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

siRNA mediated down-regulation of MUC1 does not affect intrinsic Δψm-linked constitutive hypoxia-independent VEGF secretion.

<p>Cells were mock, MUC1- or NT-siRNA transfected and 48 hours later VEGF secretion levels were determined in harvested conditioned tissue culture medium.</p

MUC1 siRNA-mediates down regulation of MUC1 expression in subcloned cell lines with different intrinsic Δψm.

<p>Subcloned cell lines with intrinsic Δψm higher (↑Δψm), lower (↓Δψm) and comparable (≈Δψm) to that of the population, and the SW620 parental population, were mock transfected, or transfected with NT- or MUC1-siRNA. Twenty-four, 48 and 72 hours later levels of MUC1 mRNA were determined by qRT-PCR, normalized to GAPDH. The level of MUC1 mRNA in the siRNA-MUC1 transfected cell line with ↑Δψm did not differ significantly from that of mock transfected SW620 cell population 48 and 72 hours after transfection (<i>P</i>>0.05).</p

siRNA-mediated down regulation of MUC1 N-ter and mt-associated C-ter levels does not affect the Δψm.

<p>Cells were mock, MUC1- or NT-siRNA transfected. Twenty-four, 48 and 72 hours later, levels of cell surface MUC1 N-ter were determined by flow cytometry (<b>A</b>); levels of mt-associated MUC1 C-ter determined by quantitative immunoblotting (<b>B</b>) and the Δψm was determined by JC-1 staining and flow cytometry.</p