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Clinical Cancer Research Vol. 12, 969-979, February 2006
© 2006 American Association for Cancer Research

Cancer Prevention

Chemoprevention of Skin Carcinogenesis by Phenylretinamides: Retinoid Receptor–Independent Tumor Suppression

Hui Xu2, Satish Cheepala1, Elisabeth McCauley3, Kevin Coombes4, Lianchun Xiao4, Susan M. Fischer3 and John L. Clifford1

Authors' Affiliations: 1 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-Shreveport and the Feist-Weiller Cancer Center, Shreveport, Louisiana and Departments of 2 Clinical Cancer Prevention, 3 Carcinogenesis, and 4 Biostatistics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: John L. Clifford, Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center and the Feist-Weiller Cancer Center, 1501 Kings Highway, Shreveport, LA 17730. Phone: 318-675-8264; Fax: 318-675-5180; E-mail: jcliff{at}lsuhsc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Fenretinide [N-(4-hydroxyphenyl)retinamide or 4-HPR] is a synthetic retinoid analogue with antitumor and chemopreventive activities. N-(4-Methoxyphenyl)retinamide (4-MPR) is the most abundant metabolite of 4-HPR detected in human serum following 4-HPR therapy. We have shown in in vitro studies that 4-HPR and 4-MPR can act independent of the classic nuclear retinoid receptor pathway and that 4-HPR, but not 4-MPR, can also activate nuclear retinoid receptors. In this study, we have compared the chemopreventive effects of topically applied 4-HPR and 4-MPR with the primary biologically active retinoid, all-trans retinoic acid (ATRA), in vivo in the mouse skin two-stage chemical carcinogenesis model. All three retinoids suppressed tumor formation but the effect of 4-HPR and 4-MPR, and not of ATRA, was sustained after their discontinuation. The tumor-suppressive effects of 4-HPR and 4-MPR were quantitatively and qualitatively similar, suggesting that the two may be acting through the same retinoid receptor–independent mechanism(s). We further explored this effect in vitro by analyzing primary cultures of mouse keratinocytes treated with the same retinoids. All three could induce apoptosis with a 48-hour treatment and only ATRA and 4-HPR induced an accumulation of cells in the G1 phase of the cell cycle. This finding is consistent with our previous results showing that the effects of phenylretinamides on the cell cycle are retinoid receptor dependent whereas apoptosis induction is not. A microarray-based comparison of gene expression profiles for mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) alone and TPA + 4-HPR or TPA + 4-MPR reveals a high degree of coincidence between the genes regulated by the two phenylretinamides. We propose that 4-HPR may exert therapeutic and chemopreventive effects by acting primarily through a retinoid receptor–independent mechanism(s) and that 4-MPR may contribute to the therapeutic effect of 4-HPR by acting through the same retinoid receptor–independent mechanism(s).

Fenretinide [N-(4-hydroxyphenyl)retinamide or 4-HPR] belongs to a class of chemical compounds called retinoids. Retinoids include active metabolites of vitamin A (retinol), as well as a diverse array of synthetic derivatives, and have been shown to modulate several essential cellular processes, including proliferation, differentiation, homeostasis, malignant transformation, and apoptosis (1). Retinoids restore regulation of differentiation and growth in certain premalignant and malignant cells in vitro and in vivo. This has led to efforts for their development as chemopreventive drugs (2). Retinoids exert their effects primarily through two families of ligand-dependent, DNA-binding, transcriptional transactivators, the retinoic acid receptors and retinoid X receptors, both members of the nuclear steroid hormone receptor superfamily (1, 3). 4-HPR has shown promise for chemoprevention in clinical studies (4–6). It has low toxicity, potent apoptosis-inducting activity, biological activity in several preclinical systems, including all-trans retinoic acid (ATRA)– and 9-cis retinoic acid–resistant neoplastic cells, and significant clinical chemopreventive activity in randomized trials for breast cancer and oral carcinogenesis (7–9).

4-HPR is one of several members of the N-phenylretinamide class of retinoids. Other N-phenylretinamides bear hydroxyl, carboxyl, or methoxyl substitutions on the terminal phenylamine ring. Among these, N-[4-methoxyphenyl]retinamide (4-MPR) is the principle metabolite of 4-HPR in rodents and humans (10–12) and, unlike the hydroxyl and carboxyl containing phenylretinamides, lacks a charged group on the terminal phenylamine ring. Our group and others have shown cell growth inhibitory activity for many of these N-phenylretinamides in human oral epithelial cells (13), bladder transitional cell carcinoma cells (14), head and neck squamous cell carcinoma cells, and non–small-cell lung cancer cells (15).

Numerous studies have shown that 4-HPR can induce apoptosis in cultured cells, many of which are resistant to the effects of ATRA, suggesting that this activity may not involve retinoid receptors (16–20). It has therefore been hypothesized that 4-HPR exerts its biological and chemopreventive effects through a retinoid receptor–independent mechanism. Studies aimed at determining the precise mechanism of 4-HPR-induced apoptosis suggest that this takes place through a wide range of mechanisms, many of which are not mutually exclusive. These include a p53-independent pathway involving elevation of intracellular ceramide levels (21), increases in intracellular reactive oxygen species (22), activation of members of the activator protein-1 (AP-1) transcription factor family (23), and activation of caspase 8 (24).

