(CANCER RESEARCH (SUPPL.):54. lW7s-l'NOs. April I. 1W4]

Suppression of Squamous Cell Carcinoma Growth and Differentiation by Retinoids1

Reuben Lotan2

Departments of Iunior lïit)log\, Tltc Univer.silv of Texas M. I). Anderson Caneer C'enter. Houston, Texas 77030

Abstract

The epithelium of the oral cavity is mostly nonkeratinizing. However, itundergoes an abnormal squamous differentiation with keratinization during vitamin A deficiency or oral carcinogenesis. Vitamin A analogues

(retinoids) were found to be effective in preventing oral premalignantlesions and second primary cancers in the upper aerodigestive tract.Further development of retinoids for prevention and therapy of squamouscell carcinoma (SCO requires a better understanding of their mechanismaction on the growth and differentiation of SCC cells. We used cultured

head and neck SCC (HNSCC) cell lines as a model system. Treatment ofHNSCC cells with ß-all-franx-retinoic acid resulted in inhibition of growth

(proliferation and colony formation) and suppression of squamous differentiation to varying degrees in the different cell lines. Because some of themalignant HNSCC cells recapitulate the main characteristics of keratino-

cyte squamous differentiation and responsiveness to retinoids, they canserve as a model for investigating the mechanism underlying the effects ofretinoids on cell growth and differentiation. It is thought that nuclearretinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediatethe above effects of retinoids by acting as DNA-binding transcription-

modulating factors. We found that HNSCC cell lines express severalnuclear RAR and that their level could be modulated by retinoids in somecell lines. An inverse relationship was found between KAU-/! expression

and squamous differentiation. An analysis of RAR mRNA expression inhead and neck cancer specimens revealed a decrease in RAR-ßin pre

malignant and malignant tissues relative to normal mucosa. The expression of this receptor increased /;/ vivo after treatment with 13-c/s-retinoicacid. These results implicate the loss of RAR-ßexpression in the devel

opment of head and neck cancer and suggest that RAR-ßcould serve as

an intermediate marker in prevention trials.

Introduction

Retinoids, a group of naturally occurring and synthetic analogues ofvitamin A, suppress carcinogenesis in various epithelial tissues (e.g.,oral cavity, skin, bladder, lung, prostate, and mammary gland) inanimal model systems (1^). More importantly, retinoids also exhibited some effectiveness in clinical trials of chemoprevention of cervical dysplasia, bronchial metaplasia, actinic keratosis, oral leukopla-

kia, second primary tumors in the aerodigestive tract, and skin cancerin xeroderma pigmentosum patients (5-10).

We are interested in elucidating the mechanism by which retinoidssuppress carcinogenesis in the oral cavity and the upper aerodigestivetract. Since premalignant lesions of the oral cavity (e.g., leukoplakias)and head and neck cancers often exhibit an aberrant squamous differentiation and retinoids are known to suppress keratinization andsquamous differentiation, we consider it important to understand howretinoids modulate this differentiation pathway. This review is focused on effects of retinoids on normal, premalignant, and malignanthead and neck epithelial cells in vitro and in vivo.

1Presented at the 4th International Conference on Anlicarcinogenesis & Radiation

Protection, April 1K-23, 1993. Baltimore. MD. Supported by USPHS grant PO I-52051from the National Cancer Institute.

" To whom requests for reprints should he addressed, at Department of Tumor

Biology-Box 108, The University of Texas, M. D. Anderson Cancer Center, 1515Holcomhe Boulevard, Houston, TX 77030. Reuben Lotan holds the Abell-Hanger Foundation Professorship in Cancer Research.

