Bioinformatics results on RXR and RAR mutations in CRC cell lines.
Retinoids are vitamin A metabolites best known for their role in embryonic development. Indeed, retinoid acid (RA) signaling plays a key role in regulating the development of the embryo body-plan by controlling embryonic stem cells (SCs). Retinoids function through their ability to induce cellular differentiation. Mutations in RA signaling pathway genes occur in most human cancers. The classic example is the chromosomal translocation involving RA receptor alpha in acute promyelocytic leukemia (APL). Because all-trans retinoic acid (ATRA) is a highly effective and often curative treatment for APL patients, determining if retinoids are efficacious for other cancer types is imperative. We review the current research on retinoids in colorectal cancer (CRC) and provide bioinformatics analyses of RA signaling. Our results show that most RA pathway genes are overexpressed and often mutated in CRC. Moreover, aberrant expression of many RA signaling proteins predicts decreased CRC patient survival. We also review aldehyde dehydrogenase (ALDH) expression in CRC because ALDH is a key enzyme in RA signaling, which regulates colonic SCs. Further investigation of RA signaling mechanisms that regulate colon SCs and how dysregulation contributes to the SC overpopulation that drives CRC growth should provide insight into strategies for designing new SC-targeted therapies for CRC.
- retinoic acid
- stem cells
- colon cancer
- adenomatous polyposis coli
- aldehyde dehydrogenase
Our goal herein is to review current research findings on retinoids in colorectal cancer (CRC), and to provide an update from our bioinformatics analysis of RA signaling components in CRC. Retinoic acid (RA) is currently being used in the treatment of specific types of human cancers . The classic example is use of ATRA as first line treatment for acute promyelocytic leukemia (APL). RA therapy has also been shown to improve survival in patients with neuroblastoma [2, 3, 4]. Additionally, RA-based agents have been evaluated for clinical anti-cancer activity in breast cancer and in lung cancer . In this review, we discuss the anti-cancer activity of retinoids using
A strong rationale to investigate RA signaling in oncology research is that ATRA is an effective drug used to treat APL patients. Indeed, ATRA effectively induces APL cells to terminally differentiate into neutrophils [9, 10, 11]. Current treatment regimens for APL also include arsenic in combination with ATRA because the combination provides a synergic drug response that cures the majority of APL patients, who would otherwise be facing a highly fatal illness. The precise mechanism involved in triggering APL cells have been extensively studied with the hope of understanding how it can be applied to trigger differentiation in other cancer types. What appears to be the basis for clinical success in treating APL is that the RA/arsenic combination not only induces terminal differentiation, but it also abrogates self-renewal of APL SCs . Thus, future retinoid-based treatments for other cancers will likely necessitate drug combinations that incorporate a RA signaling differentiation therapy and a SC-targeting therapy that inhibits cancer SC self-renewal.
2. Key components of the retinoic acid signaling pathway
To understand how the RA signaling pathway is altered in cancer and to provide a basis for designing retinoid-based treatment approaches to cancer, we provide a brief description of the key components in the RA signaling pathway. The reader is referred to Das et al.  for more detailed information. Listed below are the main proteins essential to proper functioning of the RA signaling pathway. A simplified schematic of the RA signaling pathway is illustrated in Figure 1.
2.1 STRA6 (stimulated by retinoic acid 6)
STRA6 is a cell surface protein that functions as a receptor to accept all-trans retinol from the extracellular retinol-binding protein RBP4 and to transport retinol across the cell membrane. STRA6 removes the retinol from RBP4 and transfers it to RBP1 in the cytoplasm. STRA6 does not transport RA.
2.2 LRAT (lecithin retinol acyltransferase)
LRAT is an enzyme that converts retinol to all-trans retinyl esters, which is a storage form of vitamin A. LRAT also functions to enhance cellular uptake of retinol by STRA6, which contributes to the activation of the RA signaling cascade.
2.3 RDHs (retinol dehydrogenases)
RDHs are a family of dehydrogenase enzymes involved in the conversion of retinol to retinaldehyde by catalyzing the oxidation of cis-isomers of retinol, including 11-cis-, 9-cis-, and 13-cis-retinol in an NAD-dependent manner. This family of short-chain dehydrogenases/reductases functions to catalyze the final step in the biosynthesis of 11-cis retinaldehyde.
2.4 DHRS3 (retinaldehyde reductase-3)
DHRS3 is an oxidoreductase that catalyzes the oxidation/reduction of all-trans-retinal to all-trans-retinol in the presence of NADPH. DHRS3 is essential for preventing the formation of excess RA during embryonic development.
