**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 [40] and WNT signaling, another key developmental pathway, has an opposing effect on embryonic SCs [41]. The idea that the mechanisms that regulate embryonic SCs are the same mechanisms that become dysregulated in the SC etiology of cancer [42] is intriguing because some scientists view cancer as aberrant organogenesis [43] and metastases as aberrant morphogenesis [44]. (ii) *APC* mutations occur in most CRCs (nearly 90%) during CRC development and *APC* mutation leads to constitutively activated WNT signaling. (iii) *APC* mutations that drive CRC development appear to do so by causing SC overpopulation [45]. (iv) ALDH, a key component in RA signaling, marks colonic SCs and tracks SC overpopulation during CRC development.

Indeed, our research team [39, 46–49] and others [50–52] 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 [53]. For example, ALDH+ cells possess self-renewing ability as shown by sphere-forming ability *in vitro* and tumor-initiating ability in mice [46, 51, 52, 54]. The drug resistance property of ALDH+ SCs comes from aldehyde dehydrogenase's enzymatic function, which is the cell's natural detoxification mechanism [50, 55, 56]. The ability of ALDH+ cells to differentiate comes from ALDH's functional role in the RA signaling pathway [5, 13, 57–59]. Moreover, we examined ALDH+ cells from colon tissues and observed that retinoid receptors RXR and RAR are selectively expressed in ALDH+ cells [39], which indicates that RA signaling mainly occurs via ALDH+ SCs. That RA signaling primarily occurs in ALDH+ stem cells provides a mechanism for selective treatment of SCs using RA analogues.

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, 6–8]. Because ALDH is key to retinoid acid (RA) signaling and retinoids are well known to promote differentiation of SCs [4], it follows that having ALDH in a SC provides the capacity for it to differentiate in response to retinoids.

Since *APC* mutations are known to increase WNT signaling in CRC, this raises the question: does increased WNT signaling lead to decreased retinoid signaling? Indeed, previous studies have implicated a role for APC in regulating RA biosynthesis and that *APC* mutations may lead to aberrant RA signaling [16, 36]. Notably, studies show that appropriately regulated WNT signaling is necessary for RA to induce neuronal differentiation of embryonic SCs [60]. Furthermore, not only does WNT suppress retinoid signaling, but conversely, increased RA signaling diminishes the ability of WNT signaling to block retinoid induction of the neural differentiation of SCs [61, 62]. That WNT signaling must be downregulated for neural differentiation to be inducible by RA treatment helps explain how *APC* mutation and increased WNT signaling might prevent maturation of ALDH+ colonic SCs in CRC development. Thus, it appears that *APC* mutations may alter the ability of

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**Figure 2.**

*cosmic).*

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

contributes to the development of CRC.

of the ALDH+ SC population size in CRC [39, 46].

ALDH+ SCs to differentiate in response to retinoids, which would lead to expansion

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. [63] 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,

**7. Bioinformatics analysis of retinoid signaling components in CRC**

*Bioinformatics analyses of RA signaling genes in CRC, including overexpression (a) and mutations (b). Bioinformatics data derived from cosmic catalog of somatic mutations in cancer (https://cancer.sanger.ac.uk/* *Colorectal Cancer*

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**

selective treatment of SCs using RA analogues.

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 [40] and WNT signaling, another key developmental pathway, has an opposing effect on embryonic SCs [41]. The idea that the mechanisms that regulate embryonic SCs are the same mechanisms that become dysregulated in the SC etiology of cancer [42] is intriguing because some scientists view cancer as aberrant organogenesis [43] and metastases as aberrant morphogenesis [44]. (ii) *APC* mutations occur in most CRCs (nearly 90%) during CRC development and *APC* mutation leads to constitutively activated WNT signaling. (iii) *APC* mutations that drive CRC development appear to do so by causing SC overpopulation [45]. (iv) ALDH, a key component in RA signaling, marks colonic SCs and tracks SC overpopulation during CRC development. Indeed, our research team [39, 46–49] and others [50–52] 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 [53]. For example, ALDH+ cells possess self-renewing ability as shown by sphere-forming ability *in vitro* and tumor-initiating ability in mice [46, 51, 52, 54]. The drug resistance property of ALDH+ SCs comes from aldehyde dehydrogenase's enzymatic function, which is the cell's natural detoxification mechanism [50, 55, 56]. The ability of ALDH+ cells to differentiate comes from ALDH's functional role in the RA signaling pathway [5, 13, 57–59]. Moreover, we examined ALDH+ cells from colon tissues and observed that retinoid receptors RXR and RAR are selectively expressed in ALDH+ cells [39], which indicates that RA signaling mainly occurs via ALDH+ SCs. That RA signaling primarily occurs in ALDH+ stem cells provides a mechanism for

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, 6–8]. Because ALDH is key to retinoid acid (RA) signaling and retinoids are well known to promote differentiation of SCs [4], it follows that having ALDH in a SC provides the capacity for it to differentiate in response to retinoids. Since *APC* mutations are known to increase WNT signaling in CRC, this raises the question: does increased WNT signaling lead to decreased retinoid signaling? Indeed, previous studies have implicated a role for APC in regulating RA biosynthesis and that *APC* mutations may lead to aberrant RA signaling [16, 36]. Notably, studies show that appropriately regulated WNT signaling is necessary for RA to induce neuronal differentiation of embryonic SCs [60]. Furthermore, not only does WNT suppress retinoid signaling, but conversely, increased RA signaling diminishes the ability of WNT signaling to block retinoid induction of the neural differentiation of SCs [61, 62]. That WNT signaling must be downregulated for neural differentiation to be inducible by RA treatment helps explain how *APC* mutation and increased WNT signaling might prevent maturation of ALDH+ colonic SCs in CRC development. Thus, it appears that *APC* mutations may alter the ability of

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ALDH+ SCs to differentiate in response to retinoids, which would lead to expansion of the ALDH+ SC population size in CRC [39, 46].