We have used the F9 murine embryonal carcinoma cell retinoic acid receptor/retinoid X receptor knockout system (25) to test the hypothesis that 4-HPR, 4-MPR, and several other N-phenylretinamides can exert their biological effects through a retinoid receptor–independent mechanism (26, 27). We previously determined that knocking out the most abundant retinoic acid receptor (retinoic acid receptor {gamma}) and the most abundant retinoid X receptor (retinoid X receptor {alpha}) in F9 cells, in the same cell, generates cells (abbreviated F9-KO) that are almost completely refractory to all measurable effects of ATRA (28). By comparing the action of 4-HPR, 4-MPR, and several other N-phenylretinamides on wild-type F9 and F9-KO cells, we determined that all of the them had retinoid receptor–independent growth inhibitory activity, which included induction of apoptosis (i.e., an equal effect on both cell lines; refs. 26, 27), and that all, except 4-MPR, exerted retinoid receptor–dependent differentiating effects (i.e., only on the wild-type F9 cells). Thus, 4-MPR is unique among the N-phenylretinamides tested in that it possesses the same receptor-independent cell killing effects but cannot trigger retinoid receptor–mediated effects.

The mouse skin two-stage chemical carcinogenesis protocol has been one of the best-studied models and most informative with regard to understanding mechanisms and identifying chemopreventive agents (29). Skin tumors can be readily induced by the sequential application of a subthreshold dose of carcinogen, referred to as the initiation stage, followed by repetitive treatment with a noncarcinogenic tumor promoter, referred to as the promotion stage. The initiation stage, usually accomplished by a single application of the carcinogen 7,12-dimethylbenz(a)anthracene, results in a small subset of keratinocytes carrying a mutation in a critical gene(s). The promotion stage requires repeated application of noncarcinogenic tumor-promoting agents, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which cause the initiated cells to proliferate, eventually producing papillomas, some of which spontaneously progress to squamous cell carcinomas.

Concurrent application of ATRA and TPA to initiated mouse skin reduced both the number of papillomas and carcinomas that formed, indicating that the promotion and progression stages are inhibited by ATRA (30, 31). A possible mechanism for this effect is revealed from studies of the AP-1 transcription factor complex, which is composed of heterodimers of jun and fos protein family members. AP-1 is a positive regulator of cell proliferation and transformation and its activity is stimulated by TPA (32). The promotion step is likely to involve AP-1 because the oncogenic forms of c-jun and p21ras have been shown to cooperate in converting normal keratinocytes to squamous cell carcinomas (33). As would be predicted from the antagonism between TPA and retinoids, retinoid receptors and AP-1 can antagonize each other's activity for the regulation of several genes (34, 35). It has further been shown that the tumor-suppressive effect of ATRA in the two-stage mouse skin model is mediated by blocking AP-1 activity and not through activation of retinoic acid–responsive genes per se (35).

The development of DNA microarray technology provides a very powerful tool for identifying genes expressed in a given cell type or tissue at a particular time (36). DNA microarrays have been used by numerous investigators to identify differentially expressed genes from diverse in vitro sources, such as human T cells undergoing heat shock or phorbol ester treatment (37), human keratinocytes exposed to UV light or retinoids (38), and yeast exhibiting a high temperature growth phenotype (39), and from in vivo sources, such as muscle in dystrophin deficient mice (40) and human skin (41). Of particular interest is a recent report describing the gene expression profile from mouse skin exposed to a short-term treatment with TPA using a cDNA microarray containing 5,000 genes (42). These investigators identified several previously unrecognized TPA-regulated genes, as well as genes shown to be up-regulated in advanced skin cancer. Application of DNA microarray technology for cancer includes the determination of gene expression profiles for human breast, prostate, bladder cancer, glioma, and several others including skin squamous cell carcinoma (43–47). Most of these microarray studies involve pairwise comparisons of gene expression profiles between untreated and treated cells or tissues or between normal and malignant tissues. We have attempted to use this technology to not only determine the effect of a tumor-promoting agent (TPA) on normal tissue but also to assess the effect of retinoids, which are known to suppress TPA-induced tumor promotion, in this setting.

