Retinoid Treatment for Oral Leukoplakia: Current Evidence and Future Development

. Oral leukoplakia (OLK) is one of the most common oral potentially malignant disorders (OPMDs). Preventing malignant transformation is the main purpose of OLK treatment. Te treatment approaches of OLK include surgery, chemotherapy, and other therapies, such as photodynamic therapy and CO 2 laser. Te application of genomic variation-based chemotherapy in OLK deserves further exploration. As chemopreventive drugs, drug resistance and disease recurrence have limited their use in OLK. In this review, we concentrate on the retinoid treatment for OLK, summarizing the current status of retinoids in the treatment of OLK, the mechanism of retinoid action, and themechanismsofresistancetoretinoidtherapy,andwehighlightthestrategiestoimproveretinoidefcacyinthetreatmentofOLK,such asthecombinationofretinoidsandepigeneticregulatorsormetabolism-blockingagents,newsyntheticretinoids,andnewdrugdelivery systemsofretinoids,providingnewmethodsforthesuccessfulclinicalapplicationofretinoidsinthetreatmentofOLK.


Introduction
Te cancerization of oral mucosa is a multistage and multistep process, and the risk of oral squamous cell carcinoma (OSCC) is relatively increased in patients with some oral lesions called oral potentially malignant disorders (OPMDs) [1].Oral leukoplakia (OLK) is one of the most common OPMDs, which is characterized by epithelial hyperkeratosis and dysplasia.Tis disease tends to occur in men over the age of 40, and the incidence is higher in smokers.Alcohol consumption and human papillomavirus (HPV) infection also contribute to the occurrence of OLK [2].Some patients with OLK have a trend of malignant transformation, and the overall malignant transformation rate of OLK is reported to vary between 1.1% and 40.8% [3].Risk factors for malignant transformation of OLK include female sex, long disease course, nonsmoker, location on the tongue or foor of the mouth, size over 200 mm 2 , nonhomogeneous leukoplakia, Candida albicans infection, and presence of oral epithelial dysplasia (OED) [4].
Te main purpose of OLK treatment is to prevent malignant transformation.It is recommended that OLK patients should remove irritants and receive regular followup examinations.Treatment approaches include surgery, chemotherapy, and other therapies, such as photodynamic therapy and CO 2 laser.It was previously thought that surgery was an efective means to prevent the malignant transformation of OLK.However, in recent years, the concept of feld cancerization has become widely accepted [5], which suggests that the underlying genetic alterations may be more extensive than the morphological or histological abnormalities.Terefore, even if the clinically obvious lesions can be removed, the original genetically altered mucosal tissue is still preserved; thus, local recurrence and malignant transformation cannot be prevented.Furthermore, owing to the existence of important anatomical structures in the oral cavity, surgery may not be the best treatment for OLK [6].Compared to surgery, photodynamic therapy (PDT) and CO 2 lasers are minimally invasive therapies [7], but they also fail to prevent oral carcinogenesis due to the feld cancerization efect.Surprisingly, those who are male, homogenous type, nonsmokers, and nondrinkers appeared to display a higher propensity for malignancy after laser surgery [8,9].Terefore, chemotherapy may be a more desirable approach.Vitamin A and its metabolite retinoids are the most important candidates for OLK chemotherapy.It has been shown that 13-cis-retinoic acid (13-cis-RA, isotretinoin) at high doses can prevent second primary tumors in patients treated for head and neck squamous cell carcinoma [10], and the chemoprevention of OLK deserves further study.
Vitamin A is a generic term for a group of lipophilic isoprenoids comprising a cyclic group and a linear chain with a hydrophilic polar group.Vitamin A itself cannot perform its physiological function until it is converted to its active forms which are called retinoids.Retinoids refer to a group of signaling molecules related to vitamin A, including its natural and synthetic metabolites or analogs.Te basic chemical structure of retinoid molecules consists of a cyclic end group, a polyene side chain, and a polar end group.Natural forms include retinyl esters, retinol, retinal, and retinoic acid.Vitamin A is a fatsoluble vitamin that is mainly stored in the liver and adipose tissue in the form of retinyl esters.In blood, it can bind to retinol-binding protein and transthyretin to form a protein complex that circulates in blood.Retinol can enter cells and be irreversibly oxidized to its active forms: all-trans retinoic acid (ATRA, tretinoin), 9-cis-retinoic acid (9-cis-RA), and 13-cis-RA.In addition, eforts have been made to synthesize new retinoids with less toxicity and better efciency.
Retinoids are essential regulators of many physiological processes in the human body.Tey play important roles in animal embryogenesis and organogenesis and simultaneously regulate tissue homeostasis, cell proliferation, cell diferentiation, and apoptosis.Retinoid signaling is critical for cells (especially epithelial cells) to maintain normal proliferation and diferentiation [11][12][13][14].Tus, they are widely used in treating skin diseases, central nervous system diseases, and metabolic diseases.Moreover, in the past few decades, researchers have conducted a large number of studies on retinoids in vitro and vivo and found that they can inhibit or reverse the occurrence and development of cancers, including hematological malignancies, as well as solid tumors of the oral cavity, breast, lung, prostate, bladder, liver, skin, and colon [15,16].In addition to the applications above, as chemopreventive agents, retinoids have also been used in some precancerous lesions, such as OLK, cervical dysplasia, and xeroderma pigmentosum [17].
It is worth mentioning that ATRA has an excellent efect on acute promyelocytic leukemia (APL).APL has a characteristic (15; 17) (q22; q12-21) chromosomal translocation, which leads to the promyelocytic leukemia gene (PML) fusing with the retinoid receptor gene (RARα).Te PML-RARα fusion oncoprotein acts as a transcriptional inhibitor to block normal myeloid diferentiation.Pharmacological doses of ATRA can act on RARα and induce remission of the disease.In particular, the combined application of ATRA and arsenic trioxide (ATO) allows the complete remission rate of APL to reach 90%, transforming APL from a highly fatal disease to a highly curable disease [18].
Despite the great remission results for APL, the efects of retinoids on solid cancers and precancerous lesions are still controversial, and the condition is the same in OLK.Previous clinical practice has proven that retinoids have a curative efect on OLK, but their clinical application is still limited for various reasons such as retinoid resistance.Terefore, in this review, we concentrate on the retinoid treatment for OLK, summarizing the current status of retinoids in the treatment of OLK, the mechanism of retinoid action, and the mechanisms of resistance to retinoid therapy, and we highlight the strategies to improve retinoid efcacy in the treatment of OLK, such as the combination of retinoids and epigenetic regulators or metabolism-blocking agents, new synthetic retinoids, and new drug delivery systems of retinoids, providing new methods for the successful clinical application of retinoids in the treatment of OLK.

