**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 [26]. An early study by Stopera and Bird [27] 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. [32] 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. [33], 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

**189**

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

mutation for CRC growth in humans.

this unexpected ATRA resistance.

development in *ApcMin/*<sup>+</sup>

levels and decrease RA signaling.

**5. Clinical studies**

tissues.

number of colon tumors in this rat model.

shown to reduce colonic tumor formation, and 2-CPR actually increased the

Several other animal studies to evaluate the effect of retinoids have employed the *Apc*Min/+ mouse model. Experiments using this model are important because these mice develop intestinal tumors due to *Apc* mutations and *APC* is a driver

A study of *Apc*Min/+ mice by Volate et al. [34] showed that retinoic receptors including *Rara, Rarb, Rxrb, Rxrg* were all expressed in *Apc*Min/+ adenomas. However, in AOM-treated *Apc*Min/+ mice, *Rxra* was selectively downregulated in intestinal tumors. Therefore, these findings indicate that *Rxra* downregulation occurs early in CRC carcinogenesis and is not dependent on *Apc* mutations and beta-catenin.

Another study by Mollersen et al. [35] administered ATRA to *Apc*Min/+ mice and discovered that ATRA treatment failed to prevent tumor formation. Three studies were then performed that gave results which provide mechanisms that helps explain

One line of investigation focused on C-Terminal Binding Protein 1 (CTBP1), which has been reported to inactivate retinoid dehydrogenase RDH [36]. Examination of adenomas from *Apc*Min/+ mice and familial adenomatous polyposis coli (FAP) patients showed an increased expression of CTBP1. Because CTBP1 decreases RDH levels,

In another study on *Apc*Min/+ mice, Shelton et al. [17] analyzed expression levels of CYP26A1, the major RA catabolic enzyme. They found that CYP26A1 expression was increased in tumors from *Apc*Min/+ mice, and in tumors from FAP patients. They also determined that CYP26A1 is a TCF4 target gene which explains why CYP26A1 expression is increased due to upregulated WNT signaling in *APC* mutant tissues. An increase in CYP26A1 would lead to increased ATRA degradation, which provides a mechanism that helps explain why ATRA treatment failed to prevent tumor

A recent innovative study by Penny et al. [38] involved treating *Apc*Min/+ mice with the CYP26a inhibitor Liarozole. Administration of Liarozole to *Apc*Min/+ mice increased endogenous RA signaling (presumably by blocking ATRA metabolism) and effectively reduced intestinal adenoma numbers in these *Apc* mutant mice. We also found that treatment of human CRC cells with Liarozole decreased proliferation, sphere formation and size of the ALDH+ stem cell population [39]. This suggests that Liarozole might decrease tumor stem cell numbers in *APC* mutant

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 *Apc*Min/+ model reveal it might be an effective screen for other retinoid drugs that have anti-tumor activity against *APC* mutant tissues. Perhaps a reasonable place to start would be to screen other agents for their ability to inhibit specific cellular processes upregulated in tumors that lower endogenous ATRA

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

upregulated CTBP1 will lead to lower ATRA levels in tumors [37].

mice.

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

*Colorectal Cancer*

Additionally, several studies have investigated whether RA receptors are intact

Finally, a recent study by Kropotova et al. [15] used RT-PCR to measure expres-

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

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 [26]. An early study by Stopera and Bird [27] 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. [32] 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. [33], 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

sion 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

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].

to more advanced carcinomas.

its degradation.

**4. Animal model studies**

**188**

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 *Apc*Min/+ mouse model. Experiments using this model are important because these mice develop intestinal tumors due to *Apc* mutations and *APC* is a driver mutation for CRC growth in humans.

A study of *Apc*Min/+ mice by Volate et al. [34] showed that retinoic receptors including *Rara, Rarb, Rxrb, Rxrg* were all expressed in *Apc*Min/+ adenomas. However, in AOM-treated *Apc*Min/+ mice, *Rxra* was selectively downregulated in intestinal tumors. Therefore, these findings indicate that *Rxra* downregulation occurs early in CRC carcinogenesis and is not dependent on *Apc* mutations and beta-catenin.

Another study by Mollersen et al. [35] administered ATRA to *Apc*Min/+ mice and discovered that ATRA treatment failed to prevent tumor formation. Three studies were then performed that gave results which provide mechanisms that helps explain this unexpected ATRA resistance.

One line of investigation focused on C-Terminal Binding Protein 1 (CTBP1), which has been reported to inactivate retinoid dehydrogenase RDH [36]. Examination of adenomas from *Apc*Min/+ mice and familial adenomatous polyposis coli (FAP) patients showed an increased expression of CTBP1. Because CTBP1 decreases RDH levels, upregulated CTBP1 will lead to lower ATRA levels in tumors [37].

In another study on *Apc*Min/+ mice, Shelton et al. [17] analyzed expression levels of CYP26A1, the major RA catabolic enzyme. They found that CYP26A1 expression was increased in tumors from *Apc*Min/+ mice, and in tumors from FAP patients. They also determined that CYP26A1 is a TCF4 target gene which explains why CYP26A1 expression is increased due to upregulated WNT signaling in *APC* mutant tissues. An increase in CYP26A1 would lead to increased ATRA degradation, which provides a mechanism that helps explain why ATRA treatment failed to prevent tumor development in *ApcMin/*<sup>+</sup> mice.

A recent innovative study by Penny et al. [38] involved treating *Apc*Min/+ mice with the CYP26a inhibitor Liarozole. Administration of Liarozole to *Apc*Min/+ mice increased endogenous RA signaling (presumably by blocking ATRA metabolism) and effectively reduced intestinal adenoma numbers in these *Apc* mutant mice. We also found that treatment of human CRC cells with Liarozole decreased proliferation, sphere formation and size of the ALDH+ stem cell population [39]. This suggests that Liarozole might decrease tumor stem cell numbers in *APC* mutant tissues.

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 *Apc*Min/+ model reveal it might be an effective screen for other retinoid drugs that have anti-tumor activity against *APC* mutant tissues. Perhaps a reasonable place to start would be to screen other agents for their ability to inhibit specific cellular processes upregulated in tumors that lower endogenous ATRA levels and decrease RA signaling.