We have previously established that 4-HPR and 4-MPR can induce retinoid receptor–independent cell growth inhibition in two different cell culture systems (26, 27). In this study, we have used the mouse skin two-stage chemical carcinogenesis protocol to determine if 4-HPR and 4-MPR could have a chemopreventive effect in vivo, similar to that previously shown for ATRA (31). An earlier study showed that dietary 4-HPR could inhibit tumor promotion by TPA (48) but, to our knowledge, no study has been published that directly compares topical application of phenylretinamides to ATRA in the two-stage chemical carcinogenesis protocol or that determines the in vivo tumor-suppressive effects of 4-MPR. We show here that both 4-HPR and 4-MPR have a qualitatively and quantitatively similar tumor-suppressive activity in this model, which is lasting and apparently takes place at early stages in skin tumorigenesis. ATRA has a stronger tumor-suppressive effect than either 4-HPR or 4-MPR but the effect requires the constant presence of ATRA. Finally, preliminary DNA microarray analysis indicates that 4-HPR and 4-MPR produce overlapping gene expression profiles in TPA-treated mouse skin. Several of the overlapping genes (hereafter referred to as coregulated genes) are involved in the control of cytokine signaling, apoptosis, and the cell cycle.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Two-stage mouse skin chemical carcinogenesis protocol. Young female outbred SENCAR mice were obtained from the National Cancer Institute (Frederick, MD). Mice were treated as follows: Groups of 30 mice per treatment were shaved on the dorsal side and several days later initiated with 10 nmol (2.56 µg) 7,12-dimethylbenz(a)anthracene in 200 µL acetone, followed by twice weekly applications of 1 µg TPA in 200 µL acetone for 30 weeks. Groups of 30 mice were treated with 5 µg ATRA, 4-HPR, or 4-MPR (dissolved in 200 µL acetone), or acetone alone, 30 minutes before TPA treatment for 13 weeks. ATRA and 4-HPR were obtained from Sigma Chemical Co. (St. Louis, MO). 4-MPR was obtained from the National Cancer Institute. Stock solutions for retinoids were made in DMSO at a concentration of 10 mmol/L and diluted to the appropriate concentrations in acetone. Tumor incidence (number of mice bearing tumors / total number of mice in treatment group) and the tumor multiplicity (average number of tumors per mouse) were recorded weekly.

Histologic examination of tumors and mouse skin. Tumors and tissue margins from the SENCAR mice in the two-stage experiment and from the dorsal skin from mice, in each of the treatment groups used for RNA isolation, were isolated and fixed in formalin and embedded in paraffin before sectioning. Paraffin sections of 4 µm were deparaffinized in xylene, 3 x 7 minutes, room temperature, and rehydrated by stepwise washes in decreasing ethanol/H2O ratio (100% to 50%, followed by soaking in water). Sections were cut and stained with H&E. Sections stained for cytokeratin 6 (gene symbol, Krt2-6b) expression were treated with 1% hydrogen peroxide for 30 minutes with shaking, followed by repeated washes in PBS and water. Sections were incubated in Superblock (Pierce Biotechnology, Inc., Rockford, IL) blocking reagent for 1 hour at room temperature to block nonspecific antigen sites. After washing thrice in PBS, slides were incubated for 4 hours at room temperature with a rabbit polyclonal antibody to Krt2-6b (Covance Research Products, Cumberland, VA). Secondary antibody detection was done according to the instructions for the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Duplicate control specimens receiving second antibody only were processed identically along with the antibody stained slides. Slides were photographed under oil immersion with a Spot CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI).

Isolation of primary keratinocytes. Keratinocytes were isolated from the skin of newborn mice (FVB strain) according to the method of Yuspa and Harris (49) as follows: Mice were sacrificed on the day of birth and placed on ice, rinsed twice each with betadine and 70% ethanol. Skin was removed and placed dermal side down on a sterile plastic dish to dry for 5 minutes, followed by transfer, dermal side down, to a Petri dish containing 0.25% trypsin in PBS. Skins, 5 to 10 per group, were incubated overnight at 4°C. The dermis was peeled off and the epidermis was placed in a beaker containing 10 mL of KGM-2 keratinocyte growth medium (Cambrex Bio Science, Baltimore, MD) and the pooled epidermises minced with scissors. Ten milliliters of additional KGM-2 were added to the beaker and the contents stirred for 20 minutes, filtered through sterile nylon mesh, and the rounded basal cells counted on a hemocytometer. Basal cells were plated at a density of 2 x 106 per well in six-well plates.

Apoptosis assays. The occurrence of apoptosis was visualized using a 4',6-diamidino-2-phenylindole staining assay essentially as previously described (26). Primary keratinocytes from the same primary keratinocyte cultures used for the cell cycle determination were grown on glass coverslips in six-well plates overnight before treatment with 4-HPR, 4-MPR, or ATRA for 48 hours. The medium was gently removed and replaced with medium containing 5 µmol/L 4',6-diamidino-2-phenylindole and incubated at 37°C for 15 minutes. The coverslips were removed from the wells and mounted on slides and photographed on a fluorescence microscope at a magnification of x200. The percentage of apoptosing cells was determined by the visual quantitation of apoptotic nuclei, which contained brightly staining condensed DNA characteristic of apoptosis. Triplicate fields of >100 cells were counted for each sample.

Cell cycle analysis. The cell cycle profile of wild-type and null mutant clones was determined by cell cycle flow cytometry based on cellular DNA content using an Epics Profile II cell sorter (Beckman Coulter, Inc., Fullerton, CA) essentially as previously described (26). The percentage of cells in the different phases of the cell cycle was determined from the raw data using the Epics (R) Elite Flow Cytometry software.

