Barbara G. Heerdt

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.

Mitochondria: integrators in tumorigenesis?

The mitochondrial genomes of tumor cells accumulate mutations during transformation. A new study raises questions regarding the way in which mutations accumulate and has implications for the potential role of mitochondrial function in tumor phenotype.

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.

Proteomic changes during intestinal cell maturation in vivo.

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated.

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.

Changes in the number of mitochondrial genomes during human development.

Using a cDNA probe for the mitochondrially encoded third subunit of cytochrome c oxidase (COIII) we found a progressive increase in the number of mitochondrial DNA molecules in specific human tissues during normal fetal development. The data indicate that the tissue, rather than the final number of mitochondrial genomes, apparently plays a dominant role in determining the gestational stage at which the adult complement of this DNA is established.

Cellular Mechanisms of Risk and Transformation

ABSTRACT Our early work using the first array and imaging methods for the quantitative analysis of the expression of 4000 cDNA sequences suggested that modulation of mitochondrial gene expression was a factor in determining whether colonic epithelial cells displayed a differentiated or transformed phenotype. We have since dissected a pathway in which mitochondrial function is a key element in determining the probability of cells undergoing cell‐cycle arrest, lineage‐specific differentiation, and cell death. Moreover, this pathway is linked to signaling through β‐catenin‐Tcf, but in a manner that is independent of effects of the APC gene on β‐catenin‐Tcf activity. Utilization of unique mouse genetic models of intestinal tumorigenesis has confirmed that mitochondrial function is an important element in generation of apoptotic cells in the colon in vivo and has demonstrated that modulation of cell death may be involved in intestinal tumor progression rather than initiation. Normal spatial and temporal patterns of cell proliferation, differentiation, and apoptosis in the colonic mucosa are determined by developmentally programmed genetic signals and external signals generated by homo‐ and heterotypic cell interactions, humoral agents, and lumenal contents. Mitochondrial function may play a pivotal role in integrating these signals and in determining probability of cells entering different maturation pathways. How this is accomplished is under investigation using high‐density cDNA microarrays.

Short-Chain Fatty Acids and Molecular and Cellular Mechanisms of Colonic Cell Differentiation and Transformation

There has been enormous progress in defining structural alterations of genes common in colonic cancer. Accumulation of mutations and deletions in APC, p53, DCC, and Ki-ras are the sine qua non of the disease, although the roles and interactions of these genetic alterations in the biological and clinical heterogeneity of the disease are not understood. Of even greater interest from the point of view of disease prevention are inherited mutations found in the APC gene and in genes which encode mismatch repair functions, which lead to the high frequency of colonic cancer in familial polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC) families, respectively1–6. However, unlike inherited childhood cancers, such as retinoblastoma and Wilm’s tumor, inherited colon cancer takes decades to develop. This can be partially understood in the context of what has been learned regarding genetic alterations in sporadic cancers. For example, one may assume that the inherited mutation supplies the first of a series of genetic alterations, and by so doing reduces the overall time and elevates the probability of accumulating the necessary number of alterations in genes such as those cited above. In the case of FAP, the inheritance of a mutant allele for APC provides one of the key events directly, since mutations in the gene occur somatically in over 70% of human colon tumors, and the mutation is sufficient to initiate development of intestinal tumors in mice7,8. In HNPCC, the situation is more complicated, in that the inherited mutations in genes responsible for DNA mismatch repair fail to repair errors which arise during DNA synthesis at tens of thousands of loci throughout the genome, and it must be presumed that among this plethora of changes there are key genes, again perhaps coincident with some of those mentioned above, which are eventual targets that lead to tumor formation.

Application of Gene Expression Profiling to Colon Cell Maturation, Transformation and Chemoprevention

Methods for high-throughput analysis of profiles of gene expression that assay thousands of genes simultaneously are powerful approaches for understanding and classifying cell and tissue phenotype. This includes analysis of normal pathways of cell maturation and their perturbation in transformation, the sensitivity and mechanism of response of normal and tumor cells to physiological and pharmacological agents, and modulation of tumor risk and progression by nutritional factors. However, the complex data generated by such approaches raise difficulties in analysis. We will describe some of the methods we have used in analyzing databases generated in a number of projects in our laboratories. These include: the role of k-ras mutations in colon cell transformation; the role of p21(WAF1/cip1) in intestinal tumor formation and response to sulindac; the development of the absorptive and goblet cell lineages; sensitivity of colonic cells to chemotherapeutic agents; mechanisms that regulate c-myc expression utilizing novel methods of transcriptional imaging; and interaction of nutritional and genetic factors in modulation of intestinal tumor formation.