these issues.

In addition, we summarize the relationship between only three post-translational modifications of RARα and ATRA-induced differentiation in this review. Two other modifications, methylation and acetylation, were also reported [124, 125]. However, the academic studies whether these modifications of RARα affect ATRA differentiation are still sparse. Therefore, it remains to be seen whether these two post-translational modifications could influence RARαfunction and whether other modifications of RARα would regulate its activity to enhance ATRA-based differentiation.

6. Conclusions To improve the sensitivity of ATRA in non-APL AML and solid tumors, many attempts have been made. In this review, we focused on the post-translational modifications of RARα and elucidated their important roles in ATRA-based differentiation therapy by affecting its 19

transcriptional activity and degradation levels. More importantly, we also summarized the potential combination strategies based on the regulation of RARα ubiquitination-dependent degradation and phosphorylation. The mixed evidence suggests that it is promising to develop drug combinations with ATRA for non-APL AML and even solid tumors. In the future, we should have a more comprehensive understanding of the whole picture of RARα function and its post-translational modifications during differentiation. A better understanding of RARα post-translational modifications shows promise in providing avenues for highly effective therapy against cancer.

Acknowledgments This work was supported by the grant from the National Natural Science Foundation of China (No.81973354 to M. Ying and No.81803552 to X. Shao).

Conflicts of interest The authors have no conflicts of interest to declare.

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36

Figure Captions Figure 1. Schematic representation of the RAR proteins with the functional domains. RARs have a conserved modular organization composed of 6 regions of homology (A-F, from the N-terminal to the C-terminal). The C region contains the DBD. The E region contains several domains, the LBD, the AF-2 domain, the dimerization domain and so on. AF-1, activation function 1; AF-2, activation function 2; NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand binding domain. Figure 2. The post-translational modifications of RARα protein. The major post-translational modifications of RARα and their regulators are depicted which influence its transcriptional activity and stability. Figure 3. The phosphorylation of RARα protein. Several Ser and Thr residues of RARα have been reported to be phosphorylated. Each residue site and the proteins responsible for the modification are indicated. The phosphorylation by these kinases could influence the transcriptional activity and the stability of RARα protein. Figure 4. Mechanisms involved in the proteasome-dependent degradation of RARα.

37

Table 1. Targeting PTMs of RARα enhances differentiation therapy in preclinical studies. Preclinical tumor

RARα PTM

Regulator

Therapy

Phosphorylation

PKA, MSK1

Piclamilast+ATRA

APL

(56)

P38αMAPK

PD169316/SB203580+ATRA

AML

(33)

JNK

SP600125+ATRA

Lung cancer cell line

(35)

GSK3

lithium+ATRA

AML

(36)

MDM2

HLI373, nutlin-3+ATRA

Osteosarcoma

(15)

PIN1

MG132/PiB+ATRA

AML

(46)

Proteasome

Bortezomib+ATRA

AML

(14)

Proteasome

MG132/LLnL/LC+ATRA

AML

(55)

JNK

Bortezomib+ATRA

Neuroblastoma

(16)

Ubiquitination

38

model

Reference

39

40

41

42

43