TPM4 overexpression modifies differentiated colon epithelial cell barrier integrity

Colon epithelial cell migration out of the stem cell compartment in colon crypts is essential for normal differentiation and growth arrest. Altered expression of contractility- and migration-specific genes may disrupt these processes and contribute to early stages of colon tumorigenesis. One such gene, tropomyosin-4 ( TPM4 ), is overexpressed in early and late stages of colorectal cancer. Determining how TPM4 overexpression contributes to colon cell tumorigenesis may identify novel therapeutic targets. We hypothesized that TPM4 overexpression may promote colon tumorigenesis by disrupting cell-cell junctions and reducing epithelial barrier integrity in differentiating colon epithelial cells. To test this, we utilized Caco2 cells, an in vitro model of colon epithelial cell differentiation derived from a human colon adenocarcinoma. Our approach was to determine whether artificially increasing TPM4 expression by stable transfection in differentiating Caco2 cells, when TPM4 is normally suppressed, will decrease integrity of the Caco-2 cell monolayer. To assess this, we measured transepithelial electrical resistance (TEER), an indicator of cell-cell junction integrity, in differentiating TPM4 -overexpressing Caco-2 monolayers relative to control transfectants. 100,000 Caco2 cells were seeded in 6 well filter inserts in wells of a 6-well plate at day 0. TEER measurements were recorded in triplicate every other day from day 6 to 24 using the Millicell ERS 3.0 Digital Voltohmmeter. We observed significant differences between TEER measurements in two clones of TPM4 -overexpressing Caco2 cells and control transfectants. TEER values in control empty vector stable transfectants plateaued between 1237-1445 ohm-sq. cm, while those in two TPM4 -overexpressing stable clones plateaued between 464-898 ohm-sq. cm (clone 1) and 1743-1868 ohm-sq. cm (clone 7). Clone 1 TEER values plateaued at a similar time point (day 12) as empty vector control, whereas clone 7 TEER values plateaued later (day 16). In addition, we observed alterations in expression of genes encoding junctional complex proteins in TPM4-overexpressing cells relative to controls. These results indicate that TPM4 overexpression modulates the integrity of cell-cell junctions in an in vitro model of differentiating colon epithelial cells. Future experiments will determine whether differences observed between the two distinct clones may be due to the extent of TPM4 overexpression in each clone. Our study suggests that sustained TPM4 overexpression may disrupt colon epithelial cell differentiation and promote tumorigenesis by altering normal epithelial barrier formation and junctional integrity. Therefore, we identify a potential mechanism that may link TPM4 overexpression to early colorectal tumorigenesis and elucidate possible novel approaches to prevent and treat colon cancer, particularly at early stages.