Current Status of Retinoids in the Treatment of OLK
Retinoids are among the most well-studied chemopreventive agents.Tousands of retinoids have been artifcially synthesized, but the types used in clinics are still very limited.
Tere are two types of retinoids widely used in the clinical treatment of cancer: ATRA for acute promyelocytic leukemia and 13-cis-RA for neuroblastoma.In oral diseases, clinical trials have been carried out mainly including two kinds of natural retinoids (ATRA, 13-cis-RA) and a synthetic form: N-(4-hydroxyphenyl) retinamide (4-HPR, fenretinide) (Table 1).Some early studies found that vitamin A has a signifcant antikeratosis efect, and it has been proven to have a good efect on vulvar leukoplakia and senile keratosis.Since oral leukoplakia is also a disorder of keratinization, in 1958, Mulay and Urbach [30] conducted a clinical trial in which they applied vitamin A tablets topically to 10 patients with typical OLK.Te results showed signifcant remission in 70% of patients, thus indicating the therapeutic potential of vitamin A in OLK patients.
Te researchers then began to evaluate the efcacy of retinoids.When patients with OLK were given 13-cis-RA at 1-2 mg/kg/d for three months, signifcant reductions in lesion size were observed in 67% (16/24) of the drug group, and dysplasia was reversed in 54% (13/24) of patients.However, 2-3 months after treatment, 56% (9/16) of patients who reached remission had relapsed [26].In addition to systemic administration, some researchers have also evaluated the efcacy of the topical application of retinoids, which can achieve higher drug concentrations in the target tissues and reduce general toxicity.Shah et al. [21] treated 16 patients with OLK using 13-cis-RA in the form of oral lozenges at varied concentrations; 55% (6/11) of patients demonstrated complete remission and 27% (3/11) of them demonstrated partial remission.Relapse occurred in 2 patients with complete remission, and the patient who had no recurrence also showed histologic and cytologic regression.In several later experiments, ATRA at a 0.05% concentration was often topically used to treat OLK [19,20].However, Scardina et al. [23] reported that the topical use of 13-cis-RA at 0.18% concentration is more efective and has no side efects compared to  Journal of Clinical Pharmacy and Terapeutics the 0.05% concentration.Meanwhile, it was reported that the use of oral 13-cis-RA rinses is helpful in reducing the recurrence rate of OSCC and dysplasia after using other treatments [31].Synthetic retinoids such as 4-HPR have also been examined in OLK, and several reports have shown that it can induce cell diferentiation at low concentrations and apoptosis at higher concentrations in cancer cells in vitro.However, in the treatment of OLK, increasing the concentration of 4-HPR did not improve its efcacy [29], which may be related to the low concentration in the oral target tissue.Furthermore, 4-HPR induces apoptosis by generating reactive oxygen species [32], and oral precancerous lesions have stronger antioxidant capacity; thus, increasing the dose does not produce apoptosis.Some clinical trials on the efciency of retinoids in OLK patients are summarized in Table 1.We can see that the lack of controls and the small sample size afect the reliability of some data.In the future, high-quality clinical trials should be conducted.Te sample size should be expanded, and the experimental design should be optimized (randomized, controlled, blind, multicenter, etc.).In addition, reasonable diagnostic criteria, inclusion criteria, exclusion criteria, efectiveness evaluation indices, and safety evaluation indices are needed.
Retinoids regulate cell proliferation, diferentiation, and apoptosis, so researchers hope that they can treat OPMDs, especially abnormal keratinization diseases such as OLK.However, the efcacy of retinoids in OLK is not particularly satisfactory; some OLK patients show retinoid resistance and cases with complete remission also have a high recurrence rate.Long-term research and follow-up found that they have limited efects on preventing the malignant transformation of OLK.At the same time, they exhibit obvious side efects, sometimes even more than the pain caused by the disease itself, so patients have to discontinue treatment.For example, systemic administration of retinoids can cause hypertriglyceridemia, dry skin/mucous membranes, erythema, itching, cheilitis, headache, and hair loss [22,33].A burning sensation at the application site often occurs when the agent is used topically.Furthermore, the teratogenicity of retinoids should also not be ignored [34,35].Terefore, using the existing retinoids solely to treat OLK, especially when used in large doses, is not advisable.Tus, we should seek new strategies to combat their resistance and reduce side efects [24,25,27,28].

