27 Visualise cancer cell dynamics of colorectal cancer progressionhj snippertsa rosenberg

Abstract

IntroductionCalponin 3 (CNN3) is one of the three isoforms in the Calponin family of actin-binding proteins. It is expressed in smooth muscle cells and non-smooth muscle cells and is required for cytoskeletal rearrangement and wound healing. Epithelial to Mesenchymal Transition (EMT) is important step in the development of epithelial cancers during which the cancer cell exploit the preexisting wound healing program. Therefore, it is expected that CNN3 plays a role in EMT. The few available data on the role of CNN3 in cancer shows that CNN3 is expressed in mammary cells in response to ErbB2-overexpression and in Duke’s stage C of colon cancer, but there is no comprehensive analysis of its role in cancer which is the aim of this study.Material and methodsTotal of 21 cell lines were included in this study (8 from breast cancer, 12 from colon cancer, and a cervical cancer cell (HeLa) as a positive control for EMT). CNN3 expression was examined by Western blotting, followed by siRNA silencing in the metastatic cell line SW620. The influence of CNN3 silencing on EMT was detected by EMT markers expression by Western blot and collagen invasion assay. Cell viability was performed after exposure to 5 Fluorouracil (5FU) using Sulforhodamine B (SRB) assay. Furthermore CNN3 expression was examined by immunohistochemistry in 56 formalin-fixed paraffin-embedded (FFPE) colon cancer tissue samples.Results and discussionsCNN3 showed positive expression in 6/8 breast lines and 7/12 colon lines. The primary line SW480 was negative while its metastasis from the same patient (SW620) was positive, suggesting that CNN3 was associated with metastasis. The CNN3 silencing was >98% efficient in the metastatic cell line SW620. Silenced cells were less invasive compared to the control and showed loss of mesenchymal markers and acquisition of epithelial markers. CNN3 partially restored the colon cancer cell sensitivity to 5FU. CNN3 was expressed in 20/56 (39%) of FFPE colon cancer by immunohistochemistry and it was not related to p53 stabilisation consistent with our cell line findings.ConclusionThe results suggested involvement of CNN3 in EMT and hence metastasis and also in resistance to standard chemotherapy in colon cancer. These data deserves further exploration in vivo and in clinical studies to validate the potential clinical applications of CNN3 in cancer treatment and prognosis.