2.5 ADHs (alcohol dehydrogenases)
ADHs are a family of alcohol dehydrogenases involved in retinoid metabolism via conversion of retinol to retinaldehyde by catalyzing the NAD-dependent oxidation of all-trans-retinol and its derivatives such as all-trans-4-hydroxyretinol. These enzymes metabolize a wide variety of substrates, including ethanol, retinol, aliphatic alcohols, hydroxysteroids, and products of lipid peroxidation. ADHs consist of several homo- and heterodimers of alpha, beta, and gamma subunits, which plays major roles in ethanol catabolism. For example, three genes encoding alpha, beta, and gamma subunits of ADH1 are tandemly organized in a genomic segment as a gene cluster.
2.6 RBPs (retinol-binding proteins)
RBP1 (Retinol Binding Protein 1) and RBP2 (Retinol Binding Protein 2) are cytoplasmic retinol-binding proteins, which contribute to retinol uptake, storage, and retinoid homeostasis. Specifically, RBP1 is the carrier protein for transport of retinol from the liver storage site to peripheral tissue. RBP2 also plays an important role in the uptake and intracellular transport of retinol, which is necessary for intracellular metabolism of vitamin A.
2.7 ALDHs (aldehyde dehydrogenases)
ALDHs are cytoplasmic enzymes that convert/oxidize retinaldehyde to RA. ALDHs are the enzymes that function after the alcohol dehydrogenase step in the RA signaling pathway. Nineteen ALDH isoforms encoded by 19 different genes exist in humans with as many orthologs in the mouse plus some alternatively spliced transcriptional variants. Through its role in retinol metabolism, ALDHs play a major role in the regulation of responses to RA.
2.8 CRABPs (cellular retinoic acid-binding proteins)
CRABP1 (Cellular Retinoic Acid Binding Protein 1) and CRABP2 (Cellular Retinoic Acid Binding Protein 2) are paralogous genes that encode cellular RA binding proteins. These proteins transport RA to the nucleus and function to regulate the access of RA to the nuclear RA receptors. Specifically, CRABPs are cytosol-to-nuclear shuttling proteins, which facilitate RA binding to its cognate receptor complex and nuclear transfer. These activities in the retinoid signaling pathway play an important role in RA-mediated differentiation and proliferation processes. CRABPs are structurally similar to the cellular retinol-binding proteins, but CRABPs only bind RA, which contributes to RA-directed differentiation in epithelial tissue. Diseases associated with CRABPs include embryonal carcinomas.
2.9 CYP26A1 (cytochrome P450 family 26 subfamily A member 1)
CYP26A1 is a cytochrome P450 monooxygenase that plays a key role in the metabolism of ATRA. The cytochrome P450 superfamily contains 57 members that are monooxygenase enzymes which catalyze many processes including drug metabolism and synthesis of cholesterol, steroids, and various lipids. CYP26A1 acts on ATRA by catalyzing the hydroxylation of carbon hydrogen bonds of ATRA. This includes both 4-hydroxylation and 18-hydroxylation activities. It has little activity toward 9-cis and 13-cis RA ligands. By regulating intracellular concentrations of RA, CYP26A1 can control RA signaling mediated gene expression in both embryonic and adult tissues. There are two alternatively spliced transcript variants of CYP26A1 that encode the different isoforms. This enzyme regulates the cellular level of RA which in turn regulates gene expression in both embryonic and adult tissues. Diseases associated with CYP26A1 include embryonal carcinoma and APL.
2.10 Retinoid X receptors (RXRs) and retinoic acid receptors (RARs)
The proteins encoded by RARs (
Now that we have briefly covered the key components in the RA signaling pathway that are critical to its proper function, we will discuss alterations of this pathway that occur in CRC.
3. Studies on alterations of retinoic acid signaling in CRC
Many studies have been done to identify mechanisms that explain how RA resistance occurs in solid tumors. Indeed, CRCs have been shown to lose the ability to produce ATRA and fail to growth inhibit or differentiate in response to treatment with ATRA [14, 15, 16]. Retinoic acid resistance appears to arise spontaneously in human cancers. To assess how alterations in RA signaling components effect response to RA ligands, we performed a literature search. Most of the published studies discussed below used
In a study by Jette et al. , seven CRC cell lines were evaluated for retinol dehydrogenase (RDH) enzymatic activity. They found CRC cells have decreased conversion of retinol into RA compared to normal cells. This inhibition of RDH expression appeared to be due to loss of adenomatous polyposis coli (APC) function. Interestingly, reintroduction of
Another study by Park et al.  examined the ability of retinol to inhibit the growth of CRC cell lines. They observed that some CRC cells are ATRA-sensitive (HCT-15) and other cells are ATRA-resistant (HCT-116, SW620, and WiDR). They also found that retinol inhibited the growth of both ATRA-sensitive and ATRA-resistant CRC cells through a RA receptor-independent mechanism.
