Abstract
TNS4 (Tensin 4 or Cten) is a putative oncogene in colorectal cancer (CRC) with a role in regulating cell adhesion, motility, invasion, and epithelial to mesenchymal transition (EMT). The objective is to study the role of TNS4 in CRC using more realistic models of the tumor microenvironment. CRC cells expressing TdTomato protein and shTNS4/shLUC hairpin oligos are grown in 3D spheroids with and without cancer-associated fibroblasts (CAFs). Adhesiveness to collagen I and CAFs is assessed in 2D and cell proliferation, volume, and invasion are assessed in 3D conditions. The role of TNS4 knockdown in gefitinib chemosensitivity and epidermal growth factor receptor (EGFR) and Ras protein levels are also tested. In general, TNS4 knockdown increases cell proliferation in cell lines producing compact spheroids. The addition of CAFs in spheroids supports CRC cell proliferation, whereas CAFs themselves do not proliferate, but increases ECM degradation. TNS4 knockdown reduces adhesiveness and 3D invasion and disrupts EGFR signaling which results in increased sensitivity to Gefitinib. In conclusion, in a 3D spheroid model, TNS4 inhibits cell proliferation and promotes cell invasion into the ECM, possibly by adhesion to the ECM and stromal cells. TNS4 knockdown enhances sensitivity to the EGFR inhibitor gefitinib and may be helpful for Kirsten ras oncogene homolog mutant CRC patients.
Highlights
Introduction absence ofTNS4, the effects of TGF-ß stimulation on inducing motility and invasion were abrogated, but TGF-ß-induced pro-TNS4 or Cten (C-terminal tensin-like) is a tensin local- liferation was not affected.[13]
Whereas a tendency for reduced adhesion to collagen type I could be observed for all shTNS4 colorectal cancer (CRC) cell lines, the difference to shLUC controls was only significant in HT29 (p = 0.0205) and SW480 (p = 0.0245) (Figure 2A)
The reduced adhesion to cancer-associated fibroblasts (CAFs) upon TNS4 knockdown could have an effect in the expansion and paracrine growth signaling depending on cell-to-cell contact between CAFs and CRC cells, leading to increased fluorescence signal in shTNS4 versus shLUC + CAFs spheroids
Summary
Whereas a tendency for reduced adhesion to collagen type I could be observed for all shTNS4 CRC cell lines, the difference to shLUC controls was only significant in HT29 (p = 0.0205) and SW480 (p = 0.0245) (Figure 2A). The reduced adhesion to CAFs upon TNS4 knockdown could have an effect in the expansion and paracrine growth signaling depending on cell-to-cell contact between CAFs and CRC cells, leading to increased fluorescence signal in shTNS4 versus shLUC + CAFs spheroids. A reduced spheroid volume seen in shTNS4 + CAFs compared to shLUC + CAFs could be caused by increased compactness of the CRC cells, unsupported by a CAFs scaffold. Following TNS4 stable knockdown, transduction with TdTomato lentivirus allowed us to follow the growth of the CRC cell population within the spheroids by using a conventional fluorescent plate reader to detect expression of TdTomato fluorescent protein. Whilst DLD1, HT29 and HCT116 formed compact spheroids of round uniform shape, SW480, and SW620 produced aggregates and LS1034 produced a globous irregular shape. (Figure S1, Supporting Information)
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