Epidermal RNA isolation. Epidermal total cell RNA was isolated by homogenization of fresh or snap-frozen epidermal cells using TriReagent (Molecular Research Center, Inc., Cincinatti, OH), followed by standard organic extraction and precipitation and purification on RNeasy RNA purification columns (Qiagen, Inc., Valencia, CA). Purity and yield were determined by UV absorbance over the range of 220 to 320 nm. Samples of total RNA (10-30 µg) were fractionated on 1% agarose gels containing 0.66 mol/L formaldehyde to determine its integrity. Twenty micrograms of total RNA per sample were used for fluorescent probe synthesis.

RNA labeling and DNA microarray analysis. Fluorescent probe synthesis and array hybridization were done in the M.D. Anderson Cancer Center Microarray Core Facility laboratory using established methods provided by Affymetrix, Inc. (Santa Clara, CA). Briefly, 20 µg of purified total cell RNA, pooled from three to five mice in each treatment group, were reverse transcribed into cDNA using a T7 promoter-(dT)24 primer. Following second strand synthesis, biotin-labeled cRNA was generated from the double-stranded template using T7 polymerase. The quality of the cRNA probe was verified by running an aliquot on an agarose gel. Exactly 20 µg of the labeled cRNA were hybridized to the Affymetrix Murine Genome U74v2A GeneChip for 16 hours at 45°C in 300 µL of premixed hybridization solution containing labeled hybridization control prokaryotic genes (bioB, bioC, bioD, and cre). Several duplicate spots for each control gene are present on the chip. Chips were washed in the GeneChip Fluidics Station automatic washer and scanned on the GeneArray fluorometric scanner.

Determination of gene expression differences between the sample pairs was conducted within the Bioinformatics Group of the Cancer Genomics Program at M.D. Anderson Cancer Center (Department of Biostatistics). MicroArray Suite 5.0 (Affymetrix) and the freeware software package dChip (from Dr. Wing Wong's laboratory, http://biosun1.harvard.edu/complab/dchip/) were used to analyze the raw intensity data. Gene expression data sets were further analyzed using GeneSifter (VizX Labs, Seattle, WA) and NetAffx (Affymetrix) microarray analysis software packages.

Semiquantitative reverse transcription-PCR. For confirmation of gene expression changes identified by microarray screening, semiquantitative reverse transcription-PCR (RT-PCR) was done essentially as previously described (50). Total RNA was isolated as above. RNA (1 µg/reaction) and the appropriate individual pairs of 20- to 30-mer oligonucleotides (50 pmol per reaction) for the test genes (derived from the GenBank sequence database) were combined with a 10x PCR mix [final concentrations: 50 mmol/L KCl, 10 mmol/L Tris (pH 8.3), 1.5 µmol/L MgCl2, 200 µmol/L each of dATP, dCTP, dGTP, and dTTP] to a final volume of 100 µL and subjected to the following PCR reaction variables: (94°C, 3 minutes; 94°C to 50°C slope, 10 minutes; 50°C, 22 minutes) x 1 cycle, followed by (94°C, 1 minute; 55°C, 30 seconds; 72°C, 1 minute) x 15 to 40 cycles. Five microliters of a Taq polymerase (2.5 units per tube) and avian myeloblastosis virus reverse transcriptase (4 units per tube) mix were added to each tube immediately following the 94°C to 50°C slope. Aliquots of each reaction were collected over a broad range of cycle numbers and electrophoresed in a 2% agarose gel containing ethidium bromide. RT-PCR products that were just below the visual limit of detection were blotted onto nylon membranes by capillary transfer in high-salt buffer. Blots are probed with [{gamma}-32P]-ATP end-labeled oligonucleotide probes complementary to sequences contained between the oligonucleotides used for the RT-PCR. The expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, which is ubiquitously expressed, was determined for each RNA sample to control for variations in RNA quantity.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
4-HPR and its metabolite 4-MPR suppress skin tumor formation in vivo. In this study, mice were initiated with 7,12-dimethylbenz(a)anthracene, followed by twice weekly applications of TPA, along with topical application of either acetone vehicle (200 µL) or 5 µg of ATRA, 4-HPR, or 4-MPR immediately before the TPA application. Retinoid treatment was halted 13 weeks into the experiment and TPA applications continued for an additional 17 weeks to determine whether the retinamides could produce a sustained chemopreventive effect, which was not previously observed for ATRA (31). The tumor multiplicity at week 30 of the two-stage protocol indicates that all three retinoids have tumor-suppressive activity, with ATRA being the most potent (Fig. 1A). The tumor-suppressive effect of 4-HPR and 4-MPR persisted after cessation of treatment with the tumor multiplicity reaching a plateau by 9 weeks after the last treatment. In contrast, ATRA-treated mice showed a linear increase in tumor multiplicity with time after the last treatment without reaching a plateau (Fig. 1A). The tumor incidence eventually reached nearly 100% for all treatment groups (Fig. 1B).


