Michele A. Houston

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.

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.

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.

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.

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 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.

Abstract 1708: Intestinal stem cells are sentinel cells for nutritional exposure

Lgr5+ CBC cells at the intestinal crypt bottom have been identified as canonical stem cells responsible for mouse intestinal mucosal homeostasis. The data establishing this are derived from mice fed standard diets that generate 25(OH)D serum levels well above the range well documented for the US population, with these higher levels reflecting that of “0” percent of the population. This is important because the Clevers group reported that expression of the vitamin D receptor (Vdr) is high in Lgr5+ cells, and decreased substantially in their immediate daughter cells, suggesting signaling through the Vdr is important for Lgr5+ CBC stem cell functions. Consistent with this, we showed that lower dietary vitamin D3 in the context of a purified western-style rodent diet, or lowering only vitamin D3 in control rodent AIN76A diet, or knockout of the vitamin D receptor specifically in Lgr5+ cells compromised the ability of Lgr5+ cells to lineage trace. Moreover, exposure to lower dietary vitamin D3 characteristic of a large segment of the US population substantially reduced the ability of introduction of a mutant Apc allele into Lgr5+ cells to generate tumors. However, the mucosa of mice exposed to these lower levels of vitamin D3 appears histologically normal and the mice are healthy. Consistent with this, Bmi1+ cells are mobilized to expand and lineage trace in mice in which lower dietary vitamin D3 established 25(OH)D levels similar to those found in the US population. Whether cells from other compartments reported to exhibit intestinal stem cell functions are also mobilized, and the relative contribution of cells from different compartments to intestinal homeostasis across the spectrum of 25(OH)D levels that characterize the human population, are under investigation. Thus, vitamin D3 is a critical factor in programming intestinal cells from different compartments to express stem cell functions. The extent and nature of this programming has been investigated by RNAseq of specific cell populations from mice fed different diets, and a number of important pathways identified linked to vitamin D exposure level. Moreover, the relationship of change in functional status for cells from different compartments (ie quiescent versus mobilized) to the mutational spectrum and load that accumulate in these cells has been analyzed by single cell exome sequencing. The sensitivity of intestinal stem cell function to vitamin D exposure suggests that the well-documented plasticity of intestinal stem cells may have evolved to permit adaptation to different environments. Moreover, this raises the questions of whether other nutrients key to maintaining intestinal homeostasis also modulate the relative contribution of different stem cell populations to intestinal homeostasis, and if the multiple cell populations capable of stem cell functions in the intestine can serve as sentinel cells for which analyses of their molecular programming are sensitive and responsive assays of nutritional exposures. Citation Format: Leonard H. Augenlicht, Wenge Li, Karina Peregrina, Michele Houston. Intestinal stem cells are sentinel cells for nutritional exposure. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1708.