Carcinogenesis in the Oral Cavity Is Associated with AberrantSquamous Differentiation

A major part of the oral cavity epithelium, in particular the mucosalining the soft palate, the lateral and ventral tongue, the floor of themouth, the alveolar area, the lips, and the cheeks, is a nonkeratinizingepithelium (11). However, it can undergo keratinization under pathological conditions that include injury, infection, and vitamin A deficiency and during carcinogenesis (e.g., exposure to tumor promoters,in premalignant lesions, and in SCCs3) (12-14). Many premalignant

and malignant oral lesions express higher levels of squamous differentiation markers, including transglutaminase type 1 and involucrin,than their normal counterparts in vivo (12, 13, 15, 16) and in vitro (17,18). Interestingly, the mere exposure of nonkeratinizing buccal epithelial cells in vitro to the tumor promotor, 12-O-tetradecanoylphor-bol-13-acetate, resulted in an increased involucrin level and in theformation of cross-linked envelopes characteristic of keratinizing

squamous cells (19). The expression of some squamous differentiationmarkers such as transglutaminase type I (16) and Kl keratin (13) mayincrease in early premalignant lesions and decrease in severe dysplas-

tic lesions and poorly differentiated carcinomas.

Expression of Squamous Differentiation Markers in HNSCCCell Lines

Several studies demonstrated that HNSCCs express varying degrees of squamous differentiation evidenced by detection of the markers involucrin (17, 18), Kl keratin (18), transglutaminase type I,cholesterol sulfate, and cholesterol sulfotransferase (20) and by theability to form cross-linked envelopes (18, 21). Recently, we have

studied the expression of 3 squamous differentiation markers (Klkeratin, transglutaminase type I, and involucrin) in 4 HNSCC celllines, 183, 886, 1483, and SqCC/Y 1, derived from different regions ofthe oral cavity (tonsil, larynx, retromolar trigone, and buccal mucosa,respectively). Two of the cell lines (1483 and SqCC/Y 1) expressed allthree markers. This expression appears to be aberrant since the normalcells from which these tumors derived do not express these markers.The 183 cells expressed only involucrin, and the 886 cells did notexpress any of the 3 markers. The expression of squamous differentiation markers in the 1483 and SqCC/Yl HNSCC cells appeared tobe dependent on the state of cell proliferation and cell-cell andcell-substrate interactions. Very low levels of squamous markers weredetected in proliferating cells in low-density cultures. However, as

cell density increased, and especially, when cells began to formmultilayers, the expression of squamous differentiation markers increased (18). This behavior may be related to the migration of cellsfrom basal to suprabasal positions in stratified epithelium, whicheliminates cell-substratum contact and results in cells in the upper

layers interacting with cells in the lower layers. Some of the cells inthe upper layers of SqCC/Yl and 1483 undergo terminal differentiation, form cross-linked envelopes, and slough off (17, 18).

1The abbreviations used are: SCC, squamous cell carcinoma: HNSCC', head and neck

SCC: ATRA, all-mm.v-rctinuic acid: RAR, retinole acid receptor.1987s

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Suppression by Retinoids of Cell Growth and SquamousDifferentiation in HNSCC Cell Lines

Retinoids are usually recognized for their ability to induce differentiation of various tumor cells including embryonal carcinoma, leukemia, neuroblastoma, and melanoma (22). Since these tumor cellsappear to be arrested at an early stage of differentiation, the ability ofretinoids to induce them to undergo further differentiation is therationale for using retinoids in differentiation therapy exemplified bythe treatment of acute promyelocytic leukemia patients with ATRA(23, 24). In apparent contrast to the above, retinoids inhibit squamouscell differentiation in cultured normal keratinocytes (25-28) and in

some malignant SCCs (25, 27, 29, 30), including HNSCCs (17, 18,20, 30). Treatment of several HNSCCs with ATRA suppressed trans-

glutaminase type I, Kl keratin, and involucrin at the protein level (18,20) as well at the mRNA level (31).

It is important to note that one of the physiological functions ofvitamin A and retinoids is to maintain appropriate epithelial differentiation (32). Many epithelial tissues undergo aberrant squamous metaplasia during vitamin A deficiency (Ref. 30 and references therein)indicating that vitamin A is required for prevention of aberrant kera-

tinization of nonkeratinizing epithelia like the oral cavity mucosa.Thus, the ability of retinoids to suppress aberrant squamous differentiation in premalignant and malignant head and neck tissues should beviewed as restoration of the normal pathway of differentiation. Different concentrations of retinoids may exert distinct effects on celldifferentiation. For example, at physiological concentrations ofATRA, laryngeal epithelial cells and papilloma cells cultured at anair-liquid interface formed stratified squamous epithelium, whereas at

pharmacological concentrations ATRA induced differentiation intocolumnar, ciliated epithelium (33).