Retinoid Signaling and Mechanisms of Retinoid Action
Retinoids can be divided into endogenous and exogenous retinoids.Endogenous retinoids are derived from the diet and are then transformed into their active forms (retinoic acid).Retinoic acid and other exogenous retinoids can enter the nucleus and bind to some nuclear receptors or afect the extranuclear signaling pathway to play their roles (Figure 1).

Retinoid Signaling.
As mentioned earlier, retinoids are a class of compounds linked to vitamin A. Mammals cannot synthesize vitamin A de novo and must obtain it from their diet.(1) Preformed vitamin A (retinol, retinal, retinoic acid, and retinyl esters) is found in animal products (meat, eggs).
Dietary vitamin A such as retinyl esters and β-carotene can be packaged into chylomicrons and transported into the bloodstream, where they can be taken up by target cells via lipoprotein-specifc receptors.Te most important resource of retinoids is β-carotene, which can be transformed into retinal by the action of β, β-carotene-15,15′-monooxygenase (BCMO1) in the gut or in the target cells; it is then converted into retinol by retinal reductase (RALR).Retinol can be absorbed in the proximal small intestine with the help of bile acids.Trough the action of lecithin retinol acyltransferase (LRAT), it is esterifed to retinyl esters which are the most important storage forms of retinoids in the liver.When needed, it can be transformed back to retinol by retinyl ester hydrolase (REH) [37].Retinol, the main circulating form of vitamin A, travels in the bloodstream through binding to retinol-binding protein (RBP) and transthyretin (TTR).Trough a transmembrane protein named STRA6 (stimulated by retinoic acid-6), the membrane receptor for RBP4, target cells can take up retinol.
Upon entering a target cell, retinol binds to cellular retinol-binding proteins (CRBPs), which are delivered to enzymes that transform it into retinoic acid.First, alcohol dehydrogenase or retinol dehydrogenase (ADH/RDH) reversibly oxidizes retinol to retinal.Next, acetaldehyde dehydrogenase (ALDH) irreversibly converts retinal to retinoic acid (ATRA, 9-cis-RA, 13-cis-RA), and ATRA can be isomerized to 9-cis-RA or 13-cis-RA isomers through a nonenzymatic process; it is the most transcriptionally active isomer [38].In cells, the enzyme cytochrome P450 (CYP450) can degrade retinoic acid to mostly inactive compounds, and binding to intracellular lipid-binding proteins (ILBPs) can prevent retinoic acid degradation, which is conducive to their migration to the nucleus, where they bind to specifc nuclear receptors to activate the transcription of target genes.

Te Mechanisms of Retinoid Action.
Retinoids can exert genomic and nongenomic efects.Te genomic efects are regulated by coactivators and corepressors, which can change the structure of chromatin and then afect the accessibility of retinoid receptors to target DNA, thus activating or repressing the transcription of target genes.Te nongenomic efect depends on the extranuclear signaling pathway and is closely related to genomic efects.