Figure 1
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  		Fig. 1. A, mice were treated with 7,12-dimethylbenz(a)anthracene, followed 2 weeks later by twice weekly topical application of TPA, with or without topical application of the indicated retinoids, for 13 weeks. Treatment with TPA alone continued for an additional 17 weeks. Number of tumors indicates the average number of tumors per mouse in each group (n = 30). B, tumor incidence for the same experiment shown in (A). Arrows, stopping point for retinoid application at week 13.

 
The tumors that arose in the three retinoid-treated groups appeared histologically identical to the untreated controls (Fig. 2). This would suggest that tumors that arose during the first 13 weeks of the experiment, during the retinoid treatment phase, were resistant to the effects of the retinoids. Those that arose after 13 weeks would not necessarily be retinoid resistant. Another variable that can be assayed in the two-stage chemical carcinogenesis protocol is tumor progression, the conversion of papillomas to carcinomas. Normally, the conversion rate for SENCAR mice in the mouse skin two-stage protocol is low (5-7%; ref. 29). None of the three retinoids significantly altered the conversion rate compared with the rate observed for control mice treated with TPA alone (Table 1).


Figure 2
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  		Fig. 2. Histologic examination of tumors from the two-stage chemical carcinogenesis experiment. Tumors were removed from all mice at the end of the experiment shown in Fig. 1, fixed in formalin, and processed for H&E staining. Microscopic analysis revealed no significant differences in either papillomas or carcinomas among the treatment groups with regard to tumor morphology, degree of differentiation, or extent of invasion. There were no carcinomas in the ATRA group.

 

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  		Table 1. Carcinoma incidence for the two-stage tumorigenesis experiment shown in Fig. 2

 
Retinoid-induced apoptosis and G1 accumulation in primary mouse keratinocytes. Several in vitro studies indicate that 4-HPR can induce apoptosis, suggesting that it may exert its cancer suppressive effect through this mechanism (14–20, 26, 27). In addition, we have also shown that 4-HPR, like ATRA, can induce a retinoid receptor–dependent accumulation of cells in the G1 phase of the cell cycle (26) whereas 4-MPR cannot (27). 4-MPR can, however, induce apoptosis as effectively as 4-HPR (27). We therefore attempted to explore the chemopreventive mechanism of the retinoids in this system by analyzing retinoid-treated primary keratinocyte cultures. All three retinoids caused a modest increase in the percentage of apoptosing cells after a 48-hour treatment with a 10 µmol/L concentration (Table 2). ATRA was a more potent apoptosis inducer than 4-HPR at a concentration of 1 µmol/L (9.2% compared with 4.4%; Table 2). Interestingly, both ATRA and 4-HPR induced a limited G1 accumulation whereas 4-MPR did not (Fig. 3B, compare Control with 10 µmol/L ATRA, 4-HPR, and 4-MPR). This result is consistent with our previous finding, using the F9 retinoic acid receptor/retinoid X receptor knockout system, that the cell cycle effect is receptor dependent whereas the apoptosis induction is not.


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  		Table 2. Average percentage of apoptotic nuclei determined by visual quantitation for three independent fields of cells for each coverslip

 

Figure 3
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  		Fig. 3. A, cultures of primary keratinocytes established from newborn mice (FVB strain) were grown on coverslips and stained with 4',6-diamidino-2-phenylindole after treatment with 10 µmol/L of the indicated retinoids for 48 hours. Intensely stained, condensed DNA, characteristic of apoptosing cells, is visible in all retinoid-treated samples (arrows). B, cells from same primary keratinocyte cultures were allowed to grow for 2 days, followed by treatment with the indicated retinoids for 48 hours. Cells, along with supernatant, were stained with propidium iodide and analyzed by fluorescence-activated cell sorting. X axis, integrated fluorescence intensity; y axis, particle number. Approximately 20,000 particles are represented in each histogram. % Sub-G1 is the percentage of particles containing a DNA quantity less than that of G1 cells (2N) and allows a comparative measure of apoptosis. Boxed values, percentage of cells in G1, S, and G2-M phases of the cell cycle.

 
To further determine whether induction of apoptosis may be important for the tumor-suppressive effect of the retinoids, we did the in situ terminal deoxynucleotidyl transferase–mediated dUTP end labeling assay on fixed sections of skin from mice treated for 3 weeks with TPA, with or without retinoids. The skin was harvested 6 hours after the last TPA +/– retinoid treatment. In all cases, we observed fewer than 0.1% of apoptosing cells with no noticeable difference between any of the treatment groups (data not shown).

