The nutritional environment determines which and how intestinal stem cells contribute to homeostasis and tumorigenesis.

Sporadic colon cancer accounts for approximately 80% of colorectal cancer (CRC) with high incidence in Western societies strongly linked to long-term dietary patterns. A unique mouse model for sporadic CRC results from feeding a purified rodent Western-style diet (NWD1) recapitulating intake for the mouse of common nutrient risk factors each at its level consumed in higher risk Western populations. This causes sporadic large and small intestinal tumors in wild-type mice at an incidence and frequency similar to that in humans. NWD1 perturbs intestinal cell maturation and Wnt signaling throughout villi and colonic crypts and decreases mouse Lgr5hi intestinal stem cell contribution to homeostasis and tumor development. Here we establish that NWD1 transcriptionally reprograms Lgr5hi cells, and that nutrients are interactive in reprogramming. Furthermore, the DNA mismatch repair pathway is elevated in Lgr5hi cells by lower vitamin D3 and/or calcium in NWD1, paralleled by reduced accumulation of relevant somatic mutations detected by single-cell exome sequencing. In compensation, NWD1 also reprograms Bmi1+ cells to function and persist as stem-like cells in mucosal homeostasis and tumor development. The data establish the key role of the nutrient environment in defining the contribution of two different stem cell populations to both mucosal homeostasis and tumorigenesis. This raises important questions regarding impact of variable human diets on which and how stem cell populations function in the human mucosa and give rise to tumors. Moreover, major differences reported in turnover of human and mouse crypt base stem cells may be linked to their very different nutrient exposures.

Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions.

Lgr5+ intestinal crypt base columnar cells function as stem cells whose progeny populate the villi, and Lgr5+ cells in which Apc is inactivated can give rise to tumors. Surprisingly, these Lgr5+ stem cell properties were abrogated by the lower dietary vitamin D and calcium in a semi-purified diet that promotes both genetically initiated and sporadic intestinal tumors. Inactivation of the vitamin D receptor in Lgr5+ cells established that compromise of Lgr5 stem cell function was a rapid, cell autonomous effect of signaling through the vitamin D receptor. The loss of Lgr5 stem cell function was associated with presence of Ki67 negative Lgr5+ cells at the crypt base. Therefore, vitamin D, a common nutrient and inducer of intestinal cell maturation, is an environmental factor that is a determinant of Lgr5+ stem cell functions in vivo. Since diets used in reports that establish and dissect mouse Lgr5+ stem cell activity likely provided vitamin D levels well above the range documented for human populations, the contribution of Lgr5+ cells to intestinal homeostasis and tumor formation in humans may be significantly more limited, and variable in the population, then suggested by published rodent studies.

Dynamic Intestinal Stem Cell Plasticity and Lineage Remodeling by a Nutritional Environment Relevant to Human Risk for Tumorigenesis.

New Western-style diet 1 (NWD1), a purified diet establishing mouse exposure to key nutrients recapitulating levels that increase human risk for intestinal cancer, reproducibly causes mouse sporadic intestinal and colonic tumors reflecting human etiology, incidence, frequency, and lag with developmental age. Complex NWD1 stem cell and lineage reprogramming was deconvolved by bulk and single-cell RNA sequencing, single-cell Assay for Transposase-Accessible Chromatin using sequencing, functional genomics, and imaging. NWD1 extensively, rapidly, and reversibly, reprogrammed Lgr5hi stem cells, epigenetically downregulating Ppargc1a expression, altering mitochondrial structure and function. This suppressed Lgr5hi stem cell functions and developmental maturation of Lgr5hi cell progeny as cells progressed through progenitor cell compartments, recapitulated by Ppargc1a genetic inactivation in Lgr5hi cells in vivo. Mobilized Bmi1+, Ascl2hi cells adapted lineages to the nutritional environment and elevated antigen processing and presentation pathways, especially in mature enterocytes, causing chronic, protumorigenic low-level inflammation. There were multiple parallels between NWD1 remodeling of stem cells and lineages with pathogenic mechanisms in human inflammatory bowel disease, also protumorigenic. Moreover, the shift to alternate stem cells reflects that the balance between Lgr5-positive and -negative stem cells in supporting human colon tumors is determined by environmental influences. Stem cell and lineage plasticity in response to nutrients supports historic concepts of homeostasis as a continual adaptation to environment, with the human mucosa likely in constant flux in response to changing nutrient exposures. IMPLICATIONS: Although oncogenic mutations provide a competitive advantage to intestinal epithelial cells in clonal expansion, the competition is on a playing field dynamically sculpted by the nutritional environment, influencing which cells dominate in mucosal maintenance and tumorigenesis.