Genomic Efects of Retinoids.
Te genomic efects of retinoids are mainly mediated by nuclear receptors.Retinoid receptors are involved in ligand-activated nuclear receptors (NRs), which are ligand-regulated transcription factors that bind DNA sequence-specifc motifs in enhancers and promoters of target genes to control their transcription.Tey share a common structural organization that contains a variable N-terminal domain, a DNA-binding domain (DBD), and a ligand-binding domain (LBD) [39].Tey function as monomers, homodimers, or heterodimers.Two types of retinoid receptors are known in humans: retinoic acid receptors (RARs) and retinoid X receptors (RXRs).Each nuclear retinoid receptor consists of three isoforms (α, β, c), which have distinct amino-and carboxy-terminal domains.Each RAR isoform has diferent subtypes, and their A regions are diferent.RARα and RARc have two subtypes (RARα 1, RARc 1, RARα 2, and RARc 2), while RARβ has fve subtypes (RARc 1-4 and 10).All isoforms of RXR have two subtypes (1, 2) [40].ATRA and 13-cis RA can bind to RAR, while 9-cis RA can bind to RARs and RXRs.
RAR can form a RAR/RXR heterodimer with RXR, which can bind to retinoic acid response elements (RAREs) consisting of tandem 5-AGGTCA-3 sites called DR1-DR5 [16].Te RAR/RXR heterodimers mainly function by recruiting transcriptional cofactors, including corepressors and coactivators.Tese cofactors act as adaptors to bring ligand-bound NRs to chromatin so that NRs can control the transcription of target genes [41].Te position of the H12 α-helix on the LBD determines the types of cofactors (corepressors or coactivators), and its position is determined by the ligands [42].
In the absence of ligands, RARs repress gene transcription by recruiting corepressors, including NR corepressor (N-CoR) and thyroid hormone receptor (SMRT), which promote the recruitment of complexes containing histone deacetylase (HDAC).HDACs are able to remove acetyl groups from nucleosome histones [43], which can lead to chromatin condensation, prevent binding of other factors, and cause transcriptional silencing of the target gene.
After ligand binding, RARs undergo structural changes, including the dissociation of corepressor complexes and the recruitment of coactivators such as NCOA/SRC/p16 and CREBBP/CBP [44,45].Tese coactivators can alter the chromatin structure around target gene promoters, thereby recruiting histone acetyltransferases (HATs) or histone demethylases (HDMs), histone methyltransferases (HMTs), and ATP-dependent SWItch/Sucrose Nonfermentable (SWI/SNF) chromatin remodeling complex, which is responsible for chromatin depolymerization and transcriptional activation.RXR can form a homodimer or bind to other receptors, such as the vitamin D receptor (VDR), the peroxisome proliferator-activated receptor (PPAR), and the thyroid hormone receptor (TR) [46].RXR does not directly bind coactivators, but its "subordination" relationship in heterodimers plays a critical role in avoiding confusion in signaling pathways; however, the molecular mechanism is still unclear [44].
Two ILBPs, cellular retinoic acid-binding protein-II (CRABP-II) and fatty acid-binding protein 5 (FABP5), in the body can bind to retinoids and deliver them to nuclear receptors, thereby regulating the transcription of target genes [47].Studies have found that CRABP-II transmits them to RAR, while FABP5 transmits them to PPARβ/δ.Te transcriptional activation of RAR results in the inhibition of cell growth, and the activation of PPARβ/δ promotes the expression of survival genes and stimulates cell proliferation.Terefore, the biological efects of retinoids may depend on the CRABP-II/FABP5 ratio [48].
Studies have identifed more than 500 possible target genes for retinoids.Retinoids directly regulate target genes through the binding of receptor heterodimers to RARE, and it has been proven that more than 20 genes are regulated through this pathway, while other target genes may be regulated indirectly [49].

Nongenomic Efects of Retinoids.
Retinoids can also activate kinase cascades, thereby exhibiting additional nontranscriptional efects that interact with genomic efects [50].Diferent cell lines may activate diferent kinase cascades [45].For example, studies have shown that retinoids can rapidly activate the p38 MAPK/MSK1 pathway, and the initiated phosphorylation cascade can promote the recruitment of the RARα/TFIIH complex to the target promoter to control the transcription of RARα [51].Subsequent studies found that RARα existing in membrane lipid rafts can form complexes with G protein α Q (Gα Q), and the rapid formation of RARα/ G α q complexes in lipid rafts induced by retinoids is the basis for the activation of p38 MAPK [52].Retinoids can also activate extracellular signal-regulated kinase 1/2 (ERK1/2) pathways and phosphatidylinositol-3-kinase (PI3K) and then infuence the function of RAR [50].In OSCC and oral dysplasia cell lines, ATRA can suppress STAT3 signaling by increasing JAK2 and decreasing ERK1/2 in a time-and dosedependent manner, consequently inhibiting proliferation and inducing apoptosis [53].Although the exact mechanism of the nongenomic efects of retinoids remains to be fully understood, we gradually discovered that the deregulation of certain signaling pathways in tumor cells may be associated with retinoid resistance, and it is undeniable that these efects also play important roles.

Mechanisms of OLK Resistance to Retinoid Therapy
Retinoids can promote diferentiation and apoptosis, as well as have antiproliferative and antioxidant activities.Retinoids seem promising in a subset of solid tumors and precancerous lesions, but most studies with sufcient follow-up have found merely transitional efects, even within tumor types such as gastric cancer [54] and breast cancer [55]; the condition is the same in OLK.Retinoid resistance can be divided into two types: intrinsic or acquired, and it is the result of numerous factors.Terefore, we reviewed the most common changes in retinoid function and metabolism that may be responsible for resistance to retinoid therapy (Figure 2).