Treatment of mouse skin with 4-HPR and 4-MPR produces overlapping gene expression profiles. Because both 4-HPR and 4-MPR produced a similar suppression of tumorigenesis, we hypothesized two possibilities: either they are both acting through a similar or identical mechanism or 4-HPR was being converted to 4-MPR, which then suppresses tumor formation. The two possibilities are not mutually exclusive. To gain some insight into this question, we have used Affymetrix U74v2A GeneChips to obtain the gene expression profiles of mouse skin at an early time point in the two-stage skin protocol. Mice were treated topically twice weekly for 3 weeks with TPA alone or with TPA + 4-HPR and TPA + 4-MPR, as in the previous tumorigenesis experiment, followed by RNA isolation from the skin 6 hours after the last treatment. RNA samples pooled from three to five mice in each treatment group were used as template for probe synthesis. Of the ~12,000 genes represented on the U74v2A microarray chip, 354 were found to be regulated by 4-HPR and 465 by 4-MPR, with a lower-boundary fold change of ≥1.2 between TPA treatment alone and TPA treatment plus each phenylretinamide. For a pairwise comparison of expression levels of a given gene between two U74v2A chips, a lower-boundary fold change of expression of ≥1.2 yields P < 0.05. Interestingly, a high proportion of these genes (161) were coregulated (induced or suppressed) by both 4-HPR and 4-MPR compared with treatment with TPA alone. This is represented as a Venn diagram (Fig. 4A) and graphically where the lower-boundary fold change of each of the 161 genes in the intersection is shown, with white and black columns corresponding to 4-HPR and 4-MPR treatments, respectively (Fig. 4B). Columns on the left, above the x axis, indicate genes induced by both 4-HPR and 4-MPR, relative to skin treated with TPA alone. Columns on the right, below the x axis, represent genes suppressed by both phenylretinamides. Remarkably, all 161 coregulated genes were altered in the same direction (induced or suppressed) by both phenylretinamides. Several of these genes are listed according to known functions or signaling pathways in Table 3.


Figure 4
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  		Fig. 4. Genes coordinately regulated by 4-HPR and 4-MPR. A, a high proportion of genes regulated by 4-HPR and 4-MPR in the presence of TPA are shared. Numbers indicate gene expression changes relative to skin treated with TPA alone (lower-boundary fold change ≥ 1.2; P = 0.5) for 4-HPR and 4-MPR of ~12, 000 genes represented on the microarray. The numbers in the intersection of the circles indicate changes common to both 4-HPR and 4-MPR. B, the lower-boundary fold change for each of the 161 genes coordinately regulated by 4-HPR and 4-MPR compared with skin treated with TPA alone is shown graphically. White and black columns, lower-boundary fold change for a given gene for 4-HPR and 4-MPR treatments, respectively. Columns above the x axis, genes induced by both 4-HPR and 4-MPR relative to skin treated with TPA alone. Columns below the x axis, genes suppressed by both phenylretinamides.

 

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  		Table 3. Genes coordinately regulated by 4-HPR and 4-MPR

 
We next used semiquantitative RT-PCR to independently verify the accuracy of the microarrays for several of the genes most strongly up-regulated and down-regulated by both 4-HPR and 4-MPR (Table 2, gene symbols in bold). An example of four genes confirmed to be down-regulated by both 4-HPR and 4-MPR on the Affymetrix microarrays [cytokeratin 6 (Krt2-6b), cytokeratin 16 (Krt1-16), ryudocan (SDC4), and Rras] and one gene up-regulated (an uncharacterized EST) are shown (Fig. 5A). To date, we have attempted to confirm expression of 28 genes coordinately regulated by 4-HPR and 4-MPR, relative to TPA alone, using the original source RNA that was used to generate the microarray probes as template. Of these genes, we have been able to detect 22 by RT-PCR on the first attempt. The majority of genes tested are expressed as would be predicted by the microarray expression pattern. However, this was not the case for all genes, highlighting the preliminary nature of these experiments and the need to repeat these experiments with a new set of mice using replicate arrays. Efforts are ongoing to confirm additional genes using real-time RT-PCR.


Figure 5
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  		Fig. 5. Accuracy of microarray results is confirmed by RT-PCR and immunohistochemical staining. A, RT-PCR was done for the indicated genes found to be coordinately suppressed by 4-HPR and 4-MPR using the same mouse skin RNA samples previously labeled for microarray screening. B, cytokeratin 6 (Krt2-6b) is induced by TPA and suppressed by 4-HPR, 4-MPR, and ATRA in mouse skin. Paraffin sections of skin obtained from sites adjacent to the source of the RNA used for probe synthesis were stained with an antibody to mouse Krt2-6b.

 
For further independent confirmation of gene expression, we have immunohistochemically stained skin samples that were adjacent to the tissue taken for the original RNA isolation using an antibody to one of the coordinately regulated genes, Krt2-6b. TPA strongly up-regulates the expression of Krt2-6b in the interfollicular epithelium and this expression is completely suppressed by all three retinoids (Fig. 5B, bottom). The retinoids alone have no effect on Krt2-6b (Fig. 5B, top). Note that the intense staining of Krt2-6b in the hair follicles for all treatment groups serves as an internal positive control for the antibody.

