Xiao-Han Tang

Retinoids, Retinoic Acid Receptors, and Cancer

Retinoids (i.e., vitamin A, all-trans retinoic acid, and related signaling molecules) induce the differentiation of various types of stem cells. Nuclear retinoic acid receptors mediate most but not all of the effects of retinoids. Retinoid signaling is often compromised early in carcinogenesis, which suggests that a reduction in retinoid signaling may be required for tumor development. Retinoids interact with other signaling pathways, including estrogen signaling in breast cancer. Retinoids are used to treat cancer, in part because of their ability to induce differentiation and arrest proliferation. Delivery of retinoids to patients is challenging because of the rapid metabolism of some retinoids and because epigenetic changes can render cells retinoid resistant. Successful cancer therapy with retinoids is likely to require combination therapy with drugs that regulate the epigenome, such as DNA methyltransferase and histone deacetylase inhibitors, as well as classical chemotherapeutic agents. Thus, retinoid research benefits both cancer prevention and cancer treatment.

Oral Cavity and Esophageal Carcinogenesis Modeled in Carcinogen-Treated Mice

Purpose: Squamous cell carcinoma of the oral cavity is one of the most common human neoplasms, and prevention of these carcinomas requires a better understanding of the carcinogenesis process and a model system in which cancer chemoprevention agents can be tested. We have developed a mouse model using the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in the drinking water to induce tumorigenesis in the mouse oral cavity. Experimental Design: 4-NQO was delivered by tongue painting or drinking water to two mouse strains, CBA and C57Bl/6. The incidences of oral cavity carcinogenesis were then compared. In addition, we examined the expression of some of the molecular markers associated with the process of human oral cavity and esophageal carcinogenesis, such as keratin (K) 1, K14, p16, and epidermal growth factor receptor, by immunohistochemistry. Results: After treatment with 4-NQO in the drinking water, massive tumors were observed on the tongues of both CBA and C57Bl/6 female mice. Pathological analyses indicated that flat squamous dysplasias, exophytic papillary squamous tumors (papillomas), and invasive squamous cell carcinomas were present. Immunohistochemistry analyses showed that 4-NQO changed the expression patterns of the intermediate filament proteins K14 and K1. K14 was expressed in the epithelial suprabasal layers, in addition to the basal layer, in tongues from carcinogen-treated animals. In contrast, control animals expressed K14 only in the basal layer. Moreover, we observed more bromodeoxyuridine staining in the tongue epithelia of 4-NQO-treated mice. Reduced expression of the cell cycle inhibitor, p16, was observed, whereas 4-NQO treatment caused an increase in epidermal growth factor receptor expression in the mouse tongues. Interestingly, similar features of carcinogenesis, including multiple, large (up to 0.5 cm) exophytic papillary squamous tumors and invasive squamous cell carcinomas, increased bromodeoxyuridine staining, and increased K14 expression, were also observed in the esophagi of 4-NQO-treated mice. However, no tumors were observed in the remainder of digestive tract (including the forestomach, intestine, and colon) or in the lungs or livers of 4-NQO-treated mice. These results indicate that this murine 4-NQO-induced oral and esophageal carcinogenesis model simulates many aspects of human oral cavity and esophageal carcinogenesis. Conclusions: The availability of this mouse model should permit analysis of oral cavity and esophageal cancer development in various mutant and transgenic mouse strains. This model will also allow testing of cancer chemopreventive drugs in various transgenic mouse strains.