Alterations of Retinoid-Related Receptors.
Retinoids mainly rely on binding to nuclear receptors to exert their efects.In some diseases, these receptors are altered.For example, the receptor gene may be mutated, resulting in variations in gene expression, and posttranslational modifcations (e.g., phosphorylation) can afect receptor action, all of which are able to cause receptor dysfunction and drug resistance.
Alterations of RAR typically occur in hematological malignancies.In APL, mutations in the RARα region of the PML-RARα gene are present in cases of acquired resistance and may be mechanistically involved in retinoid resistance [56].However, these patients remain sensitive to ATO therapy [57].Meanwhile, phosphorylation can also regulate RAR function.Deregulation of cytoplasmic signaling cascades can lead to aberrant phosphorylation of RAR, resulting in resistance to retinoids.As mentioned earlier, the activation of p38MAPK by retinoids is essential to the transcription of RARα [51].In ERBB2-positive human breast cancer, the p38MAPK/MSK1 pathway is deregulated, so they show no response to retinoids [58].In non-small-cell lung cancer (NSCLC) cell lines, Akt is normally activated, which can phosphorylate RARα and inhibits its transcriptional activation [59].
RARβ expression was found to be downregulated in many kinds of solid tumors and precancerous lesions.For example, researchers have used in situ hybridization to compare the expression of retinoid receptors in head and neck squamous cell carcinomas, dysplastic lesions, adjacent normal tissues, and normal volunteer tissues [60].Tey found that only approximately 70% of adjacent normal and hyperplastic lesion specimens expressed RARβ mRNA, and its expression was further reduced to 56% in dysplastic lesions and to 35% in head and neck squamous cell carcinoma (HNSCC).Tis change has also been observed in esophageal cancer [61], NSCLC [62], and breast cancer [63,64].Te expression of RARβ is lost in oral precancerous lesions and can be restored by isotretinoin therapy [65].Meanwhile, the introduction of the RARβ gene into retinoic acid-insensitive breast cancer cell lines can restore retinoic acid sensitivity [66].Toulouse et al. transfected RARα1, RARβ1, and RARβ2 into the lung cancer cell line Calu-1 and only the overexpression of RARβ2 sensitized these cells to retinoids.Te stable overexpression of RARα1 and β1 did not show the same efect [67].Terefore, it is reasonable to speculate that RARβ2 is a key receptor mediating the growth-inhibitory efect of retinoids.

6
Journal of Clinical Pharmacy and Terapeutics Later work showed that the suppressed expression of RARβ2 may be related to gene silencing due to epigenetic changes.Te concept of epigenetics was frst proposed by Conrad Waddington in 1942 [68] and is characterized by stable and heritable changes in gene expression not afecting the DNA sequence [69], including DNA methylation, histone covalent modifcation, chromatin remodeling, and the roles of noncoding RNAs and polyclonal proteins in gene expression.It has been found in many studies that epigenetic changes are the basis for the occurrence of many cancers, which may serve as a possible direction for the diagnosis and treatment of cancer [70].Youssef et al. demonstrated that 53% (66/124) of OLK specimens showed methylation of CpG islands in the RARβ2 promoter [71].Tis phenomenon was also found in breast cancer [72], cervical cancer [73], HNSCC [74], and colon cancer [75].Petty et al. determined the methylation level of the RARβ P2 promoter in retinoic acidsensitive (BEAS-2B) and retinoic acid-resistant (BEAS-2B-R1) HBE cells to investigate whether hypermethylation inhibits RARβ2 expression in HBE cells [76].Tey observed hypermethylation in the 3′ region of the RARβ P2 promoter in BEAS-2B-R1 cells, while this phenomenon was not observed in BEAS-2B cells.Moreover, the treatment of BEAS-2B-R1 cells with a DNA demethylating agent-azacitidine (5azacitidine, 5-AzaC) alone or in combination with retinoids can restore RARβ2 expression.In APL, through the recruitment of DNA methyltransferases to target promoters, the PML-RAR fusion protein induces gene hypermethylation, resulting in gene silencing [77].In t (8; 21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemia by recruiting a class I repressor complex containing histone deacetylase (HDAC) to AML1 target gene promoters [78].In addition, there has been some evidence that loss of RARβ expression was linked to abnormal histone H3 acetylation in thyroid cancer [79], lung cancer [80], and cervical cancer [81].
In addition, RARc and RXR also play a signifcant role in mediating drug resistance to retinoids.Loss of RARc predisposes mice to cutaneous squamous cell carcinoma, and knockout of RARc abrogates retinoid-induced cycle arrest and keratinocyte apoptosis [82].Te expression of RARc mRNA was signifcantly downregulated in OSCC and OLK cell lines [83].Further studies have shown that RARc can mediate the growth-inhibitory efect of retinoids and infuence retinoid turnover in HNSCC [84], while RARβ mediates the inhibitory efect of ATRA on squamous cell diferentiation in HNSCC without enhancing its growthinhibitory efect [85,86].Compared with ovarian cancer cell lines with reduced levels of single RARα or RXRα, ovarian cancer cell lines acquired the greatest retinoid resistance by reducing the levels of both RARα and RXRα [87].Wu et al. found that RXRα contributes to mediating the growth inhibition of retinoids on ovarian cancer cells [88].Te roles of RARc and RXR in OLK remain to be further investigated.4) increased degradation of retinoids, (5) low CRABP-II/FABP5 ratio, (6) epigenetic changes (e.g., DNA hypermethylation and repressive histone modifcations), ( 7) mutations in retinoid-related receptors, (8) changes in posttranslational modifcation (e.g., abnormal phosphorylation), and (9) cofactors alterations.