We next sorted the list of coregulated genes according to cell function and major signaling pathways, using GeneSifter and NetAffx microarray analysis software, in an attempt to determine which cellular processes were most likely affected by 4-HPR and 4-MPR in TPA-treated mouse skin. The coordinately regulated genes shown in Table 3 span a wide range of functions, including genes for growth factors and cytokines (Cxcl5, Cxcl2, IL-6, ligp1, Tnfsf8, and IL-1{alpha}), transcription regulators (Ifi204, Ahrr, C/EBP{alpha}, Dedd, Sox18, Notch3, and CREB-rp), other signal transduction proteins (Calm4, Nfatc, Pygm, Hras1, Rras, EMK, Fzd1, and Ctbp2), and glutathione metabolism (Gstm5, Gstt1, and Mgst3). We note that several cell cycle genes are affected, three of which are also implicated in signaling through the mitogen-activated protein kinase pathway (Table 3, shaded lines indicate genes that are found in more than one category).

A number of apoptosis-related genes are regulated (Ifi204, Plagl1, IL-6, Dedd, and Eef1a2), as would be expected if 4-HPR and 4-MPR act as apoptosis inducers in this model. Finally, in addition to Krt2-6b, two other cytokeratins are also suppressed (Krt1-16 and Krt1-17). These three cytokeratins are also known as the hyperproliferative keratins and are highly expressed in hyperproliferative skin and in inflammatory skin diseases (51).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
ATRA is a potent chemopreventive in the mouse skin two-stage chemical carcinogenesis model. It is likely that ATRA exerts at least part of this effect through activation of retinoid receptors. Previous studies from our laboratory using retinoid receptor knockout F9 embryonal carcinoma cells showed that 4-HPR and 4-MPR can induce apoptosis in the absence of retinoid receptors and that 4-HPR, but not 4-MPR, also activates retinoid receptors (27). The chemopreventive effect observed for 4-MPR is therefore likely taking place through a retinoid receptor–independent mechanism. The activity of 4-HPR was quantitatively and quantitatively similar to 4-MPR in the two-stage model, suggesting that although 4-HPR can activate retinoid receptors in F9 cells (26, 27), its action in the two-stage model may be primarily retinoid receptor independent. These findings have led us to propose that 4-HPR, as well as the other phenylretinamides bearing charged groups on the terminal phenylamine ring, may exert therapeutic and chemopreventive effects by acting primarily as chemotoxic drugs through receptor-independent mechanisms and, in addition, may provide further benefit through activation of retinoid receptors (27).

The stable reduction in the number of tumors per mouse (multiplicity) for the 4-HPR and 4-MPR treatment groups, even after their discontinuation, indicates that they exert an early irreversible effect. The tumor multiplicity for the 4-HPR and 4-MPR treated mice never reached the level seen for the mice treated with TPA alone (Fig. 1A), suggesting that the phenylretinamides may have reduced the number of initiated cells, perhaps by inducing cell death. The lasting effect of 4-HPR and 4-MPR that we observe in the two-stage model is consistent with the chemopreventive effect of 4-HPR seen in clinical trials for oral leukoplakia (7). A 5-year follow-up of patients in those trials showed that 4-HPR not only prevented relapses and new lesions during treatment but also protected against them for up to 19 months after randomization (7).

Numerous studies have explored the apoptosis-inducing action of 4-HPR and several mechanisms for this effect have been postulated (14–19, 21–24, 26, 27). These include a p53-independent pathway involving elevation of intracellular ceramide levels, increases in intracellular reactive oxygen species, activation of caspase 8, and a paradoxical activation of members of the activator protein-1 (AP-1) transcription factor family (21–24). Thus, the weight of evidence suggests that apoptosis induction contributes to tumor suppression by 4-HPR and 4-MPR in the two-stage model. We therefore attempted to determine whether retinoid treatment induced apoptosis in mouse skin using the in situ terminal deoxynucleotidyl transferase–mediated dUTP end labeling assay. We saw no enhancement in the number of apoptosing cells over the very low background levels observed in controls (≤0.1% of epidermal cells undergoing apoptosis) after 3 weeks of treatment with 4-HPR, 4-MPR, or ATRA (data not shown). From this result, we can conclude that the retinoids do not cause detectable increases in apoptosis, at least during the first 3 weeks of treatment. However, the sensitivity of the in situ terminal deoxynucleotidyl transferase–mediated dUTP end labeling method may not be sufficient to detect low but significant numbers of initiated cells killed by the retinoids.

We have previously shown that 4-HPR, like ATRA, can induce a retinoid receptor–dependent accumulation of cells in the G1 phase of the cell cycle (26) whereas 4-MPR cannot (27). 4-MPR can, however, induce apoptosis as effectively as 4-HPR (27). Because of these findings and the difficulties inherent in assaying cell behavior in intact mouse skin, we attempted to explore the chemopreventive mechanism of these retinoids using primary keratinocyte cultures derived from newborn mice. We determined the effect of retinoid treatment on the cell cycle profile and apoptosis induction. All three retinoids caused an increase in apoptosis in cultured keratinocytes, but only ATRA and 4-HPR induced a limited G1 accumulation. This result is in agreement with our previous finding, using the F9 retinoid receptor knockout system, that the cell cycle effect is receptor dependent whereas the apoptosis induction is not (26).