Intestinal stem cell aging at single-cell resolution: Transcriptional perturbations alter cell developmental trajectory reversed by gerotherapeutics.

The intestinal epithelium consists of cells derived from continuously cycling Lgr5hi intestinal stem cells (Lgr5hi ISCs) that mature developmentally in an ordered fashion as the cells progress along the crypt-luminal axis. Perturbed function of Lgr5hi ISCs with aging is documented, but the consequent impact on overall mucosal homeostasis has not been defined. Using single-cell RNA sequencing, the progressive maturation of progeny was dissected in the mouse intestine, which revealed that transcriptional reprogramming with aging in Lgr5hi ISCs retarded the maturation of cells in their progression along the crypt-luminal axis. Importantly, treatment with metformin or rapamycin at a late stage of mouse lifespan reversed the effects of aging on the function of Lgr5hi ISCs and subsequent maturation of progenitors. The effects of metformin and rapamycin overlapped in reversing changes of transcriptional profiles but were also complementary, with metformin more efficient than rapamycin in correcting the developmental trajectory. Therefore, our data identify novel effects of aging on stem cells and the maturation of their daughter cells contributing to the decline of epithelial regeneration and the correction by geroprotectors.

Vitamin D and the nutritional environment in functions of intestinal stem cells: Implications for tumorigenesis and prevention.

Sporadic colon cancer accounts for ∼80% of CRC, with high incidence in western societies strongly linked to dietary patterns. The only mouse model for sporadic CRC results from feeding mice a purified rodent western-style diet (NWD1), establishing mouse intake of several common nutrients that mimic for each its level consumed in western populations at higher risk for colon cancer (higher fat, lower vitamin D3, calcium, methyl donors and fiber). This causes sporadic colon and small intestinal tumors at an incidence and frequency similar to that of humans. NWD1 perturbs intestinal cell maturation and Wnt signaling throughout villi and colonic crypts before tumors are detected. Surprisingly, feeding NWD1 decreases mouse Lgr5hi intestinal stem cell contribution to homeostasis and tumorigenesis, associated with extensive Lgr5hi cell transcriptional reprogramming, with nutrient levels interactive in these effects. There is a key impact of the lower vitamin D3 in NWD1 and its signaling through the Vdr. The DNA mismatch repair pathway is elevated in Lgr5hi cells by lower vitamin D3 and/or calcium in NWD1, reducing accumulation of relevant somatic mutations detected by single cell exome sequencing. There are also alterations in metabolic pathways, including down-regulation of oxidative phosphorylation. In compensation for compromise of Lgr5hi cells, NWD1 also reprograms cells derived from the Bmi1+ population, defined as those cells marked in Bmi1creERT2, Rosa26tom mice following tamoxifen injection, and at least a portion of these cells then function and persist as stem-like cells in mucosal homeostasis and tumorigenesis. The data establish a key role of the nutrient environment, and vitamin D signaling, in defining contribution of at least two different stem cell populations to mucosal homeostasis and tumorigenesis. This raises significant questions regarding impact of variable human diets on which and how multiple potential intestinal stem cell populations function in the human and give rise to tumors. Moreover, genetic and epigenetic changes in long-lived stem cells have important implications for understanding the effects of vitamin D and other nutrients on intestinal homeostasis and on intervention strategies for altering probability of tumor development.

Growth properties of colonic tumor cells are a function of the intrinsic mitochondrial membrane potential.

Development of malignant transformation in the colonic mucosa includes disruption in the equilibrium between proliferation and apoptosis, decreased expression and deletions of the mitochondrial genome, alterations in mitochondrial enzymatic activity, and elevations in the mitochondrial membrane potential (Deltapsim). Focusing on the role of the Deltapsim in tumor development and progression, we generated novel isogenic colonic carcinoma cell lines that exhibit highly significant, stable differences in their intrinsic Deltapsim. Using these cell lines, we have recently shown that the intrinsic Deltapsim has a significant influence on steady state mitochondrial activity and the extent to which cells enter butyrate-mediated growth arrest and apoptotic cascades. Here, we report that the Deltapsim is also profoundly linked to important tumorigenic properties of the cells. Compared with cells with lower Deltapsim, cells with elevated intrinsic Deltapsim have an enhanced capacity to (a) respond to hypoxia by avoiding apoptosis and initiating angiogenesis, (b) escape anoikis and grow under anchorage-independent conditions, and (c) invade the basement membrane. Combined with our previous work, these data implicate the intrinsic Deltapsim of colonic carcinoma cells in determining the probability of tumor expansion and progression.

The intrinsic mitochondrial membrane potential of colonic carcinoma cells is linked to the probability of tumor progression.