Chemoattractant activity of degradation products of fetal and adult skin extracellular matrix for keratinocyte progenitor cells

Biological scaffolds composed of naturally occurring extracellular matrix (ECM) have been utilized as templates for the constructive remodelling of numerous tissues in preclinical studies and human clinical applications. The mechanisms by which ECM induces constructive remodelling are not well understood, but it appears that the degradation products of ECM scaffolds may play key roles in cell recruitment. The objective of the present study was to investigate the effects of age and species of the tissue from which ECM is harvested on the chemoattractant activity of degradation products of ECM for human keratinocyte stem and progenitor cells. Adult human skin ECM, fetal human skin ECM and adult porcine skin ECM were prepared, enzymatically digested, characterized by SDS–PAGE and evaluated for in vitro chemoattractant activity for human keratinocyte progenitor and stem cells (HEKn). Degradation products of human fetal skin ECM showed greater chemoattractant activity than human adult skin ECM degradation products for the HEKn. Degradation products of porcine adult skin ECM showed greater chemoattractant activity than human adult skin ECM. The human fetal skin ECM degradation products showed the strongest chemoattractant activity for the HEKn. The findings of this study support the concept that the mechanism of ECM scaffold remodelling involves the recruitment of lineage‐directed progenitor cells by scaffold degradation products, and that both the age and species of the tissue from which the ECM is harvested have an effect upon this chemoattractant potential. Copyright

Homeostasis of retinol in lecithin: retinol acyltransferase gene knockout mice fed a high retinol diet.

We analyzed the retinoid levels and gene expression in various tissues after wild-type (Wt) and lecithin:retinol acyltransferase (LRAT-/-) knockout mice were fed a high retinol diet (250 IU/g). As compared to Wt, LRAT-/- mice exhibited a greater and faster increase in serum retinol concentration (mean+/-S.D., Wt, 1.3 +/- 0.2 microM to 1.5 +/- 0.3 microM in 48 h, p > 0.05; LRAT-/-, 1.3 +/- 0.2 microM to 2.2+/-0.3 microM in 48 h, p < 0.01) and a higher level of retinol in adipose tissue (17.2 +/- 2.4 pmol/mg in Wt vs. 34.6 +/- 8.0 pmol/mg in LRAT-/-). In the small intestines of Wt mice higher levels of retinol (96.4 +/- 13.0 pmol/mg in Wt vs. 13.7 +/- 7.6 pmol/mg in LRAT-/- and retinyl esters (2493.4 +/- 544.8 pmol/mg in Wt vs. 8.2 +/- 2.6 pmol/mg in LRAT-/- were detected. More retinol was detected in the feces of LRAT-/- mice (69.3 +/- 32.6 pmol/mg in LRAT-/- vs. 24.1 +/- 8.6 pmol/mg in Wt). LRAT mRNA levels increased in the lungs, small intestines, and livers of Wt mice on the high retinol diet, while CYP26A1 mRNA levels increased greatly only in the LRAT-/- mice. After 4 weeks, no significant differences between Wt mice and LRAT-/- mice were observed in either the serum retinol level or in the prevalence of Goblet cells in jejunal crypts. Our data indicate that the LRAT-/- mice maintain the homeostasis of retinol as the dietary retinol increases by increasing the excretion of retinol from the gastrointestinal tract, increasing the distribution of retinol to adipose tissue, and enhancing the catabolism by CYP26A1. We show that LRAT plays a role in maintaining a stable serum retinol concentration when dietary retinol concentration fluctuates.

A DNA methyltransferase inhibitor and all-trans retinoic acid reduce oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide

The transcriptional silencing of some cell cycle inhibitors and tumor suppressors, such as p16 and retinoic acid receptor β2, by DNA hypermethylation at CpG islands is commonly found in human oral squamous carcinoma cells. We examined the effects of the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza; 0.25 mg/kg body weight), all-trans retinoic acid (RA; given at 100 μg/kg body weight and 1 mg/kg body weight), and the combination of 5-Aza and the low-dose RA on murine oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model. All the drug treatments were done for 15 weeks after a 10-week 4-NQO treatment. Mice in all drug treatment groups showed decreases in the average numbers of neoplastic tongue lesions. The combination of 5-Aza and RA effectively attenuated tongue lesion severity. Although all drug treatments limited the increase in the percentage of proliferating cell nuclear antigen–positive cells and the decrease in the percentage of p16-positive cells caused by the 4-NQO treatment in mouse tongue epithelial regions without visible lesions and in the neoplastic tongue lesions, the combination of 5-Aza and RA was the most effective. Collectively, our results show that the combination of a DNA demethylating drug and RA has potential as a strategy to reduce oral cavity cancer in this 4-NQO model.