In contrast to the phenylretinamides, the tumor-suppressive effect of ATRA required its continued administration, suggesting that it may act primarily to suppress promotion by TPA. Removal of ATRA, with continued TPA treatment, resulted in the delayed development of papillomas that continued to increase linearly (Fig. 1A). This result is consistent with the findings of other investigators who have shown that TPA activates the AP-1 transcription factor complex and that AP-1 activity is inhibited by ATRA through either direct or indirect interaction with retinoid receptors (35, 52–54). Experiments are currently under way to compare the gene expression profiles of mouse skin treated with TPA + ATRA and TPA + 4-HPR to identify differences or similarities in gene expression induced by these retinoids. These studies will focus on identifying genes that are regulated oppositely by TPA alone compared with TPA plus the retinoids. Preliminary observations indicate a low coincidence between sets of genes oppositely regulated by TPA and ATRA and genes oppositely regulated by TPA and 4-HPR. In fact, we observe very few genes to be oppositely regulated by TPA alone compared with the 4-HPR + TPA and 4-MPR + TPA combinations (Table 2, gene symbols with asterisks).

There is a substantial degree of overlap in gene expression changes induced by 4-HPR and 4-MPR relative to TPA alone [161 of 354 (45%) 4-HPR regulated genes and 161 of 465 (35%) 4-MPR regulated genes]. The somewhat surprising finding that both phenylretinamides have quantitatively and qualitatively similar effects on tumor suppression in the two-stage model and the fact that 4-MPR is the major metabolite of 4-HPR raise the possibility that the effect of 4-HPR in this model is exclusively due to the action of 4-MPR. It also cannot be ruled out that both agents, due to their similar structures, are able to produce the same effect. In any event, it seems that 4-MPR can suppress tumor formation as effectively as 4-HPR and that both of these retinoids seem to act through a mechanism that differs from ATRA. For this reason, we decided to focus our initial microarray analyses on the 161 overlapping genes (Fig. 4A and B; Table 3).

It has long been known that the mitogen-activated protein kinase pathway is activated by TPA (55). The suppression of expression of mitogen-activated protein kinase pathway genes, such as IL-1{alpha}, Hras1, and Rras, by 4-HPR and 4-MPR is consistent with a tumor-suppressive effect that involves suppression of the hyperproliferative action of TPA. However, based on our finding that the tumor-suppressive effect of these phenylretinamides is maintained for many weeks after discontinuation of treatment, it seems more likely that other mechanism(s) are playing a role.

Of interest in the context of our findings is a recent report describing the gene expression profile from mouse skin exposed to a short-term treatment with TPA using a cDNA microarray containing 5,000 genes and a suppression subtracted hybridization technique (42). These investigators identified several previously unrecognized TPA-regulated genes, as well as genes that had been shown to be up-regulated in advanced skin cancer. Although we have used a different mouse strain and duration of TPA treatment, as well as a different microarray platform for our experiments, some of the same TPA up-regulated genes identified by the other investigators, such as Krt2-6b, Krt1-16, and Slpi, are among the genes we have identified as down-regulated by 4-HPR and 4-MPR.

In summary, these results have led us to propose that 4-HPR may exert therapeutic and chemopreventive effects by acting primarily through a retinoid receptor–independent mechanism(s) and that its metabolite, 4-MPR, may contribute to its therapeutic effect by acting through the same mechanism(s). Our results raise the possibility that 4-MPR itself could be developed as a cancer therapeutic or preventive drug. Because 4-MPR does not activate retinoid receptors, as 4-HPR and other retinoids do, the hypervitaminosis A–related side effects of classic retinoids could be avoided. These microarray results indicate a strong similarity of action for 4-HPR and 4-MPR but are still considered preliminary. The present microarray analysis includes only one time point in the two-stage protocol (3 weeks), one phenylretinamide concentration (5 µg in 200 µL vehicle), and only one time point of RNA isolation after the last treatment (6 hours). As such, we could be missing important later gene expression changes or critical transient changes that occur at earlier time points. We also note that although our microarray analyses were not done on replicate chips, we did use pooled RNA samples from three to five mice for each treatment group as template for probe synthesis to control for animal-to-animal variability. Future studies are aimed at obtaining a more thorough set of gene expression profiles done on replicate chips over a range of concentrations and times for mouse skin treated with 4-HPR, 4-MPR, and ATRA in the two-stage model. These studies should provide a clearer picture of the potential mechanism of tumor suppression by these retinoids, leading to a more rational basis for their clinical use.


   Acknowledgments
 
We thank David Menter, Vemparala Subbarayan, and other members of the Department of Clinical Cancer Prevention for helpful discussions and advice; Karen Ramirez in the M.D. Anderson Cancer Center Flow Cytometry Core Facility for flow cytometry; and especially Scott M. Lippman and Reuben Lotan for helpful discussions and support throughout the course of this project.


   Footnotes
 
Grant support: NIH grant R29 CA78560 (J. Clifford), National Institute of Environmental Health Sciences Center grant ES07784, Cancer Center Support grant P30 CA016672, and National Cancer Institute grant CA77150.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/28/05; revised 9/16/05; accepted 10/12/05.


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