The onset of the normal squamous cell differentiation programrequires cessation of cell proliferation (27, 28, 34). Therefore, it is ofinterest to determine what effects retinoids exert on cell proliferationwhen investigating their effects on squamous differentiation. Retinoids were found to exert opposite effects on the growth of normalbuccal mucosa cells and on HNSCCs in vitro. Retinoids enhanced thegrowth of human buccal mucosa epithelial cells from expiants cultured in a serum-free medium (19). The growth of epidermal kerati

nocytes was likewise stimulated (30). It is possible that the growthstimulatory effect of retinoids on cultured keratinocytes is related tothe suppression of squamous differentiation in that retinoids preventgrowth arrest, a prerequisite for squamous differentiation. In contrastto their effects on normal buccal mucosa cells, retinoids inhibited thegrowth of many HNSCCs in monolayer culture (18, 20, 35-37). Since

retinoids also inhibited squamous cell differentiation, it appears that inthe malignant cells the regulation of cell proliferation and differentiation are not as tightly linked as they are in normal keratinocytes.

Based on the above results, we suggest that a relationship existsbetween the ability of retinoids to modulate the growth and differentiation of normal head and neck epithelial cells, premalignant laryngeal cells, and HNSCCs in vitro and their activity in vivo in preventionof oral premalignant lesions and second primary cancer in head andneck cancer patients. However, more direct evidence for a causalrelationship between the in vitro and in vivo effects of retinoids isrequired to place this hypothesis on a firmer footing.

Mechanism of Retinoid Action

The mechanism by which retinoids modulate the differentiation andgrowth of malignant cells or suppress the conversion of premalignantlesions to malignant ones is not fully understood. It is thought that theability of retinoids to modulate gene expression enables them to

redirect aberrant differentiation, reregulate uncontrolled proliferation,and suppress the transformed phenotype.

Cellular Retinole Acid-binding Proteins and Nuclear RetinoleAcid Receptors. Cellular retinole acid-binding proteins have been

implicated in the action of retinoids (38). However, their precise rolein mediating the effects of retinoids on gene expression has not beenelucidated. Recently, it was shown that cellular retinoic acid-binding

proteins may sequester ATRA in the cytoplasm and enhance itscatabolism, with the consequence of decreasing cell response toretinoids (39, 40). The discovery that some members of the largefamily of steroid and thyroid hormone receptors are nuclear retinoicacid-binding proteins was a major breakthrough in the understanding

of the mechanism by which retinoids modulate gene expression (32,41^4). The retinoic acid receptors are ligand-activated, DNA-bind-ing /ram-acting, transcription-modulating proteins. Three RAR subtypes (RAR-a, RAR-ß,and RAR-y) have been cloned and localized

on chromosomes 17q21, 3p24, and 12ql3, respectively (45). Eachsubtype exhibited different distributions in adult tissues as well asspecific patterns of expression in developing mouse embryo (46, 47).Several isoforms resulting primarily from alternative splicing havebeen identified for each of these receptors, and the tissue distributionof these ¡soformsis also distinct (44). Consequently, it was proposedthat different receptors activate distinct genes. Another group ofnuclear retinoic acid receptors (designated RXR-a, RXR-ß, andRXR-y) has been cloned and characterized (48, 49). These receptorsdo not bind ATRA; rather, they bind 9-c/s-retinoic acid, a natural

metabolite of ATRA (50, 51), and several synthetic retinoids (52). TheRARs and RXRs can form heterodimers, and it is thought that theheterodimerization is essential for binding to retinoic acid responseelements (43, 44, 53-56), which are specific nucleotide sequences

present usually in the promoter region of genes that are regulateddirectly by retinoids (57-59).