Accumulation of Stemness Features of OLK Epithelial
Cells.Epithelial cells typically exhibit plasticity when exposed to noxious stimuli, thereby promoting tissue regeneration.Plasticity includes the interconversion of diferent stem cell pools, the activation of facultative stem cells, and the dediferentiation, transdiferentiation, or phenotypic transformation of diferentiated cells.When it is abnormally activated, it can promote the malignant transformation and confer tumorigenic properties to epithelial cells [89].Tese cells with the potential for renewal and multidirectional diferentiation are called tumor stem cells (CSCs) [90,91].In OPMDs and HNSCC, the accumulation of cancer-deriving molecules in epithelial or tumor cells can sustain cancer stemness characteristics.For example, SOX-2 is one of the key regulators of transcription and induction of pluripotency of stem cells.Luiz et al. have found a higher expression of SOX-2 in the OLK group compared to the control group [92].Other markers of CSCs, such as NANOG, have also been highlighted in OLK [93].CSCs can induce resistance to various chemotherapeutic drugs through metabolic reprogramming, upregulated drug efux proteins, and protective autophagy [94].In APL, retinoids combined with ATO can promote the diferentiation of tumor cells and thereby inhibit cancer progression [18].Terefore, targeting these cells is a promising strategy to overcome the retinoid resistance of OLK.

Changes in Retinoid Metabolism
Pathway.Several diseases can lead to changes related to retinoid metabolism, which can accelerate its degradation, prevent it from entering the nucleus to bind to retinoid-related receptors, or even cause it to be excreted from the cells.Tese changes reduce the efective concentration of retinoids, resulting in drug resistance.

Intracellular Transport of Retinoids.
As mentioned earlier, in cells, retinoids can bind to proteins such as CRABP-II and FABP5 which can mediate their efects.Studies have found that the expression of some lipid-binding proteins is associated with retinoid resistance; for example, CRABP-II overexpression markedly increased MCF-7 breast cancer cell sensitivity to retinoid-induced growth inhibition.Te reduction in CRABP-II expression made these cells resistant to retinoids [95].In medulloblastoma cells, it was demonstrated that the downregulation of CRABP-II was due to abnormal methylation.Terefore, the authors of this study proposed detecting the expression or methylation of CRABP-II so that targeted combination therapy can be used for patients with medulloblastoma and other types of cancers [96].However, although the demethylation agent 5-Aza-2′-deoxycytidine (5-Aza-CdR) can enhance CRABP-II expression, the recovery of CRABP-II expression or the increase in the CRABP-II/FABP5 ratio does not seem to overcome the ATRA resistance of COLO 16 cells [97].Likewise, similar conclusions were drawn in glioblastoma [98].Terefore, the resistance of cutaneous squamous cell carcinoma and glioblastoma to retinoids may need to be explained by other molecular mechanisms.Conversely, studies have been found to reduce the FABP5/CRABP-II ratio in breast tissue, the transfer of retinoids from PPARβ/δ to RAR can inhibit tumor growth [99].Likewise, in APL, there is no causal relationship between CRABP-II expression (or retinoic acid-binding activity) and acquired retinoid resistance, whereas FABP5/CRABP-II may play a role in APL cell responses to retinoids [100].Te relationship between the CRABP-II/FABP5 ratio and retinoid resistance in OLK needs to be further studied.

Efux of Retinoids.
Te efux of retinoids from cells also reduces their intracellular concentration.Proteins that belong to the ATP-binding cassette (ABC) transmembrane transporter superfamily are highly conserved among all species.Using ATP as an energy source, they function as transporters to export signaling molecules and drugs.In many cases, the overexpression of ABC transporter genes contributes to multidrug resistance (MDR).For example, studies have found that in neuroblastoma (NBL) cell lines, cells overexpressing multidrug resistance protein 1 (MRP1) are more resistant to ATRA treatment [101].High expression of MRP1 has also been observed in OSCC, which promotes drug resistance to several chemotherapy drugs [102], including retinoids.A study found that a higher TM rate is observed in OLK patients who demonstrate positive expression of the ATP-binding cassette, G2 subfamily (ABCG2) [103].Considering that retinoid signaling plays an important role in maintaining normal proliferation and diferentiation of epithelial cells, we suspect that high ABCG2 may lead to changes in retinoid signaling in some patients with OLK, but this hypothesis remains to be investigated.