The molecular features of tongue epithelium treated with the carcinogen 4-nitroquinoline-1-oxide and alcohol as a model for HNSCC

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer affecting humans worldwide. To determine the potential mechanisms by which chronic tobacco and alcohol abuse lead to HNSCC of the oral cavity, we have used both the 4-nitroquinoline-1-oxide (4-NQO) murine oral carcinogenesis and the Meadows-Cook alcohol models. In this study, we treated mice with 4-NQO in drinking water for 10 weeks and then administered 20% (w:v) ethanol (EtOH) for another 10 weeks. We observed increased levels and/or activation of signaling proteins [p38 mitogen-activated protein kinase (MAPK), β-catenin and Erk 1/2] that are typically altered during HNSCC initiation in humans. We found that EtOH administration alone increased the expression of p38 MAPK but not Erk 1/2 MAPK. Total β-catenin levels in the tongues increased by 2- to 3-fold after 4-NQO treatment, with or without EtOH. However, EtOH combined with 4-NQO reduced phosphorylated β-catenin levels, whereas 4-NQO treatment alone did not. These data implicate EtOH as a regulator of β-catenin signaling in this HNSCC model. We also utilized K14-CreER(TAM); ROSA26 mice to mark permanently stem/progenitor cells in the tongue epithelia. We found that 4-NQO alone and 4-NQO plus EtOH treatment resulted in massive, horizontal expansion of stem/progenitor cell populations arising from single stem cells in the basal layer of the epithelia. This expansion is consistent with carcinogen-associated, symmetric division of stem/progenitor cells. Our data suggest that specific therapeutic targets for prevention of HNSCC of the oral cavity associated with both alcohol and tobacco use are p38 MAPK and β-catenin.

Combination of bexarotene and the retinoid CD1530 reduces murine oral-cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide

Significance Oral-cavity squamous-cell carcinoma is one of the most common human cancers in the world. About 60–70% of oral-cavity carcinoma cases are diagnosed only after the tumors have become locally advanced. Therefore, in addition to treatment, prevention of oral cancer is a very important goal. In this study, we found that the combination of the drugs bexarotene and CD1530 was more effective than either drug alone in preventing oral carcinogenesis in our mouse model of human oral and esophageal cancers. We envision that the combination of bexarotene and CD1530 could potentially be applied to humans at a high risk for oral cancer, as a very effective strategy for the prevention and treatment of human oral cancer. We investigated the effects of bexarotene (a retinoid X receptor agonist), CD1530 (a retinoic acid receptor γ selective agonist), and the combination of these two drugs for the prevention of oral carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral-cavity and esophageal squamous-cell carcinoma previously generated in our laboratory. We observed decreased numbers of neoplastic tongue lesions and reduced lesion severity in the 4-NQO plus CD1530 (4N+C) and 4-NQO plus bexarotene plus CD1530 (4N+B+C) groups compared with the 4-NQO group. RNA-Seq analyses showed increases in transcripts in cell proliferation/cell cycle progression pathways in the 4-NQO vs. the untreated group. In addition, β-catenin and matrix metallopeptidase 9 (MMP9) protein levels and reactive oxygen species (ROS), as assessed by 4-hydroxynonenal (4-HNE) staining, were elevated in tongue tissues 17 wk after the termination of the 4-NQO treatment. The 4N+B, 4N+C, and 4N+B+C groups showed dramatically lower levels of β-catenin, MMP9, and 4-HNE staining compared with the 4-NQO group. The major reduction in 4-HNE staining in the retinoid treatment groups suggests a novel mechanism of action, reduction of ROS, by which bexarotene and CD1530 inhibit carcinogenesis.

