**2.10 Retinoid X receptors (RXRs) and retinoic acid receptors (RARs)**

The proteins encoded by RARs (*RARA, RARB, RARG*) and RXR (*RXRA, RXRB, RXRG*) genes are classified as members of the steroid and thyroid hormone receptor superfamily of transcriptional regulators. Various receptor isoforms can result from differential splicing of RA receptor genes and alternate promoter usage. RXRs and RARs are nuclear receptors that are central to retinoid acid (RA) signaling through their role in RA-mediated gene activation in response to their ligands ATRA or 9-cis retinoic acid. The 9-cis RA ligand has a high affinity for RXRs. These receptors are localized to cytoplasm and sub-nuclear compartments where they can bind RA to activate cellular signaling by forming homodimers or heterodimers. These dimers

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*Retinoids in Treatment of Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.93699*

pathway that occur in CRC.

tion in ATRA-resistant cells.

primarily act as transcription factors via binding to the retinoic acid response elements (RARE) made of tandem 5'-AGGTCA-3′ sites known as DR1-DR5. When the ligand is absent, RXRA/RARB forms a multiprotein complex containing transcription co-repressors that can induce histone deacetylation, chromatin condensation and transcriptional suppression. When the ligand is present, it induces the co-repressors to dissociate from the receptors and co-activators are recruited which leads to transcriptional activation. Moreover, depending on the RARE DNA element condition, the heterodimer can act as a transcriptional repressor or transcriptional activator. For example, the heterodimer can act as a repressor on the DR1 element and as an activator on the DR5 element. RA receptors can also dimerize with thyroid hormone, and vitamin D receptors, which increases their DNA binding and transcriptional effects on their respective response elements. RA signaling regulates gene expression in various biological processes such as embryonic morphogenesis, granulocytopoiesis, and skeletal growth. It also plays an essential role in mediating the antiproliferative effects of RA by inducing cellular differentiation and apoptosis. In oncology, translocations

between RARA and other loci are associated with the development of APL. 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

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 [14–16]. 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 vitro* experiments on CRC cell lines and analysis of human CRC tissues. In a study by Jette et al. [16], 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 *wild-type APC* into an *APC*-mutant CRC cell line (HT29) increased expression of DHRS9 (RDHL) but not RDH5. Transfection of *wild-type APC* also increased production of RA. This study indicates intracellular

**3. Studies on alterations of retinoic acid signaling in CRC**

crosstalk occurs between WNT signaling and RA signaling pathways.

resistant CRC cells through a RA receptor-independent mechanism.

retinoids and reduce intracellular storage of retinol.

Another study by Park et al. [14] 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-

Other studies by Shelton et al. [17] evaluated for over-expression of CYP26A1 enzymes that could lead to increased ATRA degradation. Indeed, CYP26A1 was upregulated in *APC*–deficient CRC tissues which provides a mechanism that might explain how increased WNT-signaling might be tied to impaired RA-signaling func-

Lecithin retinol acyltransferase (LRAT), which esterifies retinol to retinyl esters, has also been evaluated by Cheng et al. [18]. Indeed, the LRAT gene promoter was hypermethylated in CRC cell lines and neoplasms compared to normal tissue [18]. A decrease in LRAT expression due to hypermethylation could lower availability of

### *Retinoids in Treatment of Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.93699*

*Colorectal Cancer*

intracellular metabolism of vitamin A.

**2.7 ALDHs (aldehyde dehydrogenases)**

major role in the regulation of responses to RA.

**2.8 CRABPs (cellular retinoic acid-binding proteins)**

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

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

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)**

**2.10 Retinoid X receptors (RXRs) and retinoic acid receptors (RARs)**

The proteins encoded by RARs (*RARA, RARB, RARG*) and RXR (*RXRA, RXRB, RXRG*) genes are classified as members of the steroid and thyroid hormone receptor superfamily of transcriptional regulators. Various receptor isoforms can result from differential splicing of RA receptor genes and alternate promoter usage. RXRs and RARs are nuclear receptors that are central to retinoid acid (RA) signaling through their role in RA-mediated gene activation in response to their ligands ATRA or 9-cis retinoic acid. The 9-cis RA ligand has a high affinity for RXRs. These receptors are localized to cytoplasm and sub-nuclear compartments where they can bind RA to activate cellular signaling by forming homodimers or heterodimers. These dimers

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.

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primarily act as transcription factors via binding to the retinoic acid response elements (RARE) made of tandem 5'-AGGTCA-3′ sites known as DR1-DR5. When the ligand is absent, RXRA/RARB forms a multiprotein complex containing transcription co-repressors that can induce histone deacetylation, chromatin condensation and transcriptional suppression. When the ligand is present, it induces the co-repressors to dissociate from the receptors and co-activators are recruited which leads to transcriptional activation. Moreover, depending on the RARE DNA element condition, the heterodimer can act as a transcriptional repressor or transcriptional activator. For example, the heterodimer can act as a repressor on the DR1 element and as an activator on the DR5 element. RA receptors can also dimerize with thyroid hormone, and vitamin D receptors, which increases their DNA binding and transcriptional effects on their respective response elements. RA signaling regulates gene expression in various biological processes such as embryonic morphogenesis, granulocytopoiesis, and skeletal growth. It also plays an essential role in mediating the antiproliferative effects of RA by inducing cellular differentiation and apoptosis. In oncology, translocations between RARA and other loci are associated with the development of APL.

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.