Increased Degradation of Retinoids.
In the early stage of using ATRA to treat APL, some patients will relapse early after ATRA treatment.Researchers have found that the body will automatically induce accelerated ATRA clearance after repeated administration, so the efective plasma concentration of ATRA cannot be maintained during continuous treatment, thus explaining part of the ATRA resistance [104].Later studies found that this autoinduction is usually mediated by CYP26A1 in the liver.Te researchers also developed a PBPK model after ATRA administration, which helped in the design of new dosing regimens and the development of a new generation of retinoid metabolismblocking agents (RAMBAs) [105].
CYP26 enzymes have been identifed as the major retinoid clearance enzymes and can also be involved in tumor progression.For instance, the upregulation of CYP26A1 expression has been observed in HNSCC [106] and breast cancer [107], which promotes rapid drug clearance after sustained oral administration and is associated with retinoid resistance.It was reported that two types of HNSCC cell lines show increased expression of CYP26 A1 mRNA, indicating the quick metabolism of retinoids, which is related to carcinogenesis [106].Te same mechanisms may occur in OLK.Several recent studies have shown that CRABP-I and CRABP-II can also afect the metabolism of ATRA by 8 Journal of Clinical Pharmacy and Terapeutics interacting with CYP26 enzymes [108,109].In myeloid and promyelocytic leukemia cells, RARα has been reported to regulate CYP26 expression, which may be related to some feedback mechanisms, and a RARα-specifc antagonist (CD2503) can completely block CYP26 mRNA expression in NB4 cells and HL-60 cells [110].

Strategies to Improve Retinoid Efficacy
Retinoids are critical molecules in biological diferentiation therapy, but patients with OLK respond diferently to retinoid therapy, which has seriously limited their wide application.Terefore, breaking through resistance and enabling their curative efects have become urgent problems to be solved.
Although there is currently no comprehensive solution, drug combinations, new synthetic retinoids, and new methods of drug administration may be useful (Figure 3).

Combination of Retinoids and Epigenetic Regulators.
As mentioned above, the downregulation of RARβ expression in solid tumors is mainly associated with some epigenetic changes that lead to gene silencing.Terefore, it is possible to consider combining retinoids with some epigenetic regulators to improve their efcacy, such as DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi).McGregor et al. found that 5-Aza-CdR can reverse the immortality phenotype of dysplasia [111].Combined use of 5-Aza-CdR and ATRA induced RARβ re-expression in immortalized OPMDs [112].Similarly, in HNSCC cells, by combining the histone deacetylase inhibitors trichostatin A (TSA) and ATRA, it is possible to enhance the growth-inhibiting efects and greatly increase transcriptional activation of the RARβ promoter, breaking resistance to retinoids [113].
Moreover, these epigenetic regulators can not only remove some unfavorable epigenetic modifcations but also help to eradicate CSCs.Studies have shown that 5-Aza-CdR can efectively inhibit prostate cancer tumorigenesis by targeting CSCs [114].Inhibition of HDAC can destroy CSCs in HNSCC [115].Terefore, the combination of these two drugs can counteract retinoid resistance in two ways.

Combination of Retinoids and Retinoic acid metabolismblocking agents (RAMBAs).
RAMBAs, including ketoconazole, liarozole, and talarozole, can increase endogenous retinoids or counteract auto-induced resistance to retinoid therapy.Liarozole may upregulate RARβ in HNSCC [116], but its efect in combination with retinoids for oral diseases remains to be evaluated.R116010, another highly specifc CYP26 inhibitor, has shown clinical benefts in breast cancer [117] and NBL [118]; however, its role in OLK is currently unknown.A detailed introduction to RAMBAs can be found in the review article by Nelson et al. [119].Although RAMBAs are not currently approved for the clinical treatment of precancerous lesions, numerous preclinical studies have observed their potential to break through retinoid resistance.Te synergies of retinoids and RAMBAs in OLK await further study.

New Synthetic Retinoids.
In addition to improving the efcacy of natural retinoids, researchers are also working to synthesize new retinoids.Because the expression of RARβ is mostly downregulated in HNSCC and part of OLK, retinoids with retinoid receptor-independent activities or target receptors other than RARβ have raised hopes.For example, researchers have evaluated the efects of bexarotene (a synthetic RXR agonist) and CD1530 (a synthetic specifc RARc agonist), and the combination of these two drugs can prevent oral cancer induced by the carcinogen 4-NQO and does not cause serious changes in blood lipids [120].Tese two novel retinoids can target RXR and RARc, so even if the expression of RARβ is lost, they can also exert a tumor suppressor efect.Moreover, LGD1550 can decrease cell proliferation in HNSCC in part by interfering with TGFα/EGFR autocrine signaling [121].
4-HPR, a synthetic retinoid, has been found to exhibit dual efects.At low concentrations, it induces diferentiation of cells that is dependent on RAR, while at high concentrations, it increases apoptosis independent of RAR [28,32].Moreover, its clearance is relatively slow in the human body.Some clinical trials have also been carried out in OLK, but their efcacy has yet to be improved.