Cell proliferation inhibition and alterations in retinol esterification induced by phytanic acid and docosahexaenoic acid

We investigated the effects of two natural dietary retinoid X receptor (RXR) ligands, phytanic acid (PA) and docosahexaenoic acid (DHA), on proliferation and on the metabolism of retinol (vitamin A) in both cultured normal human prostate epithelial cells (PrECs) and PC-3 prostate carcinoma cells. PA and DHA inhibited the proliferation of the parental PC-3 cells and PC-3 cells engineered to overexpress human lecithin:retinol acyltransferase (LRAT) in both the absence and presence of retinol. A synthetic RXR-specific ligand also inhibited PC-3 cell proliferation, whereas all-trans retinoic acid (ATRA) did not. PA and DHA treatment increased the levels of retinyl esters (REs) in both PrECs and PC-3 cells and generated novel REs that eluted on reverse-phase HPLC at 54.0 and 50.5 min, respectively. Mass spectrometric analyses demonstrated that these novel REs were retinyl phytanate (54.0 min) and retinyl docosahexaenoate (50.5 min). Neither PA nor DHA increased LRAT mRNA levels in these cells. In addition, we demonstrate that retinyl phytanate was generated by LRAT in the presence of PA and retinol; however, retinyl docosahexaenoate was produced by another enzyme in the presence of DHA and retinol.

Obesity Leads to Tissue, but not Serum Vitamin A Deficiency

Obesity negatively affects multiple metabolic pathways, but little is known about the impact of obesity on vitamin A (VA)[retinol (ROL)], a nutrient that regulates expression of genes in numerous pathways essential for human development and health. We demonstrate that obese mice, generated from a high fat diet (HFD) or by genetic mutations (i.e., ob/ob; db/db), have greatly reduced ROL levels in multiple organs, including liver, lungs, pancreas, and kidneys, even though their diets have adequate VA. However, obese mice exhibit elevated serum VA. Organs from obese mice show impaired VA transcriptional signaling, including reductions in retinoic acid receptor (RARα, RARβ2 and RARγ) mRNAs and lower intracellular ROL binding protein Crbp1 (RBP1) levels in VA-storing stellate cells. Reductions in organ VA signaling in obese mice correlate with increasing adiposity and fatty liver (steatosis), while with weight loss VA levels and signaling normalize. Consistent with our findings in obese mice, we show that increasing severity of fatty liver disease in humans correlates with reductions in hepatic VA, VA transcriptional signaling, and Crbp1 levels in VA storing stellate cells. Thus, obesity causes a “silent” VA deficiency marked by reductions in VA levels and signaling in multiple organs, but not detected by serum VA.

Basal stem cells contribute to squamous cell carcinomas in the oral cavity.

The cells of origin of oral cavity squamous cell carcinoma (OCSCC) are unknown. We used a cell lineage tracing approach (adult K14-CreER(TAM); ROSA26 mice transiently treated with tamoxifen) to identify and track normal epithelial stem cells (SCs) in mouse tongues by X-gal staining and to determine if these cells become neoplastically transformed by treatment with a carcinogen, 4-nitroquinoline 1-oxide (4-NQO). Here, we show that in normal tongue epithelia, X-gal(+) cells formed thin columns throughout the entire epithelium 12 weeks after tamoxifen treatment, indicating that the basal layer contains long-lived SCs that produce progeny by asymmetric division to maintain homeostasis. Carcinogen treatment results in a ~10-fold reduction in the total number of X-gal(+) clonal cell populations and horizontal expansion of X-gal(+) clonal cell columns, a pattern consistent with symmetric division of some SCs. Finally, X-gal(+) SCs are present in papillomas and invasive OCSCCs, and these long-lived X-gal(+) SCs are the cells of origin of these tumors. Moreover, the resulting 4-NQO-induced tumors are multiclonal. These findings provide insights into the identity of the initiating cells of oral cancer.