Nuclear Retinoic Acid Receptors in Normal Oral Mucosa andin Premalignant and Malignant Head and Neck Tissues. Nuclearretinoic acid receptors appear to be the direct mediators of actions ofretinoids on gene expression. Therefore, the determination of theirexpression pattern in normal, premalignant, and malignant tissuesmay provide important clues to their role in physiological processes,in carcinogenesis, and for the rational selection of receptor-specific

retinoids for prevention or treatment of cancer.The expression of nuclear retinoic acid receptors in cell lines

derived from normal oral mucosa, oral leukoplakia, and HNSCCs hasbeen demonstrated (60, 61). Cell lines derived from oral leukoplakiasin different regions of the oral cavity expressed RAR-a and RAR-y

constitutively. In contrast, only those cells derived from leukoplakiaof the soft palate expressed RAR-ß(60, 61). Treatment of these cellswith ATRA increased the RAR-ßlevel, but the same treatment was

ineffective in inducing this receptor in any of the cells that did notexpress it constitutively (60, 61). These results indicated that RAR-ßmRNA was expressed in non-keratinizing oral epithelial cells but not

in the keratinizing ones. All squamous cell carcinoma cell linesderived from cancers of the oral cavity expressed RAR-a and RAR-y.whereas RAR-ßwas expressed in only 2 of 7 HNSCC cell lines, and

a loss of expression relative to normal counterparts was evident in 2soft palate and one floor of the mouth tumor (61). Since no grossrearrangements were detected in the RAR-ßgene by Southern blot

analysis of DNA samples from 5 HNSCC cell lines that did notexpress RAR-ßmRNA (61), it appears that the lack of RAR-a

expression may be regulated by suppression of transcription. Theseresults raised the suggestion that abnormally low levels of RAR-ß

may contribute to neoplastic progression in stratified squamous epithelia (61). We found that all 4 HNSCC cell lines described aboveexpressed mRNAs for RAR-a, RAR-y, and RXR-a, 3 cell lines (183,

1988s

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RETINOIDS AND URAL CARONOOENESIS

886, and 1483) expressed RAR-ß,and none expressed RXR-ßorRXR-7. SqCC/Yl did not express RAR-ß.ATRA treatment increasedthe level of RAR-ß in the 1483 and 183 cells. In contrast, thetreatment had little or no effect on the expression of RAR-a orRXR-a. RAR-ßexpression in the 1483 cells decreased within 3 days

of culture and diminished further thereafter. Concurrently with thisdecrease there was an increase in the expression of squamous differentiation markers, suggesting an inverse relationship between RAR-ß

expression and squamous differentiation. It is not clear whether thereis a causal relationship between these opposite patterns of expression.

To complement these in vitro studies, we analyzed the expressionof nuclear retinoic acid receptors in vivo using surgical specimensfrom normal oral mucosa, premalignant lesions (oral leukoplakiasfrom patients without cancer and dysplasias adjacent to HNSCC), andHNSCCs by an in situ hybridization method. Normal buccal mucosaspecimens expressed RAR-a, RAR-ß,and RAR-y. In contrast, oralleukoplakia specimens, while expressing RAR-a and RAR-y in 100%of the cases, showed RAR-ßexpression in only 40% of the cases (62).

A further analysis of specimens from head and neck cancer patientsthat included adjacent dysplastic, hyperplastic lesions and adjacentnormal epithelium revealed that RAR-a and RAR-y mRNAs were

present in most of the specimens at levels similar to those in normalmucosa. In contrast, RAR-ßexpression decreased from 100% in

normal to 70% in adjacent normal and hyperplastic lesions, to 56% indysplastic lesions, and further to 35% of the carcinomas (62). Theseresults strongly indicate that the decreased expression of RAR-ßmay

be associated with the development of head and neck cancer. A recentanalysis of specimens from patients with oral leukoplakia before andafter 3 months of treatment with 13-c/s-retinoic acid revealed that thepercentage of RAR-ß-expressing cases increased from about 40 to

90%. This increase was correlated with the clinical response of thepatients to the treatment. These results indicate that RAR-ßis an

excellent intermediate marker in oral carcinogenesis because it isdecreased at early stages of carcinogenesis and is increased by treatment with a chemoprevcntive agent, and this increase is related toresponse.

Acknowledgments

I thank my collaborators Dr. Xiao-Chun Xu. Chang-Ping Zou, Dr. Scott M.

Lippman, Dr. Jae Ro, Dr. Jin S. Lee, and Dr. Waun K. Hong for contributingto the studies from my laboratory reviewed in this article.

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