New Drug Delivery Systems of Retinoids.
When administered systematically, retinoids have high afnities for multiple sites during the absorption process, so researchers are committed to designing novel drug delivery systems to increase the concentration of retinoids at the target site.Nanomedicine has been developed as an immunotherapy for oral diseases.Compared with free ATRA, PLGA-PEG nanoparticles loaded with ATRA and modifed with an anti-PD-L1 antibody can enhance the stability and chemotherapeutic efcacy in OSCC and oral dysplasia and reduce systemic side efects [122].Topical administration via the oral mucosa has many special advantages, allowing drugs to avoid problems with gastrointestinal tract degradation and hepatic frst-pass metabolism and to be absorbed faster.Owing to the continuous dilution by saliva, extending the residence time of medication in the oral cavity becomes the greatest challenge.Over the years, researchers have strived to use polymers in drug delivery systems to improve drug adhesion on the oral mucosa [123].Several traditional and novel drug delivery systems (hydrogels, polymeric nanoparticles, liposomes, electrospun nanofbers, etc.) have shown potential in the treatment of oral diseases [124].Te isoG-TA and G-TA hydrogels even have the potential to delay the malignant transformation of OLK [125].Terefore, for retinoid-based therapy of OLK, many attempts could be made to develop advanced systems to optimize retinoid bioavailability.

Other Strategies.
Given that the combination of ATRA and ATO for the treatment of APL produces a synergistic efect by enhancing the apoptotic process [16], similarly, we speculate that the poor efcacy of retinoids in solid tumors may also be related to the overexpression of several apoptosis-inhibiting factors, so combining retinoids with drugs that induce apoptosis, such as sulindac, may improve their efects [126].
Preferentially expressed antigen of melanoma (PRAME) is a competitive inhibitor of RA at RAR that can signifcantly repress RAR signaling [127].Compared to normal oral cells or tissues, prominent PRAME expression was observed in HNSCC and some OPMDs [128].Tus, combining PRAME vaccine/antibody or adoptive T-cell therapy with retinoids shows promise as an efective measure for retinoid-resistant OSCC and OPMDs [129].

Summary and Prospective
Here, we review the biochemical and molecular mechanisms by which the retinoid signaling pathway operates and describe the efcacy of retinoids in OLK.We highlight the main causes of retinoid resistance, and possible strategies for overcoming resistance are discussed to provide a theoretical basis for future research.
Resistance to retinoid therapy is still a major challenge in the treatment of solid tumors and precancerous lesions.Over the past few years, a great deal of research has been conducted, but the results are not satisfactory.Te causes of retinoid resistance remain unclear, which requires further study of the mechanisms to explain how the retinoid signaling pathway infuences cell growth or diferentiation and how the retinoid signaling pathway changes during the tumorigenesis process.Cellular heterogeneity is consistently the main reason for drug resistance.Tus, in the future, we can apply emerging technologies, such as single-cell sequencing or CyTOF, to comprehensively explore the heterogeneity among precancerous lesions, identify previously unknown cell types, and discover potentially drug-resistant cells, which will facilitate the development of more efective, targeted, and personalized retinoid treatments for OLK patients.Moreover, if we determine how retinoids are regulated, we can promote the production of endogenous retinoids instead of exogenous retinoids to enhance their specifcity and eliminate any unwanted side efects.In the treatment of OPMDs, the traditional administration methods of retinoids include systemic and topical administration.Te side efects of systemic administration are significant.When administered topically, an efective concentration cannot be guaranteed for a long time on account of the continuous dilution by saliva.Terefore, it is important to improve the drug delivery systems of retinoids to optimize bioavailability and minimize systemic toxicities.Moreover, changing the medication strategy may be benefcial to improve the long-term efcacy of retinoids.Intermittent administration or low-dose maintenance after the lesions disappear may be useful, but we must fully test the impact of the long-term application of retinoids on the body and the oral mucosa.In addition, the appropriate maintenance dose and suitable dosing interval should be determined.

Figure 1 :
Figure1: Te metabolism and mechanism of retinoids.Retinol travels in the bloodstream through binding to retinol-binding protein (RBP) and transthyretin (TTR).Trough a transmembrane protein named STRA6, target cells can take up retinol into target tissues.Upon entering a target cell, retinol binds to cellular retinol-binding proteins (CRABPs), which are delivered to enzymes that transform it into retinoic acid.First, alcohol dehydrogenase or retinol dehydrogenase (ADH/RDH) reversibly oxidizes retinol to retinal tissue.Next, acetaldehyde dehydrogenase (ALDH) irreversibly converts retinal to retinoic acid (ATRA, 9-cis-RA, 13-cis-RA).Retinoic acid can then enter the nucleus to exert genomic efects.In the absence of ligands, RARs repress gene transcription by recruiting corepressors, and HDACs can remove acetyl groups from nucleosome histones, which can lead to chromatin condensation, prevent binding of other factors, and cause transcriptional silencing of the target gene.After ligand binding, RARs undergo structural changes, including the dissociation of corepressor complexes and the recruitment of coactivators.Histone acetyltransferases (HATs) can bring about epigenetic changes and chromatin depolymerization so that target genes can be normally transcribed.Tey can also activate kinase cascades to exhibit additional nontranscriptional efects or be degraded under the action of CYP26 enzymes.

Figure 3 :
Figure 3: Possible strategies to overcome retinoid resistance in OLK treatment.Te following measures may help to overcome retinoid resistance.(a) Drug combination (e.g., epigenetic regulators, RAMBAs), (b) new synthetic retinoids, and (c) new drug delivery systems.

Table 1 :
Current status of retinoids in the treatment of OLK.