BackgroundTumorigenesis is driven by stem cell (SC) overpopulation. Because ALDH is both a marker for SCs in many tissues and a key enzyme in retinoid acid (RA) signaling, we studied RA signaling in normal and malignant colonic SCs.HypothesisRA signaling regulates growth and differentiation of ALDH+ colonic SCs; dysregulation of RA signaling contributes to SC overpopulation and colorectal cancer (CRC) development.MethodsWe analyzed normal and malignant colonic tissues and CRC cell lines to see if retinoid receptors (RXR & RAR) are exclusively expressed in ALDH+ SCs, and if RA signaling changes during CRC development. We determined whether RA signaling regulates cancer SC (CSC) proliferation, differentiation, sphere formation, and population size.ResultsRXR & RAR were expressed in ALDH+ colonic SCs, but not in MCM2+ proliferative cells. Western blotting/immunostaining of CRCs revealed that RA signaling components become overexpressed in parallel with ALDH overexpression, which coincides with the known overpopulation of ALDH+ SCs that occurs during, and drives, CRC development. Treatment of SCs with all-trans retinoic acid (ATRA) decreased proliferation, sphere formation and ALDH+ SC population size, and induced differentiation along the neuroendocrine cell (NEC) lineage.ConclusionsRetinoid signaling, by regulating ALDH+ colonic CSCs, decreases SC proliferation, sphere formation, and population size, and increases SC differentiation to NECs. Dysregulation of RA signaling in colonic SCs likely contributes to overpopulation of ALDH+ SCs and CRC growth.ImplicationsThat retinoid receptors RXR and RAR are selectively expressed in ALDH+ SCs indicates RA signaling mainly occurs via ALDH+ SCs, which provides a mechanism to selectively target CSCs.
BackgroundNeuroendocrine cells (NECs) reside adjacent to colonic stem cells (SCs) in the crypt stem cell (SC) niche, but how NECs are involved in regulation of SCs is unclear. We investigated NECs expressing somatostatin (SST) and somatostatin receptor type 1 (SSTR1) because SST inhibits intestinal proliferation. Hypothesis: SSTR1 cells maintain SCs in a quiescent state, and aberrant SST signaling contributes to SC overpopulation in colorectal cancer (CRC).MethodsThe proportion of SCs to NECs cells was quantified, by flow cytometry, in CRC cell lines and primary normal/tumor tissues based on cellular ALDH and SSTR1 levels, respectively. Doubling time and sphere-formation was used to evaluate cell proliferation and stemness. CRC cell lines were treated with exogenous SST and SST inhibitor cyclosomatostatin (cycloSST) and analyzed for changes in SCs and growth rate. Paracrine signaling between NECs and SCs was ascertained using transwell cultures of ALDH+ and SSTR1+ cells.ResultsIn CRC cell lines, the proportion of ALDH+ cells inversely correlates with proportion of SSTR1+ cells and with rate of proliferation and sphere-formation. While primary normal tissue shows SST and SSTR1 expression, CRC shows only SSTR1 expression. Moreover, ALDH+ cells did not show SST or SSTR1 expression. Exogenous SST suppressed proliferation but not ALDH+ population size or viability. Inhibition of SSTR1 signaling, via cycloSST treatment, decreased cell proliferation, ALDH+ cell population size and sphere-formation. When co-cultured with SSTR1+ cells, sphere-formation and cell proliferation of ALDH+ cells was inhibited.ConclusionThat each CRC cell line has a unique ALDH+/SSTR1+ ratio which correlates with its growth dynamics, suggests feedback mechanisms exist between SCs and NECs that contribute to regulation of SCs. The growth suppression by both SST and cycloSST treatments suggests that SST signaling modulates this feedback mechanism. The ability of SSTR1+ cells to decrease sphere formation and proliferation of ALDH+ cells in transwell cultures indicates that the ALDH subpopulation is regulated by SSTR1 via a paracrine mechanism. Since ALDH+ cells lack SST and SSTR1 expression, we conjecture that SST signaling controls the rate of NEC maturation as SCs mature along the NEC lineage, which contributes to quiescence of SCs and inhibition of proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2969-7) contains supplementary material, which is available to authorized users.
APC mutations drive human colorectal cancer (CRC) development. A major contributing factor is colonic stem cell (SC) overpopulation. But, the mechanism has not been fully identified. A possible mechanism is the dysregulation of neuroendocrine cell (NEC) maturation by APC mutations because SCs and NECs both reside together in the colonic crypt SC niche where SCs mature into NECs. So, we hypothesized that sequential inactivation of APC alleles in human colonic crypts leads to progressively delayed maturation of SCs into NECs and overpopulation of SCs. Accordingly, we used quantitative immunohistochemical mapping to measure indices and proportions of SCs and NECs in human colon tissues (normal, adenomatous, malignant), which have different APC -zygosity states. In normal crypts, many cells staining for the colonic SC marker ALDH1 co-stained for chromogranin-A (CGA) and other NEC markers. In contrast, in APC -mutant tissues from familial adenomatous polyposis (FAP) patients, the proportion of ALDH+ SCs progressively increased while NECs markedly decreased. To explain how these cell populations change in FAP tissues, we used mathematical modelling to identify kinetic mechanisms. Computational analyses indicated that APC mutations lead to: 1) decreased maturation of ALDH+ SCs into progenitor NECs (not progenitor NECs into mature NECs); 2) diminished feedback signaling by mature NECs. Biological experiments using human CRC cell lines to test model predictions showed that mature GLP-2R+ and SSTR1+ NECs produce, via their signaling peptides, opposing effects on rates of NEC maturation via feedback regulation of progenitor NECs. However, decrease in this feedback signaling wouldn’t explain the delayed maturation because both progenitor and mature NECs are depleted in CRCs. So the mechanism for delayed maturation must explain how APC mutation causes the ALDH+ SCs to remain immature. Given that ALDH is a key component of the retinoic acid (RA) signaling pathway, that other components of the RA pathway are selectively expressed in ALDH+ SCs, and that exogenous RA ligands can induce ALDH+ cancer SCs to mature into NECs, RA signaling must be attenuated in ALDH+ SCs in CRC. Thus, attenuation of RA signaling explains why ALDH+ SCs remain immature in APC mutant tissues. Since APC mutation causes increased WNT signaling in FAP and we found that sequential inactivation of APC in FAP patient tissues leads to progressively delayed maturation of colonic ALDH+ SCs, the hypothesis is developed that human CRC evolves due to an imbalance between WNT and RA signaling.
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