The anti-cancer effect of retinoic acid signaling in CRC occurs via decreased growth of ALDH+ colon cancer stem cells and increased differentiation of stem cells

Background: Tumorigenesis is driven by stem cell (SC) overpopulation. BecauseALDH 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.Hypothesis: RA signaling regulates growth and differentiation of ALDH+ colonicSCs dysregulation of RA signaling contributes to SC overpopulation and colorectalcancer (CRC) development.Methods: We analyzed normal and malignant colonic tissues and CRC cell linesto see if retinoid receptors (RXR &RAR) are exclusively expressed in ALDH+ SCs,and if RA signaling changes during CRC development. We determined whether RAsignaling regulates cancer SC (CSC) proliferation, differentiation, sphere formation,and population size.Results: RXR &RAR were expressed in ALDH+ colonic SCs, but not in MCM2+proliferative cells. Western blotting/immunostaining of CRCs revealed that RAsignaling 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 induceddifferentiation along the neuroendocrine cell (NEC) lineage.Conclusions: Retinoid signaling, by regulating ALDH+ colonic CSCs, decreases SCproliferation, sphere formation, and population size, and increases SC differentiation toNECs. Dysregulation of RA signaling in colonic SCs likely contributes to overpopulationof ALDH+ SCs and CRC growth.Implications: That retinoid receptors RXR and RAR are selectively expressed inALDH+ SCs indicates RA signaling mainly occurs via ALDH+ SCs, which provides amechanism to selectively target CSCs. © 2018 Impact Journals LLC. All rights reserved

Transcriptome profiling of spinal muscular atrophy motor neurons derived from mouse embryonic stem cells.

Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn-/-) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs

MicroRNA Expression Profiling of Normal and Malignant Human Colonic Stem Cells Identifies

MicroRNAs (miRNAs) have a critical role in regulating stem cells (SCs) during development, and because aberrant expression of miRNAs occurs in various cancers, our goal was to determine if dysregulation of miRNAs is involved in the SC origin of colorectal cancer (CRC). We previously reported that aldehyde dehydrogenase (ALDH) is a marker for normal and malignant human colonic SCs and tracks SC overpopulation during colon tumorigenesis. MicroRNA expression was studied in ALDH-positive SCs from normal and malignant human colon tissues by Nanostring miRNA profiling. Our findings show that: (1) A unique miRNA signature distinguishes ALDH-positive CRC cells from ALDH-positive normal colonic epithelial cells, (2) Expression of four miRNAs (miRNA200c, miRNA92a, miRNA20a, miRNA93) are significantly altered in CRC SCs compared to normal colonic SCs, (3) miRNA92a expression is also upregulated in ALDH-positive HT29 CRC SCs as compared to ALDH-negative SCs, (4) miRNA92a targets the 3\u27UTR of LRIG1 SC gene, and (5) miRNA92a modulates proliferation of HT29 CRC cells. Thus, our findings indicate that overexpression of miRNA92a contributes to the SC origin of CRC. Strategies designed to modulate miRNA expression, such as miRNA92a, may provide ways to target malignant SCs and to develop more effective therapies against CRC

Effect of HIP/ribosomal protein L29 deficiency on mineral properties of murine bones and teeth

Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues

Wound Healing and Cancer Stem Cells: Inflammation as a Driver of Treatment Resistance in Breast Cancer

The relationship between wound healing and cancer has long been recognized. The mechanisms that regulate wound healing have been shown to promote transformation and growth of malignant cells. In addition, chronic inflammation has been associated with malignant transformation in many tissues. Recently, pathways involved in inflammation and wound healing have been reported to enhance cancer stem cell (CSC) populations. These cells, which are highly resistant to current treatments, are capable of repopulating the tumor after treatment, causing local and systemic recurrences. In this review, we highlight proinflammatory cytokines and developmental pathways involved in tissue repair, whose deregulation in the tumor microenvironment may promote growth and survival of CSCs. We propose that the addition of anti-inflammatory agents to current treatment regimens may slow the growth of CSCs and improve therapeutic outcomes

MicroRNA Expression Profiling of Normal and Malignant Human Colonic Stem Cells Identifies miRNA92a as a Regulator of the LRIG1 Stem Cell Gene

MicroRNAs (miRNAs) have a critical role in regulating stem cells (SCs) during development, and because aberrant expression of miRNAs occurs in various cancers, our goal was to determine if dysregulation of miRNAs is involved in the SC origin of colorectal cancer (CRC). We previously reported that aldehyde dehydrogenase (ALDH) is a marker for normal and malignant human colonic SCs and tracks SC overpopulation during colon tumorigenesis. MicroRNA expression was studied in ALDH-positive SCs from normal and malignant human colon tissues by Nanostring miRNA profiling. Our findings show that: (1) A unique miRNA signature distinguishes ALDH-positive CRC cells from ALDH-positive normal colonic epithelial cells, (2) Expression of four miRNAs (miRNA200c, miRNA92a, miRNA20a, miRNA93) are significantly altered in CRC SCs compared to normal colonic SCs, (3) miRNA92a expression is also upregulated in ALDH-positive HT29 CRC SCs as compared to ALDH-negative SCs, (4) miRNA92a targets the 3′UTR of LRIG1 SC gene, and (5) miRNA92a modulates proliferation of HT29 CRC cells. Thus, our findings indicate that overexpression of miRNA92a contributes to the SC origin of CRC. Strategies designed to modulate miRNA expression, such as miRNA92a, may provide ways to target malignant SCs and to develop more effective therapies against CRC

APC mutations in human colon lead to decreased neuroendocrine maturation of ALDH+ stem cells that alters GLP-2 and SST feedback signaling: Clue to a link between WNT and retinoic acid signalling in colon cancer development.

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

Upstream regulator analysis of differentially expressed transcripts between mESC-derived Hb9 control and A2 SMA MNs.

<p>IPA of significantly modified upstream (<b>A</b>) transcriptional regulator, (<b>B</b>) endogenous signaling and (<b>C</b>) drug pathways in upregulated or downregulated transcripts in A2 SMA MNs compared to Hb9 control MNs. Significant upstream regulators were identified as those having an activation z-score greater than or equal to 2.0 for activated regulators or less than or equal to −2.0 for inhibited regulators.</p

RNA-Seq reads mapped to NCBI mouse genome build 37 using TopHat.

<p>RNA-Seq reads mapped to NCBI mouse genome build 37 using TopHat.</p