Other studies by Shelton et al.  evaluated for over-expression of CYP26A1 enzymes that could lead to increased ATRA degradation. Indeed, CYP26A1 was upregulated in
Lecithin retinol acyltransferase (LRAT), which esterifies retinol to retinyl esters, has also been evaluated by Cheng et al. . Indeed, the LRAT gene promoter was hypermethylated in CRC cell lines and neoplasms compared to normal tissue . A decrease in LRAT expression due to hypermethylation could lower availability of retinoids and reduce intracellular storage of retinol.
Additionally, several studies have investigated whether RA receptors are intact in CRC cells . We discuss below a few studies that reported loss of RAR in CRC cells. In one study by Moison et al. , epigenetic changes appeared to lead to loss of RARB expression in HCT116 cells from DNA hypermethylation . Interestingly, a DNA methylation inhibitor is able to restore RARB expression . In a second study by Nicke et al. , the RA-resistant LoVo CRC line was induced to over-express RARB, which produced responsiveness to ATRA resulting in growth inhibition. A third study by Lee et al.  had similar results. They observed that ATRA treatment of RA-sensitive and RA-resistant CRC lines induced
Finally, a recent study by Kropotova et al.  used RT-PCR to measure expression patterns of genes involved in ATRA biosynthesis. They evaluated normal human colorectal tissues, primary carcinomas, and cancer cell lines. Expression of most genes involved in ATRA synthesis was altered in CRC tumors and colorectal cell lines. Moreover, the expression of several genes, particularly ADH isoforms ADH2 and ADH3, showed decreased gene expression in adenomas when compared to more advanced carcinomas.
Overall, the studies on CRC discussed above show that RA signaling components become altered at many levels along the pathway. This includes: (i) loss of RAR expression that impairs RA response and gene transcription; (ii) decreased ability to enzymatically synthesize ATRA; (iii) LRAT alterations that impair retinoid storage; (iv) enhanced degradation of ATRA via CYP26A1. Many of these alterations appear to be a consequence of the mutations, such as
4. Animal model studies
In addition to the studies on RA signaling in cell lines and CRC tissues discussed above, other important investigations have been done using animal models. Many of these studies were done using azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH) to induce colonic neoplasms in rats to investigate the anti-tumor effects of retinoids . An early study by Stopera and Bird  found that ATRA treatment reduced the number of AOM-induced aberrant crypt foci (ACF), a precursor to CRCs. Two studies [28, 29] using the DMH-induced colon carcinogenesis model indicated that vitamin A dietary supplementation may diminish ACF formation. Other studies by Wargovich et al. [30, 31] reported that 13-cis-retinoic acid (13-cRA), 9-cis-retinoic acid, and the synthetic Vitamin A derivative 4-hydroxy-phenretinamide (4-HPR) diminished AOM-induced ACF in rats. An interesting study by Zheng et al.  screened thirteen retinoids for prevention of ACF. They found that two retinoids, 9-cis-retinoic acid and 4-HPR, reduced both colonic ACF and tumor formation. In another study by Zheng et al. , 2-(carboxyphenyl)retinamide (2-CPR) was evaluated because it prevents ACF. However, they found that this synthetic retinoid analogue increased the number of colon tumors. Thus, these studies on rats show that ATRA, retinol, 9-cis-retinoic acid, 4-HPR, 13-cRA, and 2-CPR can inhibit the formation of carcinogen-induced ACF. However, only 9-cis-retinoic acid and 4-HPR were shown to reduce colonic tumor formation, and 2-CPR actually increased the number of colon tumors in this rat model.
