**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 [14–16]. 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 crosstalk occurs between WNT signaling and RA signaling pathways.

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 ATRAresistant CRC cells through a RA receptor-independent mechanism.

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 function in ATRA-resistant cells.

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 and reduce intracellular storage of retinol.

Additionally, several studies have investigated whether RA receptors are intact in CRC cells [19]. We discuss below a few studies that reported loss of RAR in CRC cells. In one study by Moison et al. [20], epigenetic changes appeared to lead to loss of RARB expression in HCT116 cells from DNA hypermethylation [20]. Interestingly, a DNA methylation inhibitor is able to restore RARB expression [21]. In a second study by Nicke et al. [22], 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. [23] had similar results. They observed that ATRA treatment of RA-sensitive and RA-resistant CRC lines induced *RARA* expression in all cell lines, but ATRA only increased RARB expression in lines that were sensitive to RA. The DLD-1 RA resistant cells acquired sensitivity to ATRA when RARB was over-expressed. Additional studies that examine RA resistance due to alterations in RARs have also been reported [23, 24].

Finally, a recent study by Kropotova et al. [15] 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 *APC*, that drive CRC development [1, 25]. Thus, as CRC progresses, tumor cells develop resistance to ATRA by losing their ability to produce and respond to it, as well as, by causing its degradation.