We subcloned cell lines from SW620 cells establishing that, despite the dynamic nature of the mitochondrial membrane potential (Deltapsim), there are significant and stable differences in the intrinsic Deltapsim among cells within an in vitro population of human colonic carcinoma cells. Whereas more dramatic differences in Deltapsim would likely perturb essential mitochondrial functions, the differences in Deltapsim of the subclones did not affect steady-state reactive oxygen species levels, electron transport activity, or cellular viability and growth rates. However, the differences in intrinsic Deltapsim had a significant effect on the tumorigenic behavior of the cells. Subcloned cell lines with higher Deltapsim were more likely to exhibit elevated steady-state levels of vascular endothelial growth factor and matrix metalloproteinase 7, and increased invasive behavior (properties associated with tumor progression), than cells with lower intrinsic Deltapsim, whereas cells with lower Deltapsim were more likely to respond to the chemopreventive activities of butyrate, including Deltapsim dissipation, growth arrest, and apoptosis, than cells with higher Deltapsim. Therefore, these data establish that the probability for tumor development and progression is linked to stable differences in the intrinsic Deltapsim of colonic epithelial cells.

MUC2 mucin deficiency alters inflammatory and metabolic pathways in the mouse intestinal mucosa.

The mucus layer in the intestine affects several aspects of intestinal biology, encompassing physical, chemical protection, immunomodulation and growth, thus contributing to homeostasis. Mice with genetic inactivation of the Muc2 gene, encoding the MUC2 mucin, the major protein component of mucus, exhibit altered intestinal homeostasis, which is strictly dependent on the habitat, likely due to differing complements of intestinal microbes. Our previous work established that Muc2 deficiency was linked to low chronic inflammation resulting in tumor development in the small, large intestine including the rectum. Here, we report that inactivation of Muc2 alters metabolic pathways in the normal appearing mucosa of Muc2-/- mice. Comparative analysis of gene expression profiling of isolated intestinal epithelial cells (IECs) and the entire intestinal mucosa, encompassing IECs, immune and stromal cells underscored that more than 50% of the changes were common to both sets of data, suggesting that most alterations were IEC-specific. IEC-specific expression data highlighted perturbation of lipid absorption, processing and catabolism linked to altered Pparα signaling in IECs. Concomitantly, alterations of glucose metabolism induced expression of genes linked to de novo lipogenesis, a characteristic of tumor cells. Importantly, gene expression alterations characterizing Muc2-/- IECs are similar to those observed when analyzing the gene expression signature of IECs along the crypt-villus axis in WT B6 mice, suggesting that Muc2-/- IECs display a crypt-like gene expression signature. Thus, our data strongly suggest that decreased lipid metabolism, and alterations in glucose utilization characterize the crypt proliferative compartment, and may represent a molecular signature of pre-neoplastic lesions.

The intrinsic mitochondrial membrane potential (Deltapsim) is associated with steady-state mitochondrial activity and the extent to which colonic epithelial cells undergo butyrate-mediated growth arrest and apoptosis.

Transformation of colonic epithelial cells is characterized by decreased mitochondrial activity, increased mitochondrial membrane potential (Deltapsi(m)), and disruptions in the equilibrium between cell proliferation and death by apoptosis. We have previously shown that an intact Deltapsi(m) is essential for growth arrest and apoptosis induced by butyrate, a physiological regulator of maturation in these cells, suggesting a role for the Deltapsi(m) in the initiation and integration of proliferation and apoptotic pathways. To extend this work, we have generated isogenic cell lines, from SW620 human colonic carcinoma cells, which exhibit significant differences in intrinsic Deltapsi(m). These differences in Deltapsi(m) are not linked to alterations in viability, Bcl-2 levels, or the differentiation status of the cells. However, compared with parental cells and those with increased Deltapsi(m), cells with decreased intrinsic Deltapsi(m) exhibit significantly higher levels of steady-state mitochondrial mRNA and butyrate-induced p21(WAF1/Cip1) and G(0)-G(1) arrest. Moreover, despite butyrate-mediated translocation of proapoptotic Bax and Bak to the mitochondria, fewer cells with elevated intrinsic Deltapsi(m) exhibit concomitant cytochrome c release, and cells with elevated Deltapsi(m) undergo significantly lower levels of Deltapsi(m) dissipation and apoptosis than parental cells, or cells with decreased Deltapsi(m). Homeostasis of the colonic mucosa depends on balancing cell proliferation with apoptosis, and mitochondrial abnormalities are associated with disruptions in this balance. Thus, by affecting steady-state mitochondrial activity and the extent to which cells enter growth arrest and apoptotic cascades, these data establish a role for the intrinsic Deltapsi(m) in contributing to the probability of colonic tumorigenesis and progression.