Several other animal studies to evaluate the effect of retinoids have employed the
A study of
Another study by Mollersen et al.  administered ATRA to
One line of investigation focused on C-Terminal Binding Protein 1 (CTBP1), which has been reported to inactivate retinoid dehydrogenase RDH . Examination of adenomas from
In another study on
A recent innovative study by Penny et al.  involved treating
Thus, the above discussed animal model studies have provided valuable information on how the retinoid pathway might be targeted in designing treatment approaches for human CRC patients. The studies using chemical carcinogen models show that different retinoid drugs have different activities against colon tumors. The studies using the
5. Clinical studies
There have been an increasing number of clinical trials done on solid tumors using retinoids. However, our search of trials listed www.clinicaltrials.gov does not show any trials on CRCs using retinoids, Tretinoin or Liarozole. There were several trials listed for breast, lung, prostate, pancreatic, renal, cervical, brain, skin, and several hematologic malignancies. Given the pre-clinical data discussed above, it seems like it would be reasonable to develop a retinoid-based trial for CRC.
6. Prospect for retinoid-based, stem cell-targeted therapies for CRC
We have been interested in the role of RA signaling in regulation of colonic SCs and how dysregulation of RA signaling may contribute to CRC development for several reasons: (i) RA regulates embryonic SCs during development  and WNT signaling, another key developmental pathway, has an opposing effect on embryonic SCs . The idea that the mechanisms that regulate embryonic SCs are the same mechanisms that become dysregulated in the SC etiology of cancer  is intriguing because some scientists view cancer as aberrant organogenesis  and metastases as aberrant morphogenesis . (ii)
Indeed, our research team [39, 46, 47, 48, 49] and others [50, 51, 52] have been using ALDH as a marker to identify and isolate SCs from patient tissues for several years. ALDH not only marks colonic SCs, but ALDH+ cells also have SC properties of self-renewal, drug resistance, and cell differentiation potential . For example, ALDH+ cells possess self-renewing ability as shown by sphere-forming ability
ATRA is commonly used as a differentiating agent in SC research. For example, we found that treatment of ALDH+ cancer SCs (CSCs) with ATRA inhibits cell proliferation, reduces SC proliferation, sphere formation, and SC population size, as well as enhances SC differentiation [39, 47]. Others have shown that retinoids decrease proliferation of ALDH+ SCs and, conversely, that inhibitors of ALDH increase proliferation of ALDH+ SCs [4, 6, 7, 8]. Because ALDH is key to retinoid acid (RA) signaling and retinoids are well known to promote differentiation of SCs , it follows that having ALDH in a SC provides the capacity for it to differentiate in response to retinoids.
7. Bioinformatics analysis of retinoid signaling components in CRC
We extended our study of RA signaling in CRC herein by using bioinformatics to analyze expression and mutation of RA signaling genes in CRCs and identify RA pathway genes that predict CRC patient survival. We found that most genes in the RA pathway are overexpressed and many are mutated in CRC (Figure 2). This is consonant with our previous result showing that RAR, RXR and other RA signaling proteins are overexpressed in CRC, which parallels overpopulation of ALDH-positive SCs that occurs during CRC tumorigenesis [39, 46]. Moreover, we found that aberrant expression of many RA signaling proteins (10 of 27) predicted (p < 0.05) decreased survival of CRC patients (Figure 3). We refer the reader to the meta-analysis by Chen et al.  which reveals that increased ALDH also indicates a poor prognosis in CRC patients. These updated findings provide insight into the complexity of RA signaling mechanisms and how RA signaling, when dysregulated, contributes to the development of CRC.
8. Conclusion and future perspectives
Our results indicate that RA signaling, when dysregulated, plays a major role in the SC origin of CRC. Overall, our review provides a strong rationale for future exploration of retinoid therapies for CRC in precision oncology. A few clues gleaned from our review are as follows: (i) drug screens using CRC cell lines (Table 1) and knockout of RA-signaling genes in human CRC cells might identify which retinoid drugs are active against cells with specific mutations; (ii)
|CRC Cell line||APC mutation||Microsatellite instability||Beta-catenin mutation||RARA mutation||RARB mutation||RARG mutation||RXRA mutation||RXRB mutation||RXRG mutation|
8.1 Materials and methods
The bioinformatics analysis on overexpression and mutation of RA signaling component genes in CRCs was done through the COSMIC website (cancer.sanger.ac.uk/cosmic). Bioinformatics analysis to identify RA signaling genes that predict CRC patient survival was done through The Human Protein Atlas (https://www.proteinatlas.org).
Conflict of interest
The authors do not have any conflicts of interest.
This study was supported in part by the Lisa Dean Moseley Foundation (BB), Cancer B*Ware Foundation (BB), and Cawley Center for Translational Cancer Research Fund (BB, CF).