Stable differences in intrinsic mitochondrial membrane potential of tumor cell subpopulations reflect phenotypic heterogeneity.

Heterogeneity among cells that constitute a solid tumor is important in determining disease progression. Our previous work established that, within a population of metastatic colonic tumor cells, there are minor subpopulations of cells with stable differences in their intrinsic mitochondrial membrane potential (ΔΨm), and that these differences in ΔΨm are linked to tumorigenic phenotype. Here we expanded this work to investigate primary mammary, as well as colonic, tumor cell lines. We show that within a primary mammary tumor cell population, and in both primary and metastatic colonic tumor cell populations, there are subpopulations of cells with significant stable variations in intrinsic ΔΨm. In each of these 3 tumor cell populations, cells with relatively higher intrinsic ΔΨm exhibit phenotypic properties consistent with promotion of tumor cell survival and expansion. However, additional properties associated with invasive potential appear in cells with higher intrinsic ΔΨm only from the metastatic colonic tumor cell line. Thus, it is likely that differences in the intrinsic ΔΨm among cells that constitute primary mammary tumor populations, as well as primary and metastatic colonic tumor populations, are markers of an acquired tumor phenotype which, within the context of the tumor, influence the probability that particular cells will contribute to disease progression.

Intrinsic mitochondrial membrane potential and associated tumor phenotype are independent of MUC1 over-expression.

We have established previously that minor subpopulations of cells with stable differences in their intrinsic mitochondrial membrane potential (Δψm) exist within populations of mammary and colonic carcinoma cells and that these differences in Δψm are linked to tumorigenic phenotypes consistent with increased probability of participating in tumor progression. However, the mechanism(s) involved in generating and maintaining stable differences in intrinsic Δψm and how they are linked to phenotype are unclear. Because the mucin 1 (MUC1) oncoprotein is over-expressed in many cancers, with the cytoplasmic C-terminal fragment (MUC1 C-ter) and its integration into the outer mitochondrial membrane linked to tumorigenic phenotypes similar to those of cells with elevated intrinsic Δψm, we investigated whether endogenous differences in MUC1 levels were linked to stable differences in intrinsic Δψm and/or to the tumor phenotypes associated with the intrinsic Δψm. We report that levels of MUC1 are significantly higher in subpopulations of cells with elevated intrinsic Δψm derived from both mammary and colonic carcinoma cell lines. However, using siRNA we found that down-regulation of MUC1 failed to significantly affect either the intrinsic Δψm or the tumor phenotypes associated with increased intrinsic Δψm. Moreover, whereas pharmacologically mediated disruption of the Δψm was accompanied by attenuation of tumor phenotype, it had no impact on MUC1 levels. Therefore, while MUC1 over-expression is associated with subpopulations of cells with elevated intrinsic Δψm, it is not directly linked to the generation or maintenance of stable alterations in intrinsic Δψm, or to intrinsic Δψm associated tumor phenotypes. Since the Δψm is the focus of chemotherapeutic strategies, these data have important clinical implications in regard to effectively targeting those cells within a tumor cell population that exhibit stable elevations in intrinsic Δψm and are most likely to contribute to tumor progression.

Apoptotic sensitivity of colon cancer cells to histone deacetylase inhibitors is mediated by an Sp1/Sp3-activated transcriptional program involving immediate-early gene induction.

Histone deacetylase inhibitors (HDACi) induce growth arrest and apoptosis in colon cancer cells and are being considered for colon cancer therapy. The underlying mechanism of action of these effects is poorly defined with both transcription-dependent and -independent mechanisms implicated. We screened a panel of 30 colon cancer cell lines for sensitivity to HDACi-induced apoptosis and correlated the differences with gene expression patterns induced by HDACi in the five most sensitive and resistant lines. A robust and reproducible transcriptional response involving coordinate induction of multiple immediate-early (fos, jun, egr1, egr3, atf3, arc, nr4a1) and stress response genes (Ndrg4, Mt1B, Mt1E, Mt1F, Mt1H) was selectively induced in HDACi sensitive cells. Notably, a significant percentage of these genes were basally repressed in colon tumors. Bioinformatics analysis revealed that the promoter regions of the HDACi-induced genes were enriched for KLF4/Sp1/Sp3 transcription factor binding sites. Altering KLF4 levels failed to modulate apoptosis or transcriptional responses to HDACi treatment. In contrast, HDACi preferentially stimulated the activity of Spl/Sp3 and blocking their action attenuated both the transcriptional and apoptotic responses to HDACi treatment. Our findings link HDACi-induced apoptosis to activation of a Spl/Sp3-mediated response that involves derepression of a transcriptional network basally repressed in colon cancer.